INTERNATIONAL SERIES OF MONOGRAPHS ON PURE AND APPLIED BIOLOGY Division: ZOOLOGY General Editor: G. A. Kerkut Volume 6 ANIMAL HORMONES- A COMPARATIVE SURVEY Part I. Kinetic and Metabolic Hormones Vol. 1. Vol. 2. Vol. 3. Vol. 4. Vol. 5. Vol. 7. Vol. 8. Vol. 9. Vol. 10. Vol. 11. OTHER TITLES IN THE SERIES ON PURE AND APPLIED BIOLOGY ZOOLOGY DIVISION Raven — An Outline of Developmental Physiology Raven — Morphogenesis: The Analysis of Molluscan Development Savory — Instinctive Living Kerkut — Implications of Evolution Tartar — The Biology of Stentor Corliss — The Ciliated Protozoa George — The Brain as a Computer Arthur — Ticks and Disease Raven — Oogenesis Mann — Leeches BIOCHEMISTRY DIVISION Vol. 1. Pitt-Rivers and Tata — The Thyroid Hormones Vol. 2. Bush — The Chromatography of Steroids Vol. 3. Engel — Physical Properties of Steroid Hormones BOTANY DIVISION BoR — Grasses of Burma, Ceylon, India and Pakistan Turrill (Ed.) — Vistas in Botany Schultes — Native Orchids of Trinidad and Tobago Cooke— Cork and the Cork Tree MODERN TRENDS IN PHYSIOLOGICAL SCIENCES DIVISION Florkin — Unity and Diversity in Biochemistry Brachet — The Biochemistry of Development Gerebtzoff — Cholinesterases Brouha — Physiology in Industry Bacq and Alexander — Fundamentals of Radiohiology Florkin (Ed.) — Aspects of the Origin of Life HoLLAENDER (Ed.) — Radiation Protection and Recovery Kayser — The Physiology of Natural Hibernation Fran^on — Progress in Microscopy Charlier — Coronary Vasodilators Gross — Oncogenic Viruses Mercer — Keratin and Keratinization Heath — Organophosphorus Poisons Chantrenne — The Biosynthesis of Proteins Rivera — Cilia, Ciliated Epithelium and Ciliary Activity Enselme — Unsaturated Fatty Acids in Atherosclerosis PLANT PHYSIOLOGY Sutcliffe — Mineral Salts Absorption in Plants Siegel— T/ie Plant Cell Wall Vol. 1. Vol. 2. Vol. 3. Vol. 4. Vol. 1. Vol. 2. Vol. 3. Vol. 4. Vol. 5. Vol. 6. Vol. 7. Vol. 8. Vol. 9. Vol. 10. Vol. 11. Vol. 12. Vol. 13. Vol. 14. Vol. 15. Vol. 16. Vol. 1. Vol. 2. ANIMAL HORMONES A comparative survey Part I— Kinetic and Metabolic Hormones PENELOPE M. JENKIN M.A., D.Sc. Senior Lecturer in Zoology, Bristol University Associate of Newnham College, Cambridge with a foreword by John E. Harris, c.b.e., m.a., Ph.D., f.r.s. Professor of Zoology in the University of Bristol pergamon press NEW YORK • OXFORD • LONDON • PARIS 1962 PERGAMON PRESS INC. 122 East 55th Street, New York 22, N. Y. 1404 New York Avenue N.W., Washington 5, D.C. PERGAMON PRESS LTD. Headington Hill Hall, Oxford 4 & 5 Fitzroy Square, London, W.l PERGAMON PRESS S.A.R.L. 24 Rue des Ecoles, Paris V PERGAMON PRESS G.m.b.H. Kaiserstrasse 75, Frankfurt-am-Main Copyright © 1962 Miss P. M. Jenkin Library of Congress Card Number 60-8977 Set in Imprint lO/Upt and Printed in Great Britain by Cox and Wyman Ltd., London, Reading and Fakenham CONTENTS List of Tables Acknowledgements Preface CHAPTER 1 Introduction 1.1 Discovery of Hormones 1.2 Chemical Activators 1.3 Mechanical Activation 1.4 Definitions of Hormones 1.5 Types of Hormones 1.51 Kinetic hormones 1.52 MetaboHc hormones 1.53 Morphogenetic hormones 1.6 Identification 1.7 References 2 Sources of Kinetic and Metabolic Hormones 2.1 Ectodermal Sources 2.11 Secretory cells derived from the nervous system 2.12 Endocrine glands derived from ectodermal epithelium 2.2 Endodermal Sources in Vertebrata 2.21 Isolated cells in the gut 2.22 Endodermal endocrine glands 2.3 Mesodermal Sources in Vertebrata 2.3 1 Endocrine gland cells derived from coelomic epithelium 2.4 References 3 Kinetic Hormones— I. Control of Muscles and Pigmentary Effectors 3.1 Control of Muscles 3.11 Visceral muscle 3.12 Somatic muscles 3.2 Control of Pigmentary Effectors 3.21 Chromatophores with muscles 3.22 Pigmentary effectors with movable pigment granules 3.3 References PAGE vii ix xiii 1 1 2 4 5 6 9 10 11 12 16 18 18 19 38 44 44 46 50 50 53 56 57 57 69 70 72 73 110 79829 VI CONTENTS CHAPTER PAGE 4 Kinetic Hormones — II. Control of Exocrine and Endocrine Glands 115 4.1 Exocrine Glands 115 4.11 Digestive glands 117 4.12 Oviducal glands 128 4.13 Milk-secreting glands 130 4.14 Skin glands 131 4.2 Endocrine Glands 131 4.21 Ectodermal endocrine glands of Arthropoda 134 4.22 Endodermal endocrine glands of Vertebrata 139 4.23 Mesodermal endocrine glands of Vertebrata 143 4.3 General Considerations 152 4.31 Characteristics of kinetic hormones 152 4.32 Stimulation of the secretion of kinetic hormones 155 4.4 References 163 5 Metabolic Hormones 167 5.1 General Metabolic Rate 168 5.11 Respiration 168 5.12 Fat metabolism 186 5.2 Intermediary Metabolism of Carbohydrates and Proteins 189 5.21 Carbohydrate metabolism 189 5.22 Protein metabolism 199 5.3 Balance of Monovalent Electrolytes and Water 206 5.31 Balance of sodium ions (Na+) and of associated monovalent electrolytes (K+ and Cl~) 209 5.32 Water balance 219 5.4 Balance of Calcium and Phosphates 240 5.41 Balance of calcium 240 5.42 Balance of phosphates 249 5.5 General Considerations 252 5.51 Characteristics of the metabolic hormones 252 5.52 Control of the secretion of metabolic hormones 253 5.53 Hormones and the environment 259 5.6 References 260 Glossary 267 Index of Authors 271 Index of Animal names 277 Index of Subjects 283 LIST OF TABLES TABLE PAGE 1 Summary of the Main Types of Action of Vascular Hormones 8 2 Steps in Establishing Direct and Indirect Actions of Two Interacting Hormones 14 3 Ectodermal Sources of Kinetic and Metabolic Hormones 22 4 Cells in the Pars Distalis of the Adenohypophysis 44 5 Endodermal Sources of Kinetic and Metabolic Hormones in Vertebrata 46 6 Mesodermal Sources of Metabolic Hormones in Vertebrata 51 7 Kinetic Hormones Controlling Muscles 58 8 Kinetic Hormones Controlling Pigmentary Effectors 71 9 Kinetic Hormones Controlling Chromatophores with Movable Pigment 87 10 Crustacean Hormones Controlling White Pigment in Relation to Light 96 11 Crustacean Hormones Controlling Red and Black Pigments in Chromatophores in Relation to Light 98 12 Changes in Melanophore Index in Ligia 100 13 Illumination of Different Areas of the Eyes of Ligia 102 14 Kinetic Hormones Controlling Exocrine Glands in the Gut 118 15 Kinetic Hormones Controlling other Exocrine Glands 129 133 16 Endocrinokinetic Hormones Stimulating Endocrme Glands Vlll LIST OF TABLES TABLE PAGE 17 Means of Controlling the Secretion of Kinetic Hormones 156-157 18 Metabolic Hormones Controlling Respiration and Fats 169 19 Changes in Oxygen Consumption in Astacus^ follow- ing Sinus Gland or Eyestalk Removal 179 20 Changes in Fat Content of the Body of Crabs {Hemi- grapsus nudus) following Starvation and Sinus Gland Removal 188 21 Metabolic Hormones Controlling Carbohydrates and Proteins 190 22 The Effect of Asphyxia on the Concentration of Blood-Sugar 192 23 Changes in Body Composition of Crabs {Hemigrapsus nudus) following Starvation and Sinus Gland Removal 200 24 Hormones Associated with Nitrogen Excretion in Crabs 204 25 Metabolic Hormones Controlling Electrolytes and Water 208 26 Changes in Sodium and Potassium Concentration of Plasma and Muscle, following Adrenalectomy 217 27 Changes in Excretion of Water, Sodium and Potas- sium, following Adrenalectomy 229 28 Metabolic Hormones Controlling the Balance of Calcium and Phosphates 241 29 Effect on Blood Calcium of Injection of Hypophysial Hormones into Xenopus 245 30 Changes in the Calcium Content of the Blood of the Crayfish (Astacus), following Removal of either the Sinus Glands or the Whole Eyestalks 248 31 Means of Controlling the Secretion of Metabolic Hormones 254-255 ACKNOWLEDGEMENTS I ACKNOWLEDGE with gratitude the help and stimulus that I have received from many friends and experts, with whom I have discussed different sections of this book. Among these I would mention particularly Professor B. Hanstrom, who read the whole of chapter 3 ; Professor M. Thomsen and Dr. Ellen Thomsen who discussed the general plan with me ; Professor H. E. Heller who read § 5.3 on electrolyte and water balance; Dr. J. A. Kitching, who read §§ 4.1 and 5 and Sir Francis Knowles and Dr. D. B. CarUsle who gave me much information on crustacean hormones and discussed the nomenclature which has been adopted here and, in part, in their own recent book. Professor J. E. Harris went far beyond the duties of an Editor in encouraging me in every way and not least in his penetrating discussion of fundamental problems of hormone action. Though all these have saved the book from many errors, I must accept sole responsibility for any that remain, as I do for the selection, interpretation and presenta- tion of the material. I am particularly indebted to Sir Francis Knowles for allowing me to reproduce some of his beautiful coloured photographs of chromatophores in Leander and to the publishers of Endeavour for supplying copies of the blocks for these. My warm thanks for permission to reproduce or adapt their figures are also due to Dr. E. Thomsen and Dr. S. P. Carlson, who provided photographs for Figures 2-3 and 3-19; to Dr. W. Junk of the Hague, for the block of Figure 5-6, and to all the other authors and publishers indicated in the legends and references, as well as to the following pubhshers and sponsors of journals: Academic Press Inc. (Figs. 2-10, 4-10, 4-11, 5-10). American Institute of Biological Sciences, Washington (Fig. 3-23). American Physiological Society (Figs. 3-7, 4-2, 4-3, 4-4, 4-5, 5-9, 5-22). X ACKNOWLEDGEMENTS Chas. J. Branford, Co. (Fig. 3-3). Butterworths Scientific Publications (Figs. 5-14, 5-23). Cambridge University Press (Figs. 3-1, 3-5, 3-6, 3-14, 3-16, 3-20, 3-21, 3-22, 3-24, 5-1, 5-2, 5-3, 5-12, 5-13, 5-16, 5-20, 5-24, 5-25, 5-26). Professor I. Chester Jones (Figs. 5-12, 5-13, 5-20, 5-24). Colston Research Society (Figs. 5-14, 5-23). Company of Biologists (Figs. 2-ld, 2-4, 5-1, 5-2). Council of the Marine Biological Association of the United Kingdom (Figs. 3-1, 5-3, 5-26). D. C. Heath & Co. (Fig. 5-8). Koninklijke Vlaamsche Academic voor wetenschappen, Brussels (Fig. 5-11). Marine Biological Laboratory, Wood's Hole, Massachusetts (Figs. 2-lc, 2-5, 2-7, 2-9b, 3-2, 3-12, 3-15, 5-4, 5-7). Masson et Cie. (Figs. 2-le, 2-9a). Oxford University Press (Figs. 2-ld, 2-4). Springer Verlag (Figs. 2-lb, c, g, 2-2, 3-9, 3-10, 3-11, 5-18, 5-19). Stazione Zoologica, Naples (Fig. 2-6). Charles C. Thomas (Figs. 2-14c, 2-15). University of Chicago Press (Figs. 5-5, 5-17). Verlag Birkhauser (Fig. 2-3). Wistar Institute of Anatomy and Biology, Philadelphia (Figs. 3-13, 3-17). I am most grateful for the skill and understanding co-operation of Mr. W. R. B. Buchanan and his staff in redrawing many of these figures and interpreting my sketches; and for the clear photographs of my drawings, taken by Mr. Ken Wood, for Figs. 2-1 {a-f) and 2-14. I also wish to thank Miss J. McKinney for her help in completing the indices and not least Mrs. P. M. Richards for her unstinted help and encouragement in the prepara- tion of the typescript and the main part of the indices, and in correction of the proofs. Finally it is a pleasure to acknowledge my indebtedness for the many facilities aflForded me by the Director and Library staff of the Marine Biological Laboratory, Plymouth, as well as by the staff of the Zoological Department at Bristol. This book is dedicated to my Students^ whoy by their questions^ have stimulated me to write it XI FOREWORD A foreword, like an aperitif, should whet the appetite without dulling the critical appreciation of what is to follow. Many wise people therefore avoid them. Nevertheless, it is a personal pleasure for me to be asked to provide one to this volume, for the initiation of which I was, at least in part, responsible. Specialized scientific publications in these days may be broadly, and thus inaccurately, divided into scientific papers summarizing experiments, reviews summarizing scientific papers, and books summarizing reviews. Among the multitude of these last, the really interesting book is all too rare — one with a broad but scholarly treatment, which stimulates the reader to think about the subject, to produce his own ideas and to design his own experiments. Such a book must provide a sufficiently clear account of the experimental techniques for the student to appreciate the methods of study and their limitations; it must establish a theoretical background which gives coherence to the subject as a whole; finally it must tread sufficiently near to the frontiers of knowledge to provide a glimpse of what may lie beyond. Such a stimulus has already reached several generations of Bristol students through Dr. Jenkin's lectures on hormones; I hope that in its present form her book will successfully challenge a wider audience. John E. Harris PREFACE The idea of writing this book arose from lecturing on hormones to second and third year students of zoology, for whom the subject formed part of a course in comparative physiology. It was found that no introductory book covered the whole subject equally; even Hanstrom's admirable Hormones in Invertebrates (1939) dealt with only a part of the field and was already out of date in 1956, when he assured me that he would not be rewriting it and encouraged me to attempt this general survey. To do so necessitated evolving a scheme within which to consider and select suitable examples from the mass of available material. This resulted in a comparative arrangement, which should be of general application, since it is based on the actions of hormones, rather than on their sources or on their phyletic distribution. The actions of hormones were then seen to fall into three well-defined groups, the kinetic , the metabolic and the morpho- genetic, although these had not all been named nor clearly defined at that time. Subdividing these groups brought together examples acting upon similar effectors, such as muscles, chromatophores or glands, or having similar metabolic actions, such as increasing water excretion, blood-sugar or respiration. Still further sub- division brought together the hormones that stimulate a given action or facilitate a given process and separated them from those having the opposite effects. When consistently adhered to, this approach helped to give a clear picture of hormone actions, to emphasize cases where antagonistic hormones were known and to draw attention to apparent gaps in recorded knowledge. In writing the book, invertebrates and vertebrates were placed side by side to show the extent to which both are now known to have hormones with similar actions. Describing the invertebrate examples before those from vertebrates was a deliberate attempt XIV PREFACE to emphasize this fact. To have given pride of place to the verte- brates might have given a more clear-cut picture, and could certainly have provided more abundant and detailed examples; but it would have thrown the intended comparison out of perspec- tive. The search for good examples among invertebrates proved unexpectedly successful. It has been decided, therefore, to publish the book in two parts instead of one ; but the unified plan of relatively simple presentation is being maintained. The present part of the book covers only the kinetic and metabolic hormones, their sources, actions and the ways in which their secretion is controlled. The second part* will contain a similar treatment of the morphogenetic hormones, namely those concerned with growth, differentiation and reproduction ; it will also discuss such topics as the relation of the chemical constitution of hormones to the sources from which they are derived and their type of action. A consideration of the distribution of hormones in the animal kingdom may also throw some light on the possible evolution of these chemical activators, as well as suggesting prob- lems for investigation. Many of these problems must be apparent to anyone who surveys the field of hormone research; yet it is permissible to assume that few research workers have time to undertake such a survey, as their own work becomes more and more specialized and results in the pubhcation of books that are confined to single classes of animals or single endocrine organs. It is therefore hoped that the present work may be useful to some specialists as well as to the teachers and students for whom it is primarily intended. It has clearly not been possible for the writer to review the whole literature of so rapidly expanding a subject; but the main original papers on the kinetic and metabolic hormones of inverte- brates have been covered up to the summer of 1958, while the vertebrate examples have been checked by recent reviews and reports of symposia. The references at the end of each chapter show the sources used, but make no pretence to being complete, though they should provide a useful starting point for anyone wishing to go further. * Animal Hormoties, a comparative survey. Part \\. Morphogenetic Hormones ^ in preparation. PREFACE XV It is much to be regretted that these references are not more nearly up to date; but publication has been delayed by various unforeseeable causes, including the printing industry's national dispute during 1959 and the writer's serious illness. Finally a word of explanation about some of the things which have not been included in the book. Examples in which extracts of one kind of animal have been tested upon another kind have been avoided, on the grounds that they are apt to lead to unsound physiological deductions. Details of standard techniques are omitted, since the reader can refer to any physiological text- book for an account of such methods as recording muscle con- tractions by means of levers that mark a revolving smoked drum (e.g. Figs. 3-1 and 3-3). The use of commercial hormone prepara- tions and methods of quantitative estimation of hormones by biological assay are also omitted, as being primarily of clinical interest. Since the book is intended for zoologists and comparative physiologists, the mammalian examples have been chosen from species other than man, while reference to pathological and clinical material has been omitted, as being outside their chosen field. Such material is easily accessible elsewhere, and should not be difficult to fit into the present framework, if the reader so desires. Bristol P. M. J. CHAPTER 1 INTRODUCTION 1.1 Discovery of hormones The discovery of hormones v^as a late-comer in the study of physiology ; the circulation of the blood was demonstrated in the seventeenth century by Harvey (1628), but it was more than two centuries before it was realized that chemical messengers could be carried in that circulation. The first hint of this was when Berthold (1849)* showed conclusively that the morphogenetic effects of transplanting the testes of cockerels must be transmitted by some factor in the blood. It was even longer before Oliver and Schiifer (1895) found that a chemical extract of the adrenal medulla, if injected into the circulation, could induce a pronounced rise in blood pressure. In 1901 the active substance in this extract was isolated, identified and called adrenaline. The general term ^'Hormone" is derived from the Greek opfxau), meaning 'T arouse", and indicates the stimulating action of such chemicals ; it was first used by Starling (1905) for secretin, that had been discovered in 1902 and shown to induce the flow of alkali from the pancreas. Two hormones concerned with the cure of human disease, insulin for the control of diabetes mellitus, and thyroxine for cretinism, were among the more spectacular discoveries of the early twentieth century, and led to an intensive search for more hormones in man and other mammals. This resulted in the gradual discovery of some thirty kinds of endocrine cells and glands that can produce minute quantities of chemical substances which are carried in the blood, to stimulate or inhibit various specific effectors, or to control different aspects of metabolism and morphogenesis. * See Harris (1955) for a translated account of his experiments. B 1 2 INTRODUCTION The first indication of any hormone in an invertebrate was that postulated by Kopec (1922) as carrying the brain stimulus for moulting in Lymantria. Then Roller (1927) found a blood-borne factor controlling the colour changes of certain shrimps, and Perkins (1928) located its source in the eyestalk. The discovery of other hormones has followed, mainly in crustaceans and insects, where they have almost as many actions as those carried out by the better-known hormones of vertebrates. 1.2 Chemical activators During this period, when hormones were being discovered in ever-increasing numbers, different kinds of chemical activators were being found in other fields of biology. Substances akin to hormones were found in plants ; nerve transmission in vertebrates and some invertebrates was found in many unrelated species to be due to release of either acetylcholine or adrenaline, at the point of contact between one neuron and the next, or between the motor axon and its effector. The control of the pattern of development in early embryos of Amphibia was found to be due to the diffusion from cell to cell of particular chemical substances or organizers ; these substances were not specific in that they were capable of producing similar effects in a wide range of genera (Spemann and Mangold, 1924). Some order was brought into the variety and diversity of these and other chemical activators by Huxley (1935) in an important scheme of classification. Its main weakness was that it did not include neurosecretory cells derived from nerve cells and capable of yielding hormones. These cells had been recognized histologi- cally in vertebrates by Dahlgren (1914), and in some invertebrates by Hanstrom (1931); but their action in releasing hormone-like substances into the blood was first established by the Scharrers (1937). They are now well known in Annelida, Arthropoda and some other invertebrates as well as in vertebrates. Huxley's (1935) classification of chemical activators may therefore be modified as follows, to include neurosecretion: A. Para-Activators. By-products of normal and pathological metabolism with effects on correlation or differentiation, e.g. carbon dioxide in its effect on the respiratory centre. §1.2 CHEMICAL ACTIVATORS 3 B. True Activators. Chemical substances produced by the organism and exerting specific functions in regard to corre- lation or differentiation: 1. Local activators, with effects on the same cell, or cells, within which they are produced. (a) Intracellular activators ("intracellular hormones" of Goldschmidt), acting in each cell singly and being the direct expression oi gene activity, in relation to regional differentiation. (b) Regional activators, responsible for the chemodijferentia- tion of specific regions in embryos and for growth gradients. 2. Distance activators, with effects on cells other than those in which they are produced. (a) Diffusion activators, distributed by diffusion through the tissues. (i) Direction of transport restricted by structural organization. Growth hormones in plants. (ii) Diffusion restricted to tissues in direct contact. Organizers in embryos and ^'organisines'^ in animals without a circulatory system. It is possible that the cortical releasing factor, CRF, from the brain of vertebrates should also be included here (§ 4.323). (iii) Diffusion restricted mainly by chemical means. Neurohumoral secretions at nerve- and neuro- secretory cell-endings ("neurohormones" of Welsh, 1955). (iv) Diffusion unrestricted, the substances passing out of the tissues and into the surrounding medium to act on other individuals, usually of opposite sex. These include ''gamones'' and ''ectohormones'\ (b) Circulatory activators or vascular hormones, distributed to all parts of the body in the blood circulation, so that their actions must be limited by the sensitivity or com- petence of the tissues which they reach. They may be secreted by: (i) Isolated cells such as those of unknown origin in the gut mucosa of vertebrates (§ 2.21). 4 INTRODUCTION (ii) Neurosecretory cells with the swollen ends of their original axons forming storage-and-release organs ("neurohaemal organs" of Knowles and Carlisle, 1956), that make contact with blood vessels (§ 2.11). (iii) Endocrine gland cellsj which secrete internally into the blood and are formed from almost any tissue of the body, including the nervous system (in which case the distinction from neurosecretory cells is only one of the degree of their histological modi- fication). The chemical activators to be considered as ''animal hormones" in the present book are the circulatory activators, or vascular hormones (2 b). Yet since there is really no logical point at which some of them can be separated from other neurosecretions, or even from the organisines which have actions so much like those of morphogenetic hormones, reference to these will have to be made in the relevant sections, as will chemicals involved in nervous stimulation, where their functions overlap those of hormones. The complex interaction of many of these chemical activators is w^ell illustrated by considering the way in which genes initiate, and hormones complete, the differentiation of the gonad rudiment into a testis or an ovary within an embryo, the development of which starts under the general control of an organizer, continues by progressive chemcdifferentiation, and cannot be completed without the combined action of the nervous system and yet other hormones ! 1.3 Mechanical activation The only other means made use of by animals for co-ordinating their activities is purely mechanical. This can act in the absence of nerves or of any chemical activators, and may well be a primitive way of transmitting control. The action of a current of water stimulating the sponge osculum to remain open appears to be purely mechanical, but it is not certain that some chemical may not be diffusing from cell to cell. The best example is in the locomotion of the earthworm, where the muscles contracting in one segment stretch those in the adjacent segment behind, and §1-4 DEFINITIONS OF HORMONES 5 Stimulate them to contract in their turn to give a wave of con- traction passing back from segment to segment. This is a primitive method of control which may be supposed to have preceded that by the nervous system. A distinct contrast to this is afforded by the use of mechanical distention of the stomach as a means of initiating the secretion of GASTRIN, one of the hormones from the mammalian gut. For in this case mechanical stimulation, like the direct chemical stimulation which acts upon other endocrine cells in the same region, seems to be part of a highly specialized system for harmonizing the succes- sive stages of digestion, and to have succeeded the nervous control that is used for a similar purpose by cold-blooded vertebrates (§4.11). 1.4 Definitions of hormones The simplest and earliest definition of hormones as "chemical messengers" must be amplified, if it is to limit the use of the term "animal hormones" to the circulatory activators. A well-estabhshed definition of a hormone is "a physiologic organic compound produced by certain cells and carried by the blood to distant cells, the activities of which it influences" (Selye, 1947). This is still rather too loose a definition; it accords more nearly with what has been termed a "humoral mechanism", or "a process which has been demonstrated to be independent of nervous connections between the site of stimulation and the effector site, and is, therefore, considered to be transmitted by a blood-borne substance, but in which the hormonal or non- hormonal nature of the blood-borne substance may be uncertain" (Grossman, 1950). This refers particularly to substances like histamine, which may occur in the blood in the abnormal condi- tions of an experiment and yet play no part in normal physiology, and to the so-called "secretagogues". These last are substances that may be found in extracts of gut cells ; they have the capacity to stimulate enzyme secretion by the gut glands but are not natural secretions (§ 4.11). It has already been indicated that a hormone is not necessarily secreted by a gland, nor is its secretion by any means always stimulated by nerves, as some elementary definitions have 6 INTRODUCTION suggested (e.g. Yapp, 1942). A sound definition must be such as to include gastrin, which comes from isolated cells and for which the stimulus to secretion is the direct action of mechanical pressure in the gut lumen, and also insulin, which comes from small groups of gland cells and for which the stimulus to secrete is the level of glucose concentration in the blood. It must also include the hormones which stimulate the secretion of other hormones, like the interstitial-cell-stimulating hormone from the adenohypophysis, which stimulates the secretion of testosterone from the testis, as well as those hormones the action of which is inhibitory rather than activating. Huxley (1935) suggests that a hormone is ''a chemical substance produced by one tissue, with the primary function of exerting a specific efltect of functional value on another tissue"; but, as he admits, this has the teleological implications of any functional account. Moreover, it loses sight of the fact that some chemical substances, such as adrenaline, are present as by-products with no apparent function in many primitive animals, and seem only to have been salvaged for use as hormones in the more highly evolved phyla. It is also well to remember that hormones, or their active constituents, are usually rather stable compounds, able to persist for some time in the blood stream and yet composed of molecules sufficiently small to pass through the walls of blood capillaries and cell membranes to reach their targets. The vascular hormones, with which this book is mainly con- cerned, may best be defined as specific organic substances produced by isolated cells, or by a tissue which may form a gland; they activate or inhibit effects of functional value occurring in other cells or tissues, to which they are carried in the blood. 1.5 Types of hormones Although many actions in a wide range of animals can now be attributed to hormones, it cannot be expected that any functions will belong to hormones alone ; rather must they be recognized as playing but a small part within the complex co-ordination of metabolic and other processes that supply and direct the food and energy as between the multifarious daily activities of the animal §1.5 TYPES OF HORMONES 7 and its growth and reproduction. These processes must be con- trolled to fit the frequent changes both within the animal and in its environment. At different times of the day, or the tide, or the year, the energy must be directed to different purposes, to serve the needs of both individual and race survival. The greater part of this control is achieved by the nervous system ; but hormones may com.e in at almost any point, sometimes independently, but more often in direct or indirect response to nerve stimulation. It is not merely convenient to review the hormones in relation to their actions; it also allows of some interesting comparisons being made between those having similar actions in invertebrates and vertebrates, and reveals a notable degree of correlation between some of their actions and the sources from which the hormones come. It also shows certain striking gaps : some animals lack hormones with particular actions; many invertebrate phyla, or classes, lack hormones altogether. It may be supposed that in many cases the main detectable actions of a hormone represent its physiological functions within the normal animal; but other actions, which are apparent experimentally, may be accidental and without true functional significance. Their actions will be considered under three main headings : (1) Kinetic, or the control of effectors (§ 1.51 and §§ 3 and 4); (2) Metabolic, or the control of cell biochemistry (§§ 1.52 and 5) ; (3) Morphogenetic, or the control of growth and differentiation (§ 1.53, and Part II, to be published separately). Each of these groups can be further subdivided in relation to the particular organs or processes controlled (Table 1). This grouping of hormones is not yet widely used; but it has been found in practice to afford a very good working framework within which to consider the available information, with the minimum of ambiguity or overlap. It has been accepted in a recent presenta- tion of crustacean hormones (Carlisle and Knowles, 1959), together with the terms kinetic and endocrinokinetic suggested to them by the writer (Carlisle and Jenkin, 1959). The term kinetic hormone corresponds to their previous term of ''energetic hormone" (Knowles and Carlisle, 1956). INTRODUCTION Table. 1. Summary of the main types of action of vascular hormones EXAMPLES * VERTEBRATE INVERTEBRATE PART I Kinetic §3.1 Contraction of muscle Adrenaline Corpus car- diacum 3.2 Concentration and dis- persion of pigment W and Bt PLH and PDH 4.1 Secretion of exocrine glands Secretin Gonad 4.2 Secretion of endocrine ACTH Intercerebral glands neurosecre- tory cells PARTI Metabolic §5.1 Control of respiration rate Thyroxine Corpus allatum 5.2 Carbohydrate and pro- tein balance Insulin Sinus gland 5.3 Eloctrolyte and v^ater balance ADH Brain 5.4 Ca and P balance Parathormone Y-organ PART II MORPHOGENETIC §3 General growth "Growth", STH Sinus gland ? Moulting Thyroxine Y-organ Metamorphosis Thyroxine Prothoracic gland Regeneration STH "Organisine" Growth of glands Thyroxine ? (source unknown) §4 Gonad maturation FSH Corpus allatum Gamete release LH Corpus allatum Differentiation of geni- Oestrone or Testis tal ducts Testosterone Development of second- Testosterone Vas deferens ary sexual organs gland 1 * See tables in each section for complete lists of the hormones and their sources referred to in this book. t See glossary. §1.51 KINETIC HORMONES 9 1.51 KINETIC HORMONES The kinetic hormones act upon effector cells or organs, to produce repeatable reactions mainly concerned with feeding, digestion and protective colour change, and so are often related to the short-term interaction of the individual with its environ- ment. The results that they produce are relatively rapid compared with other types of hormone action, but considerably slower and more long-lasting than the nerve action which often controls similar effectors. Their action is also more widespread than that of nerves, since they are distributed by the circulation throughout the body, and cannot, as a rule, be used to cause the contraction of one muscle and not another, or to produce a pattern by con- centrating some chromatophores and dispersing others, unless the effectors themselves are differentiated. Many, but by no means all, of the hormones in this group are neurosecretory substances (2b. ii, p. 4). Most of the others come from ectodermal glands (2b. iii), except the notable group produced from the isolated cells (2b. i) in the mammalian gut. The last-named are stimulated directly (§ 4.11); but the rest are all controlled by the nervous system, apart from a few anomalous hormones which appear to have kinetic actions but to be derived from mesodermal glands (§ 4.324). There is one group of hormones within the kinetic type which calls for special mention at this stage, and for an elucidation of the rather confused nomenclature associated with it. These are the hormones for which the name endocrinokinetic is adopted here. They all stimulate the secretion of other hormones from endocrine glands (2b. iii), and it seems logical to class this action, with that of stimulating exocrine glands, as kinetic. But the situation in which a series of two hormones is involved is more complex than those with but one hormone, and it seems to warrant somewhat different treatment. These endocrinokinetic hormones are frequently desig- nated by the suffix ''trophic" or "tropic", as in thyrotrophic or gonadotropic ; but the suffix is not confined to this type of hormone, since it also occurs in chromatophorotrophic, where the effector is a chromatophore, and not an endocrine gland at all. Even the international decision taken in 1939 that the form trophic should be used in all cases has led to no uniformity of spelling! 10 INTRODUCTION Among vertebrates all the endocrinokinetic hormones are secreted by the adenohypophysis (anterior lobe of the pituitary body) ; but so also are hormones with such morphogenetic actions as stimulating the growth and maturation of the gonads. Yet the term trophic or tropic has been applied indiscriminately to both, as in the case of the gonadotrophins, of which the interstitial- cell-stimulating hormone, ICSH, is endocrinokinetic and causes hormone secretion from the gonads, but the folUcle-stimulating hormone, FSH, is morphogenetic and causes their growth. There are as yet no separate names for the similarly separable actions of the thyrotrophic hormone, TSH, or of the adrenocorticotrophic hormone, ACTH ; but there is a mounting body of evidence to show that the two types of action are often, and perhaps always, due to distinct, albeit closely similar hormones (§ 4.2). If in some cases the two actions are really inseparable, they may perhaps be likened to the motor and trophic actions of one and the same nerve. There are still a few effectors for which no example of kinetic hormone control is known: namely, luminous and electric organs among those usually controlled by nerves, and flagella and nema- tocysts among those for which no internal control is known. 1.52 METABOLIC HORMONES The metabolic hormones are concerned particularly with the control of metabolic activities, at the physico-chemical or bio- chemical level, within the cells of the animal, e.g. with adjustment of respiratory rate (§ 5.1), supply of sugars and proteins to the tissues (§ 5.2), and their electrolyte and water balance (§§ 5.3 and 5.4). Such processes often have a basic rate that seems to be an intrinsic or genetic property of the cells which carry them out. The rates are rarely under nerve control, but hormones may induce changes in them; in many instances, a pair of hormones act together, one increasing the rate and the other decreasing or inhibiting it. This is particularly clear in the control of electrolytes and water in the vertebrate kidney (§ 5.3). Many of the metabolic hormones in Arthropoda are products of neurosecretion, and stimulate the rate of the process in question but are themselves subject to nervous inhibition. Other metabolic hormones, especi- §1.53 MORPHOGENETIC HORMONES H ally in vertebrates, are secreted by endocrine glands and are sub- ject to control by endocrinokinetic hormones. The nervous system takes but a small share in their control, except in emergencies such as haemorrhage or other forms of shock. More often the equili- brium is maintained by a "feed-back" system, whereby the accumulation of some product of the hormone's action inhibits further secretion of the hormone until the accumulation is again reduced. This applies directly to some metabolic hormones, and indirectly to others, through its control of the endocrinokinetic hormones that stimulate them (§ 5.5). 1.53 MORPHOGENETIC HORMONES The morphogenetic hormones produce long-term changes that involve cell division, growth and differentiation ; in contrast to the quick-acting kinetic hormones, their effects can neither be reversed nor repeated, at least for a considerable time. Formerly, these hormones w^ere grouped under a more widely defined "metabolic" heading. There was some justification for this, in that growth is not possible without an adjustment of the metabolic processes to provide the growing cells with the necessary building materials for protein synthesis, and energy supplies in the form of glucose (cf. §§ 5.2 and 5.5). Yet these and other metabolic processes con- tinue throughout the life of the animal, and are not necessarily linked to morphogenesis, which is often intermittent, as is easily seen in moulting and metamorphosis and in the changes associated with seasonal reproduction. Morphogenetic hormones affecting growth and regeneration are present in several phyla, including Annelida and Mollusca, from which no metabolic hormones have so far been reported, and the means of controlling them is, in many cases, still unknown. It is only in Crustacea, Insecta and Vertebrata that nearly all the morphogenetic factors are secreted as vascular hormones and are controlled by endocrinokinetic hormones, which link the processes of growth, differentiation and reproduction indirectly to the nervous system, and thence to seasonal changes in the environ- ment (§ 4.232). Hormonal control of moulting and metamorphosis is now well known in Crustacea and Insecta, in both of which ectodermal 12 INTRODUCTION glands from the antennary or maxillary segments secrete moult- promoting hormones. Their secretion is stimulated by an endocrinokinetic hormone, prothoracotrophin, in Insecta (§ 4.211), and probably by a similar hormone in Crustacea. Otherwise the two classes differ in that, in the latter, moulting is restrained by a moult-inhibiting hormone; this does not occur in Insecta, in which a so-called juvenile hormone from the corpora allata inhibits metamorphosis only. The initiation of metamorphosis in Amphibia by thyroxine, with its dependence on the availability of iodine, was an early discovery; its control by the endocrino- kinetic thyrotrophin, TSH, w^as established later. The differentiation of the genital ducts and other sexual characters has also been found to depend on hormones in a number of invertebrates, as well as in vertebrates, where the pattern of control differs in detail in different classes (§ 4.234 and Part II, § 4). Only a few of the hormones producing these effects are shown in Table 1 ; their detailed treatment is reserved for the second Part of this work. The so-called "organisines", which stimulate regeneration in Platyhelminthes, are not vascular hormones, since in the absence of any circulation in these animals it must be assumed that the substances diffuse through the tissues in a way that is reminiscent of embryonic organizers, as their name suggests (Dubois and Lender, 1956). 1.6 Identification The technique of finding, testing and confirming the presence and action of a hormone is exacting, and needs many controls if the results are to be conclusive. It is difficult if only a single hormone with a relatively clear-cut effect is under investigation; for quite a long series of experiments is needed to elucidate the situation. All too often some steps in the proof are missing, either for technical reasons or because their importance was not fully realized during the early stages of hormone investigation. Histological examination of tissues that are suspected of sec- reting hormones is one side of the investigation, since cells capable of this type of chemical activity often have a recognizable cytologi- cal appearance (Figs. 2-2 and 4-7), with granular precursors of the §1.6 IDENTIFICATION 13 secretion and enlarged nuclei. Once the secreting cells have been located, it may be possible to make extracts of tissue containing them, and to compare this with extracts of adjoining tissue containing no such cells, in order to arrive at more definite results than can be obtained from extracts of whole structures like the crustacean eyestalk (§§3.12 and 3.223). A carefully planned experimental investigation is also essential if the action of the hormone is to be fully established. This usually falls into one of two categories, the pharmacological or the physiological: in the first, it is shown that certain extracts, or chemicals, have effects upon the animal, such as stimulating muscle contraction, or raising the salt output in the urine ; in the second, and usually more difficult category, an attempt is made to prove that the chemical in question is used in the normal physi- ology of the animal to control the same process. In general, it can be said that if only one hormone is concerned, its control of a certain reaction can be sufficiently proved if adequately controlled experiments show that: (i) removal of the source of the hormone is followed by loss of the reaction, (ii) injection of an extract from the source can restore the reaction, (iii) removal of any other structure does not cause loss of the reaction, (iv) injection of any other extract does not restore the reaction. It is better if the reaction is shown by an organ with no nerve connections, and if it can be interrupted by ligation of its blood supply. If the reaction can be restored by injection or transfusion of blood from another individual in which hormone secretion has been stimulated by natural means (cf. Fig. 3-3), the proof that this hormone plays a part in the natural physiology of the animal is more convincing. To complete the identification of any parti- cular hormone, it may be necessary to separate it from others which can be extracted from the same source, and the problem is never finally elucidated until the chemical constitution of the pure hormone is known. If more than one hormone is involved in the control of a reaction. 14 INTRODUCTION Table 2. Steps in establishing direct and indirect actions of TWO interacting hormones experiments on rats RESULT controls on litter mates preferably of SAME SEX RESULT 1. Operative removal of the adrenal cor- tex 2. Op: as 1 + injec- tions of pure cor- tex extract 3. Op: removal of Adhp : (including source of ACTH) leaving cortex in- tact but unstimu- lated 4. Op: as 3 + injec- tion of one frac- tion of Adhp : ex- tract (= ACTH) 5. Op: removal of Adhp: and cortex 6. Op: as 5 + injec- tions of ACTH (as 4) 7. Op: as 5 - cortex extract (as 2) Death Survival* Death \a. Mock operation 2a. Op : as 1 -f other injections t 3a. Mock operation Survival Death Survival Survival* Death Death Survival* 4a. Op: 3 + injec- tions of any other Adhp : ex- tracts 5a. Mock operations of equal severity 6a. Op: as 5 + injec- tion of other ex- tracts 7a. Op: as 5 + injec- tion of other ex- tracts Death Survival Death Death * For as long as injections are maintained. t NaCl injection can mitigate the effect of cortex removal for some time (§ 5.311). Adhp = adenohypophysis. ACTH = adrenocorticotrophin. Argument: Experiments 1 to 4. The result of removing either the cortex or the adenohypophysis is the same (1 and 3). So is the effect of injecting extracts of the gland removed, i.e. replacement of either missing hormone can maintain life (2 and 4). The operation itself and post-operative shock as causes of death are ruled out by survival of controls {\a and 3a), on which operations of comparable severity are performed, but without touching the endocrine organs. Injections of other materials are shown by controls to be ineffective {2a and Aa). By fractionation of the extracts of the adenohypophysis, and by testing §1.6 IDENTIFICATION 15 each separately (4), it is shown that ACTH is the only effective substance. At this point it still remains an open question as to whether the two hormones that have been identified have independent actions, or whether one is direct and the other indirect, as it would be if one were an endocrinokinetic hormone. Experiments 5 to 7. The results of these further experiments and their controls give the minimum of information upon which these questions can be decided. Cortex extract alone is effective in the absence of both sources (7), and is therefore the direct metabolic hormone, whereas ACTH is ineffective (6), unless the cortex tissue is present (4). Since the unstimulated cortex is ineffective (3), the action of ACTH must be to stimulate the cortex to secrete (as in the normal animal, and in experiments \a and 4). Conclusion: ACTH is, therefore, the endocrinokinetic hormone stimulating the secretion of a metabolic hormone from the adrenal cortex. the experimental investigation becomes more complex. The tabulated theoretical scheme for a set of experiments, to show the relation of a metabolic hormone from the adrenal cortex and the adrenocorticotrophic hormone, ACTH, from the adeno- hypophysis (Adhp. Table 2), is given as an indication of the mini- mum number of experiments involved. Taking ''death" or "survival" of the animal as the criterion of the hormone's action is obviously much too crude, and should be replaced by some surer physiological test of the metabolic activi- ties affected, such as the measurement of blood-sugar ; but in that case, the opposing action of insulin has also to be taken into account (§§4.2 and 5.2). For obvious reasons of space it will not be possible in the cases cited below to give full details and results of all the experiments upon which the conclusions are based; but an attempt will be made to give some of the clearer examples in enough detail to indicate how far the technique of the original work was satis- factorily controlled. 16 INTRODUCTION 1.7 References Berthold, a. a. (1849). Transplantation der Hoden. Arch. anat. Physiol. ^ Lpz. 42-46. Carlisle, D. B. and Jenkin, P. M. (1959). Terminology of hormones. Nature, Lond. 183: 336-337. Carlisle, D. B. and Knowles, F. G. W. (1959). Endocrine Co?itrol in Crustaceans. Cambridge: University Press. Dahlgren, U. (1914). The electric motor nerve centers in the skates (Rajidae). Science, 40 : 862-863. Dubois, F. S. and Lender, T. (1956). Correlations humorales dans la regeneration des planaires paludicoles. Attn. Sci. nat. (b) Zool. 18: 223-230. Grossman, M. I. (1950). Gastrointestinal hormones. Physiol. Rev. 30: 33-90. Hanstrom, B. (1931). Neue Untersuchungen uber Sinnesorgane und Nervensystem der Crustaceen. I. Z. Morph. Okol. Tiere, 23: 80-236. Harris, G. W. (1955). Neural Control of the Pituitary Gland. London: Edward Arnold Ltd. Harvey, W. (1628). Exercitatio anatotnica de jnotu cordis et sanguitiis in animalibus. Frankfort: Fitzer. Huxley, J. S. (1935). Chemical regulation and the hormone concept. Biol. Rev. \0: All -AA\. Knowles, F. G. W. and Carlisle, D. B. (1956). Endocrine control in the Crustacea. Biol. Rev. 31: 396-473. Roller, G. (1927). tJber Chromatophorensystem, Farbensinn und Farbwechsel bei Crangon vulgaris. Z. vergl. Physiol. 5: 191-246. Kopec, S. (1922). Studies on the necessity of the brain for the inception of insect metamorphosis. Biol. Bull. Wood's Hole, 42: 323-342. Oliver, G. and Schafer, E. A. (1895). The physiological effects of extracts of the suprarenal capsules. J^. Physiol. 18: 230-276. Perkins, E. B. (1928). Color changes in Crustaceans, especially in Palae- monetes. J. exp. Zool. 50: 71-106. Scharrer, E, and Scharrer, B. (1937). Uber Driisen-Nervenzellen und neurosekretorische Organe bei Wirbellosen und Wirbeltieren. Biol. Rev. 12: 185-216. Selye, H. (1947). Textbook of Endocrinology. Montreal, Canada: Acta Endocrinologica, Universite de Montreal. Spemann, H. and Mangold, H. (1924). Uber Induktion von Embryonal- anlagen durch Implantation artfremder Organisatoren. Arch. mikr. Anat. 100:599-638. Starling, E. H. (1905). The Croonian lectures on the chemical correla- tion of the functions of the body. Lancet, 2: 339-341. § 1.7 REFERENCES 17 Welsh, J. H. (1955). Neurohormones. In The Hormones, edited by G PiNCUS and K. V. Thimann. New York: Academic Press Inc 3- 97-151. ' ' Yapp, W. B. (1942). An introduction to Animal Physiology. Oxford: Clarendon Press. Young, J. Z. (1957). The Life of Mammals. Oxford: Clarendon Press. CHAPTER 2 SOURCES OF KINETIC AND METABOLIC HORMONES Before describing the actions of the various kinetic and metaboUc hormones, an account will be given for reference of the sources from which they are derived and of some of the ways in which they reach the blood stream. The cells in the animal body which are able to secrete hormones into the blood can be conveniently grouped by their embryological origins. Invertebrate examples will be given first. It will then be noted that the sources of those kinetic and metabolic hormones that are so far known from invertebrates all come from the ectoderm (§ 2.1) and that it is only in the vertebrates that the endoderm (§ 2.2) and the mesoderm (§ 2.3) also provide sources for these kinds of hormones. The sources of morphogenetic hormones, which affect growth, differentiation and reproduction, include the gonads of both invertebrates and vertebrates, as well as the ectodermal glands which secrete moulting hormones in the Arthropoda, namely, the Y-organ in Crustacea and the prothoracic glands and their homologues in Insecta. Passing references will be made to some of these morphogenetic hormones in the chapters that follow, but their main actions and details of their sources will be described in Part II. 2.1 Ectodermal sources The hormone-secreting, or endocrine, cells which are formed from the embryonic ectoderm can be divided into those which arise from the nervous system (§2.11) and those which arise from non-nervous epithelium (§ 2.12); but the distinction may be rather arbitrary, since the stomodaeal epithelium of the cephalo- 18 §2.11 SECRETORY CELLS FROM THE NERVOUS SYSTEM 19 pods gives rise to non-nervous cells, whereas that of insects gives nervous cells. 2.11 SECRETORY CELLS DERIVED FROM THE NERVOUS SYSTEM A large number of hormones in many phyla are now known to be secreted by nerve cells or their derivatives (Fig. 2-1). Some of these have become so specialized for secretion that they have lost almost all histological semblance of neurons, and their connection with them is only apparent in their development or in the quality of their secretion. Of such are the cells of the corpus cardiacum of insects and the adrenal medulla of vertebrates (Fig. 2-1^ and/). On the other hand, many secretory cells derived from neurons come within the histological definition of neurosecretory cells ; these have only recently been recognized as sources of hormones because they are less easy to discern than the compactly aggregated endo- crine glands, formed by most other cells of internal secretion. They differ from neurons in secreting microscopically visible quantities of granules or droplets, while retaining such characters as Nissl bodies in the cytoplasm and axons with neurofibrillae ; they may or may not have dendrites (Fig. 2-16, c and d). There seems to be no real need to separate neurosecretory cells, which secrete hormones into the blood, from any other endocrine cells derived from either the nervous system or any other part of the ectoderm, for their secretory activity is similar, although their histological form is different. Since, however, much attention has been focused recently upon neurosecretion, it will be well to summarize some of the main points about it. Neurosecretory cells have been identified in animals represent- ing most of the phyla with centralized nervous systems; but as yet their production of vascular hormones has only been demon- strated in a relatively small proportion of these phyla. In some of these phyla, and notably in Annelida, they are only known to yield morphogenetic hormones (Part II); but in the MoUusca, Arthropoda and Vertebrata neurosecretory cells are known to secrete vascular hormones with kinetic and metabolic actions. Neurosecretory cells can usually be recognized within the ner- vous system by their large size (often 30 [i or more in diameter), 20 SOURCES OF KINETIC AND METABOLIC HORMONES with large nuclei and secretory granules in the cytoplasm ; but the latter may vary with the phase of the secretory cycle. This will affect both the microscopic appearance of the cells (Fig. 2-2) and their reaction to histochemical tests which can be applied to the secretion. Some of these cells appear to release their secretion, or neurohormone, where it can only diffuse through the closely adjacent tissue without reaching the circulation. It is then difhcult to distinguish the action experimentally from that of a normal motor nerve, especially as the release of secretion is probably accompanied by electrical changes in the axon similar to those accompanying the nerve impulse. The distinction between such cells and ordinary neurons seems only to be one of degree, for it depends upon the presence or absence of ''granules". This in turn depends rather arbitrarily upon the limits of resolution of the ordinary light microscope. Since the abundant fine granules of adrenaline, which stain brown with chromates, can be readily seen with the light microscope in cells of the adrenal medulla, there seems little reason to doubt that the minute quantities of the same substance, secreted at sympathetic nerve endings, could also be seen by using the greater magnification that can now be achieved by the electron microscope. Yet these nerves are not usually con- sidered to be neurosecretory. Other neurosecretory cells have simple axon endings that discharge their secretion into blood vessels, thereby clearly acting as a source of a vascular hormone. The secretion is formed as granules or droplets either in the cytoplasm immediately surrounding the nucleus, or within the nucleus itself. Thence the granules have been seen to move slowly along the axon and are probably carried in the axoplasm current, which flows at a rate of about 3 mm per day (the movement of endoneural fluid is about 20 times as fast). They accumulate at the unbranched ends of the axons, which become swollen and are often aggregated together to form a storage-and-release organ at the point where the hormone is passed into the blood. Such structures have been called neurohaemal organs (Carlisle and Knowles, 1953). The secretion can sometimes be detected for a short distance even after its discharge into the blood vessel. It is probable, however, that the visible secretion often acts as a "carrier", to which is attached the chemical substance that acts §2.111 SECRETORY CELLS FROM THE NERVOUS SYSTEM 21 as the hormone. The carrier may be some large molecule, like a protein, which helps to anchor the smaller hormone molecule in the cell until the time for its release. The hormone is then separ- ated from the carrier, and apparently becomes free to enter the blood and be passed on to the tissues. The carrier is usually visible in the living cells by dark-ground illumination because of its highly refractile granules which show up as bright spots (Fig. 2-3) ; in fixed preparations the carrier often stains, in a character- istic but not specific way, with Gomori's chrome haematoxylin phloxin and other stains, such as Mallory's triple stain for connec- tive tissue (Scharrer and Scharrer, 1954a). There is increasing evidence that neurosecretory cells not only secrete a greater quantity of some active chemical substance than do typical nerve cells, but that they may also be specialized to produce a greater variety of substances than just the acetylcholine or adrenaline and noradrenaline of nerve endings. Recently, five distinct staining reactions have been found among the neuro- secretory cells terminating in the sinus gland of a crab (§2.112; Potter, 1954), and it seems likely that eventually these will be found to be related to separate hormones. The occurrence of neurosecretory cells, which release hormones that have either kinetic or metabolic actions, is given with the other sources in Table 3. The last column shows the later sections of the book in which examples of these actions are des- cribed. A more detailed summary of the occurrence of neuro- secretion in invertebrates can be found elsewhere (Gabe, 1954). 2.111 Epistellar body of Cephalopoda In most octopods there is a small compact body on the outer surface of the stellate ganglion in the mantle cavity. In Eledone moschata it is yellow and about the size of a pin's head. Micro- scopic examination shows this epistellar body to contain a group of neurosecretory cells (Fig. 2-\d) with their axons converging on a central cavity, which contains secreted granules in a homo- geneous ground substance. The granules presumably release a hormone into the adjacent artery; but its ability to stimulate muscle tone in the mantle (§ 3.12) has only been postulated from extirpation experiments 22 SOURCES OF KINETIC AND METABOLIC HORMONES Table 3 . Ectodermal sources of kinetic and metabolic hormones SOURCE OF HORMONE store of hormone type of section (or name) action* no. 2.11 Cells from the nervous system 2.111 Epistellar body of Cephalopoda Stellate ganglion lEpistellar body K 3.12 2.112 Neurosecretory systems of Crustacea Ganglionic-X-organ Sinus gland K 3.222 and brain ,, >> >) K 3.223 >> >' M 5.112 )> >> M 5.122 >> )> M 5.211 j> >> M 5.321 5> >> M 5.422 Hanstrom's sensory EK 4.211 pore organ Commissures p K 3.223 Pericardial organs ? K 3.111 Eyestalk tip ? M 5.223 2.113 Neurosecretory systems and glands of Insecta Protocerebrum Corpora cardiaca EK 4.211 ? M 5.321 Suboesophageal — K 3.221 ganglion >) >' — M 5.112 Corpora cardiaca — K 3.111 2.114 Neurosecretory systems and glands of Verteb rata Paraventricular and Neurohypophysis K 3.114 supra-optic nuclei n >> M 5.312 of hypothalamus >> )) >> >> M 5.322 Supra-renal tissue (Adrenaline) K 3.112 Adrenal medulla (Adrenaline) K 3.112 )> jj ,, K 3.116 2.12 Glands from ectodermal epithelium 2.121 Salivary glands of Cephalopoda Salivary glands l(Tyramine) K 3.21 2.122 Corpora allata of Insecta Corpora allata | — M 5.111 2.123 Adenohypophysis of Vertehrata Pars distalis (TSH)t EK 4.221 >> >> (STH) EK 4.223 >> >> (ACTH) EK 4.231 )) J) (ICSH) EK 4.232 " . " (LSH) EK 4.232 Pars intermedia (B or MSH) K 3.223 Pars tuberalis (W) K 3.223 * K = kinetic, t See glossary. EK = endocrinokinetic. M == metabolic. Fig. 2-1 (a-/) ( For legend see over) (g) Fig. 2-1 (5^) Fig. 2-1 . Cells derived from the nervous system, (a) Typical motor nerve cell with branched dendrites (de), cell body with Nissl bodies (n.b.) in cytoplasm, nucleus (nu) w4th nucleolus, and long axon (ax) branching to motor end-plates (mot.e.p.) on muscle fibres (m). (b — d) Neurosecretory cells with stainable granules (gr) : (b) with dendrites, from supraoptic nucleus of dog (after Scharrer and Scharrer, 1954ft); (c) wdthout dendrites, the blunt axon is swollen with secretory granules that pass to a blood vessel (b.v.) from gang- lionic-X-organ of crab, Sesarma (after Enami, 1951). {d) cell with shorter axon, from epistellar body of Eledone (after Young, 1936). All drawn roughly to upper scale, ie) and (/) Gland cells with secre- tory granules (s.g.) but no histological characters of neurons (drawn to lower scale) : {e) cells from corpus cardiacum of beetle, Hydrous (cf. Fig. 2-9 after de Lerma, 1956); (/) cells round blood space (b.v.) from adrenal medulla of a tetrapod, with "chromaffin" gran- ules (e.g.) (after Maximow and Bloom, 1942). The differences in quantity of secretion are not characteristic of these cells, but indicate different phases of secretion (cf. Fig. 2-2). {g) Electron micrograph of a highly enlarged section across the axon of a neurosecretory cell from the neurohypophysis of a cat, showing fine granules (Gr) 0.1 to 0.3 [x in diameter, and mitochondria (Mit) that are larger (from Bargmann, 1958). _x* Fig. 2-2. Neurosecretory cells, with axons cut short, from the suboesophageal ganglion of the cockroach, Leucophaea maderae. The differences probably correspond to phases in a secretory cycle : (A) laying down fine granules in the cell-periphery in the position of Nissl bodies at the onset of secretion ; (B) abundant larger granules passing into the swollen axon above; (C) empty vacuoles replacing secretory droplets; (D) an almost empty cell body, with only a few granules left at the base of the axon. The cycle then probably starts again at (A) ; but the sequence has not yet been proved. Cells, X 320, fixed in Zenker-formol and stained in Masson (from Scharrer and Scharrer, 19546). Fig. 2-3. Living median neurosecretory cells in the protocere- brum of the blowfly, Calliphora, photographed by dark-ground illumination. The secretory granules in the cells are highly refrac- tile and therefore show as bright areas filling the cell bodies ; only the nuclei remain dark. Other brain cells without granules can be seen faintly outlined in the background (from E. Thomsen, 1954). col.. Fig. 2-14. Endocrine cells from the endoderm and the mesoderm of mammals, (a) Follicle of the thyroid gland, enclosing colloidal store (col.) of secreted diiodotyrosine ; this precursor substance is later reabsorbed by the cells, converted to thyroxine and passed to the blood vessel (b.v.) through the outer cell surface (cf. Figs. 4-7 and 4-8). (b) Three kinds of cells in an islet of Langerhans stained with Mallory-azan : a cells that secrete glucagon and have coarse granules that stain red; pale j8 cells that secrete insulin and have fine granules that stain red ; D cells that have no known function and stain blue; b.v., blood vessel, c, capsule of con- nective tissue, (c) Mesodermal cells, forming part of adrenal cor- tex; they are shown in close contact with each other, and with capillaries of the blood supply (cf. Fig. 2-15). The vacuolated cytoplasm (v.c.) is shown after removal of all fat, which is abundant in living cortical cells (after Maximow and Bloom, 1942 and Pauly. 1957). § 2.112 SECRETORY CELLS FROM THE NERVOUS SYSTEM 23 The epistellar body is of interest, not only because the term neurosecretory was first used in this country to describe its cells, but also because it provided some of the earliest evidence for the conversion of neurons into secreting cells in an invertebrate (Young, 1936). The corresponding nerve cells in the stellate gangha of decapod cephalopods still retain the form of neurons, but have their axons fused to form giant fibres to the mantle muscles. They are scattered in Sepia, but collected together in the same position as the epistellar body in Loligo (Fig. 2-4). These neurons, as well as the neurosecretory cells of the epistellar body, are innervated by axons coming from the pedal ganglion of the brain. Fig. 2-4. The stellate ganglia of Sepia (A) and Loligo (B), and the epistellar body of Eledone (C). In (A) the giant fibres (g.f.) and the stellate nerve (st.n.) arise from nerve cells that are scattered throughout the ganglion; in (B) they are collected into a lobe (g.f.l.). In the octopus (C) there are no giant fibres, but instead there are neurosecretory cells (n.s.) whose axons end blindly in the central space of the epistellar body (ep.); their secretion passes in the blood to the mantle muscles. A nerve (n.) to the epistellar body replaces the preganglionic fibres (p.g.f.) to the nerves in (A) and (B), and presumably controls the release of secretion in (C). (From Young, 1936). 2.112 Neurosecretory systems of Crustacea There are four neurosecretory systems in crustaceans. Two of these have their nucleated cell bodies in the brain and in the^optic 24 SOURCES OF KINETIC AND METABOLIC HORMONES lobes; the ends of their secreting axons are aggregated into distinct storage-and-release organs, known as the sinus gland and Hanstrom's sensory pore organ respectively. A third group of neurosecretory cell bodies has been found in (a) Fig. 2-5. Neurosecretory cells (n.c.) in decapod Crustacea, (a) Brain, connectives and fused ventral ganglia of a crab, Gecarcinus, and (6) eyestalk of Gecarcinus, cut open dorsally to expose the § 2.112 SECRETORY CELLS FROM THE NERVOUS SYSTEM 25 the commissures and connectives arising from the brain, and extracts showing hormonal activity have been obtained from them (§ 3.223); but the natural point of release for their secretions into the blood stream is uncertain. It may be the post-commissure organs (Knowles, 1953). A fourth system has been found in the pericardial organs of various decapod crabs and of Stomatopoda; these also yield an active extract (§ 3.11), but a natural secretion has not been fully established. Brain, ganglionic- X-organ and sinus gland Some of the neurosecretory cells, that release their secretion in the sinus gland, have their cell bodies in the protocerebrum of the brain and others in its extension into the terminal medulla of the optic lobe. The details vary for different orders and species. For instance, in the crab, Gecarcinus, the cells extend from the protocerebrum into the deutero- and tritocerebral lobes of the brain (Fig. 2-5^-^; Bliss and Welsh, 1952). The relatively large group of neurosecretory cells (Fig. 2-7) that lies in the terminal medulla is best known as the ganglionic- X-organ (pars ganglionaris X organi of CarHsle, 1953). It is important to remember the positions of these neuro- secretory cells in experimental work. Removal of the whole eyestalk removes the ganglionic-X-organ but leaves the cell bodies outer part of the optic stalk and nerves to the retina (R) (after Bliss and Welsh, 1952); (c) post-commissure organ (Post, com.) of a prawn, Leander, attached to tritocerebral commissure and having neurosecretory granules, (after Knowles, 1955). Protocere- brum (PRO), carrying optic lobes (OP), and deuterocerebrum (DEU), both joined by internal commissures, not shown; tritocere- brum (TRI) with its commissure (Tr. com.) behind oesophagus (Oes); circumoesophageal connectives (Conn.) join brain longi- tudinally to fused suboesophageal (SUBOES) and thoracic ganglia (THOR). Eyestalk contains continuation of optic lobe ending in terminal medulla (MT) with ganglionic-X-organ (GXO), and neurosecretory axons that end in sinus gland (SG) on internal medulla (MI); the external medulla (ME) also has some neuro- secretory axons. Nerve roots to antennae (A i and A ii), viscera (Vis), mandible (Mdb), maxillae (Mx i and Mx ii) and thoracic appendages (Th i, etc.). 26 SOURCES OF KINETIC AND METABOLIC HORMONES in the brain still in place; removal of the sinus gland only (by means of a minute punch, like an apple corer; Kleinholz, 1947) leaves both sources undamaged and able to continue their secretion. Dorsa SP HSPO ON SN X-SP Fig. 2-6. Eyestalk of prawn, Lysmata, cut open in the vertical plane (cf. Fig. 2-5). Some neurosecretory cells in the ganglionic- X-organ (GXO) in the terminal medulla (MT) have axons which pass in a bundle (X-SG) to the sinus gland (SO) on the dorsal surface of the external medulla (ME) ; others have axons (X-SP) that pass ventrally to hanstrom's sensory pore organ (HSPO) and end in "onion bodies" (ON), Both sinus gland and sensory pore organ also have axons (B-SG and B-SP respectively) from neurosecretory cells in the brain. The sensory pore retains some sense cells (SP) connected by a sensory nerve (SN) to the brain (from Carlisle, 1953). In the Malacostraca with long eyestalks, the sinus gland is usually on the dorsal surface of the optic ganglion, either on the internal or external medulla (Figs. 2-Sb and 2-6). It is largely com- posed of an aggregate of swollen ends of the long neurosecretory cell axons from the brain and the ganglionic-X-organ ; they con- § 2.112 SECRETORY CELLS FROM THE NERVOUS SYSTEM 27 verge on a half open cavity in communication v^ith the adjacent blood sinus. In the blue crab, Callinectes, there is evidence that the sinus gland is composed of as many as five groups of axon endings, each with a distinctive staining reaction, which can be traced back along the axon (Potter, 1954). It seems highly prob- able that these groups are responsible for secreting and releasing most of the specific chemicals with different hormonal actions, which can be found by experiment in the sinus gland. NPMT' Fig. 2-7. Neurosecretory cells (NSC) in the ganglionic-X-organ of a crab, Sesarma. Stained granules of secretion (G) pass down the axons (SGN) to the sinus gland. Some cells (CB) have few granules, others (RC) are almost depleted of granules. Small ganglion cells and nerve fibres (NPMT) of the surroundmg ter- minal medulla are also shown (from Enami, l^.-^l). 28 SOURCES OF KINETIC AND METABOLIC HORMONES It has been suggested that, in addition to acting as a storage- and-release organ for the neurosecretion, the walls of the sinus gland may include some secretory cells, but of this there is no clear evidence (Knowles and Carlisle, 1956). It seems more likely that active hormones are being set free here from the inactive carrier substance that travels down the axons. In the sessile-eyed Malacostraca, such as the Isopoda, both the ganglionic-X-organ and the sinus gland itself lie within the head capsule (Amar, 1948). Hanstrom^s sensory pore organ The sensory pore organ, like the sinus gland, is the storage-and- release organ for neurosecretory cells some of which are situated within the brain and others in the ganglionic-X-organ. All the axons have their endings swollen into characteristic onion- shaped bodies in Hanstrom's sensory pore organ, situated on the ventral surface of the eyestalk in Malacostraca (Fig. 2-6). In addition to these axon endings and to the sensory cells, which give their name to the organ, there are some small secretory cells confined within the organ, at least in Lysmata (Carlisle and Passano, 1953). This structure was originally called the "X-organ" and was identified in several species by Hanstrom (1939) and his pupils; but since then, many workers have used the name X-organ for the neurosecretory cell bodies located in the terminal medulla. The modified name of ganglionic-X-organ is here used for this latter group of cells, from which in fact axons run to both the sinus gland and to Hanstrom's organ (Knowles and Carlisle, 1956). Commissures and connectives Active extracts have been obtained from many other parts of the crustacean nervous system besides the supraoesophageal "brain"; but as yet there is little indication of where any natural hormones, corresponding in their actions to these extracts, may be released into the blood stream. The tritocerebral (or antennal) commissure, which is a cross connection passing below the oesophagus between the tritocerebral parts of the brain (Fig. 2-5«), is particularly rich in chromactivat- § 2.113 SECRETORY CELLS FROM THE NERVOUS SYSTEM 29 ing materials (§ 3.2) in many Decapoda. In the prawns, Leander and Pefiaeus, it has been shown (Knowles, 1953 and 1954) that the attached post-commissure organs are the swollen bases of nerves, some motor fibres of which pass to dorsoventral muscles ; the two nerves have a cross connection and contain many neurosecretory fibres and secretory droplets and yield an active extract. Some of the cell bodies are in the commissure (Fig. 2-Sc). The circumoesophageal connectives and the thoracic and abdominal ganglia also yield extracts of varying activity, particu- larly in forms such as Palaemonetes, in which the ganglia are not fused in one mass. Pericardial organs In Decapoda and Stomatopoda there are some rather unusual neurosecretory axons in the pericardium. They are supported by larger nerve trunks and end blindly in networks of very fine branches spread over the venous openings from the gills; they are therefore exposed to the blood stream and appear to secrete into it one or more chemicals that increase the rate of the heart beat (§3.111). The position of the cell bodies of these pericardial organs has not yet been found ; but recent evidence indicates the presence of very fine granules in the fibrils, and experimental evidence for their secretory activity is good. Similar structures may also be present in Isopoda (Alexandrowicz, 1953). 2.113 Neurosecretory systems and glands of Insecta The nervous system of Insecta has given rise to both neuro- secretory cells and simple secretory cells which have lost any morphological signs of their nervous origin. The former occur within the central nervous system, mainly in the suboesophageal ganglion and the brain, and the latter in the corpora cardiaca. The tritocerebral commissures and the circumoesophageal connectives, though similar in form to those of crustaceans, have not been shown to yield active extracts in insects. Suboesophageal ganglion Neurosecretory cells have been identified microscopically in the suboesophageal ganglia of several insects, including some 30 SOURCES OF KINETIC AND METABOLIC HORMONES Ephemeroptera (Arvy and Gabe, 1953) and Plecoptera, where their axons are connected anatomically to the ventral gland (Fig. 2-8). Experiments have shown that in higher insects the suboesophageal ganglion is the source of a kinetic chromactivating HEAD Ocelli PRO- PROTHOR An.ii DEU' Fr. gang — Aorta Mouth Lbr Mdb Mx i Mx ii Vent, gl Leg I Fig. 2-8. Central nervous system and sources of hormones in the head and prothorax of a hemimetabolous insect, in lateral view. The nervous system, with optic lobes (OP) cut off, resembles that of the crab (Fig. 2-5) and is similarly lettered and named, except that Mx ii is here the labium, and ganglia of the ventral chain are § 2.113 SECRETORY CELLS FROM THE NERVOUS SYSTEM 31 hormone (§ 3.221) and of the metabolic diapause hormone (§ 5.112). In the cockroach, Leiicophaea, these cells present a variety of appearances in fixed preparations (Fig. 2-2). These probably represent phases in secretion ; but this has not been confirmed in the living animal. Neurosecretory cells of the brain Paired groups of neurosecretory cells are to be found in the brains of insects, as in crustaceans ; but in most insects they appear to be confined to the protocerebrum and not to extend into other parts of the brain or optic lobes. The neurosecretory cells usually lie in groups: the median neurosecretory cells (m.n.c, Fig. 2-3) lie anteriorly near the mid-line and other cells lie ventrally or laterally (l.n.c). The former are connected to the corpus cardi- acum of the opposite side by an internal nerve, and the latter by an external nerve (Fig. 2-8). Their axons lead into the corpora cardiaca, where their secretion is stored (Fig. 2-9). The lateral neurosecretory cells may represent the frontal organs of Aptery- gota and may even be homologous with the cells of Planstrom's sensory pore organ (§ 2.112). It is interesting to note that these groups of neurosecretory cells secrete endocrinokinetic hormones that stimulate endocrine glands in both Crustacea and Insecta (§ 4.21 ; Scharrer and Scharrer, 1954 b). not fused. The stomatogastric, or visceral, nervous system arises from the stomodaeal ectoderm, and has paired nerves from the tritocerebrum, a median frontal ganglion (Fr. gang), a branch to the labrum (Lbr) and a recurrent nerve to the hypocerebral ganglion (Hy) and the paired ventricular ganglia on the gut (near Oes). Hormones are secreted by median (m.n.c), lateral (l.n.c.) and suboesophageal neurosecretory cells (s.n.c); perhaps also by cells in paired corpora pedunculata (C.ped.). Secretions from these pass in two paired nerves to be stored in the corpora cardiaca (CC), which arise from stomodaeal ectoderm (dashed arrow). Hormones are also secreted by two paired glands that arise as ectodermal invaginations: the corpora allata (CA from Mx i), which migrate (dashed arrow) above the oesophagus, where they receive axons from the corpus cardiacum (cf. Fig. 2-9); and the VENTRAL GLANDS (Vent. gl. from Mx ii), which persist in primi- tive insects, but form prothoracic glands (Proth. gl.) in most other orders. (Based on two diagrams by Weber, 1949). 32 SOURCES OF KINETIC AND METABOLIC HORMONES Storage of the neurosecretion from the brain in the corpora cardiaca has been conclusively shown in the cockroach, Leuco- phaea, where the system is paired (B. Scharrer, 1952). Unilateral section of the axons from the median neurosecretory cells results ax.b (a) Corpus cardiacum (b) Corpus allatum Fig. 2-9. Sections of parts of {a) the corpus cardiacum of a beetle, Hydrous piceus (after de Lerma, 1956), and (b) the corpus allatum of a grasshopper, Melanoplus differ entialis (after Mendes, 1948). Axons (ax.b) from neurosecretory cells in the brain carry granules ; some end in swellings (s.ax), like Herring bodies, and release masses of neurosecretion (ns) in the corpus cardiacum. Cells in different phases of secretion (sc and sc') release masses of granules (mg) that stain with phloxin, and may be the intrinsic secretion of the organ. Tracheoles (t) and non-secreting nerve cells (en) also occur. Other axons (ax.c) pass to the cells of the corpus allatum, where their granules disappear. It has undifferentiated cells (uc), secreting (sc") and giant secreting cells with polyploid nuclei (gsc) ; acidophil granules pass with some fluid into intracellular vacuoles and are then extruded (v). in the depletion of all stainable secretion from the corpus cardia- cum of that side. At the same time, increased quantities of the secretion appear in the axons proximal to the cut (Fig. 3-2). The corpora cardiaca are nearly always fused with the dorsal blood vessel or aorta, into which they presumably pass the stored secre- tion as required (Hanstrom, 1940). § 2.114 SECRETORY CELLS FROM THE NERVOUS SYSTEM 33 Gland cells of the corpora cardiaca The CORPORA CARDIACA are not only storage organs for neuro- secretion from the cerebrum but they are also endocrine organs in their own right. Together with the corpora allata (§ 2.122) they lie dorsal to the oesophagus and form the retrocerebral system (Fig. 2-8), the form of which varies in different insects; both organs may be paired, or one or both may be fused in the mid-line. Together with some nerve cells, connective tissue and tracheae, loosely packed secretory cells form the bulk of the corpora car- diaca and produce the intrinsic secretion of this organ (§ 3.111). Like the sympathetic cells of the hypocerebral ganglion, these cells arise from the stomodaeal ectoderm and are undoubtedly nervous in origin, although they have become so much specialized for secretion that they have lost most of the characters of neurons, notably the axons of typical neurosecretory cells (Figs. 2-\e and 2-9a). These cells are rich in ribonucleic acid and reveal a Golgi apparatus after suitable treatment. With haematoxylin chrome phloxin, their secretion stains quite distinctively from the neuro- secretion, the former having a greater affinity for phloxin and the latter for haematoxylin (de Lerma, 1956). 2.114 Neurosecretory systems and glands of Vertebrata There are two main sources of hormones that are derived from the vertebrate nervous system. One is a set of neurosecretory cells in the hypothalamus of the brain with their storage-and-release organ (akin to the sinus gland of the Crustacea) in the neuro- hypophysis. The other is an aggregation of gland cells that are derived from sympathetic ganglia and form the suprarenal body of fish and the adrenal medulla of tetrapods. Neurosecretory cells of the hypothalamus and the neurohypophysis It is now well established that the hormones of the neuro- hypophysis (or posterior lobe of the pituitary body) are not secreted within that body but by neurosecretory cells in the hypothalamus of the brain. The cell bodies are grouped together in the preoptic nucleus in fish and amphibians and separated into two groups, the supraoptic and paraventricular nuclei, in reptiles, birds and 34 SOURCES OF KINETIC AND METABOLIC HORMONES .Nucleus porQventricuiaris Tractus suproopticohypophyseus Optic chiosmo Accumulation of neurosecretory material Plane of stalk section. Fig. 2-10. Diagram of the pituitary body of a dog, Cams; (a) before and (b) after the pituitary stalk has been cut. Neuro- secretory cells in the paraventricular and supraoptic nuclei of the hypothalamus of the brain pass secretory granules down their axons to the neural lobe of the neurohypophysis (pars nervosa), where they are stored in Herring bodies, or swollen axon endings, and thence pass into the surrounding blood vessels (Fig. 2-12). After cutting across the stalk, the neurosecretion accumulates in the proximal part of the axons and the supply previously accumulated in the neurohypophysis becomes depleted after a time and is not replenished. The pars distalis of the adenohypophysis (and pars tuberalis) lie in front of the pars nervosa, separated from it by the pars intermedia (from Scharrer and Scharrer, 1954a). § 2.114 SECRETORY CELLS FROM THE NERVOUS SYSTEM 35 mammals (Fig. 2-10; Scharrer and Scharrer, 1954a). Unlike most neurosecretory cells of the arthropods, those of the hypothalamus of vertebrates possess dendrites (Fig. 2-1^). In fish and aquatic Urodela their axons are not well developed and their function is uncertain ; but in all terrestrial vertebrates from the terrestrial Urodela and Anura upwards the axons pass down the infundibular stalk to end in an enlarged neural lobe (pars nervosa). This becomes distinct from the median eminence at the base of the infundibulum and is not present in the lower forms (Fig. 2-11). In the course of evolution, the neural lobe has acquired an independent blood supply from the internal carotid arteries, forming a relatively rich vascular network (Fig. 2-12) with which the axons of the neurosecretory cells make contact by their swollen endings, called Herring bodies, similar in appearance to those of the comparable cells in the crustacean sinus gland. They can be shown by appropriate staining to be filled with neuro- secretory granules. In mammals the secretory granules first become visible in the embryo, where they appear to be carriers for the actual hormones released from the neurohypophysis. The relation between secretion and hormone formation has now been as clearly shown here as anywhere, although the proofs were obtained later than in the invertebrates. In any vertebrate from fish to mammals, section of the axons in the infundibular stalk results in accumulation of the secretion in the parts proximal to the cut and its depletion beyond ; and the possibility of obtaining active extracts from the different parts of the system follows the same pattern. It has also been possible to grow^ cells from the supraoptic nucleus of a dog in tissue culture and to observe (and even to make a cine film of) the secretory granules passing from the cell body down the axon. The neurohypophysis therefore acts as a storage-and-releasc organ for the hypothalamic secretion or secretions. Two distinct hormones have been recognized, the antidiuretic hormone and oxytocin, but there does not seem to be any constant arrangement of the neurosecretory cells which produce them. In the dog, for instance, both substances can be obtained from both supraoptic and paraventricular nuclei, although the proportion of oxytocin to the antidiuretic fraction is always small; but in the camel, oxytocin 36 SOURCES OF KINETIC AND METABOLIC HORMONES |^>;vj Primary capillary net ^^ Secondary capillary net Vascular plexus Portal vessels Fig. 2-11. Pituitary body in diagrammatic sagittal section with its circulation shown by thin arrows : a to d in fish, c to ^ in amphi- bians and reptiles. A.H., adenohypophysis, divided into pars distalis (P.D.), pars tuberalis (P.T.), and pars intermedia (P.I.) in tetrapods; N.H., neurohypophysis, divided into median eminence (M.E.) and neural lobe (N.L.) with separate circulation § 2.114 SECRETORY CELLS FROM THE NERVOUS SYSTEM 37 is the more abundant in the paraventricular nucleus (Van Dyke, Adamsons and Engel, 1957). These hormones are concerned with counteracting thirst and the desiccation that is the main risk accompanying the migration from water to land. The antidiuretic hormone facilitates reabsorp- tion of water from the urine (§ 5.322) and oxytocin increases the excretion of Na+ and CI" (§ 5.312). It is therefore understandable that the neural lobe, where these hormones can be quickly released into the blood, should be best developed in terrestrial animals (Harris, 1955). The correlation has been confirmed by the observa- tion that natural thirst, or an equivalent state caused by injecting rats with saline, is followed by depletion of the secretory granules within a few minutes, to be slowly replaced in a day or so after giving the animals water to drink. Depletion of secretory granules in fish has been observed in response to immersion in hypertonic sea water, which would have the same effect as desiccation (Arvy, 1957). Gland cells of the suprarenal tissue and adrenal medulla The peripheral neurons of the sympathetic nervous system all secrete adrenaline, or noradrenaline, at their motor nerve endings. In most vertebrates some of these neurons become modified to secrete relatively enormous amounts of either or both of these substances ; at the same time the cells lose all histological resemblance to ganglion cells (Fig. 2-1/). Their origin and func- tion is nevertheless the same as that of neurosecretory cells ; but opinion is divided as to whether they should be regarded as such (Welsh, 1955). They are often referred to as "chromaffin" cells, because the contained adrenaline gives a characteristic olive-brown colour with any chromic salts used either as fixative or stain. Staining shows that the adrenaline is secreted by the cytoplasm as a mass of very fine granules. In fish, where these cells form the ''suprarenal" tissue, they in most tetrapods. Heavy arrows indicate the presence of portal veins between primary venous plexus in median eminence and secondary plexus in pars distalis (cf. Fig. 2-12) in fully terrestrial forms. O.C, optic chiasma; S.V., saccus vasculosus; V.L., ventral lobe (from Green, 1951). 38 SOURCES OF KINETIC AND METABOLIC HORMONES remain in their original paired positions and are innervated by the pregangUonic fibres of the visceral motor or sympathetic system. They secrete mainly noradrenaline. From Amphibia to Mammalia this tissue, having migrated towards the anterior ends of the kidneys, becomes progressively enveloped in the interrenal or cortical tissue, derived from coelomic epithelium (§ 2.311). The chromaffin tissue forms the adrenal medulla, or core, and secretes mostly adrenaline in mammals. Together, the cortex and medulla form the complex adrenal gland.* The cells of the medulla are innervated in. the same way as in fish. Some unmodified ganglionic cells may be seen among them ; but the chief characteristic of the tissue is the net- work of blood spaces with which every cell is in contact and into which their secretion can be passed with great rapidity in response to nervous stimulation in an emergency (§ 3.11). 2.12 ENDOCRINE GLANDS DERIVED FROM ECTODERMAL EPITHELIUM A number of endocrine glands are derived from ectodermal epithelium, without having any apparent connection with the nervous system. These seem to be more frequent in invertebrates than in vertebrates, and include the Y-organ and the prothoracic glands, which are the main sources of the morphogenetic moulting hormones of Arthropoda (§ 4.21 and Part II). The salivary glands of Cephalopoda may be included here (§ 2.121), although it is doubtful if their secretion is a true hormone. The corpora allata of Insecta (§ 2.122) and the adenohypophysis of Vertebrata (§ 2.123) are both important ectodermal sources of kinetic and metabolic hormones. 2.121 Salivary glands of Cephalopoda Salivary glands of cephalopods are primarily used for the external secretion of tyramine, a poison for immobilizing the prey ; * Unfortunately, in medical terminology, this compound gland is usually referred to as the "suprarenal", from its position "above" the kidney in the upright posture. It must not be confused with the supra- renal of fish, which is homologous with the medulla only. § 2.122 ENDOCRINE GLANDS FROM ECTODERMAL EPITHELIUM 39 but this substance also passes into the blood, to affect the chroma- tophore muscles (§ 3.21), probably by an indirect action. The glands, of which there are two in decapod and four in octopod cephalopods, arise from the stomodaeal ectoderm, with which they retain their connection as a duct to the mouth. It is the posterior (or dorsal) pair which secretes tyramine in Eledone moschata and in the two species of Octopus which have been investigated (Bacq and Ghiretti, 1951). 2.122 Corpora allata of Insecta The CORPORA ALLATA are endocrine glands, the cells of which arise in development as a pair of small ventrolateral invaginations near the base of the first maxilla (Fig. 2-8). Thence the tissue migrates inwards to lie between the oesophagus and the aorta ; it may remain paired, or the two parts may fuse more or less com- pletely. Each part is supplied with neurosecretory axons passing on from the corpora cardiaca, which lie just in front of them (Fig. 2-9). There is evidence that these axons must remain intact if they are to control secretion by the corpora allata, as though their action were either nervous or due to a neurohormone diffusing from cell to cell rather than being carried in the circulation. The corpora allata are also connected with the stomatogastric system by nerves from the hypocerebral ganglion. The densely packed cells of the corpora allata show cyclic phases of secretion and multiplication, coinciding with their cyclic activity in controlling the "juvenile" character of nymphal and larval moults (Part II, § 3). A new phase of activity starts very soon after each moult is completed: at first the gland is small and the cells are all alike ; then there is a burst of mitotic activity followed by an increase in size of some cells, leading to increase in gland size. The larger cells, which have much larger nuclei than the undiffer- entiated cells, then begin to form their secretion. This appears first as granules in the cytoplasm ; it then accumulates in vacuoles, which can later be seen to lie outside the cells in intercellular spaces. From this position the secretion, or the hormone released from it, presumably passes into the blood stream at the critical period for controlling the next moult. Some of the secreting cells 40 SOURCES OF KINETIC AND METABOLIC HORMONES become polyploid by division of their nuclei without cell division. They form giant cells (g.s.c, Fig. 2-9). When secretion is com- pleted, the gland returns to the initial undifferentiated state shortly before the actual moult (Mendes, 1948). The glands also secrete metabolic (§5.11) and morphogenetic hormones (Part II, §3) during the adult life of the insect, v^hen the histology of secretion appears to be the same as that in younger stages, and gives no indication of the hormones being different at different ages. 2.123 Adenohypophysis of Vertehrata The adenohypophysis, or the anterior lobe of the pituitary body, is the only endocrine gland which arises from non-nervous ecto- derm in vertebrates. Its origin is the hypophysis, an upgrowth from the roof of the stomodaeum. It meets the brain and induces a downgrowth from the hypothalamus or floor of the diencephalon ; together they form the pituitary body (Fig. 2-13). Throughout the vertebrates the contribution from the nervous system forms the neurohypophysis which has already been described (§ 2.114). The subdivisions of the ectodermal adenohypophysis are difficult to homologize in different groups. It has recently been recommended (Pickford and Atz, 1957) that the noncommittal terms pro-, meso- and meta-adenohypophysis should be used for fish and that these should only be very tentatively homologized with the three parts recognizable in most tetrapods, namely the PARS tuberalis, the pars distalis and the pars intermedia (Fig. 2-10).* Of these, the pars distalis is usually the largest and appears to be the main source of hormones. It lies in front of the original hypophysial cavity, or cleft, if this persists, whereas the pars intermedia forms behind the cleft and often comes into close contact with the adjacent neural lobe. The pars tuberalis surrounds the infundibular stalk. The shapes and relative sizes of these parts * Unfortunately, the old nomenclature is still to be found in many books; in this, the pars intermedia, although formed from the same tissue as the rest of the adenohypophysis or "anterior lobe of the pituitary", is included with the pars nervosa in the so-called "posterior lobe of the pituitary"; this is due to its morphological position in some mammals. Other writers use posterior lobe as synonymous with the neural lobe, or pars nervosa, only (Fig. 2-10). The names used here follow those recom- mended by the International Congress of Anatomists (Woerdeman, 1957). § 2.123 ENDOCRINE GLANDS FROM ECTODERMAL EPITHELIUM 41 vary greatly in different animals (Fig. 2-11). The pars distalis becomes increasingly important in land animals, and especially in mammals, whereas the pars intermedia decreases in size and may even be wholly lacking, as in the chick and whale. The blood supply of the pituitary also shows important differ- ences in different classes of vertebrates (Fig. 2-11). In fish, hypophysial arteries from the internal carotids break up into a vascular plexus, which penetrates the whole organ and eventually drains into venous sinuses under the skull. (The saccus vasculosus may have a separate supply; but it is not part of the endocrine gland.) In Urodela, the arterioles pass first to the pars tuberalis and thence to the capillary plexus of the pars distalis and pars nervosa. The pars intermedia hardly has any share in the blood supply. In the Anura and amniotes, the plexus in the pars tuberalis penetrates into the median eminence and then the vessels join up to form a number of portal veins which break up into a secondary venous plexus in the pars distalis (Fig. 2-11 g and h). It might be thought that this portal system was less important in mammals than in lower forms, since in them alone the pars distalis acquires a direct arterial blood supply from the internal carotids (Fig. 2-12); but it may allow the passage of CRF (§ 4.323). In all land animals the newly developed neural lobe of their neurohypophysis also acquires an independent arterial supply from the same source. These vessels from the neural lobe, together with all those from the pars distalis, drain into the same venous sinus and eventually join the internal jugular veins. Nerve axons to the pituitary fall into two distinct categories: those of neurosecretory cells, and those of sympathetic nerves. The neurosecretory cells are confined to the neurohypophysis, and most of them end in the neural lobe; but some of them make contact with the primary venous plexus in the median eminence. In this way, secretions from the latter can pass into the portal circulation and so to the pars distalis. How much they do so, and whether they affect the rates of hormone secretion from the adeno- hypophysis, is still a matter for discussion. The vasomotor fibres of the sympathetic nerves follow the course of the hypophysial arteries (Fig. 2-12); they can therefore 42 SOURCES OF KINETIC AND METABOLIC HORMONES Fig. 2-12. Diagrammatic sagittal section of the pituitary gland and hypothalamus of a rabbit, Oryctolagiis, with the circulation super- imposed and simplified. The neurosecretory cells (NS^ and NS") have been disproportionately magnified to show their axons con- necting with the primary plexus of veins (VP) in the median eminence (ME) and (VI) in the neural lobe (NL). The only other nerves are sympathetic vasomotor fibres (S^, S^ and S^). Blood comes from the internal carotids (IC) by hypophysial arteries; the upper right (HY^) takes an independent supply to the neural LOBE (NL), which is drained by the main vein (V). The pars INTERMEDIA (PIN) lias practically no circulation. The upper left hypophysial artery (HY-) supplies the primary venous plexus in § 2.123 ENDOCRINE GLANDS FROM ECTODERMAL EPITHELIUM 43 control the blood supply to the neural lobe and the pars distalis, but do not appear to make any contact with the secretory cells. There are no nerves to the pars intermedia in mammals, thou^^li secretions from this part appear to be under nerve control in fish and amphibia (§ 4.3). The PARS DISTALIS of the adenohypophysis contains at least three distinct types of cells: chromophobe (gamma) cells which have no stainable granules in their cytoplasm and seem to have no secretory function, though they may give rise to one or both of the secreting types ; basophil cells, which contain secretory granules of glycoprotein that stain blue by the Mallory or Azan trichrome methods, and acidophil cells, which contain phospholipid granules that stain selectively by an acid haematin method. The last two types can also be distinguished by staining with safranine-acid violet (cf. Maximow and Bloom, 1942, Fig. 261.-2) and show changes which are clearly associated with the secretory activity of the gland. For some time it was claimed that only these two types of secreting cells could be identified histologically although the gland was known to secrete six or seven distinct hormones; but recently more sensitive tests have been applied and tentative subdivisions of the two types have been proposed (Table 4) ; further details have recently been summarized by Pickford and Atz (1957). The PARS TUBERALis has not been studied so fully but appears to consist of columns of cells separated by blood spaces, and to be closely similar to the adenohypophysis in appearance. A secretory activity has only been claimed for it in some fish and amphibians (§3.23). The PARS INTERMEDIA may consist of cells with basophil granules and non-granular cells that form follicles filled with a colloid, similar in appearance to that in the thyroid gland but containing the PARS TUBERALIS (PT), from which loops penetrate deeply into the median eminence and can receive neurosecretions. These vessels join again to give the hypophysial portal system (HP) which breaks up into the secondary venous plexus (VS) in the PARS DISTALIS (PD) of the adenohypophysis. This part also receives blood directly from the lower hypophysial arteries (HY=^). (Original, based on Scharrer and Scharrer, 1954^, and Harris, 1955). 44 SOURCES OF KINETIC AND METABOLIC HORMONES Table 4. Cells in the pars distalis of the adenohypophysis CELLS STAIN SECRETION SECTION NO. Basophil cells beta delta »> p PAS] and aldehyde fuchsin PAS only, peripheral ? ,, ,, central ? TSH* FSH LH ICSH? 4.221 4.232 4.232 4.232 Acidophil cells alpha epsilon • Orange G Fuchsin STH LSH ACTH 4.223 4.232 4.231 * See glossary. t PAS = Periodic acid-Schiff method, with which these cells give a positive reaction. no iodine. This region is well known to secrete a melanophore dispersing hormone, intermedin, in many fish and most amphi- bians, but not in Agnatha. It has also been claimed that this lobe may act as a storage-and-release centre for adrenocorticotrophin, ACTH (Mialhe-Voloss, 1955). The claim may be due to the chemical identity of intermedin with a large part of the chain molecule that makes up ACTH. 2.2 Endodermal sources in Vertebrata Hormones secreted from endodermal sources have so far only been recognized in vertebrates. They fall into two categories: isolated cells in the stomach and intestinal mucosa (§ 2.21) and well-developed endocrine glands in the pharynx and pancreas (§ 2.22). The former secrete kinetic hormones which control gut muscles and the secretion of digestive enzymes from exocrine glands ; the latter secrete various metabolic hormones (Table 5). 2.21 ISOLATED CELLS IN THE GUT A large number of hormones can be located in the stomach and intestine of mammals ; some of these also occur in birds, but they are not known to be active in cold-blooded vertebrates. § 2.21 ISOLATED CELLS IN THE GUT 45 Since no recognizable endocrine glands or groups of secreting cells have been found in the gut mucosa in those regions from which the gastrointestinal hormones, such as gastrin and sec- retin, can be extracted (§ 4.11), it must be concluded that all these hormones arise from isolated cells (Table 5). So far, however, these cells have not been found, despite intensive search; it is possible that they may not be histologically distinct from other cells in the gut lining, such as those secreting mucus, but it seems more likely that they will eventually be revealed by more sensitive or selective staining techniques. It has been suggested that secretin may be produced in certain "argentafhne" cells, which can be stained with silver; but this seems unlikely since similar cells are abundant in the vermiform appendix from which no secretin can be extracted (Grossman, 1950). The origin of the secretory cells certainly deserves further investigation. The clues that would seem to be the most worth following are the facts that (1) the action of this whole group of hormones is kinetic, (2) all other kinetic hormones come either from modified nerve cells or at least from the ectoderm and (3) the action of these hormones in stimulating the secretion of stomach, pancreas and intestinal glands is carried out in lower vertebrates by the parasympathetic nerves in the vagus. The parasympathetic nerves form part of the general visceral motor system and like the sympathetic nerves have peripheral ganglia, connected to the brain by preganglionic fibres. The latter are of central nervous origin ; but there is considerable doubt as to the origin of the peripheral cells, though the neural crest has been plausibly postulated. If so, crest cells might be expected to migrate to the intestine from the cranial region to form ganglia; and it seems possible that some of them might also form argentaffine cells, and others secretory cells. Although there is evidence that the argentaffine cells of the gut can differentiate in grafts of chick intestinal epithelium, even if this is separated from the embryo before any trunk neural crest material is formed (Van Campenhout, 1946), it is not so clear that cranial neural crest cells were excluded in these experiments. On the other hand, there is one other curious feature about all the hormones secreted by the gut wall in mammals: unlike kinetic hormones derived from neurosecretory cells, they are not secreted 46 SOURCES OF KINETIC AND METABOLIC HORMONES in response to nervous stimulation, but always depend upon a direct stimulus, either mechanical or chemical (§ 4.321). The positions from which the various hormones have been found to be secreted in the gut are summarized in Table 5. . Table 5. Endodermal sources of kinetic and metabolic hormones in vertebrata TYPE CLASS source of hormone NAME OF HORMONE OF ACTION * SECT. NO. 2.21 Isolated cells in the gut Mammalia Stomach Gastrin K 4.111 >> Duodenum Cholecystokinin K 3.112 >> »> Secretin K 4.111 M >> Pancreozymin K 4.111 >j >> Duocrinin K 4.111 >> Intestine Enterocrinin K 4.111 >« )> Enterogastrone K 4.112 2.22 Endodei =(MAL ENDOCRINE GLANDS 2.221 Glands of the pharymx Agnatha to Thyroid Thyroxine M 5.111 Mammalia Teleostei Ultimobranchial body Parathormone M 5.411 Tetrapoda Parathyroid ,, M 5.411 >j „ >> M 5.422 2.222 Gland cells in the pancreas Agnatha to Islets of Langerhans Insulin M 5.212 Marnmalia Aves and )) Glucagon M 5.211 some Mammalia * K = kinetic. M = metaboHc. 2.22 ENDODERMAL ENDOCRINE GLANDS Recognizable endocrine gland cells are derived from the gut epithelium in two regions: the pharynx, where there are at least § 2.221 ENDODERMAL ENDOCRINE GLANDS 47 two groups (§ 2.221), and the pancreas, where they form the islets of Langerhans (§ 2.222). These glands all secrete metabolic hormones, and their structure has long been known; it is well described in most text-books of vertebrate anatomy, embryology and histology, and little need be said here, except to emphasize the fact that they can be identified in most classes of vertebrates and are not confined to the warm-blooded forms, like the sources of the gastrointestinal hormones. 2.221 Glands of the pharynx These are the thyroid and parathyroid glands and the ULTIMOBRANCHIAL BODIES, which are the homologues of the para- thyroids (Table 5). The thymus glands also arise here (Fig. 2-13); but their endocrine nature is uncertain. It will be considered in relation to growth (Part II, § 3). Thyroid glands In Agnatha, the endostyle in the floor of the larval pharynx can accumulate iodine, and even synthesize thyroxine, albeit in small quantities, even before its transformation to the adult thyroid gland. The endostyle of the amphioxus, Branchiostomay also accumulates iodine ; but it does not appear to synthesize thyroxine (Barrington, 1958). It is then possible to argue either that the endostyles of the Protochordates and the Agnatha are homologous with each other and with the thyroid gland, because the former are structurally similar although only the two latter have acquired the ability to synthesize thyroxine, or that a true homology should be marked by similar chemical activities, and is in this case limited to the vertebrates. The thyroid gland retains its position as a single median organ in the floor of the pharynx in lower vertebrates (Fig. 2-13) where the gland itself may be diffuse, as in many teleost fish, or relatively compact and enclosed in a capsule of connective tissue, as in Elasmobranchii and a very few Teleostei, such as Pseudoscarus, from which it can therefore be relatively easily removed. In tetrapods, in which the gills are lost, the gland often becomes paired and may even be further subdivided; but each part is 48 SOURCES OF KINETIC AND METABOLIC HORMONES Pin Fig. 2-13. Diagrammatic sagittal half of the head and pharynx of a tadpole, Rana. An early stage in development of the brain and stomodaeum (St) shows the adenohypophysis (Hyp) growing up to meet the infundibulum (Inf), or neurohypophysis, from the floor of the fore brain (F) ; together they form the pituitary body. Optic chiasma (OC), pineal organ (Pin), spinal cord (CNS) and notochord (Nch). The pharynx, leading to oesophagus (Oes), is shown at a later stage \ I to VI, visceral arches; III to VI with branchial arteries running behind gill slits to dorsal aorta (DA) ; THYROID (Th) is mid-ventral; a series of ventrolateral epithelial thickenings form the carotid gland (C) on III, the parathyroids (P) on IV and V, and the ultimobranchial body (U) on VI. Dorsolateral thickenings on II and III (and also on IV and V in other animals) form the thymus gland (Thym). compact and encapsuled. It receives a rich blood supply from the carotids, and its nerve supply is mainly vasomotor. In most vertebrates the histological character of the gland is fairly constant. Its cells form a cubical epithelium surrounding spaces, which become filled with a colloidal material secreted § 2.222 ENDODERMAL ENDOCRINE GLANDS 49 into it as droplets from the cells (Figs. 2- 14a and 4-7). This appears to be a precursor of the hormone, usually in the form of diiodo- tyrosine ; it is later reabsorbed into the cells, converted to thyroxine, and passed into the blood as the hormone (Fig. 4-8). This process is described more fully in relation to its control by the thyrotrophic hormone, TSH (§ 4.221). Ultimohranchial bodies These structures arise ventrolaterally from the epithelium of the last gill slit and may be seen in development to give rise to a pair of small glands. In fish they appear to replace the parathyroid glands ; but in tetrapods they may be present in addition to them (Fig. 2-13). In function they appear to be similar to the para- thyroids (§ 5.4). Parathyroid glands These glands are serially homologous w^ith the ultimohranchial bodies behind (and with the so-called carotid gland of Amphibia in front). They arise ventrolaterally from the epithelium lining the gill slits on the anterior surfaces of the fourth and fifth visceral arches (Fig. 2-13). The individual cells of the parathyroid glands form a densely packed mass amid ramifying blood vessels, and are not arranged in follicles. They may be of two kinds: the more numerous have clear cytoplasm and relatively large nuclei ; the rest are acidophil with granular cytoplasm. The former are thought to be the main source of parathormone (§ 5.4). 2.222 Gland cells in the pancreas In most vertebrates, groups of endocrine cells occur among the exocrine cells (secreting enzymes and alkali) in the pancreas ; they form the islets of langerhans. In larval lampreys these endo- crine cells lie adjacent to the rest of the pancreatic tissue, and only become embedded within it in the adult. The endocrine cells are distinguishable histologically because they stain much less readily than the surrounding exocrine gland cells. This is due to the constant presence of so-called beta (^) cells that secrete the anti- diabetogenic hormone, insulin (§ 5.212). These cells have very 50 SOURCES OF KINETIC AND METABOLIC HORMONES fine granules only, and have little affinity for any cytoplasmic stains. In at least some of the higher vertebrates, and especially in such mammals as the cat and dog, two other types of cell can also be distinguished in the islet tissue. Of these, the alpha (a) cells secrete the diabetogenic hormone glucagon (Table 5); they contain large granules that stain bright red with Mallory-azan stain, but their cytoplasm also has little affinity for any stains.The D type of cell in the islets stains blue in the same preparation, but its function is unknown (Fig. 2-146; Maximow and Bloom, 1942). 2.3 Mesodermal sources in Vertebrata Metabolic hormones secreted by mesodermal sources have so far only been found in vertebrates. These come from the adrenal cortex and its homologue, the interrenal tissue. Other hormones from the mesoderm are all morphogenetic in their actions, or predominantly so; their sources, including the source of pro- gesterone (despite the possible kinetic activities that have been attributed to this hormone in § 4.12), will be described in Part II. 2.31 endocrine gland cells derived from coelomic epithelium In all vertebrates the coelomic epithelium in the region of the kidneys gives rise to characteristic yellow gland cells, filled with fat and secreting cortical sterolic hormones. The cells are homolo- gous in all classes; but they are differently named according to their positions. In fish they often retain their median position between the kidneys, where they form interrenal tissue, or they may become paired, as in the perirenal organs of Dipnoi. In the tetrapods the tissue forms the adrenal cortex; it is always paired, lies in front of the kidneys, and becomes closely associated with the adrenal medulla during development. The cortex finally encloses the medulla completely in mammals (Table 6). 2.311 Interrenal tissue In Elasmobranchii, cortical gland tissue occurs as one or more median yellow masses, the interrenal tissue, so-called from its position between the kidneys. It is widely separated from the paired §2.311 KNDOCRINE CELLS FROM COKLOM 51 groups of suprarenal cells which represent the adrenal medulla of higher forms. In Teleostei and related bony fish, the homologous tissue is called the anterior interrenal body, to distinguish it from the corpuscles of Stannius, or posterior interrenal bodies. The former resembles the adrenal gland of tetrapods, in containing a mixture of typical cortical and medullary cells. Of these, the cortical cells Table 6. Mesodermal sources of metabolic hormones in vertebrata TYPE CLASS SOURCE OF NAME OF OF SECT. hormone HORMONE ACT- ION* NO. 2.31 Endocrine GLANDS FROM COELOM IC EPITHELIUM Elasmohranchii Interrenal tissue Cortical hormone M 5.311 Teleostei Anterior ,, ,, (& corpuscles of n M M 5.311 Stannius ?) >> j> M 5.321 Tetrapoda Adrenal cortex >> M M 5.112 >> j> 5> >> M 5.211 n n >> >) M M 5.223 5.412 > > >> „ M 5.421 >) )> Aldosterone-like M 5.311 )> )> Hydrocortisone-like M 5.321 M = metabolic. respond to stimulation by the adrenocorticotrophic hormone, ACTH, from the pituitary. Like the interrenal cells of the elasmobranchs, this tissue yields an active extract which is inter- changeable with that of any tetrapod adrenal cortex. Injection of any of the extracts can keep alive a mammal or bird from which the cortex has been removed. The corpuscles of Stannius are small, paired globules of tissue, embedded in the mesonephric kidneys; there are numerous pairs 52 SOURCES OF KINETIC AND METABOLIC HORMONES in Amia, but most teleosts have only one pair. Histologically their cells look like cortical tissue ; but they do not seem to respond to ACTH, and their function is uncertain. Their homology with true interrenal tissue is also in doubt, since they appear to arise from the pro- and meso-nephric ducts, and not from coelomic epithe- lium (cf. Pickford and Atz, 1957). 2.312 Adrenal cortex Like the interrenal tissue, the adrenal cortex is a small mass of conspicuous yellow tissue ; but it is paired and situated in front of, or below, the kidneys, w^hether these are mesonephric or meta- nephric. The colour is due to large amounts of fat enclosed in the cells and associated with the formation of the sterolic hormones secreted by the gland. The presence of stored hormone in the gland is also related to the presence of ascorbic acid, which becomes depleted when the hormone is passed into the blood stream in response to some form of stress (§ 4.231 and Fig. 4-9), and to release of ACTH. Three layers can usually be recognised in the cortex : an outer layer (just within the connective tissue capsule enclosing the whole gland), where active cell multiplication follows the frequent nuclear mitoses ; a thicker middle region of actively secreting cells ; and the innermost layer, next to the medulla, where the cells become degenerate and are eventually consumed by macrophages from the blood. It seems that throughout the life of the gland, cells are formed near the outer surface, migrate inwards during their secretory phase and are then destroyed as they reach the inner surface. The main secreting cells of the cortex form a compact mass or continuum, in which the individual cells tend to be polyhedral, from contact with adjacent cells (Fig. 2-14c). In the rat, the mass is tunnelled through by a network of blood sinusoids, so abundant that every cell has a facet in contact with a blood vessel into which its secretion can be passed (Pauly, 1957; Fig. 2-15). The structural details of the gland, and the proportion of connective tissue to gland cells, varies from species to species; but this has not been shown to have any effect upon the functional activity of the gland. No nerves have been detected. §2.4 REFERENCES 53 Fig. 2-15. Stereogram of part of the adrenal cortex of Rattus; the cells form a continuum through which blood capillaries tunnel to make contact with each cell. The fibres on the outer (upper) surface represent the connective tissue capsule surrounding the gland; below this, the blood vessels branch freely and then run directly inwards through the main secretory region (zona fascicu- lata) of the gland and anastomose at the inner (lower) surface, next to the medulla (which is not shown). (From Pauly, 1957). 2.4 References Alexandrowicz, J. S. (1953). Nervous organs in the pericardial cavity of the decapod Crustacea, jf. mar. hiol. Ass. U.K. 31 : 563-580. Amar, R. (1948). Un organe endocrine chez Idotea (Crustacea isopoda). C. R. Acad. Sci., Paris, 111'. 301-303. Arvy, L. (1957). In discussion of I. Chester Jones. Colston Pap. 8: 273-274. 54 SOURCES OF KINETIC AND METABOLIC HORMONES Arvy, L. and Gabe, M. (1953). Donnees histophysiologiques sur la neurosecretion chez quelques Ephemeropteres. La Cellule, 55 (2): 203-222. Bacq, Z. M. and Ghiretti, F. (1951). La secretion externe et interne des glandes salivaires postdrieures des Cephalopodes Octopodes. Arch. int. P/zj'^fo/. 59:288-314. Bargmann, W. (1958). Elektronenmikroskopische Unter suchungen an der Neurohypophyse. In Zweites Internationales Symposium Uber Neuro- sekretion, edited by W. Bargmann, B. Hanstrom and B. Scharrer. Berlin: Springer-Verlag. 4-12. Barrington, E. J. W. (1958). The localization of organically bound iodine in the endostyle of Amphioxus. J. mar. biol. Ass. U.K. 37: 117-126. Bliss, D. and Welsh, J. H. (1952). The neurosecretory system of brachy- uran Crustacea. Biol. Bull. Wood's Hole, 103: 157-169. Campenhout, E. van, (1946). The epithelioneural bodies. Quart. Rev. Biol. 21: 327-347. Carlisle, D. B. (1953). Studies on Lysmata seticaudata Risso (Crustacea Decapoda) VI. Notes on the structure of the neurosecretory system of the eyestalk. Pubbl. Staz. zool. Napoli, 24: 435-447. Carlisle, D. B. and Knowles, F. G. W. (1953). Neurohaemal organs in crustaceans. Nature, Lond. 172: 404-405. Carlisle, D. B. and Passano, L. M. (1953). The X-organ of Crustacea. Nature, Lo?id. 171: 1070-1071. De Lerma, B. (1956). Corpora cardiaca et neurosecretion protocerebrale chezle Coleoptere Hydrous piceus L. Ann. Set. nat. (b) Zool. 18 : 235-250. De Robertis, E. (1949). Cytological and cytochemical bases of thyroid function. Ajin. N.Y. Acad. Sci. 50: 317-335. Enami, M. (1951). The sources and activities of two chromatophoro- tropic hormones in crabs of the genus Sesarma. II. Histology of incretory elements. Biol. Bull. Wood's Hole, 101 : 241-258. Gabe, M. (1954). La neuro-secretion chez les invertebres. Annee biol. Ser. 3, 30: 5-62. Green, J. D. (1951). The comparative anatomy of the hypophysis, with special reference to its blood supply and innervation. Amer. J. Anat. 88: 225-312. Grossman, M. I. (1950). Gastrointestinal hormones. Physiol. Rev. 30: 33-90. Hanstrom, B. (1939). Hormones in Invertebrates. Oxford: Clarendon Press. Hanstrom, B. (1940). Inkretorische Organe, Sinus Organe und Nerven- system des Kopfes einiger niederer Insektenordnungen. Kungl. Svenska Vetenskap. Handl. Ser. 3, 18, No. 8: 3-266. Harris, G. W. (1955). Neural Control of the Pituitary Gland. Monogr. Physiol. Soc. (3). London: Edward Arnold. § 2.4 REFERENCES 55 Kleinholz, L. H. (1947). A method for the removal of the sinus gland from the eyestalk of crustaceans. Biol. Bull. Wood's Hole, 93 : 52-55. Knowles, F. G. W. (1953). Endocrine activity in the crustacean nervous system. Proc. roy. Soc. B. 141 : 248-267. Knowles, F. G. W. (1954). Neurosecretion in the tritocerebral com- plex of crustaceans. Pubbl. Staz. zool. Napoli, 24, Supplemento: 74-78. Knowles, F. G. W. (1955). Crustacean colour change and neurosecre- tion. Endeavour, 14: 95-104. Knowles, F. G. W. and Carlisle, D. B. (1956). Endocrine control in the Crustacea. Biol. Rev. 31: 396-473. Maximow, a. a. and Bloom, W. (1942). A Textbook of Histology. Phila- delphia and London: W. B. Saunders Company. Mendes, M. V. (1948). Histology of the corpora allata of Melanoplus differentialis (Orthoptera: Saltatoria). Biol. Bull. Wood's Hole, 94: 194-207. MiALHE-VoLOSS, C. (1955). Activite corticotrope des lobes anterieur et posterieur de I'hypophyse, chez le rat et le canard. J. Physiol., Paris, 47:251-254. Pauly, J. E. (1957). Morphological observations on the adrenal cortex of the laboratory rat. Endocrinology, 60 : 247-264. PiCKFORD, G. E. and Atz, J. W. (1957). The Physiology of the Pituitary Gland of Fishes. New York: New York Zoological Society. Potter, D. D. (1954). Histology of the neurosecretory system of the blue crab Callinectes sapidus. Anat. Rec. 120: 716. ScHARRER, B. (1952). Neurosecretion. XI. The effects of nerve section on the intercerebralis-cardiacum-allatum system of the insect Leucophaea fnaderae. Biol. Bull. Wood's Hole, 102: 261-272. ScHARRER, E. and Scharrer, B. (1954a). Hormones produced by neuro- secretory cells. Rec. Prog. Horm. Res. 10: 183-240. Scharrer, E. and Scharrer, B (19546). Neurosekretion. In Handbuch der mikroskopischen Anatomic des Menschen, edited by W. von Mollen- DORFF and W. Bargmann. Berlin: Springer- Verlag. 6 (5): 953-1066. Thomsen, E. (1954). Darkfield microscopy of living neurosecretory cells. Experientia, 10: 206-207 Van Dyke, H. B., Adamsons, K. Jr. and Engel, S. L. (1957). The storage and liberation of neurohypophysial hormones. Colston Pap. 8: 65-76. Weber, H. (1949). Grundriss der Insektenkunde. Jena: Gustav Fischer- Verlag. Welsh, J. H. (1955). Neurohormones. In The Hormones, edited by G. Pincus and K. V. Thimann. New York: Academic Press Inc. 3: 97-151. WoERDEMAN, M. W. (1957). Nomina anatomica parisiensa (1955) et B.N.A. (1895). Utrecht: A. Oosthoek Publishing Co. Young, J. Z. (1936). The giant nerve fibres and epistellar body of cephalopods. Quart. J. micr. Sci. 78: 367-386. CHAPTER 3 KINETIC HORMONES I. CONTROL OF MUSCLES AND PIGMENTARY EFFECTORS The term "kinetic" (§ L51) brings together a large group of hormones, which act upon certain effectors in the organism in ways which often resemble the effects of nerve stimulation. Kinetic hormones acting upon muscles and pigmentary effectors are considered in this chapter, and those causing the secretion of glands, both exocrine and endocrine, in the next (§§ 4.1 and 4.2). The similarity in action between these kinetic hormones and some nerves is not entirely accidental, since many of the kinetic hormones are neurosecretions from modified nerve cells (§2.111), and some are chemically akin to the acetylcholine or noradrenaline secreted by cholinergic and adrenergic nerves respectively. An important difference lies in their means of distribution; the hor- mone reaches the effector through the blood circulation and is therefore widespread in its effect, whereas the nerve cell releases its chemical in contact with a single effector only. There are, however, other kinetic hormones that are not derived directly from nervous tissue. Some of these, like those from the corpus allatum of insects or the adenohypophysis of vertebrates, are likewise derived from the ectoderm. A few others, such as secretin, appear to be derived from neither nerve cells nor ectoderm, but from the endoderm (§ 2.22). The kinetic hormones therefore form a wider group than either ''neurohormones" (Welsh, 1955) or the secretions of "neurohaemal organs" (Carlisle and Knowles, 1953) ; yet they show quite sufficient functional similarity among them- selves to warrant their inclusion in one group. The means of stimulating their secretion may be mechanical, as 56 §3.111 CONTROL OF MUSCLES 57 in the case of gastrin, or chemical, as in that of secretin (§ 4.1 1 1) ; but it is usually nervous (§ 4.32). Few are under the control of any other hormones, and this is probably a question of speed. Hormone action is slower than nerve action; and, whereas the delay due to one hormone may not be significant for the effectors in question, a chain of two hormones might well be so. The hormones dealt with in the following sections are shown in a series of tables, where they are arranged according to their actions. The hormones of vertebrates have names which are widely accepted and are known to occur in a variety of animals; the example quoted is usually the one described in the text, but in no way implies that the hormone is limited to the genus named. Hormones of invertebrates for the most part have no names and can therefore only be referred to by the organ from which they are secreted. If they have a name, or an abbreviation that is used in the text, this is given in a separate column. Each invertebrate example in the tables is also referred to in the text ; it is often the only example from which the hormone has so far been identified. 3.1 Control of muscles Muscles can be grouped according to their functions or their histology, and there is some evidence that all types can be influ- enced by hormones. It will be convenient to take the involuntary muscles of the viscera and heart first, because these are the most commonly subject to hormone control and react similarly, although most visceral muscle of vertebrates is smooth, or unstriated, and that of arthropods and of the hearts in both phyla is striated. 3.11 VISCERAL MUSCLE In nearly all cases the action of hormones is to stimulate both the rate and amplitude of the contraction of visceral muscle ; only rarely is a hormone known to inhibit muscle action (Table 7). 3.111 Heart muscle CRUSTACEA. Control of heart muscle by a hormone has been shown experimentally in the crab. Cancer pagtinis, and the lobster, Homarus vulgaris. Extracts of the neurosecretory "pericardial organs" (§2.112) added to fluid perfusing isolated hearts, 58 KINETIC HORMONES — I to Q K Q g 2-t2 r^ iS J3 i3 ll I I I i I I :3 a CD a o .2 '3 u O O a u O U I I I I I I I I I I I I SIM K^ I G^OOOfc,ttl I I jij D c C -K3 o 0) o << a o J « CO ;d "^ S ^^ > 1 rO C3 CO ^2 1 ^5 .22 B c/3 C3 c« '1 <4j Jo c ^ ^ Wi -^ ■-Si 5 g^a •S ►-^ a a Q ^-XJ O rt" LO \0 vO CO ro CO CO o W) +i o C -M «« n <^ ?^ W c/3 c 2 be -M O w Uh O a -I CA! Op w 5 S 1 CO CO ' Oh § 3.111 VISCERAL MUSCLE 59 increase the amplitude and frequency of the heart beat (Fig. 3-1) This action is beUeved to be due to an or^/zo-dihydroxytryptamine and is closely similar to the action of adrenaline and noradrenaline, which are chemically not widely dissimilar. Results on other species are rather contradictory, and different doses are needed at different times of year to get similar results. This may be compared with the effects that adrenaline and noradrenaline have on vertebrates, where contraction or relaxa- tion can be obtained, according to conditions in the organs resulting from previous treatment, size of dose, etc. (Welsh, 1955). "It is assumed that the function of the pericardial organs in these Crustacea consists in liberating, through fine neuropile-like terminations of the nerve fibres, some hormone passing with the blood into the heart and producing on it a stimulating effect" (Alexandrowicz and Carlisle, 1953). Blood taken from the peri- cardial cavity before reaching the heart gave the same reaction as the extracts ; but that taken from the leg arteries after leaving the heart did not, presumably because the chemical was destroyed before it reached the legs. Earlier statements (e.g. Welsh, 1937) that the sinus glands of Crustacea provided a heart- accelerating extract might have been unreliable, because insufficient care was taken to avoid the presence of histamines in the extracts (the same is probably true of extracts of sub-neural glands of ascidians credited with similar activity). A heart-accelerating hormone from sinus gland extracts has, however, now been obtained from the freshwater shrimp, Paratya, and also an inhibiting extract from the brain (Hara, 1952). They are thought to be distinct from the chromactivators from the same sources (§ 3.223). INSECTA. The frequency of beat of the isolated heart of the cock- roach, Periplaneta americanay perfused with a suitable Ringer solution, can be increased some 50 per cent above normal, and the amplitude of the muscle contraction increased also, if an aqueous extract of one pair of corpora cardiaca (§ 2.111) of the same species is added to each 10 ml of the perfusate (Cameron, 1953). At concentrations one-tenth as strong, the amplitude is still increased, but not the frequency. If the extract is separated by paper chromatography, one spot at a time can be eluted and added 60 KINETIC HORMONES — I Fig. 3-1. Kymograph record of the heart beat of the crab, Cancer. Changes in frequency are shown as abscissae in relation to the time scale in minutes (below); changes in amplitude are shown as ordinates. The record reads from left to right. The arrows indicate the times at which different reagents reach the heart, which is the § 3.111 VISCERAL MUSCLE 01 to the perfusate. Only one of these shows the same action as the crude extract; this active constituent is thought to be an ortho- diphenol (and therefore related to adrenaline). Beautiful as this technique is, it only demonstrates, like most experiments on organ extracts, the pharmacological fact that the animal produces a chemical which has an effect upon the heart. It would be another matter to show conclusively that in life the heart beat is physio- logically controlled in any way by this substance, or that in the absence of the corpora cardiaca the animal is unable to control its heart beat to suit the conditions under which it is living. The stimulus which might cause the secretion of the chemical is unknown. The corpus cardiacum acts as a storage organ for a neurosecretion from the brain, as can be seen in Leiicophaea^ where severing the nerve on one side prevents the passage of the secretion (Fig. 3-2). Yet if a similar operation is performed on Periplaneta and separate extracts of the corpora cardiaca of the two sides are tested after 5 and 17 days, that from the severed side is still as active as the other. This eliminates the neurosecretion as a source of the heart- accelerating hormone, which must be the intrinsic secretion of the corpus cardiacum itself. These cells are of ectodermal, rather than nervous, origin (§ 2.112), so that their secretion is one of the few kinetic hormones that is not a vascular "neurohormone" (Welsh, 1955), although its action is akin to that of adrenaline, secreted from cells of the vertebrate sympathetic nervous system (Mendes, 1953). Adrenaline itself has a similar stimulating effect upon the heartbeat of many invertebrates, whether it is neurogenic or myogenic; but there is as yet no clear evidence of its being secreted by any gland in an invertebrate. In only a few inverte- brates is adrenaline used as a chemical transmitter at any nerve ending. Vertebrata. Adrenaline (§2.111) increases the amplitude same specimen throughout the series : {A) extract of pericardial organ of Cancer ; {B) adrenaline (1 :10«) ; (C) noradrenaline (1 :10«) ; {D) the same extract as in {A). Between exposures to these sub- stances the heart is restored to seawater and the beat slows down with a reduced amplitude (from Alexandrowicz and Carlisle, 1953). 62 KINETIC HORMONES — I Pors intercerebrolis of brain Accumulation of neurosecretory material proximal to site of nerve section Nervus corp. cardiaci with unobstructed flow of neurosecretory material Depletion of neurosecretory material in corp.cardiacum of operated side Storage of neurosecretory moterial in corp. cordiocum of operated side Larger corp.allatum of operoted side Normal corp. ollatum Fig. 3-2. Diagram of the dorsal aspect of the brain, corpora car- diaca, corpora allata and the nerves connecting them in the cock- roach, Leucophaea maderae. On the left side the nerve from the neurosecretory cells in the brain has been cut, so that the neuro- secretion accumulates in the proximal part of the nerve and does not reach the corpus cardiacum of that side (from Scharrer, 1952). and frequency of the heart beat in Vertebrata. This can only be demonstrated with difficulty in normal mammals, although as little as 1 part of adrenaline in 1,400,000,000 (Turner, 1955) can increase the beat of a denervated heart, freed from the ''depressor" action of parasympathetic fibres of the vagus nerve. It is likely, however, that the action of this drug on the heart is usually through the sympathetic nerve, rather than through the circula- tion, as the drug, or hormone, is quickly destroyed in the tissues by an enzyme. 3.112 Gilt 7nuscle Insecta. The striated muscles in the gut of P^;7/)/^/z^/rt (Cameron, 1953) and Locusta will record peristaltic movements for at least ^_l"ilH Fig. 3-3. Rhythmic peristaltic contractions of intestinal muscle of the cat, Felis; frequency is recorded as abscissae from left to right and amplitude as ordinates. From a to b, the muscle is in Ringer's solution; then at b, and again at/, blood from an "excited" cat, that had been barked at by a dog for some time, is added and the contained adrenaline causes almost immediate and quite prolonged inhibition of peristalsis. At d, peristalsis is restored by changing the perfusing fluid to one containing blood from a "quiet" cat, in which adrenaline secretion had not been stimulated (from Cannon, 1915). Fig. 3-4. Smooth muscle contraction in isolated rat uterus, showmg frequency as abscissae, and amplitude as ordmates. The t res h extract of the neurohypophysis, containing °f ™^;^' ^"J^^'^^, at the time indicated by the arrow on the l-^J'/f-^/X' rhythmic contractions. The marked time mterval is 10 mm. (alter Trendelenburg in Buddenbrock, 1950). §3.112 VISCERAL MUSCLE 63 24 hours, if perfused with a suitable Ringer sohition. In Pcri- planeta, an extract of one pair of its own corpora cardiaca to 10 ml Ringer solution causes the rate of peristalsis in the hind-gut to be doubled, and stronger solutions have more marked effects; but the peristalsis of the fore-gut seems to be inhibited. It is certain, at least in Periplaneta and in the locust, that increase in peristalsis of the Malpighian tubules can also be induced by a substance from the corpus cardiacum, as well as possibly one from the brain. The latter observation requires confirmation, in view of Cameron's finding that the neurosecretion from the brain does not contain the active principle causing heart muscle contraction. Vertebrata. The visceral muscle of the vertebrate gut is mainly under the dual control of the nerves of the sympathetic and parasympathetic systems working in opposition ; but the effect of the former can also be brought about in emergency by adrenaline from the adrenal medulla. Cold-blooded vertebrates. The effects of adrenaline appear to depend upon dosage. In the frog, Rana, small doses stimulate the contraction of gut muscles ; but larger doses inhibit it (Budden- brock, 1950). Mammalia. Adrenaline, secreted in response to excitement or fear, contracts the sphincter muscles of the gut and inhibits peristalsis, as can be shown by adding blood from an excited cat to the Ringer solution in which isolated gut muscle is contracting rhythmically (Fig. 3-3). Both reactions tend to stop digestion and fit in with the emergency mobilization of the blood supply in the somatic muscles. As its name implies, cholecystokinin contracts the muscles of the gall-bladder in frogs and in most mammals. It may also relax the sphincter muscle. The hormone therefore causes dis- charge of the bile down the cystic duct, and appears to be the only means of stimulating this reaction, for which there is no nerve control (Grossman, 1950). It can be extracted from the duodenal mucosa, and in nature is secreted in response to the presence of fat, or fatty and other acids, in the duodenum. Its histological origin has not been determined, but it appears to be derived from endodermal cells, like secretin, and therefore not 64 KINETIC HORMONES — I to be a neuro-secretory product. It is absent in the horse, EquuSy which has no gall-bladder. Gastrin, secreted by the stomach, can induce the contraction of stomach muscles, forcing food into the duodenum as the gastric phase of digestion is completed; but this action of gastrin follows, and is subsidiary to, that of stimulating the acid-secreting gland cells of the stomach (§4.111). The contraction is said to be inhibited by enterogastrone ; but this action is not purely hor- monal, since it is more effective if the vagus nerves are intact (Grossman, 1950). The nervous inhibition can be stimulated by acid in the duodenum. 3.113 Muscles of the genital ducts No cases seem to have been recorded so far in which the muscles of any part of the genital ducts of an invertebrate are controlled by hormones. Mammalia. Isolated uterine muscle of many mammals, such as the rat, Rattus, reacts to oxytocin from the neurohypophysis by strong rhythmic contraction (Fig. 3-4). At the end of pregnancy these contractions force the embryo out of the uterus. The fact that this does not normally occur until the embryo is fully devel- oped seems to be due, not to a lack of oxytocin during earlier stages of pregnancy but to changes in the level of sensitivity of the uterus. In the rabbit, Oryctolagus, the relative insensitivity of the uterus to oxytocin during the 30 days of pregnancy, as compared with variability at other times, is due to the abundant presence of progesterone. As this decreases and oestrogen increases in the circulation towards the end of pregnancy, the uterus becomes increasingly sensitive to the oxytocin until finally it reacts sufficiently strongly to bring about parturition Earlier experiments, in which it was found that rats from which the hypophysis had been removed were still able to produce their litters successfully, have now been explained on the grounds that the neurohypophysis is only a storage organ for hormones secreted in the hypothalamus of the brain (§ 2.111), and the experimental technique therefore failed to remove the source of the oxytocin. In those cases where the source was also destroyed by hypothala- 65 § 3.114 VISCERAL MUSCLE mic lesions, parturition was not achieved, both the mother and the Ktter dying in the attempt. It has also been suggested that oxytocin may play some part in driving sperm upwards into the fallopian tubes after copulation, 0-002 r- 0-03 0-5 >2-0 70 20 30 40 Days after mating Fig. 3-5. Graph showing schematically the sensitivity to oxytocin of strips of rabbit uterus tested at various stages of pregnancy. Days after mating are shown as abscissae, with the normal term for parturition at P ; ordinates (on a logarithmic scale) show reactivity expressed as the minimal number of units of oxytocin which causes a motor effect when added to 100 ml of Ringer-Locke solution (from Robson, 1933). by affecting the tonus differentially in different parts of the oviduct (Fitzpatrick, 1957). 3.114 Myoepithelial cells of mammary glands There is no known case of a hormone activating the duct muscles or myoepithelial cells of a gland in invertebrates. Mammalia. Branched myoepithelial cells (Fig. 3-6), which surround the alveoli and smaller ducts of the mammary glands, can, by their contraction, drive the milk from the ductules of the gland 66 KINETIC HORMONES — I down into the teats, achieving what is known to farmers as "let- down" of the milk. Figure 3-7 shows that injection of oxytocin into an anaesthetized bitch, Canis, stimulates the contraction of these cells, and increases the amount of milk available to the puppies. In the normal state, the act of suckling (or milking) stimu- lates the brain to induce secretion of oxytocin for this purpose. Fig. 3-6. Diagram of a generalized exocrine gland to show the location of factors which may influence the flow of secretion. Duct activity influenced by: 1, smooth muscle sphincters; 2, longi- tudinal smooth muscle shortening ducts or producing peristalsis ; 3, myoepithelium; 4, reservoirs in large ducts, or cisterns; 5, vasodilatation pressing on ducts or reservoirs; 6, vasoconstriction shortening inter-lobular vessels and squeezing adjacent ducts; 7, secretion from duct epithelium. Lobule activity influenced by: 8, smooth muscle bundles in inter- lobular septa squeezing lobules as a whole; 9, smooth muscle interspersed between alveoli; 10, vasodilatation or vasoconstriction affecting alveoli mechanically; 11, myoepithelium; 12, elastic fibre recoil in stroma, when pressure in distended alveoli is released; 13, nervous stimuli to secretory epithelium and smooth muscle; 14, hormonal stimuli to epithelium. In mammary glands the myoepithelial cells, 11, round the lobules, contract in response to OXYTOCIN. The secretory epithelium, 14, is stimulated by prolactin (§4.13). (From Richardson, 1949). § 3.115 VISCERAL MUSCLE 67 3.115 Muscles of blood vessels No case of hormone control of muscular contraction in the vascular system (vasoconstriction), apart from the contraction of heart muscle, has been reported for any invertebrate except, possibly, in the cephalopoda (p.416). Vertebrata. The arteries of vertebrates react to a number of hormones: for instance, to vasopressin*, ADH, and to a lesser extent to oxytocin, both from the neurohypophysis (§ 2.1 14). This effect, v^hich may be merely pharmacological, can best be shown by intravenous injection of vasopressin into any tetrapod after the peripheral blood vessels have been expanded by hypophysectomy (Buddenbrock, 1950). Certain doses of pure adrenaline dilate the peripheral blood vessels when first injected into the rabbit's ear, whereas subsequent injections of similar doses cause contractions (Fig. 3-8). This seems to be unexplained. 3.116 Other visceral muscles Vertebrata. Visceral muscles attached to the hair follicles in mammahan skin cause erection of the hair (as on a dog's neck or a cat's tail) in response either to sympathetic nerve stimulation or to ADRENALINE sccrction. Thcsc also cause the radial muscles of the iris of the eye to contract, thereby greatly enlarging the pupil. The release of adrenaline into the circulation, as a result of fear, shock or rage (cf. secretion in response to excitement, Fig. 3-3), is therefore accompanied by at least the sensation of the hair standing on end, and by the appearance of staring eyes with expanded pupils, and blanching of the face, due to the contraction of the peripheral blood vessels (§ 3.115). More useful features of this "emergency" syndrome due to adrenaline are the increased rate of heart beat (§ 3.111), and the enlarged blood flow bringing a greater supply of sugars to the body muscles (§ 5.211), which enable the animal to achieve a very high output of muscular effort for a time— probably long enough to effect an escape from the predicament causing the original fright. * This hormone would be better named antidiuretin from its main action (§ 5.32). 68 KINETIC HORMONES — I - 100 90 80 " *f y -\ 70 j // 60 / # / 50 - / / / 40 30 20 - / C-- 'If / Oxytocin 10 -^T^ 1 I I ! I 1 1 I 111! I I 2 3 4 5 6 7 8 9 10 II Time, min 12 13 14 15 16 17 18 Fig. 3-7. Effect of oxytocin on milk "let-down" in the nursing bitch, Canis. The time in minutes is given as abscissae and the amount of the milk yield (as indicated by the increase in weight of the pups) as ordinates. In the normal case, the pups get all the milk that the mammary glands can yield in about 8 min, there being an initial latent period before milk becomes available to them and a falling off as the gland becomes exhausted. In the right-hand curve, starting again at time 0, the pups were put to the anaesthetized mother and failed to obtain more than 30 per cent of the milk, after a very slow start. After 7 min an injection of 0.5 ml Pituitrin containing oxytocin enabled the pups to obtain almost all the rest of the milk, presumably by causing contraction of the myoepithelial cells round the alveoli. The injection of oxytocin has no effect upon the amount of milk secreted, as can be seen by the lack of effect of similar injections made when the curves had already exceeded 90 g (from Gaines, 1915). In the vertebrates, most types of visceral muscle which are innervated by the sympathetic fibres of the autonomic nervous system are thus seen to react also to secretion from the adrenal medulla; but the nerves of mammals are now known to secrete mostly noradrenaline at their end-plates, whereas the greater part of the gland secretion (derived from neurosecretory cells which originated in sympathetic ganglia) is adrenaline itself, although it is admixed with larger amounts of noradrenaline in the lower vertebrates, e.g. in the secretion of the suprarenal bodies of the elasmobranchs. Fig. 3-8. Changes in volume, increasing upwards as ordinates, of the perfused ear of a rabbit, Oryctolagus, with time in 30 sec intervals as abscissae. In the upper tracing increase in volume is due to dilation of the blood capillaries following the first injection of adrenaline. The lower tracing shows the opposite effect of two later injections of the same dose. This is an example of reversal from a dilator response of the capillaries to a constrictor response with repeated doses (from Burn and Robinson, in Welsh, 1955). § 3.12 SOMATIC MUSCLES 69 3.12 SOMATIC MUSCLES Cephalopoda. Ablation of the epistellar body of the octopod, Eledone moschata (§ 2.111, Fig. 2-4), is followed by general loss of muscle tone, with even the tentacles hanging limply ; but recovery starts about a w^eek after the operation (Young, 1936). In one case the tone gradually returned to normal during the 186 days for which the animal survived, although there was no trace of regene- rated epistellar tissue. No attempt seems to have been made to restore the tone by injecting extracts during the first post-operative week; but control experiments made it clear that the effect cannot have been due to shock, which is an important factor in these very sensitive animals with their highly developed nervous systems. The loss of muscle tone extended to the chromatophores, so that animals without the epistellar body became abnormally pale (cf. §3.21). Crustacea. Well-controlled experiments (Roberts, 1944) show that exposure of the crayfish, Cambarus virilis, to relatively bright light releases an unidentified eyestalk hormone that reduces loco- motion. It is possible that this hormone may act either by raising the stimulation threshold of the skeletal muscles of the legs or by lowering the strength of the nerve impulses from the brain ; but it seems more probable that the result is due to an indirect metabolic efltect (§5.1). The reaction may have adaptive value because the animals normally feed by night and escape from predators by remaining hidden by day. Insecta. The normal rhythm of nocturnal activity of the cock- roach, Periplaneta, is stimulated by a hormone, from the suboeso- phageal ganglia, the secretion of which is controlled through the ocelli. If the illumination is kept constant, or the ocelli are painted over, the rhythm disappears. The action of the hormone has been shown by implants and in parabiotic experiments, in which two cockroaches are so joined that blood can flow from one to the other. If, for instance, a specimen in which the ocelli have been occluded has another specimen, with no legs, joined to its back, then normal diurnal changes in illumination acting upon the upper specimen cause a correlated rhythm of locomotor activity in the lower (Harker, 1956). 70 KINETIC HORMONES — I Vertebrata. There are no specific hormones in vertebrates to control somatic muscles, although these are noticeably affected by the hormones from the gonads and also to some extent by thyroxine and adrenaline. For instance, the loss of testosterone in castrated mammals, such as cart-horses, results in lowered spontaneous activity and muscle tone, and is shown by the whole stance of the animals, as compared with a stallion, Equus. In females, the pres- ence of oestrogen in the circulation during oestrus (§ 4.234 and Part II, § 4) is accompanied by a great increase in activity, as has been shown by attaching a pedometer to a sow. Similar variations in activity are shown during the 4-day oestrus cycle of rats (Beach, 1948) ; a loss of 82 per cent in muscular activity can follow ovari- ectomy. It seems probable that all these effects and those of thyrox- ine may be the result of metabolic changes caused by changes of hormone balance, rather than of any direct kinetic action of the hormones on the muscle contractions, or even on the tonus. The action of adrenaline is slightly to prolong the active state of the muscle fibres and to increase the tension accompanying a twitch initiated by the nervous system; it does not itself cause contraction of skeletal muscle as it does in the case of visceral muscle (Goffart and Ritchie, 1952). 3.2 Control of pigmentary effectors Pigmentary effectors of a variety of animals bring about colour changes mainly in two distinct ways: one in which extrinsic muscle fibres change the shape of the colour-containing cells, and the other in which the pigment granules themselves are moved within the confines of a stationary cell. Rarely, the cells change shape and may even move their position. These processes cause so-called physiological colour changes (Tables 8 and 9). "Morpho- logical colour change" (Sumner, 1940, and Dawes, 1941) produces similar effects by slow alteration in the total amount of pigment present, rather than by its redistribution. This reinforces the adaptive physiological changes when the external conditions remain relatively constant for days or weeks. Together they play an important part in providing "protective coloration" for the animal. §3.2 CONTROL OF PIGMENTARY EFFECTORS 71 [^ 5 s •w <;j ft o o g ^ S ^ « .^ Q ^^^^ 2 S 9 bo a o o -^ CO w CO TJ Ji c 3 n1 CO hO '■§ C/3 3 i cn U ^1 I I M I I I I I I I "t- ^ «3 '^ a -ts • S-S ^ ?^ rt i3 ^3 >4 ft5 72 KINETIC HORMONES — I 3.21 CHROMATOPHORES WITH MUSCLES Cephalopoda. This type of chromatophore occurs only in cephalopods, and forms a convenient link between those muscular structures which have been considered in the previous section, and the chromatophores with movable granules which follow. Fig. 3-9. Chromatophores with muscles from a cephalopod, Loligo; on the left the muscles are relaxed and the chromatophore cell is elastically contracted so that it looks pale, with the pigment in a small mass at the centre; on the right the muscles have con- tracted and stretched the cell body to which they are attached so that the chromatophore shows the maximum amount of colour (from Bozler, 1928). The cephalopod chromatophore (Fig. 3-9) consists of a central pigment-containing cell with a highly elastic wall, and from 4-24 single muscle fibres, attached radially around the circumference ; when the fibres all contract, they increase the area of exposed pigment. Contraction of muscles, therefore, corresponds to ex- pansion of the pigment and a darkening in appearance of the animal. Although each muscle fibre is under direct nerve control, the fibres to any one chromatophore usually contract together; but adjacent chromatophores can be separately stimulated to produce the very rapid and varied patterns of colour change which are characteristic of cephalopods and appear to be connected with their emotional states, as well as related to the colour of their environment. § 3.22 EFFECTORS WITH MOVABLE PIGMENT GRANULES 73 If this were the whole story, cephalopod chromatophores would deserve no place in the present context; but overall changes of colour can also be produced by chemicals in the blood. Tyramine from the posterior salivary glands is normally used as poison for paralysing the prey, but is also associated with pigment dispersion, so that animals usually become much darker in colour when the salivary glands are active ; betaine in the blood is associated with pigment concentration. Moreover, of the three Mediterranean octopuses, Eledone moschata and Octopus macropus are normally well-coloured species, but Octopus vulgaris is pale and habitually has less tyramine in its blood. Transfusion of blood from either of the first two into the last of these species results in darkening. Denervated chromatophores, on the other hand, are completely insensitive to these chemicals (Bacq and Ghiretti, 1951). Tyramine and betaine can therefore be better compared to "para-activators" (§ 1.2) than to true hormones, in that they seem to act through the nervous system and not directly upon the effectors. Their mode of action is still uncertain; Sereni (1930) postulated that they might act directly upon the inhibitory and excitatory centres in the brain, but this has not been fully established. 3.22 pigmentary effectors with movable pigment granules Although varying in form and situation, these effectors all contain pigment granules which move to and fro within them, dispersing widely to give a large coloured area, or concentrating into a limited space to give only a small spot. Dispersal here gives the same effect as contraction of the muscles around a cephalopod chromatophore. The most plausible explanation of how this granule movement is brought about is that given by Marsland (1944) for the branched chromatophores of fish; but it seems probable that the same principle underlies all cases. The pigment granules are attached to a partially gelated system of long protein molecules in the cyto- plasm of the cell, and as the cytoplasm gelates fully the molecules contract, drawing the granules ''as on a string bobbing through the current" towards the centre of the cell, while squeezing the 74 KINETIC HORMONES — I "plasmosol phase" out into the tips of the cell branches. Dispersal may then be a re-stretching of these proteins on solation, and not a matter of Brownian movement. High pressures (up to 8000 Ib/in^) can be shown to cause solation, and to inhibit the concen- tration of the granules. This is on a par with amoeboid movement and particularly with muscle contraction ; but the problem is still unsolved of whether either nerve stimulation or hormone action can affect the state of the chromatophore proteins in the same way as in the muscles, or whether changes in osmotic pressure (Abramowitz and Abramowitz, 1938) or in permeability of the cell membrane play a part. As a rule chromatophores react much more slowly than muscles ; but there is a great difference in the reaction speeds of the similar-looking chromatophores of Amphibia, Reptilia and various Crustacea. This may be a question of the strength of stimulation. The effectors with movable pigment fall into three types. (1) The epidermal cells of certain Insecta are relatively un- specialized, except for the presence of pigment granules, which may be of more than one colour and may move in different direc- tions (Fig. 3-10, § 3.221), (2) The retinal pigment cells of certain Crustacea and Insecta occur in two positions, proximal and distal, round the ommatidia of the compound eyes (Fig. 3-12, § 3.222), and contain moving granules of black pigment. Movement of reflecting, or white pigment, granules in cells round the retinulae also occurs. (3) The branched and specialized chromatophores of Crustacea and some other invertebrates, as well as of lower Vertebrata, may be epidermal, but are usually mesodermal, and may contain more than one pigment; but if so, each pigment remains in its own branch of the cell (Plate 3-1, § 3.223). 3.221 Pigment movement in epidermal cells Insecta. The cell-boundaries are reputed to disappear between moults, and the pigment granules can then migrate to an extent comparable with that in chromatophores. In green specimens of the stick insect, CarausiuSy the colour change is obscured by stationary yellow-green pigment; but in brown specimens the red pigment moves parallel to the surface of the epidermis, and the § 3.221 EFFECTORS WITH MOVABLE PIGMENT GRANULES 75 black melanin, which has been more fully observed, moves almost at right angles to it, to cause darkening in appearance (Fig. 3-10). The upward, or dispersing, movement of the melanin depends on both moisture and light, the effects of which are transmitted by the nervous system to the brain, and thence, either by nervous stimulation or by neurosecretion through the circumoesophageal connectives, to the suboesophageal ganglion (§2.111). This releases a Car^w^w^-DARKENING hormone which passes in the blood to disperse the melanin. The ganglion cells only release the hormone if the connectives from the brain are intact (Dupont- Raabe, 1956). If moisture is maintained constant, the melanin granules con- centrate in the light and disperse in the dark, and tend to maintain mm ?v m^^KA^sh* ^W^^^'^^^^ Fig. 3-10. Two diagrammatic sections through the skin of the stick-insect, Carausius, to show the movement of pigment granules in epidermal cells. The clear space above indicates the position of the cuticle. The pigment in the upper section is concentrated m the light-adapted position, and in the lower dispersed, in the dark- adapted position. The green pigment (1) remains stationary; the red pigment (2) disperses laterally above the nuclei; the dark, melanin-like pigment (3) disperses mainly outwards (from Giers- berg, 1928). 76 KINETIC HORMONES — I this diurnal rhythm for a time under constant conditions. They show a Umited background response, becoming darker on an illuminated dark background than they are on a white or yellow background. In constant light, the melanin disperses in response to moisture, under the control of what is probably the same darkening hormone, since its secretion is also stimulated through the nervous system (Giersberg, 1928). This was established by putting the insect into a humid box, with its head projecting through a diaphragm into the dry air outside (Fig. 3-11); this induces darkening of the whole animal from head to tail in about half an hour. If a ligature is put round the body to prevent the circulation of the blood and hormone to the tail end, the darkening only affects the part in front of the ligature. If the ventral nerve cord is cut at a level just outside the humid box, no darkening takes Fig. 3-11. The stick-insect, Carausius, with the hinder part of the body in a moist chamber and the head and thorax projecting through a membrane. The pigment dispersion, caused by the moisture, is transmitted by a darkening hormone from the suboesophageal ganglion. This acts only upon the head and pro- thorax because of the ligature just behind them (from Giersberg 1928). place, because the stimulus from the damp skin is not conducted to the brain. But if the same animal, with the nerve cord cut but the body unligatured, is then reversed, with its head in the box and the tail left out, the whole body darkens, because the stimulus from the skin of the head can reach the brain, which therefore stimulates the secretion of the hormone. This then circulates freely to all parts of the body. Secretion of the darkening hormone has been located histologically in the suboesophageal ganglia and confirmed experimentally by injection of extracts into animals from which the brain had been removed. Headless animals, lacking the source of the darkening hormone, have their pigment fully con- centrated, as in the normal light-adapted animals ; they therefore § 3.222 EFFECTORS WITH MOVABLE PIGMENT GRANULES 77 form good test subjects for extracts containing the darkening hormone, and show that extracts from other parts of the brain are also active, but those from the corpora allata and cardiaca are inactive (Dupont-Raabe, 1954). A Hghtening hormone has not yet been located, partly because of the lack of a suitable preparation for testing extracts. It is not clear why damp-adapted insects with dispersed pigment are not used. One source, at least, of the concentrating hormone must be situated in the body, since it is present in headless animals from which the corpora cardiaca are almost certainly absent (Dupont-Raabe, 1956). Otherwise, it might perhaps have been supposed that the corpora cardiaca would yield a Carausius- lightening hormone, since they yield a neurosecretory substance (Cameron, 1953) which concentrates the melanophores of Crago (Knowles, Carlisle and Dupont-Raabe, 1955, § 3.223), and some, at least, of the erythrophores of Leander. It seems significant that the concentration of both these crustacean chromatophores are light-adaptations, and it is a light-adaptation hormone that appears to be missing from Carausius. 3.222 Pigment movement in retinal cells Crustacea. The compound eyes of most Crustacea have three groups of pigment-containing cells round each ommatidium; distal and proximal retinal cells that surround the cone (Fig. 3-12), and reflecting cells that extend below the basement membrane but are not shown in the figure. These last contain white pigment, which behaves like the pigment in white chromatophores (§ 3.223) and is probably under similar hormone control. The proximal retinal cells contain dark pigment granules, which may be fixed in appositional eyes, such as those of Eupagurus, but which in many species can be dispersed outwards to isolate the sensitive rhabdomes and convert superpositional eyes into a temporarily appositional type for accurate vision in bright light (Bruin and Crisp, 1957). The movement of the granules in these cells is sometimes a direct effect of light, as in some chromatophores (§ 3.223); but in others it is controlled by hormones, that appear to be the same as those controlling the distal cells (Knowles and Carlisle, 1956). 78 KINETIC HORMONES — I I ■crn. -con.. -d.r.c. — rh. o/\o d.r.Cr p.r.c— Fig. 3-12. Three ommatidia from the compound eye of the cray- fish, Camharus hartoni\a-c in longitudinal section and d-f in surface view, (a) Ommatidium of a typical dark- adapted eye with pigment concentrated at two levels : round the cone (con.) in the distal retinal cells (d.r.c), and below the basement membrane (b.m.) in the proximal retinal cells (p.r.c). The corresponding surface view (d) § 3.222 EFFECTORS WITH MOVABLE PIGMENT GRANULES 79 The distal retinal cells contain dark, melanin-like pigment granules. The pigment movement can be observed in the intact eye (Fig. 3-12, d-f), and is accurately adapted to the light intensity, especially over the lov^er ranges, such as inshore, under- water animals are most likely to encounter. The adaptive move- ment is, however, achieved in two different ways : (i) by the usual migration of pigment granules in stationary cells (Fig. 3-12), and (ii) by contractile fibres which change the shape of the cells, thereby causing redistribution of the pigment in relation to the rhabdome (Fig. 3-13). This latter process overrides any sign of pigment migration (Parker, 1932). Migration of pigment granules in stationary distal retinal cells In the crayfish Astacus and Camharus and the crabs Dromia and Maia^ the pigment granules move inwards to meet the out- ward movement of the proximal pigm.ent, as they become light- adapted; they move outwards, towards the surface of the eye, to reach the dark-adapted position, which occurs in dim light, rather than in complete darkness. The latter movement appears to be comparable to concentration of pigment in the cell body of a chromatophore ; the former inward movement is like dispersal of the pigment into the stationary but outlying cell-processes (Fig. 3-12C). In Cambarus, inward migration, or dispersal, of retinal pigments to their light-adapted position is induced by injection of an eye- stalk extract, the extent of the migration being dependent on the quantity of the retinal-pigment-dispersing hormone, RPDH, used. The threshold for response of the distal pigment is lower than that for the proximal pigment (Fig. 3-12, b and c). No shows a bright orange centre to the cornea (cm), when seen by reflected light, because the rhabdome (rh) is unscreened by the proximal pigment, (b) and (e) In response to the injection into a dark-adapted animal of retinal-pigment-dispersing hormone extracted from one eyestalk, the distal pigment has partially dis- persed inwards but the proximal has not moved towards the light- adapted position, (c) Extract from two eyestalks has been injected and pigment in both sets of retinal cells has dispersed to the fully light-adapted position. In surface view (/) this eye appears black all over, as in a naturally light-adapted animal (from Welsh, 1939). 80 KINETIC HORMONES — I evidence of the action of a concentrating hormone has been reported (Welsh, 1939). Movement of pigment due to change in shape of distal retinal cells Change in cell shape in the prawns Palaemonetes and Leander and in a Bermudan shrimp Anchistioides is brought about by con- tractile fibres (Welsh, 1936). The distal retinal cells here lie in the same position round the cones of the ommatidia, as in Cam- barus; but if the usual pigment migration occurs, it is obscured by such a surging inwards or outwards of the protoplasm that, even the nucleus is moved as well as the pigment granules, and the whole cell appears to change shape (Fig. 3-13fl). If the pigment is dissolved away, fibres in these cells can be seen to cause the inward pigment movement by their contraction (Welsh, 1930; Fig. 3-13^). It may be noted that physiologically the same effect is produced by this fibre contraction as by pigment dispersal in Cambarus (Fig. 3- 12c), yet these would appear to be opposite reactions in terms of contraction of protein molecules. Much work on Palaemonetes and Leander has confirmed the action of a retinal-light-adapting hormone, from the sinus GLAND. This has been found to be similar to that in such Brachyura as Cancer and Uca (Kleinholz, 1936); but it is not clear if it is the same as RPDH of Cambarus. Evidence for an antagonistic, RETINAL-DARK-ADAPTING HORMONE has been found in Palae- monetes, and the richest extracts have been obtained from the TRiTOCEREBRAL COMMISSURES (§ 2.112; Brown, Hiucs and Finger- man, 1952). A persistent diurnal rhythm of movement of distal retinal cells has been shown, at least in continuous darkness, for Anchistioides (Welsh, 1936), Palaemonetes (Webb and Brown, 1953), and Leander, Praunus and Pandalus (Bruin and Crisp, 1957); for this a hormonal control has been postulated. It is not inhibited by sinus gland removal ; but it is probable that the sinus gland is, as in other cases, only the storage organ for the hormone, of which sufficient is still secreted from the source in the ganglionic-X- organ (§ 2.112) to maintain the rhythm. Changes in distal retinal pigment of grapsoid crabs (R. I. Smith, 1948) are very similar to those in prawns, with a marked § 3.222 EFFECTORS WITH MOVABLE PIGMENT GRANULES 81 Fig. 3-13. Two ommatidia from the compound eye of the prawn, Palaemonetes , in longitudinal section to show pigment cells which change shape, (a) In Z), the ommatidium is shown with the pigment cells in the extreme dark-adapted position; in L, in the extreme light-adapted position. As in Fig. 3-12, the pigment in the distal retinal cells (d.r.c.) surrounds the cone (con.) in the dark position, and the rhabdome (rh.) in the light. In L, the pigment in the proximal retinal cells (p.r.c, not shown in D) has dispersed outwards from below the basement membrane (just out of the figure), (b) The same ommatidia with the pigment removed to show contractile fibres (c.f.) in the distal retinal cells (d.r.c). They are attached to a mass of accessory pigment (ac.p.). In D, the fibres are relaxed in the dark-adapted position; in L, the fibres are contracted in the light. Most of the cell protoplasm and the nuclei (d.r.n.) have moved inwards with the pigment, leaving only an attenuated distal cell process (d.p.). The nuclei of the proximal or retinula cells (r.t.n.) remain stationary (from Welsh, 1930). diurnal rhythm that persists in constant darkness; but this is reduced or absent in continuous light. The rhythm can be induced experimentally to become out of phase with the time of solar daylight. About one-third of the dark-adapting hormone of the crab eyestalk is in the sinus gland, and two-thirds in the ''optic 82 KINETIC HORMONES — I ganglion", which presumably indicates the ganglionic-X-organ as its source. The light- adapting hormone for the distal retinal pigment comes from the same source as that which concentrates the red chromato- phores of Palaemonetes (PLH § 3.223), and the two may be the same, since they both appear to cause contraction of protein molecules. Yet it requires 20 times as much crude extract to be effective on the retinal cells as on the chromatophores (Kleinholz, 1942). Insecta. The distal retinal cells in the compound eyes of in- sects can be divided into the same two types as in Crustacea; but the means of controlling their pigment migration is not known. 3.223 Pigment movement in chromatophores There is an embarrassing w^ealth of detail about the so-called chromatophorotrophic or chromactivating hormones from which it is difficult to select representative examples. They are interesting because they have similar physiological functions in both Crustacea and the cold-blooded vertebrates, and because similar methods of investigation have been applied to both groups, so that they can be directly compared. Chromatophores are usually elaborately branched cells which apparently remain stationary in the tissues, although they become Plate 3/1. Coloured photographs of the prawn, Leander serratus. (a) Dorsal view of the cephalothorax to show the pattern formed by the differentiation of the chromatophores into large red ones form- ing the stripes, with small red and white ones between ( X 4). (b) Part of the same, enlarged to show that there is more than one pigment in each chromatophore ; the yellow component in the red chromato- phores can be seen faintly and the central red component of the reflecting, white chromatophore is clear. All are fully dispersed ( X 50). (c) Two eyestalkless specimens kept on a white background, on which red pigment becomes fully dispersed. Half an hour before the photographs were taken each was injected with a different fraction of an extract of the sinus gland. That given to the upper specimen caused strong red pigment concentration; that given to the lower specimen had no effect, {d) Part of the tail fan of a specimen like the last, in which the maximum dispersion of red pigment is shown (from Knowles, 1955). Plate 3-1 («) (b) (c) id) J» :■■•■ .- % # : 9- ''■ 1 2 3 4 5 Fig, 3-14. Photomicrographs of melanophores from the wch of the foot of the clawed toad, Xenopus laevis, with pigment in progressive stages of dispersion corresponding to the melanophorc indices 1 to 5. Values such as 15 are sometimes recorded directly; but they usually represent the mean of several readings (from Hogben and Slome, 1931) Fig. 3-18. Photomicrographs of chromatophores exposed by remov- ing a scale mid-dorsally from the killifish , Fundidus. {a) Black-adapted with melanophores dispersed and showing iridosomes at the centre of some ; guanophores are concentrated and xanthophores appear grey, {b) The same after injection of adrenaline. The melanophores are concentrated, and the guanophores widely dispersed as they would be in a light-adapted specimen (from Odiorne, 1933). Fig. 3-19. Photographs of the male fiddler crab, Uca piigilator, in the light. The normal specimen on the left is dark ; that on the right had the eyestalks removed 2 hours previously and the pigment has concentrated in the legs; the pallor shows best in the large asymmetric chela. The carapace is too thick to allow the colour change to be seen through it (from Carlson, 1936). P'iG. 3-20. Photographs of the head of the sea-slater, Ligta oceanica, with the antennae cut off short, (a) Face-view; (b) lateral view of the head and eyes, differential illumination of which controls the release of chromactivating hormones (from Smith, 1938). § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 83 difficult to see (Plate 3-1) when their pigment granules "con- centrate" at the centre. The branches reappear as the pigment ''disperses" to their extreme tips. The cause of this pigment migration has already been referred to (§ 3.22). The cells are sometimes named according to their pigments: "erythrophores" for those with red pigment; "guanophores" with white guanin; "melanophores" with black melanin, and "xantho- phores" with yellow pigment. Some cells have black pigments other than melanin. All these colours are to be found in vertebrates ; the Crustacea, especially the Malacostraca, have other lipophores, or coloured cells, with a blue pigment as well. Both may have iridosomes, with a movable reflecting material giving a blueish- white appearance (Fig. 3-18). The control of chromatophores in Crustacea is by hormones only, but can be either by nerves or hormones or a combination of the two in vertebrates. The type of control affects the response somewhat, since hormones, which reach all the chromatophores through the blood, tend to produce a slow and similar response in all parts of the animal, whereas nerve control can quickly stimulate individual chromatophores to produce a colour pattern that may match the background closely, as in Pleuronectidae and cham- eleons. Such an effect can only be simulated in those Crustacea in which the chromatophores themselves are much differentiated and each type responds to distinct hormones. This seems to be the case in the prawn, Leander, in which some chromatophores are large and together form dark bands, while others supply a stippled and variable body colour. They include as least four colours and can be adapted to a variety of backgrounds (Plate 3/1), but their control is not yet fully elucidated (Knowles and Carlisle, 1956), and is too complicated to use as an example here. Among vertebrates, it is the chromatophores of all Agnatha, Elasmobranchii and Amphibia, but of only some species of Teleostii and Reptilia, that are under hormone control (Fig. 3-24). The chromatophores of the leeches, Hirudinea, concentrate in light and disperse in the dark, but show no background response. They are probably all under nerve control, and need not be con- sidered here. A typical background (or albedo) response is related to the 84 KINETIC HORMONES — I amount of light reflected from the background to the eyes. It helps to afford protective coloration to the animal, so that on a light background the result is a pale appearance; but to achieve this the dark pigments must be concentrated and the white dis- persed. It is a wide-spread phenomenon in both Crustacea and Vertebrata, and more often than not it is controlled by a pair of antagonistic hormones, which between them can maintain the pigment in the chromatophores at any position between full concentration and full dispersion. Observation on the behaviour of chromatophores has been facilitated by the introduction of a chromatophore or melanophore index, by which five stages of pigment dispersion are defined, from 1, fully concentrated, to 5, maximally dispersed (Fig. 3-14). Half stages can be used ; but it is important to use chromatophores in the same region of the body for successive measurements, as different groups of cells can show considerable differences (Hogben and Slome, 1931). It must also be remembered that intervals on the chromatophore index scale are not quantitatively equal nor exactly related to the dosage of hormone needed to shift the pattern from one stage of dispersion to the next. Their representation by equal intervals on a graph can, therefore, be somewhat misleading. Before considering hormonal control of responses in more detail, it must be emphasized that several extraneous factors, other than "morphological colour change" (§ 3.2), can also affect chromatophores. The direct effect of light on chromatophores usually causes dispersion (rarely concentration) in the absence of either nerve connections with a light receptor, or of any hormones in the circulation. Stephenson (1932) showed this clearly in the hermit crab, Eupagurus prideauxi, which has functional red and yellow chromatophores, not only on the exposed limbs, but also on the abdomen, where they are usually hidden within the whelk shell that it carries. The direct effect of bright light causes pigment dispersion, whereas the secondary effect, due to hormones stimu- lated by the eye, is to cause concentration of pigment. On an illuminated light background in a dull light, only this secondary response is elicited, and the animals are pale all over ; but in bright light the pigment on the limbs disperses considerably. That this §3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 85 is due to antagonism between the direct dispersion and the second- ary hormonal concentration can be seen by quickly removing the crab from its sheher; at first, the newly exposed abdominal chromatophores are concentrated by the hormone effect only, but rapidly darken to match those on the limbs, as a result of exposure to the direct effect of the light. A similar direct effect occurs among reptiles. When a cUmbing lizard, AjioUs, is blinded, its chromatophores show dispersal in the light and concentration in darkness (Brown, 1950^). The degree of dispersion usually in- creases with increase in the light intensity. Temperature increase can concentrate the pigment in the white chromatophores of Palaemonetes (Fingerman and Tinkle, 1956) and thereby counteract its dispersion in response to a bright white background. This may help to keep the state of dispersion under control in very bright, hot conditions. In reptiles, such as Phryno- somaj concentration of dark pigments at high temperatures may help to prevent excessive heat absorption by the body. Moisture disperses some pigments, like those of Caraiisius (§ 3.221) and that in the melanophores of the frog, Rana, where the concentration due to a light background may be enhanced by dry conditions or almost completely overcome by total immersion. Other degrees of moisture give intermediate results. A rhythm of colour change related to the tides is said to persist for some time, under still water conditions in the laboratory, in inter- tidal forms such as the fiddler crab, Uca pugnax. A diurnal rhythm of colour change persists under constant conditions of either light or darkness among other Crustacea, Insecta and Amphibia, and may be mediated by hormones in some cases, such as Ucapugilator. In experiments designed to show the action of hormones in controlling physiological colour change, care must be taken to control or eliminate these extraneous factors, affecting the reac- tions of the chromatophores. Once this has been done, two main lines of attack on the problem can be followed, though a combina- tion of the two is needed for its full elucidation. The first method is that due to Hogben and Slome (1931 and 1936), who studied the behaviour of the chromatophores in relation to changing environmental factors in intact Xenopus. This, which may be called the physiological method, they later 86 KINETIC HORMONES — I extended by the second, or pharmacological, form of investigation, using injections of organ-extracts and purified hormones. The second method has been that chiefly exploited in the search for crustacean hormones by Brown and his co-workers in America; they inject extracts of suspected endocrine organs into variously prepared animals, for which environmental changes are as far as possible ruled out. This method can eventually lead to the location of the source of the hormones, and since about 1942 it has been shown (Brown, 1944), for a steadily increasing variety of pigment cells with moving granules, that two hormones are in control (Table 9), although, for technical reasons, one is usually easier to demonstrate than the other. This, perhaps, accounts in part for the controversy which centred for so long around the problem of whether or not a second antagonistic hormone was necessary to account for crustacean colour change, or whether all the observed changes in any given animal could be accounted for by quantitative differences in a single hormone (Parker, 1948). The following account of the reactions of chromatophores and their control by hormones in Arthropoda and Vertebrata is only limited to red, white and black pigments in order to simplify the picture (Table 9) ; this is perhaps to oversimplify it, but it should suffice to give a frame of reference within which to consider further data, such as those relating to the complex system in Leander (Carlisle and Knowles, 1959). Much more information is still needed to extend the knowledge of most species from the realm of mere pharmacology towards an understanding of their natural physiology. Red pigment in chromatophores Crustacea. Physiological colour change in Crustacea is rela- tively slow in reaching equilibrium; but it can bring about an almost complete reversal of the state of some chromatophores in two or three hours. For instance, the following observations can be made on the red chromatophores of Palaemonetes, or of the fiddler crab, Uca (Figs. 3-15 and 3-19), if in the latter case it is remembered that the animals show a diurnal rhythm of colour change in daylight, so that observations must all be made over the same few hours of the day. One group of animals can be adapted § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 87 t/5 a I a, ijq b a, cq b CI, b a. >^bo fi( W U) Pu W P hJP C3 o-. u u n3 CO CO '^' rt CO CO r-i C/2 a a o o 3 C CO r>.. cC n., CO •— I •3 tao m C (n m ■5 'i t •2 -S •- c * I? 13 S u 7. -T3 PQ 1 1 I ■SJ c« ^ iJ (U -Si C! CO 1 § -^ ^O u Ct^ CO ). The first conclusive evidence that such changes in the chromato- phores were controlled by a hormone was obtained by Perkins (1928), who showed for the prawn, Palaemonetes, that, although cutting the nerve supply to any part of the body does not interfere with its colour responses to a change of background, a ligature occluding the blood supply to that part stops the response, as in the stick insect Carausius (§ 3.221). Release of the ligature restores the response. Concentration of red pigment in Palaemonetes. The source and action of the red-concentrating hormone that causes the response to a white background has been identified in Palaemonetes by the pharmacological method. In the first place it is found that, in prawns and other Decapoda, except the Brachyura, removal of the eyestalks destroys the background response and leads to permanent dispersion of the pigment in the red chromatophores ; extracts of the eyestalks can overcome this dispersion and lead to a temporary concentration of the pigment. Within the eyestalk the most potent source of the red-concentrating or Palaemonetes- LiGHTENiNG HORMONE, PLH, is in the sinus gland. Moreover eyestalk extracts from most Decapoda, except crabs, produce this pigment concentration, if injected into a dark, eyestalkless prawn ; PLH is therefore not specific to any particular genus (Brown, 1950^). Much contradiction in earlier work was due to the fact that crude eyestalk extracts, containing PLH, were admixed with varying amounts of a second type of hormone that has a darkening effect on crabs. These two kinds of hormones were first separated § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 89 by fractionation in alcohol (Brown and Scudamorc, 1940); but much greater elegance and precision in separating pure substances from the extracts is now possible by paper electrophoresis (Knowles, CarHsle and Dupont-Raabe, 1955). So far the substances cluted Backgrounc chanaes .'-•' -•-Intact -o Intact (a) , , 1 Connective injection V y >< Intact Eyestalkless 1 I i!Sk!i=gWjDJi£tion Sinus g land froctions Intact / > '+ insol. / Eyestalkless Eyestalkless"^® ^^Z" / sol. (d) 2 3 12 3 Time, hr Fig. 3-15. Reactions of the red chromatophores of the fiddler crab, Uca. id) The natural background response: the rising curve (dotted line) shows the rate of change of red chromatophores after a light-adapted specimen is transferred from a white to a black background; the falling curve (full line) shows the converse change from black to white, in constant illumination. All measure- ments were made over the same time of day to avoid the influence of diurnal rhythmic changes. (6) The response of intact and eye- stalkless specimens from black backgrounds to injections of Uca~ RED-DISPERSING HORMONE, URDH, from the sinus gland. Prompt dispersal occurs in the eyestalkless specimen; the dark intact specimen shows pallor first, presumably due to operative shock, (c) The response of similar specimens to injections of an extract of circumoesophageal connectives. The result indicates a short-lived action of t/ca-RED-CONCENTRATING HORMONE, URCH, but is ambiguous, {d) The responses of eyestalkless specimens to in- jections of alcohol insoluble (above) and alcohol soluble (below) fractions of a sinus gland extract, to show that the former contains most of the dispersing hormone, URDH (all from Brown, 1950). 90 KINETIC HORMONES — I from different spots appearing on the paper have only been tested upon eyestalkless prawns kept on a white background ; in these the red chromatophores are fully dispersed owing to the lack of PLH, reinforced by the dispersing effect of direct light. It follows that only light-adapting substances causing concentration of red pig- ment can be satisfactorily identified, though by using sufficiently concentrated extracts relatively rapid reactions can be obtained (Fig. 3-16). Other parts of the nervous system yield extracts with an action similar to that of PLH, but it is more than likely that time (min.) Fig. 3-16. Effect of extracts of the tritocerebral commissures on the dispersed red pigment in the chromatophores of eyestalkless prawns, Penaeus hraziliensis . The effect of sinus gland extracts would be similar. The abscissae represent time in minutes after the injections; the ordinates, the state of dispersion of the pigment on the same scale as the melanophore index. The dotted lines show that readings taken at night are closely similar to those by day, shown in full lines. The extracts from the post-commissure region ip.c.) are rather more active than those from the main commissure (c.) in yielding a pigment-concentrating hormone, the effect of which is to cause rapid pigment concentration. This wears off as the hormone is destroyed in the tissues and the pigment returns to its original state of dispersion (from Knowles, 1953). § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 91 these are precursor substances, rather than the natural hormone, since they often have mixed effects or need to be "activated" in some way, such as boihng or extraction in alcohol, before becoming effective (Figs. 3-15-17). Dispersal of red pigment in Palaemonetes. The red-dispersing or Prt/«^mo;z^^^5-DARKENiNG-HORMONE, PDH, that acts antagonistic- ally to PLH, has been difficult to locate because it requires a test animal that is pale, yet contains no source of an overriding, concentrating hormone. This has been achieved by starting with eyestalkless specimens of Palaemonetes in which the red pigment is fully dispersed (index 5); then at time (Fig. 3-17) an extract containing PLH from either sinus gland or tritocerebral commis- sures is injected, and pigment concentration becomes almost complete in 15 min (index 2 or even 1.5). After this, the effect wears off slowly as the hormone is destroyed in the tissues and the chromatophores gradually return towards full dispersion. The rate of this return is unaffected by an injection of sea water at 30 min (Fig. 3-17, curve i). If, instead of sea water, an injection of an extract of the darkening hormone, PDH, from either abdomi- nal ganglia or circumoesophageal connectives is given, the rate of dispersion is greatly increased (index 4 is reached in 15 min), and may continue until full dispersion is achieved, within an hour from the start of the experiment (Fig. 3-17, curve ii). This shows conclusively the presence of PDH in the extract, whereas testing this extract on eyestalkless animals which had not been pre- treated and were therefore dark, though consistent with this interpretation, does not by itself prove the activity of the extract (Fig. 3-17, curve iii). If PDH is injected into normal, eyed animals with pigment concentrated in the light, only a slight dispersion is produced and this is quickly followed by a return to full concen- tration, showing that the action of PDH is unable to overcome the naturally secreted PLH (Fig. 3-17, curve iv). To allow for the natural reversal of the chromatophore response within a relatively short time, a further elaboration of the two- hormone hypothesis has been postulated, namely, that in response to a black-to-white background change, a large amount of PLH is discharged suddenly into the blood, but that "as adaptation becomes complete there would be a reduction of the hormone 92 KINETIC HORMONES — I level to some lower maintenance one". Conversely, a white-to- black change would result in an abrupt discharge of PDH, which would become similarly reduced to some lower level as adaptation became complete. "The introduction of large amounts of either factor, in the presence of a somewhat lower titre of the second, o ( ^cr-^ — '"l II' • Eyestolkless '/ / Injections: 4 / orTritocerebral (PLH) = Abdo:gang: (PDH) 1 / • = Seawater i 3 . / Q. / O / 1 O / / !^ \ .• 1 Eyestolkless <- I. / ^ , ^-~,f iv Intoct 1 12 3 Time, hr Fig. 3-17. Effects of extracts containing Palaemonetes-iAGYiT^i^Y^G and -DARKENING HORMONES, PLH and PDH, on the red chromato- phores of the prawn, Palaemonetes. Pigment dispersion is shown with time in hours, on a much less extended scale than Fig. 3-16; the extracts are less concentrated and act more slowly. Curves i, ii and iii show effects in eyestalkless specimens with initially dis- persed pigment: curve i: the effect of PLH injected at time wears off and is unaltered by subsequent injection of seawater at the time marked by the arrow; curve ii: PLH injected at time followed by PDH, instead of sea water, injected at the arrow, shows the rapid dispersing effect of PDH; curve iii: PDH injected at time pro- duces a slight concentration due to shock and a rapid return to full dispersion. This last is compatible with a dispersing effect of PDH, but does not prove it alone. Curve iv shows that injection of PDH only partly overcomes the natural supply of PLH in a normal specimen exposed to light on a white background. PLH was obtained from sinus glands and tritocerebral commissures. PDH was obtained in least contaminated form from the abdominal ganglia (from Brown, Webb and Sandeen, 1952). § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 93 effects an initial response which is practically that which would be achieved in the complete absence of the second. Such a mechanism would assure rapid response in either direction, provided the time elapsing between the responses is adequate for the normal reduc- tion in titre after the initial secretory burst" — i.e. about 15-50 min in the black-to-white change, and 30-60 min in white-to- black (Brown, Webb and Sandeen, 1952). Eupagurus. The red pigment in the hermit crab, Eupaounis, (Table 11) behaves like that in Palaemonetes ', it is concentrated in bright light on a pale background, and dispersed on a dark back- ground. It is clear that both reactions in Eupagurus are under hormone control, but the actual substances and their exact sources have not been determined, although removal of the eyestalks again results in persistent darkening. Uca. The background responses of the red pigment in the chromatophores of the fiddler crab, UcUy show well and have already been referred to (Fig. 3-lSa). Both red-concentrating and -dispersing substances can be extracted from nearly all parts of the nervous system; but, as in most brachyuran crabs, the sinus gland is the richest source of t7c«-RED-DiSPERSiNG hormone, URDH (Fig. 3-15^ and d). As the same is true of the hormone dispersing the melanophores, removal of the eyestalks of crabs results in permanent pallor (Brown, 1950^). This is unlike the situation in prawns and most other Malacostraca that have been examined, where the sinus gland provides a concentrating hor- mone, like PLH, and removal of the eyestalks therefore results in permanent darkening. The main source of the Uca-BED-CONCEN- TRATING HORMONE, URCH, is in the circumoesophageal connec- tives (Fig. 3-1 5c) and this is again the opposite site from that in the prawns (Tables 9 and 11). The sinus gland also yields small quantities of URCH. On a constant white background Uca also shows a diurnal rhythm of colour changes that are comparable to the direct effects of light and darkness on most dark chromatophores. The crabs become dark by extensive pigment dispersal by day, and pale at night; but these changes in Uca are not merely direct effects (p. 84), since their rhythm persists for four days in constant dark- ness, and it is not altered, although the amplitude of the pigment 94 KINETIC^HORMONES — I dispersion is reduced, by reversal of day and night illumination (Webb, Bennett and Brown, 1954). Vertebrata. Relatively little is known of the control of red pigment in vertebrates. Teleostei. The red (and also the yellow) chromatophores of the minnow, Phoxinus, appear to be under purely hormonal con- trol and to show the usual background responses. The dispersing action of intermedin, B, from the pars intermedia (§ 2.123), is well established (Giersberg, 1930 and 1932), but the source of a red-concentrating hormone has only been rather uncertainly located in the epiphysis or pineal organ. Adrenaline has no effect. Other red pigment cells of fish, such as Holocentrus, are controlled by nerves only. Reptilia. The erythrophores of the chameleon, Lophosauray are probably under nerve control, like their melanophores, but they do not seem to have been fully investigated (Brown, 1950«). White pigment in chromatophores Crustacea. The hormonal control of other pigments in chromatophores is essentially similar to that already described for red pigments, though many minor variations have been found (such as those between Palaemonetes and Crago)^ and much remains unknown (Tables 10 and 11). In bright light on a white background the response of guano- phores is usually dispersion of their white pigments, which reinforces the pallor produced by the concentration of dark pig- ments in the other chromatophores. In all the Decapoda exam- ined (except the Brachyura), the richest source of extracts which induce these light-adaptations is the sinus gland, and this is almost certainly the organ where the natural hormones are liber- ated into the blood; for eyestalk (and therefore sinus gland) removal causes the opposite effect. Like the natural response to an illuminated black background, it results in the white pigment becoming concentrated and the dark pigments being dispersed. Extracts causing the latter effects can usually be obtained from the commissures (which include circumoesophageal connectives and tritocerebral commissures) ; but it is not known for certain at what point the natural hormone passes into the blood. § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 95 Palaemonetes. The white chromatophores (Table 10) reinforce the protective colour change provided by the red pigments of this prav^n (Brown, 1950a). The concentration of white pigment, which occurs naturally on a dark background, can be induced by injecting an extract from the commissures. This contains a Pfl/^^mow^fg^-w^HiTE-coNCENTRATiNG HORMONE, PWCH. The dis- persion, that occurs on a white background, can be induced by a Pa/«^mO«^^^5- WHITE-DISPERSING HORMONE, PWDH, from the sinus glands. The direct effect of light on the skin is to cause pigment disper- sion in these white chromatophores, as it does in erythrophores ; but here it reinforces the hormone reaction controlled by the eyes, instead of counteracting it (Tables 10 and 11). Crago*, The hormonal control of white chromatophores in the shrimp follows the same pattern as that in Palaemonetes. The sources of the two hormones, CWCH and CWDH, are probably similar, but this is not certain. There appears to be no direct effect of light upon the guanophores in the skin, for they remain con- centrated, whatever the light intensity. Uca. In Brachyura, the control of the white chromatophores, like that of the red and black cells of the same animals, follows a pattern distinct from that of the other Decapoda. Although the background response is the same as in the prawns, the source of the L^cfl-WHITE-DISPERSING HORMONE, UWDH, Cannot be mainly in the sinus gland, as theirs is, since the pigment remains perma- nently dispersed in eyestalkless animals. The latter observation suggests that, like the f/^a-red-dispersing hormone, the source of the C/Cfl-WHITE-CONCENTRATING HORMONE, UWCH, might be in the sinus gland; but extracts have not so far given any concen- trating effect. There is apparently no direct effect of light upon the white chromatophores of Ucay which remain permanently dispersed, even in the dark, in the absence of hormonal control. Teleostei. The white chromatophores, or guanophores, of the killifish, Fundulus, show the same adaptive reaction to background colour as do those of the Crustacea, dispersing on a white back- ground and concentrating on black (Fig. 3-18 a and h). Their *This spelling of Crangon is commonly used in this context in America, and is retained here for simplicity. 96 KINETIC HORMONES — I X •M- ,1 # # 1 9 • • 1 ^ # * * * # # u # # 1 & • • # # # # * # o o On p ^ w o 23 K PI O f_, Q < o Q rj Z, n-. w M O ffi K (/) W '^* r\-. w bp tf # # Q 1 • • 1 o ^ •• • • • • W <3 < O * # B' • • 1 ^ Ui < 5 • • S M <=^ ^ H « ^ < -1— g 1 2 K ffi # # Q 1 • • o I • • * * • • ■^ # # ^ 1 • • ^ 1 • • # * • • ^ Ph fu C3 c^ w ^ o « § '0 *4_> a 2 ^ C3 u C o 1 1 1 ;2 CO S c o c g S "cJ^D U UP Q Z < S §1 1 •M ^ 9 ►< -^ ^fti J2 i •? < K .t>o .5p ^ &i5 .£? •<»> 2 g S 1 •Si i:3 a •->~i ^ 1 "i ^ •^ 1 >-] 0) 0^ ^ 05 Q c CO C 00 § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 97 normal control may be by nerves, as is that of the melanophorcs of this fish; but both types of chromatophores react rapidly to injections of either adrenaline, or Antuitrin, that supplies a MELANOPHORE-CONCENTRATING HORMONE, MCH (Odiornc, 1933) to give the "light reaction" in which melanophorcs concentrate, and guanophores disperse (Fig. 3-186). Although denervatcd melanophorcs of Fundulus are known to disperse in response to injections of B (or MSH), there is only slight evidence that this causes the expected concentration of the guanophores. Black pigment in chromatophores Crustacea. The shrimp, Crago, is the only member of the Decapoda Natantia so far investigated that has black pigment akin to melanin in cells that may be called melanophorcs. These melano- phorcs can produce the beginnings of a pattern, because they are differentiated into two sizes, larger on the body and smaller on the tail. These both react to one Cr«^o-DARKENiNG hormone, CDH, from the commissures, but are concentrated independently (Brown, 1946 and 1950«); those on the tail by Crago-TAiL- LIGHTENING HORMONE, CTLH (alcohol-insolublc extract from the SINUS gland), and on the body by Cr^^o-BODY-LiGHTENiNG HORMONE, CBLH (water-soluble extract from the brain), both of which are dominant to CDH. In nature, this means that the shrimp can go wholly dark if only CDH is acting, or the tail or body only may be lightened, while the rest remains dark, accord- ing to which of the concentrating hormones are present with the CDH. Finally, if both CTLH and CBLH are present, the shrimp becomes completely pale. An eyestalkless specimen lacks CTLH, and is at first pale with a dark tail, but after a time this effect dis- appears. The natural stimulation of pattern forming changes in the shrimp is not known ; but in prawns, like Leander, the exten- sion of a similar system to a large number of different types of chromatophores, apparently each controlled by separate hormones, must give them adaptive advantages by increasing their ability to match a variety of backgrounds (Plate 3-1). Uca. The background responses of Uca melanophorcs (Fig. 3-19) are more difficult to demonstrate than those of the red chromatophores, because they are almost completely overridden 98 KINETIC HORMONES — I 1 ^^ ii3 » « 1 |S • • « * •• « « ' ' Q • • * * •• "" P ^1 g z dM * * rv. §11 1 1 c; * * S 1 1 o * * .^ • • C^ • • a 1 * * 8" M * * 5 b • • '^ • • S- w fe_ § ^-^ J H Q s X ffi ffi ^ hJ 1 S O 1 o * * I 1 Q < • • * * rx-. J ^ ' ci * * ' ' s >* •• Q * * D P p Fh Q ^ < Z ffi 1 1 * « iS 1 * * * * •• § S^ 3 1 ' « « ' R ' * * * * •• ti^ w H S a a 1 • • K 1 1 * * ffi 1 1 * * * * •• • • J 1 1 * * Q 1 1 * * * * •• u Z o ^ ^ §§ 2: ffi d 2 fe H O _o ^3 2i T> < u 2 1 g d o g o d o s-si ^ lis c« •S 2 c CO o c.c U c/2eqD 5 OPm •^ ►ftT Q 's » CO 5.2 o o d d aa C5 QQ. § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 99 by diurnal changes, which operate in the opposite sense. If, however, allowance is made for the diurnal changes, it can just he seen that, for any given intensity of light incident upon the background, the degree of dispersion of the black pigment is greater on a black background than on a white background (Brown and Sandeen, 1948). The Uca-DARKENiNG hormone, UDH, controlling dispersion of the melanophore pigment, like the Uca-red-dispevsing hormone, URDH, is secreted from the sinus gland; eyestalk removal therefore results in relative pallor. The dispersing activity of UDH can be neatly demonstrated by using the two sexes of these crabs (Fingerman and Fitzpatrick, 1956). Melanophores in the female are normally more dispersed than in males in the same situation. If the large, hollow, asym- metrical chela, which distinguishes the male from the female, is removed before the crabs are exposed to light, the operated male becomes as dark as the female. If more legs are removed he be- comes even darker. This can best be interpreted by assuming that the same amount of dispersing hormone, UDH, is released in each animal in response to similar stimuli ; but that the degree of disper- sion of the chromatophores depends upon the concentration of the hormone in the blood. This is increased as the blood volume is decreased by removing successive appendages. The natural secretion of [/crt-LiGHTENiNG hormone, ULH, has been demonstrated by using the blood of crabs, in which the melanophores were maximally concentrated, to perfuse isolated limbs on which the melanophores were dispersed. These show slow pigment concentration even if perfused with sea water ; but the rate of concentration is increased if perfused with blood containing ULH (Fingerman, 1956). The source of ULH has not been located ; but, unlike CTLH, it cannot be in the eyestalk. Ltgia. Apart from the Decapoda so far described, the only crustacean that has had its melanophores investigated in detail is the sea slater, Ltgia, among the Isopoda. In nature it changes colour from black, when lurking in damp and shady crevices, to pale mottled grey, when exposed to light. On any black background in bright light they show the usual response, by which the melano- phores become fully dispersed (index 5). On an experimental white background the pigment can become concentrated to an unnatural 100 KINETIC HORMONES — I pallor (index 1.5, Figs. 3-14 and 3-20). In darkness the melano- phores assume an intermediate condition (index 2.7). These reactions are similar to those of the red pigment of Palaemonetes (Table 11). Blinded animals show a direct effect by which the melanophores are more expanded the brighter the light, but never as much as in normal animals on a black background in the same light (Table 12). If different groups of the ommatidia in the sessile compound eyes of these animals are illuminated separately, either by painting over part of the eye with opaque varnish (which affords a. good class demonstration) or by exposing the animals in specially constructed boxes which admit light only to certain narrowly delimited retinal areas (D, L, and V, Figs. 3-20 and 3-21), the effects shown in Table 13 can be obtained. It is concluded from these and other observations that two antagonistic hormones are involved : a Lz^/<2-darkening hormone, LDH, normally stimulated by direct light on area D to cause dispersion; and a Lz^/a-lightening hormone, LLH, stimulated by reflected light on areas L and V to cause full concentration. When the whole eye is illuminated both are secreted, but LLH overrides LDH to produce almost complete concentration. A Table 12. Changes in melanophore index in ligia All index values are an average of measurements made on the posterior part of the body of 24 specimens (from H. G. Smith, 1938). ANIMAL BACKGROUND BRIGHT LIGHT DIM LIGHT DARKNESS Normal Norfjial Blinded Black White White 5-0 ± 1-7 ± 0-08 4-2 ± 0-06 4-6 + 0-08 1-4 ± 0-06 3-9 ± 0-09 2-7 ± 0-1 2-7 ± 0-1 2-7 ± 0-1 balance between the two hormones produces an intermediate effect (Fig. 3-21^). The sources of the two hormones have not been fully determined, but Kleinholz (1937) showed that removal of the whole head is followed by dispersion, and extracts of the head cause concentration of the melanophores. The source of § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 101 Light activating LDH No LLH secretion Reflected light activating some LDH Light activating LLH No LDH seaetion Light activating LLH only Fig. 3-21. Diagram of Ligia, in face view (cf. Fig. 3-20«), to show a method of illuminating separately the dorsal CD), lateral (L) and ventral (V) areas of the compound eyes. Arrows indicate the direc- tion of light falling on the eyes, which are exposed by cutting short the antennae (An.ii). The animals are confined in shallow cells allowing horizontal movement only. Screens, indicated by black- ened areas, prevent the entry of light. Stippling represents the chromatophore reaction to secretion of LLH, L?'§'/a -lightening hormone and LDH, Lz^/«-darkening-hormone. (a) Illumination from above only, (b) Illumination by direct light from below, with reflected light from above, (c) Illumination from below only (Original diagram, based on Smith, 1938). 102 KINETIC HORMONES — I LLH is therefore probably in the organ equivalent to the sinus gland, since isopods lack eyestalks and the gland lies within the head capsule, in the vicinity of the optic centres, where it is associated with a blood sinus (Hanstrom, 1939). The source of the darkening hormone is unknown, but must be, in part at least, behind the head. Table 13. Illumination of different areas of the eyes of ligia Blue light is most effective in eliciting these responses in Ligia. The index values are averages (from H. G. Smith, 1938). AREA MELANOPHORE CONVERSE AREA MELANOPHORE ILLUMINATED INDEX COVERED INDEX D 4-7 L -1- V 4-6 D + L 2-5 (dark) 2-7 D + V 2-4 — — V* 1-8 — — V + L* 1-4 D* 1-5 D + L + V 1-7 none 1-7 (normal) * illuminated from below. Insecta. The black pigment cells of the phantom midge larva of Chaohorus ( = Corethra) differ from those of Carausiiis in being mesodermal. They normally cover the surface of the two pairs of air sacs and show a background response of the usual protective type; but concentration of pigment on a white background is brought about by an amoeboid change in shape of the cells which become spherical instead of elongated. The cells also tend to aggregate in small groups instead of being evenly spread out to show the maximum amount of colour, as they do on a dark back- ground. Extracts of the brain cause pigment dispersion, and therefore darkening, as in Carausiiis (Dupont-Raabe, 1956). Vertebrata. Melanophore control varies in different fish; among Teleostei it is only in some species that it is wholly under hormone control or even partially under hormones and partially under nerves. Amphibia, where the control is purely hormonal, will be considered first. § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 103 Amphibia. The background responses of Amphibia are similar to those of the Crustacea in so far as the animals become pale on an illuminated white background and dark by melanophore dispersion on an illuminated black background. Early investiga- tions of these colour changes were made on the African clawed toad, Xenopus and attempted to use the different rates of change of the chromatophores, following changes of background or changes to and from darkness (Hogben and Slome, 1931), to indicate the number of hormones that were necessary to control the changes. The observations were good in that they did not interfere with the integrity of the animals, but recorded their natural physiological reactions. But the changes were so slow and were interfered with by extraneous factors like diurnal rhythms and unsuitable temperatures, so that the results were unsatis- factory. Recourse to the pharmacological method of injecting extracts into variously prepared specimens was therefore necessary to obtain conclusive evidence for the presence of two hormones. The dispersing or melanophore-stimulating hormone, MSH, (known as B to the earlier writers) can be easily established by injection of extracts of the posterior lobe of the frog hypophysis, although the early experiments made use of mammalian extracts. The source is the pars intermedia (§ 2.123). It has not been possible to prepare active extracts of the antago- nistic concentrating hormone, known as W and believed to be secreted from, or controlled by, the pars tuberalis (§ 2.123). The best evidence for its presence is obtained by injecting equivalent extracts of active B substance into variously prepared test toads, and comparing results on a white background (Hogben and Slome, 1936). Injecting a standard dose of B into the normal pale animal produces a temporary increase in melanophore dispersion that wears off in about 10 hr, when the toad's normal response again takes charge (Fig. 3-22, curve B). If the whole hypophysis is removed, including both the known source of B and the postulated source of W, a much greater effect (Fig. 3-22, curve A) is produced for the given dose of B than in the intact animal. The effect also persists longer, just as if the natural supply of W were absent. 104 KINETIC HORMONES — I If the posterior lobe of the pituitary is removed (taking with it the pars intermedia, which is the source of B, while the pars tuberalis is left intact) a similar dose of B results in the least and shortest response of the three (Fig. 3-22, curve C). If there were no V: Total removal 8 10 12 Hours 18 20 Fig. 3-22. Effects upon the melanophores of Xenopus of extracts, containing equal amounts of the melanin dispersing hormone, B (intermedin). (A) Six toads from which the whole hypophysis, including sources of both B and W, has been removed. (B) Six normal intact toads, in which production of both hormones is stimulated by the white background. (C) Six toads from which the posterior lobe and the pars intermedia have been removed, taking with them the source of B only. For further explanation see text. B was obtained from extracts of ox ''posterior pituitary", freed from pressor and oxytocic activity, but including intermedin from the pars intermedia to provide a dispersing effect. Although this is not the natural amphibian hormone, the facts of the dose being the same in each case and producing slow dispersion, and the fact that the magnitude of the effect is altered by prior operations on the toads make the results appear significant. The specimens used for the experiments were "selected for the same degree of pallor", and all were exposed to an illuminated white background (from Hogben and Slome, 1936). active hormone secreted by the pars tuberalis, this curve should be the same as curve Ay since the source of B is absent in both § 3.223 EFFECTORS WITH MOVABLE PIGMENT GRANULES 105 cases. The difference may therefore be attributed to the secretion of a large amount of the concentrating hormone, W, by the pars tuberaUs in response to the white background (Hogben and Slome, 1936). The toads always remain pale on a black background after removal of the source of B in the posterior lobe of the hypophysis. If the whole adenohypophysis, including the pars tuberalis, is removed, the toads become permanently dark. That this is due to the loss of the source of W is claimed from finding that, after slightly incomplete hypophysectomy of Xenopus, the pars tuberalis may regenerate, in which case the lost response to a white back- ground is regained (Waggener, 1930). As in Ligia^ it is claimed that the adaptive colour changes are controlled by the secretion of the two hormones in different pro- portions in response to illumination of different parts of the retina of the eye by direct and reflected light (Fig. 3-23). The eyes of Xenopus are on the dorsal surface of the head and direct light stimulates the "floor" of the retina; on a black background no other light reaches the eye and the secretion of the dispersing HORMONE, B, is induced. Scattered light from a white background stimulates the ''peripheral" part of the retina and induces secretion of the concentrating hormone, W, whatever position the toad adopts (Hogben and Slome, 1936). In the eel, Angiiilla, where the eyes are lateral, the ventral and dorsal parts of the retina are as- sumed to play the same role as floor and peripheral parts in the toad. These results seem to be conclusive; but they lack control injections of saline or of some other non-active substance, and they have not been confirmed by more recent work using better techniques. It is possible, therefore, that there is no real difference between Xenopus and the frog, Ranapipiens, in which there appears only to be the one melanophore-dispersing hormone, B (Parker and Scatterty, 1937), concentration of melanin resulting merely from absence of B. Elasmobranchii. The background responses of the melano- phores of the rough dogfish, Scyliorhinus, and other elasmo- branchs are similar to those of the Amphibia, but they seem to be even slower (Waring, 1938). There is no doubt that the melano- phores are dispersed by B, since this can be extracted from their 106 KINETIC HORMONES — I own pars intermedia and can also be detected in effective quantities in the blood of dogfish that have been kept on a black background. There is still uncertainty about the presence of the antagonistic, concentrating hormone, W, which is always more difficult to Light Light Dispersed Concentrated Fig. 3-23. Diagram of the nerves and hormones concerned in mixed control of melanophores in the eel, Anguilla. Similar hormones act alone in an amphibian such as Xenopus. An illuminated black background is represented on the left and an illuminated white background on the right. On black, only the floor of the retina is stimulated by light and causes pigment dispersion, either by stimulating the secretion of B (MSH) from the pars intermedia (p.in.), or by adrenergic nerves (a.n.). On white, both the floor and the upper part of the retina are stimulated and pigment is concen- trated, either by secretion of B as before and of overriding W (MCH) from the pars tuberalis {p.t.), or by cholinergic nerves {cm.). In either case nerves from the eye pass through the brain (C.N.S.) to transmit stimuli from the retina to the chromactivating hormone or nerve (from Parker, 1943). detect. A full review of the literature relating to the chromatophores of these and other fish has been given recently by Pickford and Atz (1957). Teleostei. The control of melanophores in the teleosts is more § 3.224 EFFECTORS WITH MOVABLE PIGMENT GRANULES 107 complicated and varied than in the other groups considered, because there appear to be at least two distinct types of response to hormonal stimulation, as well as the differences in reaction introduced by mixed control by both hormones and nerves. In some of the more primitive forms, such as the pike, Esox, and the carps, eels and Ameiiirus, the control appears to be purely hor- monal. The melanophores disperse in response to the melano- PHORE-STIMULATING HORMONE, MSH, from the pars intermedia, when it is injected into pale specimens, whether the source of the hormone is from the same fish, from amphibia or even from mammals. The majority of other teleosts like FunduluSy are in- sensitive to MSH, at least until the melanophores are denervated. This second group is, however, found to be more sensitive to a MELANOPHORE-CONCENTRATING HORMONE, MCH, which is present in most extracts of fish adenohypophyses. It is present in Antui- trin and has no action on Amphibia. A few teleosts, like Phoxinus, although having their melano- phores normally under nerve control, can be induced to respond appropriately to both MSH and MCH under suitable conditions. Hormonal control in Phoxinus apparently serves to maintain the melanophore reactions over long periods when the nerve control becomes fatigued (Healey, 1948). Many other teleost fish have melanophores which are controlled only by nerves (for further details see Pickford and Atz, 1957). Reptilia. Hormones, especially intermedin, B, play a part in the dispersal of the melanophores of certain of the older genera of lizards, such as Phrynosojna, Hemidactylus and Anolis; but con- centration appears to be controlled by adrenaline and not by a pituitary secretion. Dual nerve control replaces this in the more highly evolved chameleons and others, producing varied patterns of colour change. 3.224 Discussion of the hormonal control of pigmentary effectors Looking back over the pigmentary effectors just considered (Tables 9, 10, 11), it seems possible to make some generaliza- tions, although the more complex cases have been left out, and the evidence is not always complete for those that have been included. Two hormones are concerned in the control of nearly all types of 108 KINETIC HORMONES — I pigmentary effectors with movable granules, including some which have nerve control as well. But in nearly all cases the actions and sources of one hormone are much more fully established, than those of the other; the more certain of the two is the light-adapting hormone for Crustacea, and the fishes Phoxiniis and Fundiilus, whereas it is the dark-adapting hormone for the Amphibia, Elasmobranchii and some Teleostei, such as Ameiurus. It seems plausible to expect that in the stick insect, where so far only the Car flw^/w^- darkening hormone has been found (§ 3.221), an antagonistic pigment-concentrating hormone wdll be revealed in due course. In Crustacea there is evidence from Isopoda and from many Decapoda, other than Brachyura, that the hormones producing the light background response are normally stored in, and pre- sumably discharged from, the sinus gland or its equivalent; this is true for the concentration of dark pigments such as black and red, and also for the converse dispersion of the white pigment (Tables 10, 11). Similar converse reactions of the pigment in coloured and white chromatophores in fish may also be due to one and the same hormone, as in Fundulus. The situation in the Brachyura is peculiar. There is a background response, which though slight is similar to that in prawns; but, instead of the concentrating hormone, it is the dispersing hormone which is released from the sinus gland in the eyestalk. In many crabs, such as Uca, the background response is overridden by an opposing reaction causing darkening in bright light. It is tempting to interpret this as being either (i) a morphological accident the result of which is that, when light stimulates the sinus gland via the eye, the gland releases its hormone, as in other decapods (but this produces the opposite effect in crabs because the secretion is a dispersing instead of a concentrating hormone); or (ii) the effect of a single hormone controlling dispersion of pigment in both the melanophores and the retinal cells; for dispersion of retinal pigment, as in Cambariis, is the adaptive response to light and the daytime dispersion of the crabs' melanophores would then be but following in the wake of their retinal pigment. It must be emphasized, however, that none of the cases consid- ered (except Crago) have more than one colour of pigment in § 3.224 EFFECTORS WITH MOVABLE PIGMENT GRANULES 109 the same chromatophore, unlike the complex pattern-forming chromatophores of Leander and Penaeiis (Knowles, 1955 ; Knowles, Carlisle and Dupont-Raabe, 1955, and Knowles and Carlisle, 1956), in which there may be as many as four. It is to be hoped that the refined technique developed by these authors for separa- ting pure substances from tissue extracts by paper electrophoresis will soon be extended to the circulating blood of these and other prawns, with their chromatophores in different states of dispersion in response to different states of illumination or background colour. When extracts of tissues yield the same substance as that found to be active in the blood, it should be possible to identify the sources of hormones actually used by the animal, and to distinguish these from other substances, like acetylcholine, pro- duced at nerve endings in the central nervous system ; for these can react upon effector systems experimentally, and yet never reach them through the circulation in the living animal (Knowles, 1955). Modern techniques might also throw light upon the rate of chromatophore reaction, which seems normally to be so much slower in the vertebrates than in the Crustacea, and to be controlled to a considerable extent in the latter by the concentration of the hormone reaching the cells. The rates could be compared with the high speed of reaction of nerve-controlled chromatophores, to see if the differences were due to the more concentrated dose of the chemical which can be supplied at a nerve ending, rather than to differences in sensitivity of the chromatophores (Waring, 1942). Nevertheless, Waring's idea, that an evolution in either the sensitivity of the chromatophore or in its speed of reaction was a necessary precursor of the evolution of nerve control with adaptive value, is interesting. It appears to be borne out to some extent by his table showing that nerve control is only of importance in vertebrates of more recently evolved families, and has not been achieved in the Elasmobranchii and Amphibia, which are both classes with a much longer fossil history than either the Teleostei or the Reptilia (Fig. 3-24). The persistence of purely hormonal control in Crustacea would then be expected, and could be looked upon as having evolved along lines of chromatophore differenti- ation with multiple hormone control, instead of being replaced by nerve control. 110 KINETIC HORMONES — I 1 ■ ■ XI ,_ !S - 2 « a: s -^2_ *?_ •S- Jl in Z3 o yIIiidae'{ScyIlium rchariidae (Muste nacidae (Acantii uotinidae (Rhina jidae( Raja) guiHidae(Anguil jridae(Ameiurus uronectidae prinidae (Phoxini lnrionidae(Saimo sterosteidoeXGo prinodontidae (F ura(Rana : Xeno ^ _i 1 1 1 i O O cnoc/)(/)a: 55 O « o 1111 CO CO z < -§ < C3 a <: ^ S ^ B ^ a o < 1 P^ ■S o 5 1 §^ P ?^ Co t^ ^ o Z -^ »-~^ , (N CO Tt- T— < CO 7—1 y—i .—1 (N o J-?^ CO O ,^ O O 300 < en 2 200 Saline Histamine 30 Minutes 45 60 Fig. 4-11. Effects of injections of saline, epinephrine (adrenaline) and histamine on the ascorbic acid content of the adrenal cortex of rats, in mg/100 g gland (ordinates). The saline causes no change in 60 min (abscissae) ; the reduction due to adrenaline and histamine is by then the same, showing the two doses to be equivalent. The effect of histamine is much the more rapid, and must therefore evoke secretion of ACTH directly and not through any inter- mediary action of adrenaline (cf. Fig. 4-10, based on similar experiments). (From Munson and Briggs, 1955). many animals, including an unusually wide range of invertebrates as well as vertebrates. So far, it is only in the vertebrates that the secretion of these gonadial hormones is known to be controlled by endocrinokinetic hormones. Like the other hormones of this type in vertebrates these are all secreted from the adenohypophysis ; they are often referred to as gonadotrophins, in common with § 4.232 MESODERMAL ENDOCRINE GLANDS OF VHRTK BRA TA 149 Other hormones from the same source which have marked morphogenetic effects upon the growth of their target organs. Only those causing secretion of endocrine glands need be treated in the present section; ahhough all the gonadotrophic hormones play an important role in relating breeding cycles to seasonal stimuli from the environment (§ 4.232 and Part II, § 4). The gonadial hormones themselves are mainly morphogenetic in action (§ 1.53); but they are peculiar in having some subsidiary kinetic effects (§§ 3.12, 4.12 and 4.324). In their latter capacity they pro- vide the only examples of kinetic hormones which are controlled by endocrinokinetic hormones. Information regarding the gonadotrophic hormones of the cold- blooded vertebrates is scanty, compared with that concerning birds and mammals; but in general they appear to have similar effects upon the secretion of the gonadial hormones (Pickford and Atz, 1957). Further reference will be made to them in relation to the latter (Part II, § 4). Interstitial cells in the testis stimulated by ICSH In birds and mammals, and probably in most other vertebrates, the release of testosterone, or other androgenic hormones, from the testis is brought about by an endocrinokinetic interstitial- cell-stimulating HORMONE, ICSH, secreted from the adeno- hypophysis. The control of its secretion, like that of TSH, comes from the hypothalamus of the brain and can reflect the effects of environmental changes, transmitted to the brain either directly or by the eyes. The secretion of ICSH, by its release of testosterone, fires off the whole chain of events associated with the breeding cycle in the male. In birds these can include migration, courtship and nest-building and the appearance of secondary sexual characters of plumage and wattles, as well as the essential develop- ment of the genital ducts. In mammals corresponding changes in behaviour and structure are brought about by similar hormones; in those with a limited breeding season there is a slow feed-back mechanism, whereby the accumulation of testosterone inhibits further ICSH secretion after a few months. In those species with continuous breeding this inhibition is not effective, at least until extreme old age. 150 KINETIC HORMONES — II Follicle cells in the ovary stimulated by ICSH The release of oestrone, or other oestrogenic hormones, from the vertebrate ovary appears also to be stimulated primarily by the interstitial-cell-stimulating hormone, ICSH. Its action may depend in some cases on the synergic effect of the follicle-stimulating hormone, FSH; but the most important action of the latter is to cause the growth of the follicle cells, along with that of the ovum that they enclose. There are differences in the relative importance and effects of these hormones between different species and even more between different classes of vertebrates. The release of oestrogens by ICSH results in the development of the female genital ducts, but not as a rule in that of any secondary sexual characters ; the external appearance of the female is usually distinguishable from the male by the absence of male characters, rather than by any positive features due to oestrogenic hormones. In the female mammal, whether the breeding season is short or continuous, there is always an alternation, or cycle, of phases within the breeding period and of these it is the oestrus phase which results from the secretion of oestrogens (§ 4.323). The feed-back reaction, whereby the accumulation of oestrogens decreases ICSH secretion, is relatively rapid in the female and makes way for the alternate phase of the cycle, whether this is dioestrus, pregnancy or pseudopregnancy. In most species oestrus only lasts for a few days each time. Corpora lutea in the ovary stimulated by LSH Mammalia. The endocrinokinetic hormone luteotrophin, LSH, from the adenohypophysis, stimulates the secretion of progesterone from the corpora lutea that form in the ovarian follicles after the shedding of the ova. LSH is probably the same as PROLACTIN, which causes the secretion of milk from crop and mammary glands (§ 4.13) ; if it is the same it is the only one of the endocrinokinetic hormones which has the power to stimulate any exocrine glands as well as an endocrine gland (§ 4.323). It can only affect the corpora lutea after their growth has been stimulated by the morphogenetic luteinizing hormone, LH, also from the § 4.232 MESODERMAL ENDOCRINE GLANDS OF VERTEBRATA 151 adenohypophysis. Incidentally, LH was for many years confused with ICSH ; but there is now good reason to beheve that they are distinct and that while ICSH occurs in both sexes, LH is normally present in the female only. Its occurrence throughout life can he completely inhibited if a testis, either natural or implanted, is present during the development of an animal of either sex (Witschi 1955). The action of LSH, by inducing the secretion of progesterone, initiates the alternate, or dioestrus, phase of the female cycle, a phase which makes pregnancy possible and for which there is no hormonal counterpart in the male. This may not have been the primary function of progesterone, for it occurs in association with viviparous development in many vertebrates besides mammals ; it is probably under the same endo- crinokinetic control throughout (Matthews, 1955). Interaction of gonadial and endocrinokinetic hormones in repro- duction In the sexual reproduction of all vertebrates there is need for the gametes to ripen simultaneously in males and females of the same species, and for the young of most land forms to be pro- duced at a suitable season of the year for their early growth. The endocrinokinetic hormones play a vital role in bringing this about. Mammalia. In placental mammals the viviparous development of the young embryo in the maternal uterus and its subsequent nourishment from the mammary glands increase the complexity of the situation, so that several more hormones are needed to control reproduction in the female than in the male. In the male, the secretion of testosterone is induced by ICSH, and maintains the reproductive ducts and the accessory organs in a steady, active state, as well as developing secondary sexual characters, such as antlers. At the same time the morphogenetic hormone, FSH, ensures the continuous ripening of sperm. In species with a limited breeding season, like deer, the onset and ending of these hormone-controlled changes appears to be related by the brain to environmental factors, such as temperature and length of daylight. 152 KINETIC HORMONES — II In the female placental mammal at least three pairs of hormones are involved, the two members of each pair acting alternately to control the changes associated with the alternation of oestrus and dioestrus phases. The latter group are also active during pregnancy. In the oestrus phase the endocrinokinetic hormone is ICSH and the morphogenetic hypophysial hormone is FSH, as in the male; but the gonadial hormone is an oestrogen. In the dioestrus phase these are replaced respectively by LSH, LH and progesterone, for which there are no counterparts in the male. Their detailed actions, interactions and variations will be discussed in relation to reproduction (Part II, § 4). Oxytocin from the neurohypophysis also plays a part in the reproductive processes in the female ; but its main actions have already been mentioned, being concerned with muscular contraction in parturition (§ 3.113) and with the action of the myoepithelial cells in bringing about ''milk let-down" from the mammary glands (§ 3.114). Prolactin that stimulates the secretion of the mammary glands seems to be identical with LSH, although its action here is so different (§ 4.13). Even this list of seven or eight hormones involved in the reproductive activities of the female takes no account of the hormones which must actively adjust the maternal metabolism to meet the demands of gestation and lactation. 4.3 General considerations As the account of the kinetic hormones is now as complete as space allows, it will be well to review their general characteristics and the means by which their own secretion is induced, before passing on to a consideration of the metabolic hormones, which fall into so different a category (§5). 4.31 characteristics of kinetic hormones The kinetic hormones controlling the reactions of muscles and glands in vertebrates have much in common with each other and they may even be identical, whereas those controlling the pigment- ary effectors are usually distinct and differ from the former in various ways which may in part be due to the nature of the effectors concerned. Gastrin, progesterone and adrenaline are §4.31 CHARACTERISTICS OF KINETIC HORMONES 153 among the hormones which can control the activities of both muscles and exocrine glands. Among invertebrates there are several examples of hormones controlling muscles, but as yet none have been found to control secretion of any exocrine glands in these animals, so that there is no overlap; its investigation might be interesting. The control of the secretion of endocrine glands is usually separate from that of other glands; but even here there is an exception in that prolactin can stimulate the exocrine mammary glands as well as the endocrine corpora lutea of the ovary. Apart from adrenaline, the hormones controlling pigmentary effectors do not overlap with the foregoing at all, unless the hormone that stimulates dispersion of pigment in the stick insect, Caraustus, should prove to be the same as that which stimulates locomotor activity in Periplaneta^ since both come from the suboesophageal ganglia. There is also a difference in the number of hormones involved; the control of pigmentary effectors, and particularly of chromatophores with movable pigment, is nearly always achieved by the interaction of a pair of antagonistic hor- mones (§ 3.24), but the same can rarely be said of muscles or glands. The only unequivocal case of a pair of hormones is gastrin and enterogastrone, which have opposing actions on both the peristaltic muscles and the acid-secreting cells of the mammalian stomach (§§3.112 and 4.11). Even the apparently opposed actions of progesterone and oxytocin on uterine muscle may not be direct inhibition and stimulation, but rather a case of the presence of progesterone rendering the muscle insensitive to oxytocin throughout gestation. Nearly all the other kinetic hormones, acting upon muscles, myoepithelial cells and glands, serve to stimulate these effectors, for which there are no known inhibitors. A general inhibition of locomotion seems to be the action of an eyestalk hormone in some Crustacea, and for these no stimulator is known (§3.12). Elsewhere different effectors react differently to the same hormone, some being stimulated and others inhibited. This occurs with cholecystokinin, which causes contraction of the main muscles of the gall bladder but relaxation of the sphincter muscles, and with adrenaline, which contracts the intestinal sphincters and inhibits the peristalsis of the longitudinal muscles. In the frog, adrenaline 154 KINETIC HORMONES — II has been reported to have opposite effects upon the same muscle at different dosages; this suggests that the other cases might be accounted for by different threshold levels of sensitivity. It seems more likely, however, that there may be some specificity in the muscles themselves, akin to the differentiation of many visceral muscles into those which are sensitive to sympathetic stimulation and those which are inhibited by it, but are usually sensitive to parasympathetic stimulation (Rosenblueth, 1950). Otherwise the apparent lack of inhibitor hormones may partly be because muscles and myoepithelial cells give an all-or-none reaction to stimulation and remain indefinitely quiescent in its absence ; no intermediate state of contraction of any given cell can be achieved by a balance betw^een stimulating and inhibiting factors, such as can arrest the pigment in a chromatophore at any position between the extremes of dispersion and concentration. Graded responses might seem more probable for glands; but it may well be that a similar all-or-none reaction to that in muscles holds good also for the individual secreting cells within the gland, and that changes in rate of secretion depend upon the number of cells induced to release their secretory products into the gland lumen. When kinetic hormones control chromatophores the effect is usually rather slow ; but the rate differs in different organisms and may depend chiefly upon the concentration of the hormone reaching the effector in any given case. In achieving and main- taining a protective background response, or in adapting an eye to changes in daylight intensity, a slow response may be an advantage in preventing sudden and conspicuous changes. Muscles and glands often show rapid responses to kinetic hormones, and may complete their reactions in a matter of minutes or even seconds, like the response to heart-accelerators (§ 3.111), or to oxytocin inducing milk "let-down" (§ 3.114), or to ACTH stimulating the release of ACH (§ 4.231). In such cases the only limitation on the speed of reaction seems to be the time that the hormone takes to pass in the circulation from its source to the effector. The effector, once stimulated, can react as quickly to a hormone as to a nerve impulse. In the case of rhythmically con- tracting muscles, like those of the heart or those causing gut § 4.32 CONTROL OF THE SECRETION OF KINETIC HORMONES 155 peristalsis, the increase in rate of contraction caused by a hormone can persist for some time without further release of hormone, if the latter is only slowly destroyed in the tissues. This type of effect can clearly relieve the nervous system from a considerable expenditure of energy, on repeated impulses. This control of the level of activity may be one of the most important general functions of kinetic, or perhaps of all, hormones. 4.32 STIMULATION OF THE SECRETION OF KINETIC HORMONES Most kinetic hormones are secreted in response to stimulation that is either direct or nervous. As far as the present evidence goes it is rare for the stimulus to come from another hormone or to be absent altogether. 4.321 Direct control of isolated cells in the gut Direct stimulation can be brought about by mechanical or chemical stimuli without any intervention by the nervous system. The hormones from the gut mucosa of mammals are stimulated in this way, either by mechanical distension of the stomach walls or by the presence of acids or the products of digestion in the lumen (Table 17). The integrated, self-regulating control of digestion, which is brought about by a succession of these hormones has already been pointed out as a specialized feature of mammals (§ 4.113). The cells which secrete these hormones have not been identified ; but they are assumed to occur singly in the mucosa from which the hormones can be extracted, as there is no sign of any endocrine glands there. They might be neurosecretory cells, such as secrete so many other kinetic hormones; but no other neuro- secretory cells have been shown to be directly stimulated as these cells are, without any connection with the nervous system. On the other hand, if the cells are endodermal they would be like the only other hormone-secreting cells which can be directly stimulated, i.e. those which secrete metabolic hormones from the parathyroids and the islets of Langerhans (§ 5.521). The fact that the action of these hormones is performed by the parasympathetic nerves in cold-blooded vertebrates is in favour of the former postulate. 156 KINETIC HORMONES — II Table 17. Means of controlling SOURCE OF NAME OF MEANS OF KIND OF SECTION HORMONE HORMONE CONTROL CONTROL* NO. 4.321 Direct control of isolated cells in the gut Mammalia Duodenum Cholecystokinin fats + 3.112 Stomach Gastrin distention + 4.111 Duodenum Secretin acid + 4.111 Duodenum Pancreozymin peptones + 4.111 Duodenum Duocrinin acid + 4.111 Intestine Enterocrinin food? + ? 4.111 Duodenum Enterogastrone fats + 4.112 4.322 Nervous control of secretory cells of nervous origin Cephalopoda Epistellar body Muscle tone increaser nerve p 3.12 Crustacea Pericardial Heart- nerve p 3.111 organ accelerator Sinus gland RPDH, etc. nerve + 3.222 Sinus gland PLH, etc. nerve + 3.223 Commissures RPCH nerve ? 3.222 Commissures PDH, etc. nerve p 3.223 Hanstrom's Moult- nerve ? 4.211 sensory pore accelerator organ Insecta Suboesophageal Pigment- nerve + 3.221 ganglion disperser Corpora Heart- p p 3.111 cardiaca accelerator Brain Prothoraco- trophin nerve + ? 4.213 Vertebrata Adrenal Adrenaline nerve + 3.11 medulla (muscles) >> >> (chromato- phores) nerve + 3.223 M ,y ,, (skin glands) nerve + ? 4.14 + stimulation of hormone secretion inhibition of hormone secretion § 4.32 CONTROL OF THE SECRETION OF KINETIC HORMONES 157 THE SECRETION OF KINETIC HORMONES SOURCE OF NAME OF MEANS OF KIND OF SECTION HORMONE HORMONE CONTROL CONTROL* NO. Neuro- Oxytocin nerve + ? 3.113 hypophysis (uterine muscle) ! »> >» ,, (myoepithel- ial cells) nerve + 3.114 >» >> ADH (pressor action) nerve 1 3.115 4.323 Nervous or other control of ectodermal glands Vertebrata Adenohypo- physis, pars intermedia B (MSH) nerve + 3.223 pars tuberalis W (MCH) nerve + 3.223 pars distalis LSH, prolactin nerve ? 4.13 >> >> LSH, luteo- trophin nerve P 4.232 >> j» TSH, thyro- trophin nerve or CRF + ? 4.221 >> >> >> )> thyroxine — 4.221 )> 5> STH, somato- trophin ? p 4.223 >> ') ACTH, adreno- corticotrophin nerve, CRF ov histamine + 4.231 >> >> ACTH ACH — 4.231 >» >> ICSH, intersti- tial-cell-stim- ulating nerve p 4.232 4.324 Hormonal c ontrol of endocrine glands Vertebrata Gonad, testis Testosterone (somatic motor muscle tone) ICSH + 3.12 Gonad, ovary Oestrogen (somatic motor muscle tone) ICSH + 3.12 Gonad, ovary Progesterone (oviducal gland secre- tion) LSH + 4.12 control uncertain 158 KINETIC HORMONES — II 4.322 Nervous control of secretory cells of Jiervous origin Many hormone-secreting cells derived from the nervous system are neurosecretory cells. These seem always to retain an intimate contact with it (§ 2.11); positive evidence of nervous control of the release of secretion from these cells is limited to a few cases only, but it may well be true of all. The main doubt is as to how the control is achieved ; perhaps it is only a question of degree which determines at what point in a series of neurones the transmission of a stimulus from one to another ceases to be a nerve impulse, with a minute release of the appropriate chemical substance at the nerve endings, and becomes the release of a microscopically visible amount of neurosecretion, passing down the axon and activating the cells with which the axon is in contact. Technically the two are hard to separate, since section of the axon stops the flow of the chemical as completely as it stops the passage of the nerve impulse. Since in either case activation only passes to the immediately adjacent neurosecretory cell, a hormone (in the sense of a substance circulating in the blood) is not produced, though a neurohormone might be. Nervous stimulation of hormone secretion from the neurosecretory cell is most likely; but in any case the process remains distinct from the stimulation of gland cells by endocrino- kinetic hormones in the circulation (§ 4.323). All too often the action of kinetic hormones has only been shown in extracts, and the effect of the nervous system upon their natural secretion has been neglected. Yet there is often evidence, presump- tive or actual, for an action of the nervous system on the effectors, although they are under the intermediate control of kinetic hormones; for they produce responses that are so closely related to environmental factors that it seems as though the nervous system must be involved (Table 17). Examples of this are to be seen in both crustaceans and vertebrates, the chromatophores of which respond to changes of background that can only stimulate hormone secretion indirectly through the eyes (§3.222); or in CarausiuSy the pigment cells of which only respond to the effect of moisture on the skin, if the ventral nerve cord is intact (§ 3.121). The neurosecretory cells in the brain of insects, which are the sources of the endocrinokinetic hormones stimulating secretion by §4.322 CONTROL OF THE SECRETION OF KINETIC HORMONES 159 the prothoracic glands, and possibly by the corpora allata, arc probably also under nervous control. The action of climatic and other factors upon the brain certainly determines whether or not these activating hormones are released; but this is best seen in relation to the morphogenetic actions of the moulting hormones. There the brain hormone, prothoracotrophin, determines the time of larval moults by stimulating simultaneous secretion from both the prothoracic glands and the corpora allata; but it deter- mines the change-over to metamorphosis by activating the pro- thoracic glands alone (Part II, § 3). In the crustacean brain little is as yet known of the activation of the neurosecretory cells ending in Hanstrom's sensory pore organ, but they may be assumed to be influenced by the nervous system like other cells of the same sort. Their endocrinokinctic activity is not yet fully established (§ 4.211). In vertebrates nervous stimulation of secretory cells of nervous origin is well authenticated for the adrenal medulla, which maintains its connection Vv^ith the sympathetic nervous system, from which its cells are derived (§ 2.114). The situation in the neurohypophysis (§ 2.114), which derives its secretion from the neurosecretory cells in the brain, is less clear. There is good presumptive evidence for nervous control of the secretion of OXYTOCIN to cause contraction of myoepithelial cells (Fig. 3-7). Moreover, it is clear that aff"erent nerve pathways lead through the brain to the neurosecretory cells in the supraoptic and para- ventricular nuclei in the hypothalamus. These cells release oxytocin, which is then stored in the neurohypophysis; but the action of the nervous system on the neurosecretory cells is natur- ally difficult to investigate. Section of the neurohypophysial stalk provides no more answer to the problem than removing the neurohypophysis altogether, since it merely separates the store of the secretion from its source and leaves the source in contact with the rest of the brain. It has already been observed that after removal of the neurohypophysis, sufficient oxytocin can be released from the hypothalamus to ensure parturition (§ 3.113). An investigation of VASOPRESSIN, ADH, from the same nuclei in the hypothalamus would suffer from the same difficulties; but the action of ADH in contracting blood vessels may well be accidental, and the means 160 KINETIC HORMONES — II for Stimulating its secretion be linked to its metabolic purposes, for which it is certainly under nervous control (§ 5.322). It has been maintained that oxytocin and ADH are always secreted in the same proportions and may either be combined in one molecule or be linked to the same inactive matrix. More recently it has been claimed that the ratio between the two hormones can be appreci- ably changed in the neurohypophysial secretion, since electrical stimulation of the supraoptic nucleus releases a secretion with a ratio of oxytocic to ADH activity of 4:1, whereas suckling releases a secretion with a ratio of 100:1 (Harris, 1955). Nothing is known of the way in which the endogenous heart- accelerating hormone from the gland cells of the insect corpora cardiaca (§ 2.113) is stimulated, since its presence does not depend upon either the neurosecretion or the nerves from the brain ; but neither is it known if this substance from the corpora has a true physiological action (§ 3.111). 4.323 Nervous or other control of ectodermal glands There are no kinetic hormones in invertebrates secreted from ectodermal cells other than nerve cells. In vertebrates, they are confined to the adenohypophysis, which is derived from the stomodaeum (§ 2.123). This is the anterior lobe of the pituitary and includes the pars tuberalis, the pars intermedia and the pars distalis. The first is the source of the melanophore concentrating hormone, W, in Amphibia, and the second is the source of the antagonistic dispersing hormone, B. Both are secreted in response to stimulation of the eyes and must have a direct nerve connection with the brain. The pars intermedia has no nerves in mammals (Fig. 2-12); but it appears to secrete intermedin and may affect morphological, rather than physiological, colour changes. The kinetic hormones secreted from the pars distalis of the adenohypophysis are all endocrinokinetic, apart from prolactin, LSH, which stimulates the exocrine mammary glands as well as the endocrine corpora lutea. In most mammals the stimulation of these two glands would seem to alternate rather than to be linked, in that the mammary secretion of milk follows at the end of pregnancy, when the secretion of progesterone from the ovary § 4.323 CONTROL OF THE SECRETION OF KINETIC HORMONES 161 is fading out and being replaced by a return to secretion of oestrone. Many theories have been put forward to explain these effects of prolactin. The effects may perhaps be determined in part by the state of the glands upon which the hormone acts, the corpus luteum, for instance, only secreting when it has reached a certain level of growth, under the action of the luteinizing hormone LH. If this fails, as gestation ends, the corpus luteum may lose its power to secrete, despite the continuing presence of LSH. Equally the mammary glands do not grow enough to be able to secrete until they have been stimulated by oestrone as well as progesterone, and this does not begin until the end of pregnancy is near. In this way, the action of prolactin might appear to be switched from one type of gland to the other at the time of parturition, but proof is lacking (Cowie and Folley, 1955). Such a situation would not necessitate a control of LSH different from that exerted by the brain over the other endocrinokinetic hormones ; but what this is remains uncertain. One view is that these hypophysial gonadotrophins and also ACTH and TSH (acting upon the adrenal cortex and the thyroid) depend for their stimulation upon one or more chemical substances that diffuse or circulate from the brain, rather than on nerve impulses; but the evidence relates chiefly to the morphogenetic effects of these hormones rather than to their endocrinokinetic actions. Recent experiments on rats have shown that if the adenohypophysis is removed from the brain and implanted in, say, the kidney, it undergoes partial degeneration in about 4 weeks, and ceases to secrete any of its hormones except LSH. By re- implanting it in contact with the median eminence of the brain, whence it originally came, the various cell types are induced to differentiate and regain much of their secretory capacity. The effect is first apparent on the growth-promoting fractions of ACTH and TSH (§ 4.221), and later upon the morphogenetic gonadotrophins, FSH and LH ; there is also some slight evidence for the re-activation of the secretion-inducing action of TSH (§ 4.221). Re-implanting the adenohypophysial tissue in contact with other parts of the brain does not have this re-activating effect. Since there is no question of renewed nervous connection, it is claimed that the effect is due to a chemical cortical-releasing 162 KINETIC HORMONES — II factor, CRF^ from the median eminence of the brain (Nikitovitch- Winer and Everett, 1957). The substance is clearly not a true vascular hormone, since it does not act upon the hypophysis when the latter is out of contact with the brain, but supplied by the same blood. It may perhaps be a neurohormone (§ 1.2), or a short- lived vascular hormone, since some neurosecretory cells of the hypothalamus normally make contact with the portal blood supply of the adenohypophysis in the median eminence (Fig. 2-12). It is claimed that after re-implanting the hypophysis in contact with the median eminence, these cells may again pass their secretion for a short distance in the portal circulation, rather than just allowing it to diffuse between the hypothalamus and the hypophysis. One curious result has been reported. Implantation of an adenohypophysis from a male rat, under the median eminence of a hypophysectomized female, is able to maintain her normal oestrus cycle, and even to support pregnancy. This must mean that the implanted hypophysis is secreting the luteinizing hormone, LH, the secretion of which is normally totally inhibited in the male (§ 4.232). It seems necessary to conclude that "the hypothalamus supplies not only a general stimulus to anterior pituitary function, but also sets the pattern of this function''^ (Harris, 1955). It would clearly be of interest if this type of experiment, and those on CRF, were to be related more specifically to effects upon the release of endocrinokinetic hormones from the hypophysis. 4 . 3 24 Hormojial control of endocrine glands Four exceptional hormones, among those classed as kinetic, remain to be considered ; they all come from the gonads and are therefore mesodermal in origin. Of these, the vertebrate gonads secrete three : oestrone and testosterone, the two sex hormones which affect the tone and perhaps the activity of somatic muscle (§ 3.12), and progesterone, which is said to stimulate the secretion of oviducal glands in Amphibia and Mammalia (§ 4.12). Unlike any other kinetic hormones, the secretion of these vertebrate hormones is induced by endocrinokinetic hormones from the adenohypophysis (Table 17); such stimulation is characteristic of morphogenetic hormones, and it is noticeable that all the other * My italics. P. M. J. §4.4 REFERENCES 163 activities of these hormones are morphogenetic (§ 4.232). Progesterone, in the course of preparing the uterus for the implantation of the embryo, causes growth of the uterine glands to the stage when secretion from them becomes possible ; it may be that it does not actually stimulate the release of their secretion in the strict kinetic sense. The kinetic actions of oestrone and testos- terone on somatic muscles may also be considered as incidental; they cannot in any case be claimed as the sole actions of these hormones. The last gonadial hormone in this group occurs in Gastropoda and controls the secretion of mucus from the oviduct as well as having far-reaching morphogenetic effects; it is, there- fore, similar in function to progesterone, but nothing is known of the means whereby the secretion of the hormone is controlled. 4.4 References Adams, A. E. (1946). Variations in the potency of thyrotrophic hormone of the pituitary in animals. Quart. Rev. Biol. 21 : 1-32. Arvy, L. and Gabe, M. (1954). Donnees histophysiologiques sur la neuro-secretion chez les Insectes Paleopteres (Ephemeropteres et Odonates). Pubbl. Staz. zool. Napoli, 24, Supplemento: 54-55. AsTwooD, E. B. (1955). Growth hormone and corticotropin. In The Hormones, edited by G. PiNCUS and K. V. Thimann. New York: Academic Press Inc. 3: 235-308. Bacq, Z. M., Fisher, P. and Ghiretti, F. (1952). Action de la 5-Hy- droxytryptamine chez les Cephalopodes. Arch. int. Physiol. 60: 165-171. Barker, S. B. (1955). Thyroid. Ann. Rev. Physiol. 17: 417-442. Bastian, J. W. and Zarrow, M. X. (1954). Stimulation of the secretory glands of the skin of the South African frog (Xenopiis laevis). Endocrin- ology, 54: 116-117. Bates, R. W. and Cornfield, J., etc. (1957). An improved assay method for thyrotrophin using depletion of P^^ from the thyroid of day-old chicks. Endocrinology, 60: 225-238. Bayliss, W. M. and Starling, E. H. (1902). The mechanism of pan- creatic secretion, jf. Physiol. 28 : 325-353. Bidder, A. M. (1950). The digestive mechanism of the European squids, Loligo vidgaris, Loligo forbesii, Alloteuthis media and Alloteuthis subulata. Quart, jf. micr. Sci. 91: 1-43. BoGDANOVE, E. M. (1957). Selectivity of the effects of hypothalamic lesions on pituitary trophic hormone secretion in the rat. Endocrinology, 60:689-697. 164 KINETIC HORMONES — II CowiE, A. T. and Folley, S. J. (1955). Physiology of the gonadotropins and the lactogenic hormone. In The Hormones, edited by G. PiNCUS and K. V. Thimann. New York: Academic Press Inc. 3: 309-387. Day, M. F. and Powning, R. F. (1949). A study of the processes of digestion in certain insects. Aust.J. sci. Res. B.2: 175-215. De Robertis, E. (1949). Cytological and cytochemical bases of thyroid function. A?in. N.Y. Acad. Sci. 50: 317-335. EcHALiER, G. (1956). Influence de I'organe Y sur la regeneration dss pattes, chez Carcinides moenas L. (Crustac6 decapode). C. R. Acad. Sci., Paris, 242: 2179-2180. Gabe, M. (1953). Quelques acquisitions recentes sur les glandes endo- crines des Arthropodes. Experientia, 9: 352-356. Galli-Mainini, C. (1951). Secretions des glandes de I'oviducte du crapaud par Taction de la progesterone. C R. Soc. Biol., Paris, 145: 436-437. Greer, M. A., Scow, R. O. and Grobstein, C. (1953). Thyroid function in hypophysectomized mice bearing intraocular pituitary implants. Proc. Soc. exp. Biol., N.Y. 82: 28-30. Grossman, M. I. (1950). Gastrointestinal hormones. Physiol. Rev. 30: 33-90. Grossman, M. I., Robertson, C. R. and Ivy, A. C. (1948). Proof of a hormonal mechanism for gastric secretion — the humoral transmission of the distention stimulus. Amer.J. Physiol. 153: 1-9. Harris, G. W. (1955). Neural Control of the Pituitary Gland. London: Edward Arnold Ltd. HiNTON, H. E. (1951). The structure and function of the endocrine glands of the Lepidoptera. Proc. S. Land, ent, nat. Hist. Soc. 1950-1 : 124-160. HiRSCH, G. C. and Jacobs, W. (1930). Der Arbeitsrhythmus der Mittel- darmdruse von Astacus leptodactylus. II. Wachstum als primarer Faktor des Rhythmus eines polyphasischen organigen Sekretions- systems. Z. vergl. Physiol. 12: 524-558. Karlson, p. (1956). Chemische Untersuchungen iiber die Meta- morphosehormone der Insekten. Ann. Sci. nat. (b) Zool. 18: 125-137. Keeton, R. W., Koch, F. C. and Luckhardt, A. B. (1920). Gastrin studies. III. The response of the stomach mucosa of various animals to gastrin bodies. Amer.J. Physiol. 51 : 454-468. Knowles, F. G. W. and Carlisle, D. B. (1956). Endocrine control in the Crustacea. Biol. Rev. 31 : 396-473. Krijcsman, B. J. (1928). Arbeitsrhythmus der Verdauungsdriisen bei Helix pomatia. II. Sekretion, Resorption und Phagocytose. Z. vergl. Physiol. 8: 187-280. Laviolette, p. (1956). Role de la gonade dans la maturation glandu- laire du tractus genital chez quelques Gasteropodes. Ann. Sci. nat. (b) Zool. 18: 171-173. § 4.4 REFERENCES 165 LuscHER, M. and Engelmann, F. (1955). Uber die Steuerung der Corpora allata-Funktion bei der Schabe Leucophaea maderae. Rev. Suisse Zool. 62: 6^9-657. Matthews, L. H. (1955). The evolution of viviparity in vertebrates. Mem. Soc. Endocrin. 4: 129-148. MuLLER, J. (1953). tjber die Wirkung von Thyroxin und thyreotropem Hormon auf den Stoffwechsel und die Farbung dcs Goldfisches Z. vergl. Physiol. 35 : 1-12. MuNSON, P. L. and Briggs, F. N. (1955). The mechanism of stimula- tion of ACTH secretion. Rec. Prog. Horm. Res. 11 : 83-107. Nalbandov, a. N. (1959). Role of sex hormones in the secretory function of the avian oviduct. In Comparative Endocrinology, edited by A. GoRBMAN. New York: John Wiley and Sons Inc. 524-532. NiKiToviTCH-WiNER, M. and Everett, J. W. (1957). Resumption of gonadotrophic function in pituitary grafts following retransplantation from kidney to median eminence. Nature, Lond. 180: 1434-1435. Pavlov, I. P. (1910). The Work of the Digestive Glands. London: Chas. Griffin & Co. Ltd. PiCKFORD, G. E. and Atz, J. W. (1957). The Physiology of the Pituitary Gland of Fishes. New York: New York Zoological Society. Prosser, C. L. (1950). Comparative Animal Physiology. Philadelphia and London: W. B. Saunders Company, 1-208. Rasquin, p. and Rosenbloom, L. (1954). Endocrine imbalance and tissue hyperplasia in teleosts maintained in darkness. Bull. Amer. Mus. nat.Hist. 104: 359-426. RiNFRET, A. P. and Hane, S. (1955). Presence of ACTH in pituitary gland of Pacific Salmon (O. keta). Proc. Soc. exp. Biol., N.Y. 90: 508- 510. RoMijN, C. (1935). Die Verdauungsenzyme bei einigen Cephalopoden. Arch, neerl. Zool. 1 : 373-431. Rosenblueth, a. (1950). The Transmission of Nerve Impulses at Neuro- effector Junctions and Peripheral Synapses. Massachusetts : Institute of Technology. Saka, M. O. (1952). Hyperglycemic-glycogenolytic factor in diabetic man and alloxan-diabetic animals. Amer. J. Physiol. 171 : 401-406. Salter, W. T. (1949). The metabolic circuit of the thyroid hormone. Ann. N.Y. Acad. Sci. 50: 358-376. Sayers, G. and Sayers, M. A. (1949). The pituitary-adrenal system. Ann. N. Y. Acad. Sci. 50 : 522-539. Scharrer, B. (1952). Neurosecretion. XL The effects of nerve section on the intercerebralis-cardiacum-allatum system of the insect Leuco- phaea maderae. Biol. Bull. Wood's Hole, 102: 261-272. Simpson, S. A. and Tait, J. F. (1955). Recent progress in methods of isolation, chemistry and physiology of aldosterone. Rec. Prog. Horm. i^e5. 11:183-210. 166 KINETIC HORMONES — II Slusher, M. a. and Roberts, S. (1957). Fate of adrenal ascorbic acid relationship to corticosteroid secretion. Endocrinology, 61 : 98-105. Smith, C. L. (1955). Reproduction in female Amphibia. Mem. Soc. Endocrin. 4: 39-56. Taurog, a., Tong, W. and Chaikoff, I. L. (1958). Thyroid P^i meta- bolism in the absence of the pituitary: the hypophysectomized rat treated with thyrotropic hormone. Endocr'mology, 62: 664-676. Thomsen, E. (1952). Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blow-fly, Calliphora erythrocephala Meig. J. exp. Biol. 29: 137-172. Turner, C. D. (1955). General Endocrinology, 2nd Edit. Philadelphia and London: W. B. Saunders Company. Wang, C. C. and Grossman, M. I. (1951). Physiological determination of release of secretin and pancreozymin from intestine of dogs with transplanted pancreas. Amer.J. Physiol. 164: 527-545. Wastl, H. (1922). tjber die Wirkung des Adrenalins auf die Driisen der Krotenhaut. Z. Biol. 74: 77-80. Williams, C. M. (1952). Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the Cecropia silk- worm. Biol. Bull. Wood's Hole, 103 : 120-138. Winton, F. R. and Bayliss, L. E. (1955). Human Physiology. London: J. & A. Churchill Ltd. WiTSCHi, E. (1955). Vertebrate gonadotrophins. Mem. Soc. Endocrin. 4: 149-165. Young, F. G. (1945). Growth and diabetes in normal animals treated with pituitary (Anterior Lobe) diabetogenic extract. Biochem. J. 39: 515-536. CHAPTER 5 METABOLIC HORMONES The term metabolic hormone is in general use, unlike the term kinetic introduced above; but some authors extend it to include morphogenetic hormones (Knowles and Carlisle, 1956). It is here applied to those hormones activating, inhibiting, or controlling the rate of certain biochemical processes of metabolic importance, occurring within the cells of the animal body, even w^hen these processes can only be measured by their end-products, or by the accompanying changes in oxygen consumption. It is not used for those morphogenetic processes which, although they depend upon and are often limited by cell metabolism, yet manifest themselves in growth and differentiation. Metabolic hormones in this limited sense differ sharply from kinetic hormones, because they do not act upon specific effectors or replace a type of control that is otherwise exerted by nerves. They control the rates of some cell activities which are often under no other apparent control, but appear to be determined genetically and to persist unaltered throughout the animal's life, at least until senescence intervenes. For instance, the absorptive activity of the gut cells of most animals seems to be as uncoordinated as the flagellar beat of sponge collar cells! The hormones which introduce variability and control into some of these biochemical systems are further differentiated from kinetic hormones, at least among vertebrate examples, in that their secretion is not directly induced by nerves. In many cases the stimulus to secretion, apart from general nervous ''stress", comes from an endocrinokinetic hormone (§ 4.2) intervening between the metabohc hormone and any possible nerve control. It follows that the action of these metabolic hormones is usually long-term in character, the most rapid probably being those concerned with 167 168 METABOLIC HORMONES water balance and response to stress. They will be considered in relation to their main actions, namely, the control of general metabolic rate and fat deposition (§5.1), intermediary metabolism of carbohydrates and protein (§ 5.2), and the balance of monovalent electrolytes and water (§5.3) and of calcium and phosphates (§ 5.4). 5.1 General metabolic rate Changes in the general metabolic rate are reflected in the respiration rate of resting animals and also, less directly, in the deposition of fat, since the latter tends to increase with reduction in oxygen consumption (Table 18). 5.11 RESPIRATION Hormones that increase respiration, and therefore increase oxygen consumption, are well-established in Insecta and Verte- brata; but are less clear in Crustacea. Hormones that decrease respiration are best known in Arthropoda. 5.111 Increase in oxygen consumption Crustacea. Extracts of brain and nerve cord increase respira- tion in the freshwater crayfish, Cambarus immunis. Injection into eyestalkless crayfish of an extract of as little as one-tenth of a brain in 0-05 ml saline results in an increase of 22-8 per cent in oxygen consumption (Scudamore, 1947). This increase is in addition to that produced in the test animals by eyestalk removal (§ 5.12). Similar injections of an extract of the nerve cord and its ganglia can result in an increase of 29 per cent. Cautery of the eyestumps of eyestalkless crayfish, causing injury or stimulation of the brain, also produces a temporary increase of 56-8 per cent in oxygen consumption. Insecta. The corpus allatum secretes a hormone which significantly increases the respiratory rate of insects. A series of experiments by E. Thomsen (1949, and Thomsen and Hamburger, 1955) on the blowfly, Calliphora, are noteworthy for the use of extensive controls. Uniformity of material was maintained by using only 7- day-old adults cultured at 25 °C and starved for 24 hr before the males and females were tested separately. Since allatectomy, or removal of the corpus allatum, §5.111 < M W H < K m w H K W W > Z O « O RESPIRATION 169 -C c Q C3 (U u O CO a D 03 U Q c -a " >< » J ! 1 L 5 10 15 20 25 30 35 40 45 50 Oxygen consumption mm^/lOOmg fly/10 min, in class intervols Fig. 5-2. Diagram, similar to Fig. 5-1, to show the effect of implanting three extra corpora allata into CalUphora. The mean oxygen consumption for the flies with implanted corpora allata (below) is increased as compared with that of the mock-operated controls (above). (From Thomsen, 1949). produced when glands with no nervous connection are implanted in the body cavity. Although it is now known that an active extract of the corpora allata can be obtained with ether, it has not yet been tested for its action on respiration (Hasegawa, 1957). § 5.111 RESPIRATION 173 It is possible that the effect measured here should be associated with the diabetogenic action of the corpora allata (§ 5.211). The action of the corpus allatum that increases oxygen consump- tion is not stimulated by nerves ; nor is there any definite evidence of an endocrinokinetic effect (§ 4.213) upon them from either the median or lateral cerebral neurosecretory cells or their storage organ, the corpus cardiacum (E. Thomsen, 1952). Chordata. Thyroxine secreted by the thyroid gland (§ 2.221) is the hormone that is effective in many chordates in raising the basal metabolic rate and increasing oxygen consumption ; but it is best known in birds and mammals. The hormone contains iodine, and is not highly specific, since extracts from the thyroid glands of dogfish, Scyliorhinus, have almost the same effect upon mammals as do extracts of their own glands (Fig. 5-3). Although thyroxine is obtainable from many of the cold-blooded vertebrates, it is not always clear that it has any physiological function in them, or has at best more than seasonal significance in regard to metabolic rate. In many cases thyroxine acts mainly as a morphogenetic hormone (Part II, § 3). Protochordata. The Urochordata and Cephalochordata have a ciliated endostyle in the floor of the pharynx, the primary function of which is the secretion of mucus ; but on embryological grounds the structure was, at one time, believed to be homologous with the thyroid of vertebrates. Urochordata. The ascidian, Perophora, has been examined to see if any of the tissues would accumulate radioactive iodine, as the thyroid gland does. This examination gave the anomalous result that the endostyle did not accumulate iodine, although the stolon did so (Gorbman, 1941). Cephalochordata. Unlike the ascidians, the amphioxus, Branchiostoma, does accumulate quite appreciable quantities of iodine in the endostyle (Thomas, 1956), but this is associated with mucopolysaccharides, and not with glycoproteins, as it is in thyroid glands. There is no evidence of any action of the iodine compound upon the amphioxus itself, though extracts provide iodine that can be utilized by the thyroid glands of Amphibia (Harrington, 1958). Agnatha. Lampreys undergo a well-defined metamorphosis 174 METABOLIC HORMONES from a larval ammocoete, which has a pharyngeal endostyle resembling that of the Protochordata, to an adult form with a thyroid comparable with that of other vertebrates. The endostyle of ammocoetes is capable of accumulating and storing iodine, associated with a glycoprotein, but the organ does not secrete the colloid, typical of a thyroid gland, until the adult stage. Leach (1946) found no evidence of changes in oxygen consumption accompanying these secretory changes ; but he did not remove the gland nor try the action of extracts. Elasmobranchii. Dogfish and skates have well-defined thyroid glands, which can be removed relatively easily. Extracts of thyroid from the dogfish, Scyliorhinus {= Scyllium), increase the oxygen consumption of mammals (Fig. 5-3) ; but the presence of thyroxine in the blood of these fish seems to have no discernible effect upon their own metabolism. For a period of 42 days after thyroidectomy, the fish showed no appreciable changes in their oxygen consump- tion, as compared with mock-operated controls (Matty, 1954). Teleostei. Most teleost fish have diffuse thyroid tissue which is recognizable histologically, but almost impossible to remove surgically, except from the parrot-fish, Pseudoscariis, in which the gland is in a compact capsule. Like the thyroxine of elasmobranchs, that of teleosts is active in mammals (Fig. 5-4; D. C. Smith and Brown, 1952). Earlier reports claim that the bony fish are as insensitive to the metabolic effects of thyroxine as the foregoing groups, at least when the thyroxine is of mammalian origin (Root and Etkin, 1937). More recently a slight increase in oxygen consumption has been induced in Bathystoma, by injecting extracts of thyroid from another teleost fish, Pseudoscariis (Smith and Matthews, 1948). It has also been found that synthetic thyroxine and thyroid stimulation by a thyrotropic preparation (§4.221) both increase the oxygen consumption of goldfish, Cyprinus, by as much as 100 per cent for as long as 5 hr (Miiller, 1953). Despite the well-known sensitivity of fish to handling, which makes experimentation extremely difficult (Hoar, 1957), control fish showed only 20 per cent increase in oxygen consumption, lasting only 40 min, as a result of saline injections. Amphibia. Seasonal changes in size and activity of the thyroid § 5.111 RESPIRATION 175 have been recorded for several amphibia (as well as for some fish and lizards) living in temperate climates (Lynn and Wachowski, 1951). It would seem as if the increase in thyroid activity in the summer enables these cold-blooded species to attain a high enough rate of basal metabolism even in temperate climes to become active despite the relatively low and variable temperature. Fig. 5-3. Percentage changes in oxygen consumption of the rat, Rattus, (ordinates) with time in days (abscissae) when thyroid EXTRACTS are injected intra-peritoneally. There is little difference in effect between injection of 100 mg mammalian thyroid powder (black circles) and of 95 mg dogfish, Scyliorhinus, thyroid (white circles). Both cause considerable increase for about a week as compared with either normal controls (white triangles) or even controls injected with 2-0 ml 0-9% NaCl (black triangles). (From Matty, 1954). Increase in oxygen consumption due to thyroxine is never very great in Amphibia. Tadpoles and axolotls seem to be more responsive than adult frogs to thyroid treatment, whether this is given by feeding or injection. Thyroidectomy might make the frogs more sensitive, especially if they were tested in the winter with amphibian rather than mammalian thyroid extracts. The fact 176 METABOLIC HORMONES that increased respiration can also be obtained in adult salamanders by thyroid injection (Taylor, 1939) shows that the action is not merely a reflection of the stimulus towards metamorphosis in the larval forms (cf. Part II, § 3), as might otherwise be supposed. 140 120 100 80 Control 6 onimals "Respiratory quotient" .Oxygen consumption Ttiyroxine 5 animals 140 I 2 3 4 5 6 7 Mammalian ttiyroid 2 animals 10-14 10-14 140 1^0 Fish ttiyroid 7 animals ^^ ^^ 100 an ^ 1 1 1 1 I 1 I 12 3 4 5 No. of days after injection 10-14 Fig. 5-4. Effects of thyroxine and thyroid extracts on the oxygen consumption (full line) and respiratory quotient (as percentages of the average values for the controls, shown by broken line) in male w^hite rats, Rattus. Time in days as abscissae. The effects of purified thyroxine and the extract of a mammalian thyroid are similar. The extract of parrot fish, Pseudoscarus, thyroid causes less increase in oxygen consumption, but the effect lasts longer than the others (from D. C. Smith and Brown, 1952). §5.111 RESPIRATION I77 AvES. Judging by their high normal temperature of 39 °C (as compared with about 37 °C in man), the basal metaboHsm and heat output of birds must be the highest of any vertebrates. Yet evidence for the control of heat production and oxygen consumption by the thyroid is scanty. The basal metabolic rate drops by 20 per cent in pigeons within a week of thyroidectomy (Marvin and Smith, 1943). Heat output and respiration have also been shown to vary in different races of pigeons, and this is believed to be due to genetic variations in the activity, but not necessarily in the size, of their thyroid glands (Riddle, 1947). Mammalia. Hormone control of oxygen consumption is well- established in mammals, marked increase accompanying hyper- function of the THYROID GLAND or injection of thyroxine (Fig. 5-3). A small injection of saline gives control evidence that the increase in oxygen uptake by the rat, due to the experimental procedure, is barely significant and of a different order of magnitude from the increase due to thyroxine of whatever origin (Matty, 1954) ; the converse effect, of thyroid removal, is not shown in the figure. An important feature of the results is the close similarity in effect produced by injections of roughly similar amounts of thyroid extract from a mammal and from an elasmobranch or a teleost fish (Fig. 5-4). There has been much discussion as to the exact role of thyroxine in mammalian metabolism, for measurements of its effect upon oxygen consumption only show the end stage in what may be a long chain of events, any or all of which may be sensitive to the hormone. Although thyroxine has been shown to act upon a very large number of enzyme systems in the body, the only consistent reaction that has been demonstrated in vitro is the effect of thyroxine in increasing the phosphorus turnover in oxidative phosphorylation (§ 5.211 ; Rawson et al.y 1955). In vertebrates the thyroid gland is apparently always under endocrinokinetic control by thyrotrophin, TSH, from the adenohypophysis. In goldfish, Cyprinus (Miiller, 1953), as well as in mammals, there is now definite evidence that secretion of the thyroid gland in relation to its metabolic action is stimulated by thyrotrophin, when this is injected as a purified extract (§ 4.221). This will probably be found to occur in all vertebrates. 178 METABOLIC HORMONES 5.112 Decrease in oxygen consumption Crustacea. One of the hormones from the ganglionic-X- organ/sinus gland complex in the eyestalk of decapod crustaceans decreases oxygen consumption. This may either be a direct action or the hormone may inhibit the secretion, or action, of the brain hormone which increases oxygen consumption (§ 5.111); but no decision between these ahernatives is possible at present. The experimental evidence is not very satisfactory in the case of individual species; but taken together the results are consistent for all except Leander. ;; /■' .500 400 L_^'^ /^. 300 :_^_-c-_^ - 1 1 1 200 -1 1 1 I 1 6 :. — 4 5 6 Time , days Fig. 5-5. Effect of eyestalk removal on the rate of oxygen con- sumption in the crayfish, Cambarus immunis. N, average for six normal crayfish; E, average for six crayfish from which both eyestalks were removed at X (there is no explanation of the high value for the previous 4 days); S, results for one crayfish from which removal of one eyestalk at 2 days gave no effect, but removal of the second at 4 days caused a marked increase in the one subsequent measurement. It is claimed that the eyestalks supply a RESPIRATION-INHIBITING HORMONE (from Scudamore, 1947). In Cambarus immunis there is no significant change in oxygen consumption after the removal of only one eyestalk, and very little after removing both sinus glands ; but removal of both eyestalks allows the oxygen consumption to increase by about 60 per cent (Scudamore, 1947). Extract of sinus glands alone, injected into eyestalkless animals, is said to decrease oxygen consumption by §5.112 RESPIRATION 179 16 per cent; but when the glands are stored for some time before making alcoholic extracts, it is doubtful whether the extracts contain a substance that is normally released into the blood (Edwards, 1950). Control injections of saline and of muscle extracts did give negative results ; but extracts of other parts of the eyestalk or even of whole eyestalks were not tested in this case (Fig. 5-5). When such extracts are made and injected into eyestalkless crabs, such as Uca, oxygen consumption is reduced to nearly normal (Fig. 5-6). Removal of sinus glands from Astacus (Frost et al., 1951) has practically the same effect as a mock operation in which the glands are exposed but not removed, whereas removal of the whole eyestalk has a much greater effect (Table 19). This last experiment Table 19. Changes in oxygen consumption in astacus, following sinus gland or eyestalk removal Individual values for oxygen consumption in cmVhr/lOOg body weight (from Frost et al. 1951). SPECIMEN Pre-operative values After mock operation After sinus gland removal After eyestalk removal 3-06 4-56 4-77 5-57 5-13 5-33 617 3-02 5-38 6-26 5-16 7-77 was not well controlled, but in Camharus the removal of both antennae, which would be an operation of comparable severity to eyestalk removal, had no effect upon oxygen consumption. ' Bliss (1953) interprets similar but more detailed experiments to mean that although the respiration-inhibiting hormone is stored in the sinus gland, and can therefore be supplied by injected extracts, its source is in the ganglionic-X-organ, which supplies sufficient hormone for control of respiration after removal of the sinus glands. Eventually, after removal of both sinus glands. 180 METABOLIC HORMONES which probably regulate the release of hormone into the blood, the crab, Gecarcinus lateralis, loses "its normal ability to vary the type and rate of metabolism". Indications of this are seen in a O 0.12- 0.10- 008- 0.06- 0.04- 0.02- PUGILATOR WOODS HOLE PUGILATOR (FLORIDA] PUGNAX Fig. 5-6. Effects of eyestalk removal and subsequent injection of eyestalk extract on the average rate of daytime oxygen consump- tion of two fiddler crabs, Uca pugilator and U. pugnax, over a period of some weeks after treatment. Removal of one eyestalk removes less of the respiration-inhibiting hormone than does that of two; the injected extract is not strong enough fully to replace it (from Edwards, 1950). normal respiratory quotient and a low and relatively invariable respiratory rate, compared with normal crabs (Fig. 5-7^). The greatest increases in oxygen consumption in eyestalkless crabs are §5.112 RESPIRATION 181 associated with the onset of moulting, which follows eyestalk removal (Part II, § 3) and tends to obscure other effects. At Naples, Leander serratus shows no increase in oxygen consumption after eyestalk removal, neither does this operation lead to moulting, as in the other decapods so far considered (Scheer and Scheer, 1954). Nevertheless, successive stages in N |IES| 2ES moult 10 20 30 10 20 30 2SG I 2ES moult Fig. 5-7. Effects of eyestalk and sinus gland removal on the oxygen consumption of land crabs, Gecarcinus lateralis, at 10-day intervals after the treatments indicated, a and b show results from two different crabs; N, when normal; 1 ES, after removal of one, and 2 ES, after removal of two, eyestalks; 2 SG, after removal of two sinus glands only; arrows mark the time of ecdysis, or moulting, preparation for which must have occupied several previous days during which there is a great increase in oxygen consumption (from Bliss, 1953). moulting are associated with changes in respiratory rate, and the writers postulate control by two or three hormones acting in turn ; but these have not been located. A further counterclaim that the oxygen consumption of muscle homogenates from a number of Crustacea could be decreased by removing the eyestalks of the specimens shortly before taking the muscle samples seems to be unfounded (Scheer et al., 1952). 182 METABOLIC HORMONES Insecta. a marked decrease in respiration accompanies the phenomenon of diapause, or arrested development and inertia, which occurs seasonally in many insects. During diapause the cytochrome c system in the cells becomes inactive, except in some muscles, and the cyanide-stable respiration accounts for most of the remaining low rate of oxygen consumption. The phenomenon is best known in the embryos of the Japanese silkworm, Bombyx, (Fukuda, 1952 and 1953) and in the pupae of the Cecropia silkworm of America, Hyalophora (= Platysamia, Williams, 1952). There is a certain amount of disagreement as to whether the hormonal control is the same in both cases (Hinton, 1953) or different (e.g. Lees, 1955). The action of a diapause hormone, D, secreted by the sub- OESOPHAGEAL GANGLION and reducing the oxygen consumption, is most clearly established in Bombyx. The situation is peculiar, as compared with other examples of hormonal control, in that secretion of the hormone D is determined by environmental factors in one generation, but only takes effect upon the next. It appears that the hormone secreted into the haemolymph of the female moth passes into her ovary, where sufficient D is absorbed into the eggs to put the resulting embryos into diapause within 28 hr of their being laid. The eggs containing the hormone can be recog- nized by their brown colour and are referred to as diapause eggs. They can be made to continue their development beyond the 28 hr by being put into Ringer solution after being stripped of their enclosing chorion and cuticle. It seems probable that this treatment allows the diapause hormone D to diffuse out of the embryonic tissues. Some races of Bombyx have only two generations in the year. That w^hich develops during the long, hot days of summer lays diapause eggs, which then over-winter in a dormant state. When the embryos begin to develop in the short, cool days of spring, they are not affected by sufficient daylight to induce the later secretion of D. So they lays eggs which develop directly, without diapause, during the summer, and the cycle starts again. Long daylength has been found to be the most effective environmental factor in determining the summer generation of these Bombyx to lay diapause eggs. It acts most strongly on the females just after § 5.112 RESPIRATION IN DIAPAUSE 183 the 18-somite stage in their development; but the effect is delayed until the adult stage, when the diapause hormone, D, is secreted. Other (univoltine) races of Botnbyx have only one generation per year, and this always undergoes diapause, unless the source of D in the suboesophageal glands is removed at the pupal stage (Fukuda, 1953). Much experimental work in Japan has proved that the source of the diapause hormone is in the suboesophageal ganglion of the adult, and that the brain can inhibit its secretion if the circum- oesophageal connectives remain intact. It is possible that the brain may even stimulate secretion of D, after the moth has been influenced by long day length to lay diapause eggs (Fukuda, 1953). The following series of experiments are typical : (1) If the suboesophageal ganglia are removed from late larvae after they have been exposed to long summer days (which normally induce the laying of diapause eggs) all the resulting females lay eggs which will not diapause, owing to the lack of D. (2) If the brain is removed at pupation from specimens that have been exposed as embr3^os to short spring days (which should result in their all laying non-diapause eggs) they turn into moths of which only 14 per cent lay such eggs, while 36 per cent lay diapause eggs, and the rest lay mixed batches. The increase in diapause eggs is interpreted as being due to removal of the inhibit- ing action of the brain, which leaves the suboesophageal ganglia free to secrete D. It may also show the lack of sufficient stimulus to cause all the females to lay only diapause eggs. (3) If isolated abdomens of specimens that should lay non- diapause eggs are used as hosts, the type of eggs actually formed in them can be influenced by transplants, as follows : (a) Brain or prothoracic gland transplanted alone has no effect. (b) Brain and suboesophageal ganglia, joined by their con- nectives, have no effect if taken from a moth which has been climatically determined to lay non-diapause eggs, and in which the brain was therefore inhibiting the secre- tion of D. (c) Suboesophageal ganglia transplanted alone, or severed from the brain and therefore not inhibited by it, result in the laying 184 METABOLIC HORMONES of diapause eggs in 70 per cent of cases. This effect can, in fact, be produced by ganglia from males as well as females, and even from moths of other species, including Antheraea pernyi (which does not itself lay diapause eggs but has a so-called "pupal diapause"; Lees, 1955). These experiments have been substantiated by extraction of pure diapause hormone from the suboesophageal ganglia of Bombyx (Hasegawa, 1957). Another very unusual feature has been postulated for the hormones concerned with diapause in Bombyx (Hinton, 1953). Since the adults are apparently unaffected by the diapause hormone which they secrete, and the eggs they lay continue to develop for over a day before the hormone D acts upon them, it is suggested that the moult-promoting hormone, ecdysone (Part II, § 3), from the prothoracic glands, in some way protects the tissues of the adults from the action of the diapause hormone, present at the same time. Moreover, some ecdysone must be passed into the eggs with D before they are laid, and this postpones the onset of dia- pause. It is assumed that ecdysone is broken down in a few hours in the young embryo, whereas the break-down of D is extremely slow, so that diapause lasts until the following spring when the supply of D is eventually eUminated. This might also be true for the embryos of Locustana pardalina, in which diapause ends and growth begins again before any organized endocrine glands have developed (Jones, 1956). In the Cecropia silkworm, Hyalophora^ the climatic determina- tion of diapause acts on the late embryo or early larva, which, unlike that of Bombyx^ itself undergoes diapause some months later in the pupal stage, when the suboesophageal ganglion is already well developed. Hinton (1953) maintains that the hormone D is again responsible for causing diapause ; but others, especially Williams (1952), claim that it is due to the lack of the prothoracic HORMONE, rather than to the presence of an inhibitor, D, acting on the brain. The somewhat scanty evidence can be interpreted either way. It is agreed that the end of diapause is due to the re-activation of the prothoracic glands. This is due to renewed neurosecretion of prothoracotrophin from the brain (§4.211), § 5.112 RESPIRATION 185 as in moulting; it can be induced artificially by chilling the brain. Injection of extracts of prothoracic gland can also break diapause. Even if D is the cause of diapause here, it is still not known whether the action of the prothoracic hormone in bringing diapause to an end is to inhibit the further secretion of D, or to release the tissues from its action, as in the adult Bomhyx. The fact that the diapause of hosts and their parasites is normally synchronous provides further evidence that diapause is determined hormonally (Hinton, 1957). For instance, an ichneumon, Diplazon fissoriuSy parasitizes several species of syrphids, although some of them diapause and others do not. If an active parasite is trans- planted (Schneider, 1950) from an active host to one that is diapausing, the parasite becomes immobile ; but in the reciprocal case, when the diapausing parasite is transplanted from a dia- pausing larval host to an active pupal host, the parasite resumes active growth. It therefore seems that the hormones of the host are able, not only to control the metabolic level of its own tissues, but also to override those of the parasite. Vertebrata. There is not much conclusive evidence for hor- mones that decrease oxygen consumption in vertebrates, except in amphibians and possibly mammals. Amphibia. A significant decrease in oxygen consumption, of about 30 per cent, occurs in starved frogs in the 4th and 5th weeks of treatment with A.C.E., an extract of the adrenal cortex, if 0-36 ml/Kg body weight is injected on alternate days (Calhoon and Angerer, 1955). There is no difference in loss of body weight during this period as between injected frogs and untreated controls. The authors do not specify which cortical steroid is dominant in Upjohn's extract which they used. It might be expected that one of those concerned with carbohydrate metabol- ism, rather than one of the ''mineralocorticoids", would decrease oxygen consumption. Mammalia. Decrease of basal metabolic rate and of oxygen consumption occurs chiefly in hibernating mammals; at other times the possible need for a hormone having this action would seem to be confined to maintaining the normal equilibrium in opposition to the thyroid (§ 5.111). There has been much work on the effect of injecting extracts of adrenal cortex into mammals; 186 METABOLIC HORMONES but the results reported have been contradictory. It is probable that in physiological doses the cortical hormones cause no significant change in the oxygen consumption oi normal mammals. In hibernation the presence of active adrenal cortex seems to be essential, in conjunction with relatively inactive thyroid glands. It is therefore possible that cortical secretion helps to reduce oxygen consumption in hibernating mammals, as it does in normal frogs ; but the adrenal cortex is by no means the sole cause of all the changes in metabolism and heat regulation that occur in hibernation (Lyman and Chatfield, 1955). 5.12 FAT METABOLISM Changes in fat stores in the body, like changes in respiration, are often indicative of the general rate of metabolism, so that increased fat consumption and decreased storage may accompany increased oxygen consumption, and may be controlled by the same hormone. Periodic changes in activity, such as moulting, may be preceded by an increase in the rate of fat con- sumption, and others, such as hibernation, by an increase in fat storage. 5.121 Increase in fat consumption or decrease in storage Crustacea. Fat consumption increases after the removal of a moult-inhibiting factor in the sinus gland (§ 5.122) ; but, as yet, no hormone or extract has been found that stimulates fat consump- tion. It might be sought in the brain, v^hich yields an extract that stimulates respiration (§ 5.111). Insecta. The presence of the corpora allata, with their stimulating effect on the metabolic rate, tends to reduce the store of fat in the fat bodies of most insects ; in adult females, when yolk is being deposited in the ripening eggs, the corpora also facilitate transport of fat to the ovary. This occurs in all insects except in Bombyx, where the eggs are matured before the adult emerges, and in Carausius. A detailed study of the production and transport of fat in the grasshopper, Melanopliis dijferentialis^ shows that the corpora § 5.121 FAT METABOLISM 187 allata cause a change in metabolism in normal females, from an early phase of fat accumulation in the fat body, to a later phase when the fat body becomes depleted and all the fat is passed into the Qgg yolk (Pfeiffer, 1945). If the corpora allata are removed near the beginning of the adult stage (whether the ovaries are present or not), the fatty acid content of the fat bodies continues to rise at the same rate as before. If adult females retain their corpora allata, the usual depletion of the fat bodies follows, even in castrated speci- mens, showing that the ovary has no effect. Normally the corpora allata begin to secrete sufficiently to cause this change some time after the emergence of the aduh, as they are inhibited during metamorphosis (Part II, § 3). Vertebrata. Thyroxine causes decrease in fat stores, at least in mammals. Treatment of rats with thyroxine causes an increase in the synthesis and turnover of phospholipids in the liver and in the secretion of cholesterol in the bile (Rawson et al.y 1955). Conversely, hypofunction, or reduction, of the thyroid is accom- panied by increase in fat deposition, provided the food supply is riot limited. The secretion of the thyroid is stimulated by thyro- trophin from the hypophysis (§ 4.221). 5.122 Decrease in fat consumption or increase in storage Crustacea. A sinus gland hormone seems to help in maintain- ing the amount of fat stored in the body. When the sinus gland or the whole eyestalk is removed, there is a rapid disappearance of fat, as well as an increase in oxygen consumption (§ 5.111), followed by the onset of moulting. It is not known if the same hormone controls all three processes. The effects of starvation, with and without sinus gland removal, have been examined in the crab, Hemigrapsus nudus. The sinus glands were removed by drilling through the eyestalk and aspir- ating out the gland tissue. Male and female crabs were compared with normal controls in the same inter-moult stage, but no figures are given for mock-operated animals. Female crabs normally have more fat than males, and 23 days of starvation makes no significant difference ; but sinus gland removal, followed by starvation for 23 days, results in the fat content dropping by nearly a half in both sexes (Table 20). 188 METABOLIC HORMONES Table 20. Changes in fat content of the body of crabs {hemic rap sus nudus) following starvation and sinus gland removal All values are given on a wet weight basis and are means of measure- ments on two to four individuals. The differences in fat, due to sex, and the losses after sinus gland removal are significant (from Neiland and Scheer, 1953). NORMAL STARVED 23 DAYS STARVED WITH SINUS GLAND REMOVED Sex Body weight, g Fat index/g 10-5 4-31 F 9-3 6-13 M 9-5 4-76 F 7-4 6-4 M 10-3 2-82 F 7-4 3-79 This, in conjunction with evidence on glycogen and chitin content (Table 23), suggests that "the eyestalk principle of crustaceans restrains . . . metabolism, but especially those processes connected with preparation for a molt" (Neiland and Scheer, 1953). But this finding is, perhaps, somewhat sweeping, since the authors appear only to have tested removal of the sinus glands, and not to have compared this with removal of the whole eyestalk, nor to have replaced either organ by injection of its extract. This is important, since the sinus glands are usually only storage organs for neurosecretory cells in the brain or the ganglionic-X-organ, both of which were left intact in these experiments and might have been expected to continue to supply some of the fat-preserving hormone as well as of the moult-inhibiting hormone. It has already been noted that the comparable hormone that restrains oxygen consumption is more abundant in the whole eyestalk than in the sinus glands alone (§ 5.112 and Fig. 5-7). Vertebrata. No hormones have been specifically associated with fat storage invertebrates, apart from lack of thyroxine (§5.121) or of thyrotrophin (§ 4.221). Relative inactivity of the thyroid seems to be the main factor in allowing accumulation of the fat stores that are necessary for hibernation in mammals (§5.112). It has been claimed that, in the brown trout, Salmo truttay most § 5.211 CARBOHYDRATE METABOLISM 189 fat is deposited in the gut wall around midsummer, when the thyroid gland reaches the single peak of its activity, as estimated by its accumulation of radioactive iodine (Swift, 1955). This seems to be anomalous ; but it may be noted that fat is also deposited in January, before the spring growth period. There may, therefore, be no causal connection between fat deposition and thyroxine secretion. 5.2 Intermediary metabolism of carbohydrates and proteins The biochemistry of intermediary metabolism is complex, but it need not be considered in detail here, since it is possible to measure the end-products of anabolism or catabolism, rather than the series of chemical transformations within these processes. These measurements can then be related to hormone treatment. Evidence is accumulating to show that in arthropods and in vertebrates the metabolism of carbohydrates and of proteins is often linked; but diiferent hormones may be concerned in con- trolling the two types of metabolism, which will therefore be considered separately. 5.21 carbohydrate metabolism Among the carbohydrates, sugars are most easily traced and can be measured quantitatively in the blood, as they increase in quantity after a meal, or after hormone treatment, and then decrease as they pass into the tissues to be stored or assimilated. Their distribution between the blood and the tissues is normally maintained at a relatively stable level by a balance of hormones, the diabetogenic type increasing the blood-sugars and the anti- diabetogenic decreasing them. Evidence of both types have been obtained in Arthropoda, as well as in Vertebrata (Table 21). 5.211 Increase in blood-sugar by ''diabetogenic'' hormones Crustacea. The sinus gland is the main source of a diabeto- genic hormone in both the crayfish, Astacus, and the blue crab, Callinectes. This has been shown in carefully controlled compari- sons (Table 22) between the effects of "stress" caused by asphyxia in normal and in operated animals, from which either the whole eyestalk or the sinus gland alone had been removed (Kleinholz 190 METABOLIC HORMONES P ,, o 5 s ^ '^ <3 « <3 ~ H " " 5 g « i:§,3.i ^ X ai o < « o a ';: -^ Ti £ - ^ ^ B c u 3 ^ Eyesta (MIH Y-Org (MPH Brain 6 B^.s g §:§ .to £ < O Q:; f^ e) O O C^ ^ C3 I I I o K O o o Hi 13 c o o C >> g H a d G U e C < w 1 I 1 H o o 5 :: b o o s^ o ■^ ;< >l •ft, K to z •S^ «o ^ [2 O - TI (3 K 1^ ^ o; ^ in lo §5.211 CARBOHYDRATE METABOLISM 191 et al, 1950). It is clear that an increase in blood-sugar, or hypcr- glycaemia, is produced in these crustaceans as a result of stress, very much as in mammals, as long as the sinus gland has its innervation intact; the converse experiment of injecting sinus gland extract raises the blood-sugar content as much as 400 per cent in Callinectes (Abramowitz et al, 1944). The comparable effect of excitement or pain in inducing hyperglycaemia in Callinectes (but not in the less pugnacious Astaciis) was shown by injecting plain saline ; this produced as great a rise in blood-sugars as injections of adrenaline, but in either case the effect could be almost completely inhibited by cutting the nerve to the sinus gland. Injection of an eyestalk extract, not identified more exactly, increases blood-sugars in a number of other Crustacea, including the freshwater shrimps, Paratya and Palaemon (Nagano, 1951). An eyestalk hormone also increases the concentration of glucose in the blood, and apparently decreases its utilization in the tissues of two species of spiny lobsters, Panulirus (Scheer and Scheer, 1951). However, since C^^ when used to label injected glucose, does not reappear in the respiratory CO 2 during the following 24 hr, but 30 per cent of it can eventually be recovered from the skeleton, it seems that glucose must be mainly concerned in chitin formation. It is therefore arguable that the hormone of the eyestalks w^hich increases blood-sugars should not be considered as comparable with the diabetogenic hormones of vertebrates. The hormone secreted from the sinus gland is presumably a neurosecretion derived as usual from either the brain or the ganglionic-X-organ ; but the exact origin has not yet been decided. There appears to be direct nervous control of the release of the diabetogenic hormone, with no intervention of any endocrino- kinetic hormone. Insecta. Reduction in blood-sugar follows the removal of the CORPORA ALLATA in the stick insect, Carausius (= Dtxippus; L'Helias, 1955), indicating a diabetogenic function for their secretion. There appears to be no direct evidence of the corpora allata being controlled by a hormone from the brain in this case, any more than there is in relation to oxygen consumption. Vertebrata. Recent information on diabetogenic hormones m 192 METABOLIC HORMONES be u .o C (11 ■'r v! -o c ° § 8 ^i^-^ ^- c -6 CO ^ - fJ tyn - C2 o S c S "^ ^ •§ r C C O ^ ^ IDX) ^ r^ 'r? c« a» I> be "^^ -^ g .t: '-i !U o «« ^ C ^ ^ S ^ - ^ C beo as CO 13 N CO s j < 1 ^00 ^00 SSw v^^-^^ -w ^^ T-. t^ Tj- T^ CO (N lO < o < T-i 00 tN lO O CO vb -H-H-H 1 o ^ O S O < ON ON IT) 00 t^ 00 ON « o r^ CO sb i^ 4-1 -H -H +1 T^ CO -4- -IH -H-H < t^ CO CO \0 ^ i- o T-l T-l CO CO rq (N cs K s* cu ^ ^ iz; < ^ Q w C/3 O ON SO O Tj- vO ^ T-< 6 c« ;z; hJ < z < § H — , ^ S-- !^ 1 •. c c § gc/^0 ^ £ W c/: •So,, U 5i o , o , ^z 1 ^ 1 =§^ 1 1 1 ^ c3 §5.211 CARBOHYDRATE METABOLISM 193 cold-blooded vertebrates and birds has shown that they resemble those in mammals. There are two different diabetogenic systems that can increase the blood-sugars in different species: either glucagon from the pancreas or a hormone from the adrenal cortex (Table 21). Teleostei. The tunny, Thunnus germo, and other fish secrete a substance like glucagon from the a cells of the pancreas, but the action of an extract has only been tested on rats (Mialhe, 1952). Amphibia. In frogs, Rana^ the adrenal cortex probably plays a part in the control of the sugar balance by secreting a diabeto- genic hormone ; for adrenalectomy and the consequent lack of this hormone results in hypoglycaemia, or a low level of blood-sugar. Adrenalectomy is also said to reduce the frog's capacity to absorb glucose and galactose from the gut, and to cause loss of glycogen reserves from the liver (Chester Jones, 1957a). The first effect is due to the unopposed action of insulin, the presence of which has now been recorded in frogs. The last effect is probably indirect; for the hypoglycaemia induced by the insulin would in due course inhibit further insulin secretion and so prevent the building up of glycogen reserves, even if it would not cause their depletion (§5.212). In Urodela the diabetogenic hormone is also cortical, but comes from the interrenal tissue. Reptilia. There is recent evidence for glucagon being the diabetogenic hormone in some lizards (Miller and Wurster, 1959). AvES. The total blood-sugars are normally about twice as high in birds as in mammals, and show a cyclical change, increasing by 14 per cent near the time of egg-laying. It is possible that, like mammals, different birds have different diabetogenic hormones. In the intact pigeon, Columba, injections of adrenocorticotrophin, ACTH, cause a great increase in blood-sugar, lasting about 10 hr. Pancreatectomy does not influence this reaction, but adrenalectomy eliminates it (Riddle et al, 1947). This is the same as in the rat, where the adrenal cortex is the source of the main diabetogenic hormone. In the chick, on the other hand, blood-sugar can be raised by STH, the so-called growth hormone of the anterior pituitary (Hsieh, Wang and Blumenthal, 1952). This seems to be 194 METABOLIC HORMONES like the dog, where STH acts as an endocrinokinetic hormone stimulating the secretion of glucagon, the actual diabetogenic hormone, from the pancreas. No mention of glucagon has been made in relation to this work on the chick; but the pancreas of some other birds contains large amounts (Miller and Wurster, 1959). Mammalia. A striking difference between different mammals occurs in respect of their diabetogenic hormones. In some carni- vores, including the dog and cat, but not the ferret, the main hormone that increases the blood glucose is glucagon, secreted by a cells in the islets of langerhans (§ 2.222). In the rat and in GLYCOGEN ^ Thyroid TSHf ACTHj* Ad. cortex* GLUCOSE ATP Insulin? Insulin > FATS and ^ GLUCOSE PHOSPHATE Ad. cortex ACTHf GLUCOSE - 6 -PHOSPHATE PYRUVIC ACID LACTIC ACID Fig. 5-8. Diagram to show some of the hormones believed to facilitate chemical transformations in the intermediary metabolism of the rat, Rattiis. In the dog, Canis, and some other carnivores, the diabetogenic action of the adrenal cortex, Ad. cortex*, stimu- lated by ACTH*, is replaced by that of glucagon stimulated by STH. ATP is adenosine triphosphate, an enzyme and not a hor- mone (from Ebling, 1951, and Fieser and Fieser, 1950). man, the main hormone having this action is hydrocortisone from the adrenal cortex. This has interesting repercussions in experimental work, since the cortex is stimulated by ACTH from the adenohypophysis (§ 4.231), and glucagon by the "growth" hormone, STH, from the same source (§ 4.222) ; it follows that § 5.212 CARBOHYDRATE METABOLISM 195 carnivores may become diabetic if treated with growth hormone, but that the rat can continue growing under the same treatment, with Uttle disturbance of its sugar balance, except in extreme cases. Glucagon secretion can also be stimulated directly by a low content of sugar in the blood (cf. § 5.521 ; Saka, 1952). The action of glucagon is usually estimated from its effect upon the glucose level in the blood; but it is claimed that the hydro- cortisone acts at two stages in the process of gluconeogenesis ; by stimulating the transformation of fats and proteins to pyruvic acid, and also by increasing the blood-glucose at the expense of glucose- 6-phosphate in the tissues and liver. The transformation of glycogen stores to free glucose via phosphorylated glucose is facilitated by thyroxine (§ 5.111); but the latter hormone alone is not able to release glucose into the blood stream (Fig. 5-8). As has been mentioned, hydrocortisone secretion is stimulated by the endocrinokinetic hormone ACTH, and glucagon by STH. Adrenaline can also have a diabetogenic effect in liberating sugar from liver glycogen into the blood. This is presumably an indirect effect, since adrenaline causes a slow release of ACTH (§ 4.231). The latter must be the main cause of the hyperglycaemia associated with stress and excitement, and (like the comparable sinus gland reaction in Callinectes) is also under direct nervous stimulation. 5.212 Decrease in blood-sugars by antidiabetogenic hormones Arthropoda. a hormone of this type has not so far been reported for the Crustacea. Insecta. It has long been postulated that an antidiabetogenic hormone is released just prior to moulting, and it is now believed to be the same as the moult-promoting hormone, ecdysone, from the prothoracic glands, or their equivalent in the ring gland of Calliphora (Dennell, 1949). The secretion of the mouhing hormone is under endocrinokinetic control from the neuro- secretory cells of the intercerebrum. Vertebrata. The main antidiabetogenic hormone of vertebrates is insulin, secreted from the ^ cells of the pancreatic islets of langerhans (§ 2.222). This hormone lowers the level of sugars in the blood by facilitating the supply of glucose to the tissues ; but 196 METABOLIC HORMONES there are two possible sites for this action. One is the cell mem- branes of the blood vessel walls and the tissues, where there is evidence that insulin stimulates the transfer of glucose from the blood stream to the tissue fluids. The other is the seat of chemical transformation within the tissues, where intracellular enzymes, particularly hexokinase, hasten the phosphorylation of glucose by adenosine triphosphate (ATP). This results in the formation of Fig. 5-9. Changes in distribution of injected galactose caused by INSULIN injection in eviscerated and nephrectomized dogs, Cams. Galactose was injected at the beginning at the rate of 1 g/Kg ; ordin- ates show the amounts recovered in the blood in mg per 100 g (not %); abscissae show time in hours after the injection; vertical hnes show the range of variation of all values in 6 or 8 specimens. (a) The upper curve shows that when equilibrium is reached after about 1 hour, in absence of insulin, the galactose concentration is such that it occupies 45 % of the body weight, or the volume of the blood only. The lower curve, for similar measurements made in presence of insulin, shows that the galactose is distributed in a larger volume, amounting to 70% of the body weight and equiva- lent to all the body fluids, (b) A similar record in which insulin is added after equilibrium in the blood has been reached in 2^ hr and causes a drop to the 70 % body weight distribution, as before (from Levine et al., 1950). glucose-6-phosphate, which is the starting-point both for utilizing glucose and for converting it to glycogen (Fig. 5-8). It has recently been postulated (Levine and Goldstein, 1955«) that stimulation of the transfer system is the main action of insulin, and that it merely § 5.212 CARBOHYDRATE MHTABOLISM 197 allows of a more rapid movement of glucose both in and out of the cells. Since the cell membranes are not permeable to the phos- phorylated hexose, the formation of this within the cell would automatically have the role of trapping the glucose and rendering the transfer virtually a one-way system. Even though insulin had no effect upon the rate of phosphorylation, such a system "would be consistent with the fact that glucose utilization [in contrast to galactose distribution, Fig. 5-9] is a summated phenomenon of both insulin-stimulated cellular entry and of metabolic disposal". Fish. In some teleosts and elasmobranchs insulin maintains a relatively low level of sugar in the blood; but the results of pan- createctomy are inconsistent, presumably because the source of glucagon (§ 5.211) is removed as well as that of the insulin. Amphibia. In Rana and Bufo the loss of insulin by pancre- atectomy results in hyperglycaemia, or increased blood-sugar, because the source of the diabetogenic hormone in the adrenal cortex is not removed (Houssay, 1959). AvES. Injected insulin is effective in lowering the level of blood- sugar in pigeons, which can survive higher doses of this hormone than can mammals (Riddle et al., 1947). Mammalia. Experiments have been carried out on anaesthetized and eviscerated dogs and rats to test the effect of insulin on the level of blood-sugars, in the absence of utilization and storage in the liver or loss by excretion (Levine et ah, 1950). Glucose is steadily consumed in the tissues; but another monosaccharide, galactose, is not utilized. When a known quantity of the latter is injected, it is found to distribute itself in a volume equivalent to about 45 per cent of the animal's weight. This is equal to the volume of circulating blood. After injection of insulin the con- centration of galactose in the blood drops, as though it were distributed in a volume equal to 70 per cent of the body weight (Fig. 5-9). This is equivalent to the volume of all the body water, and it is assumed, therefore, that the sugar has been allowed to pass into the intracellular fluid of the cells. By comparing a number of monosaccharides, it can be further shown that insulin only facilitates their passage into the tissues from the blood if the configuration of their molecules is such that the side-chains attached to the first three carbon atoms are the same as those of 198 METABOLIC HORMONES glucose. The optical isomers are unresponsive to insulin (Fig. 5-10). This suggests a point of further chemical attack upon the problem of the nature of the hormone action in lowering blood- sugars and making them available to the tissues ; but the relation Insulin responsive D- Glucose D- Galactose L-Arabinose Insiitin unresponsive ! CHO j CH2OH I CHO i HO — C — H 0=0 HO — C — H __j. [ , I 1 HO — C — H I I HO — C — H I H — C — OH H — C — OH H — C — OH H C — OH H— C — OH H — C — OH CH2OH CH2OH CH2OH D-Monnose D-Fructose D-Arobinose Fig. 5-10. The structure of some of the sugar molecules which have been found to be responsive to insulin and to resemble glucose in having the same side chains on the three terminal carbon atoms ; and (below) three molecules of sugars which differ, however slightly, from glucose, and are found to be unresponsive to insulin (from Levine and Goldstein in Stetten and Bloom, 1955). of these facts to the known chemical structure of the insulin, which is a protein, is not yet within sight. The rate of secretion of insulin is self-regulating, in that a high level of blood-sugar increases insulin ; and, as this lowers the level, so the insulin secretion itself is reduced. § 5.22 PROTEIN METABOLISM 199 There is no known hormone that stimulates the secretion of insuHn, unless secretion is found to be related to the increased growth of the gland, which is said to occur as a result of injecting extracts of the posterior pituitary (Staszyc, 1956). This is as yet unconfirmed. The claim that STH increases insulin secretion is based on an indirect effect, since it only occurs in those species where STH stimulates glucagon to raise the level of blood-sugars, and this in turn stimulates the insulin secretion (§§ 4.222 and 5.521). 5.22 PROTEIN METABOLISM Hormones play a part in protein metabolism in Arthropoda and Vertebrata, but the results so far reported are not always clear. Noble (1955) points out that, in vertebrates, most attempts to assess nitrogen, which is a characteristic element in proteins, have been based on measurements of the over-all balance in the body, whereas a more realistic picture might be obtained by following reactions in different organs of the body separately. Increased protein catabolism in one organ may be accompanied by an increase in nitrogen excretion (§ 5.222), or it may be mainly responsible for supplying materials for protein anabolism in some other organ. Arthropoda. The same difficulties of interpretation are prob- ably true for Arthropoda. Perhaps the best that can be suggested at present is that an eyestalk hormone in Crustacea tends to re- strain protein breakdown and nitrogen excretion and that the Y-organ hormone may inhibit them (Table 24). In Insecta a brain hormone appears to be associated with an actual increase in new protein formation. No hormones are yet known to stimulate catabolism in either Crustacea or Insecta. 5.221 Restraint of protein catabolism Crustacea. It has been claimed that an eyestalk hormone restrains protein catabolism and that removal of the sinus gland, which is the main storage organ of the hormone, results in protein breakdown and loss of nitrogen ; but the evidence is ambiguous, partly because the results seem to vary with stages in the moult and intermoult cycle. Positive evidence shows that sinus gland removal causes a loss 200 METABOLIC HORMONES of nitrogen in the crab, Hemigrapsus. Sinus gland removal also leads eventually to moulting, because of the absence of the moult- inhibiting hormone (Part II, § 3); but it is probable that the time required for moulting w^ould be more than the 23 days of the experiments summarized in Table 23 (Neiland and Scheer, 1953). Table 23. Changes in body composition of crabs (hemigrapsus nudus) following starvation and sinus gland removal All values are given on a wet weight basis and are means of measure- ments on two to four individuals (cf. Table 20). The changes in protein content, following the operation, are significant and are shown in italics (from Neiland and Scheer, 1953). NORMAL STARVED 23 DAYS STARVED WITH SINUS GLAND REMOVED Sex M F M F M F Body weight, g 10-5 9-3 9-5 7-4 10-3 7-4 Glycogen, mg/g 0-69 1-24 0-8 0-8 0-76 1-02 Protein, mg N/g 12-71 14-96 12-08 12-08 1039 10-40 Chitin, mg glucose equivalent/g 3-91 4-23 3-89 4-09 3-49 4-14 The results may be taken to represent the intermoult situation. The effects of fasting and the technique of sinus gland removal in Hemigrapsus have been referred to already in relation to reduc- tion of fat (§ 5.112). Their effect upon glycogen "which might be regarded as the most logical source of glucose and of chitin", is not appreciable, and supports the suggestion that the crabs w^re not near moulting, v^^hen new chitin is formed. There appears to be a certain weight loss, which is reasonable in starved animals ; but it would not occur in those from which removal of eyestalks was inducing moulting, as this is accompanied by increased water uptake (§ 5.321). It is claimed that in these conditions the reduction in total nitrogen is significant, and represents the effect of removing an eyestalk hormone that normally restrains protein catabolism. § 5.221 PROTEIN METABOLISM 201 This hormone may be the same as the moult-inhibiting hormone. Increased loss of proteins following eyestalk removal is also claimed for Carcinus from measurement of nitrogen excretion. This is partly due to the stress of wounding, since a similar effect, but of only half the magnitude, results from leg amputation (Needham, 1955). As no hormone injections were used to counter- act this eifect, the evidence is not conclusive ; but it again suggests that in non-moulting crabs an eyestalk hormone restrains protein breakdown. During moulting the situation is different. Although the protein content of the plasma is unusually high because of resorption from the old skin, nitrogen excretion is particularly low. Needham (1957) concluded that ''within limits the animal is able to control its nitrogen output, whatever the external conditions". As moulting proceeds most of the protein from the plasma is presumably deflected into certain anabolic processes, such as the formation of the new integument. These protein transfers would not of them- selves alter the total proteins in the body. Nor does starvation affect the issue, since most crabs do not feed for some days before, during or after moulting. Koch (1952) confirms the view that the nitrogen and protein content of the body remains remarkably constant during moulting, and is not affected by the absence of any eyestalk hormone. Incidentally, the moult-inhibiting hormone is not released from the eyestalk at this stage (Part II, § 3). Koch examined the nitrogen content of the mitten crab, Eriocheir sinensis, and found that the eyestalks have no effect upon the nitrogen metabolism, at least during the first experimentally induced moult. There is no difference in the ratio of nitrogen content to size, in the cast skins of crabs moulting normally and those induced to do so by eyestalk removal. The ratio of total nitrogen content to carapace width measured before moulting is the same, within limits, for moulting and non-moulting control crabs, and for operated crabs (between curves. Fig. 5-11). After an induced mouh, the ratio of nitrogen in body and cast skin to carapace width, measured after moultmg in the operated crabs, is significantly lower (B, Fig. 5-11) than before, by an amount that seems to be proportional to the increase in water content that follows from the absence of the diuretic 202 METABOLIC HORMONES hormone of the eyestalk (§ 5.321); but this is not shown quantita- tively. Excessive water uptake cannot afford an explanation for the contrary findings in Hemigrapsus, where there is no weight in- 800 600 - .o 400 - 200 30 40 50 60 Carapace breodth 70 Fig. 5-11. Relation of the total nitrogen content of the crab, Erio- cheir sinensis, to its size before and after moulting. The recorded values fall into three categories, (i) Total nitrogen in body (ordin- ates) measured before moulting and plotted against carapace breadth (abscissae) : % for non-moulting control crabs, (ii) Total nitrogen in body plus that in cast skin measured immediately after moulting and plotted against carapace breadth of old shell: O for control crabs moulting naturally; Q for eyestalkless crabs after forced moult, (iii) Same total nitrogen as in (ii) plotted against carapace breadth of neiv shell : © for controls that still fall within normal range shown by curves; B foi* eyestalkless crabs after forced moult due to loss of moult-inhibiting hormone, MIH. These show markedly low ratios and it is assumed that though loss of MIH does not affect nitrogen metabolism (ii), the loss of the DIURETIC HORMONE in the eyestalk allows increased water imbi- bition and swelling that increases the crab's size relative to its nitrogen content (from Koch, 1952). crease (Table 23). Whether the abnormally large volume increase observed during the first moult, following eyestalk removal in Eriocheir, might be followed later by a correlated increase in tissue synthesis remains to be investigated. Recent evidence on moulting hormones makes a tentative interpretation of these events possible, § 5.222 PROTEIN METABOLISM 203 if the apparent difference in nitrogen excretion in the intermoult and moulting periods may be taken as vaHd. The intermoult period is characterized by secretion of the moult-inhibiting hormone, MIH, and the lack of any Y-organ secretion. Removal of the eyestalk, or even of the sinus gland, allows the Y-organ to come slowly into action. Normal moulting occurs when the secretion of MIH stops naturally and the moult- promoting hormone, MPH, from the Y-organ becomes active (§4.211 and Table 24). From this it appears that during intermoult, the amount of protein catabolism, probably accompanied by steady synthesis, is restrained by MIH (as has been postulated) since lack of MIH results in increased catabolism. During moulting, catabolism and N-excretion are practically inhibited ; since this cannot be attributed to the lack of MIH, it may perhaps be correlated with the presence of MPH from the Y-organ, which was absent during the intermoult period but is now active. The lack of excretion at this stage is accompanied by evidence of transfer of proteins to the new integument, and is a process so clearly related to moulting that it might well be under the control of the moult-promoting hormone. 5.222 Increase in protein synthesis Insecta. Evidence from both Periplaneta (Bodenstein, 1953) and Calliphora shows that removal of either the median neuro- secretory CELLS OF THE BRAIN or of their stored products in the CORPORA CARDIACA rcsults in reduced protein synthesis ; in Peri- planeta this is show^n rather indirectly by the disappearance of urates from the fat bodies, and their re-formation after re- implantation of the corpora cardiaca. In Calliphora an effect upon protein synthesis is deduced from the reduction or cessation of growth of the ovaries, accessory glands, oenocytes and corpora allata after ablation of the neurosecretory cells (E. Thomsen, 1952 and 1956). The effect is similar to that of keeping the flies on a protein-free diet of sugar and water. The effect of removing the corpus cardiacum is similar to, but not so profound as, removing the source of the neurosecretion. The removal of the corpora allata of Carausiiis is followed by an increase in amino acids in the tissues, which is interpreted as 204 METABOLIC HORMONES o o absent (inhibited) absent (still not secreted during experiment) present present o O 1— 1 present absent (operatively) absent (no secretion) absent (operatively) z o :z; Intermoult medium increased Moult low low (no change) o 3 intact removed intact removed m s Hemigrapsus and Carcinus Eriocheir and Carcinus % *o u o .S '^ 'c >> *^ C« "3 H S ^ ii a a s H W S ^ .2 Cfl "i X §. o.. ■^ o e^ "1 -1^ K o c o 05 75 O "^S 3 u o ^ .—1 (U > ^ is C) B s 73 w- Q < C <" < r'H (/) 3 J5 2 CL, a ^ Ph o c z •;;+i O K U z o o o 0) .2 S < z, « Q < < O X, 3^ f^ z 12 X o ? •pH Q o z l-l o ^E ^n § p p^ C3 Q a^ g '/, O u rr. :3 ^ ag ES < 6i3t3 ffi Ul CO O ^x \o C o CN ^'^ w i ^ J P3 c« ^ bc o c a C3 >. o o s C^ CO 00 ON O ON o t^ V H ^ ^ r» (N vO CO O so ^^ < ITi O lO o so O sb o ^ ^ +1 f" +1 ^ -i-i ^ -i-l w J • VO ON \0 CO 00 (N 00 (^) ^ p ^1^ 04 ON - VO ON GO vO 00 OS lo in P o ^ VO CN ON lO VO LO 2 +1 S-n 22+1 2 -H Cfl tf Z H « pq D b % . •£ 2 o U 1% ■q "o 00 «3 S Q 218 METABOLIC HORMONES Aldosterone is secreted in response to adrenocorticotrophin, ACTH, in rats, but not in the human (§4.231). 5.312 Decrease o/Na+ in the blood There is no evidence for any hormones stimulating Na+ and Cl~ excretion in invertebrates, and so far it is rather tentative in vertebrates, especially in the cold-blooded classes. Amphibia. It has been shown that injection of a neurohypophy- sial extract containing oxytocin increases Cl~ excretion in the axolotl (Sawyer, 1956). Teleostei. Most marine teleosts maintain their body fluids at a lower level of salt concentration than that of the sea, by means of specialized salt excreting cells on the gills. If any of the endocrine organs are concerned in stimulating this process, their action has not yet been proved. Extracts of mammalian adrenal cortex, ACH, have been injected into trout in sea water, and have been found not to increase their survival time in the excessively saline medium. Despite the fact that in other vertebrates ACH causes an increase in salt reabsorption rather than its excretion, the author suggests trying even larger doses (D. C. W. Smith, 1956). A single test of mammalian neurohypophysial extract likewise failed to increase the survival time of these trout, although it increases salt excretion in mammals. It is known that other teleostean hormones are very specific; also that the neurosecretory store in the neuro- hypophysis of Callionymiis is depleted when this teleost is exposed to a hypertonic medium (Arvy, 1957). It might, therefore, be expected that extracts of fish neurohypophysis might be more successful than mammalian hormones in increasing salt excretion. On the other hand, salt excretion by the gills may be under quite other control than that by the kidneys. Aves. The nasal glands of the cormorant, Phalacrocorax, and probably of other oceanic birds* secrete salts, enabling them to eliminate excess Na+ and Cl~ taken up from any hypertonic media in which the birds may feed (Schmidt-Nielsen et al., 1958). It is not yet known if this active transport is under hormonal control, as might be expected. * The need for some such mechanism for flamingoes feeding in highly alkaline water had already been indicated (Jenkin, 1957). § 5.32 WATER BALANCE 219 Mammalia. Removal of the neurohypophysis, and therefore of the supply of oxytocin from rats (last line of Table 26), mitigates considerably the effects of adrenalectomy, at least as far as plasma sodium is concerned. Plasma sodium is practically equivalent to that of the control, and potassium is lowered, as compared with specimens that had had the adrenals only removed; plasma potassium is, however, not fully restored to normal, and the balance of ions in the muscles is decidedly abnormal. A series of such experiments shows that the neurohypophysis is at least partially responsible for the loss of sodium in adrenalectomizcd animals, and that its action must be to inhibit the active tubular reabsorption of sodium ions (Fig. 5-156). Its action on chloride ions may be similar to that on sodium ; but that on potassium is, as yet, far less clear (Chester Jones, 1957^). The interesting if tentative suggestion has been made that the effect of neurohypophysial removal may be due to the loss of the oxytocin secretion, rather than the antidiuretic fraction, ADH. Injections of oxytocin, at least in pharmacological doses, are more effective than ADH in increasing sodium excretion in dogs with a low rate of urine flow (Brooks and M. Pickford, 1957). If this last suggestion is further substantiated, the situation would be that sodium as well as chloride ions, at least in rats (Dicker and Heller, 1946), are controlled by the balance between aldosterone, favouring their reabsorption, and oxytocin, favouring their excretion. Potassium ions are excreted during periods of aldosterone activity. Secretion by the adrenal cortex is usually stimulated by ACTH, but this has less effect upon aldosterone than on other cortical hormones ; the only control of neurohypophysial secretion is nervous. 5.32 WATER BALANCE The direction of movement of water through a cell surface is determined by osmotic forces ; but the rate of movement can be affected by hormones, which vary the permeability of the surface. Decrease in permeability, which is associated with diuresis, will be considered first (§ 5.321), as it tends to concentrate the blood salts and to accompany salt reabsorption (§ 5.311); increase m permeability associated with antidiuresis will be taken second 220 METABOLIC HORMONES (§ 5.322), since, like salt excretion (§ 5.312), it tends to dilute the blood-salts (Table 25). Such hormones are well-established in Arthropoda as well as in Vertebrata. In the latter, the hormones tending to concentrate the blood come from the adrenal cortex, and those tending to dilute it from the neurohypophysis, as they do for the control of Na+ and CI" (Fig. 5-15). 5.321 Decrease in cell permeability and diuresis leading to con- centration of the blood Crustacea. Water uptake is a normal accompaniment of moult- ing, and probably plays a crucial part in helping to force off the old cuticle. In the crayfish, Cajnbarus, eyestalk removal induces both precocious moulting and abnormally great water uptake (from 23 52 f 12 10 1 r 8 - / / 6 - ° / / 4 : ^y^^ / 2 ^/l--^"'^" "l , 1 , 1 , 1 1 1 , 1 , 1 , 1 , 1 , 1 , -1 8 10 12 Time, days 14 16 18 20 Fig. 5-17. Percentage increase in water content (ordinates), as measured by changes in body weight in the crayfish, Cambarus immunis and C. propinquas, during 16 days (abscissae) after eye- stalk removal. Black circles show values for one specimen with two SINUS GLAND implants (curve A). This specimen shows less water uptake than the mean for eight eyestalkless controls with no implants (open circles, curve B). (From Scudamore, 1947). to 50 per cent of the body weight) during premoult ; but this can be reduced again to about the normal level by implantation of two SINUS GLANDS (Scudamore, 1947; Fig. 5-17). Opinions diflPer as to whether eyestalk removal has a significant eflFect upon the water § 5.321 WATER BALANCE 221 uptake of the fiddler crab, Uca (Scudamore, 1947 and Guyselman, 1953); but in the shore crab, Carcinus, as in Camharus, eyestalk removal before moulting results in a volume increase of 180 per cent instead of 80 per cent in the normal crab. Injected sinus gland extracts reduce the former value to 80 per cent and the latter to 50 or 60 per cent (CarHsle, 1956). At premoult, w^hen the natural secretion of moult-inhibiting hormone begins to fade out, there is known to be an increase in internal osmotic pressure, owing to the mobilization in the blood of materials resorbed from the old skin. In forced moults, following eyestalk ablation, water absorption is greater than in natural moults, although this mobilization proceeds more slowly. The change in internal osmotic pressure in forced moulting cannot therefore be the only cause of increased water uptake. The loss of eyestalks and their hormones must increase the permeability of the tissues to water, as well as inducing the moult and the increase in osmotic pressure. Swelling at the time of natural moulting must then be due to a decrease in the secretion of this hormone, but not to its complete cessation. This would allow the skin, and particu- larly the branchial epithelium, to become more permeable than in the intermoult stage, but not as permeable as in forced moults. Increased permeability of the excretory tissue would also allow of increase in reabsorption of water from the urine, but there seem to be no figures relating to urine flow during moulting. Even under natural conditions in sea water, there must be considerable water endosmosis in Carcinus to maintain the high rate of urine flow, amounting to 14 per cent of the blood volume daily. The necessary internal osmotic pressure to achieve this, (unless active transport of water is to be postulated) must be partly due to active inw^ard transport of such ions as Na + , Ca++ and CI" ; but this is small, though it may, perhaps, be aided by the presence in the haemolymph of ionized proteins, which do not pass into the urine. At the same time the endosmosis must be limited by a certain degree of impermeability of the tissues, maintained by the eyestalk hormone, otherwise eyestalk removal would not resuh in increased water uptake. It therefore seems reasonable to postulate that the action of one of the eyestalk hormones is the same as that of other diuretic hormones, namely, 222 METABOLIC HORMONES to decrease the permeability of the skin and of the excretory organs. In a dilute medium such as 50 per cent sea water, active transport of ions is increased, gill permeability is decreased, and urine flow is increased to about 24 per cent of the blood volume daily. At the same time there is a limited swelling of the tissues (Webb, 1940). The increased diuresis is presumably achieved by an increased secretion of the diuretic hormone from the sinus gland causing increased impermeability of the kidney tissues, as well as of the gills. Carlisle (1956) has claimed that the diuretic hormone of Carcinus differs from the moult-inhibiting hormone that is also obtained from the eyestalk, because only in the winter, at least at Plymouth, could he extract a moult-inhibiting substance, even from the sinus glands of the same species ; extracts of sinus gland from a number of other Crustacea were effective at all times of the year in reducing water content. The diuretic extracts could not be obtained from the cerebral ganglia, although these yield a strong moult-inhibiting extract. Passano (1953) claims that the differ- ences are quantitative and that the tissues have a lower threshold value of sensitivity to the water-balance effect than to the moult- inhibiting hormone. The observation that eyestalk removal reduced deaths of Carcinus from exposure for 24 hr to conditions of lowered salinity (Knowles and Carlisle, 1956) remains obscure. Survival would seem to require the presence, rather than the absence, of the diuretic hormone in the eyestalks. (Carlisle, in lit. 4.3.1957, agrees that the situation is far from clear, and thinks that perhaps in Crustacea, as in vertebrates, the balance of water, or of water and salts, is under the control of two hormones. The moult-promoting hormone from the Y-organ may be one of them.) Insecta. a diuretic hormone has been claimed for the beetle larva, Afiisotarsus cupripenniSy and perhaps for other insects such as Blaptica (Nunez, 1956). It appears to be secreted by the brain, and is possibly stored in the corpora cardiaca, which were not separated from the brain in the experiments. The larva lives in a damp environment and can take up water by endosmosis through the integumental cells lining the tracheoles ; but it normally remains constant in size and weight by excreting the excess water into the § 5.321 WATER BALANCE 223 Malpighian tubules (Fig. 5-18, upper specimen, and Fig. 5-19a, dotted curve). If either the neck is Hgatured or the head is cut off, preventing any flow of hormone to the body, or if the source of hormone is removed by ablating the upper part of the brain, the operated specimen swells steadily by retention of water (Fig. Fig. 5-18. Water uptake in larvae of the beetle, Anisotarsus cupri- pennis. a. Normal larva, and h. operated larva to show the swelling due to water uptake after removal of the upper part of the brain, including the cerebral neurosecretory cells, and the corpora cardiaca (from Nuiiez, 1956). 5-18, lower specimen, and Fig. 5-19a, full curve). Injection of brain extract restores the water balance, presumably by increasing excretion (Fig. 5-19^, full curve); but extracts of suboesophageal ganglion have no such diuretic effect (Fig. 5-196, dotted curve). The seat of action of this diuretic hormone does not seem to have been established, but it acts on excretion rather than on water uptake. Probably it inhibits the reabsorption of water as it passes from the Malpighian tubules through the intestine. If so, a decrease in cell permeability in the intestine could be responsible, and w^ould be comparable with that occurring in the skin of Carcinus, or in the distal kidney tubules of vertebrates, during diuresis. One curious feature is that the secretion of this metabolic 224 METABOLIC HORMONES Operated with / saline / With brain 0'^^' extract injected (b) Fig. 5-19. Water uptake in larvae of Anisotarsus. a. The dotted line gives the mean weight (ordinates) in 3 days (abscissae) for five normal larvae, which remain constant in weight by diuresis. The full line gives the mean weight of five initially rather smaller, operated larvae (as Fig. 5-186) over the same period. Vertical lines show the range of weights between the extremes in each group. These do not overlap after the first day, b. Effect of injected extracts made at the times marked by arrows. The curves give weights of indi- vidual larvae, in which secretion of their own hormone was prevented by section of the circumoesophageal connectives. The full line shows an arrest in water uptake, i.e. facilitation of diuresis, following injection of an extract of brain and corpora cardiaca in 1 % NaCl. The dotted line shows continued water uptake after injection of a control extract of suboesophageal ganglia in 1 % NaCl ; salt solution alone gave a similar result (from Nuiiez, 1956). § 5.321 WATER BALANCE 225 hormone is stimulated nervously from the ganglionic network round the gut; if this is damaged, or if the circumocsophagcal connectives to the brain are cut, hormone secretion is not induced when the abdomen begins to swell. This nervous stimulation of hormone secretion recalls the activation of the pigment-controlling hormone of Caraiisius (§ 3.221), but is unusual for a metabolic hormone (§ 5.522), unless it be for the neurohypophysial hormones (§ 5.322). Vertebrata. Hydrocortisone*, from the adrenal cortex (§ 2.31), has been found to have a diuretic effect on the kidneys, and also on the skin, of some vertebrates ; but the evidence is most definite for amphibians and mammals (Table 25). Amphibia. When amphibians enter their normal freshwater environment, excess water tends to enter through any per- meable tissues and to dilute the salt concentration in the blood. No observations on the action of hormones on the skin have so far been recorded in connection with limiting this endosmosis ; it would clearly be of interest to know whether the relative imper- meability that is usual for the skin (controls. Fig. 5-20 a-c^) results merely from the lack of antidiuretic hormone, (§ 5.322), or whether a positive, pore-contracting action of an adrenocortical hormone may also be involved. The similarity in behaviour between isolated skin and that in whole animals would lend no support to the latter supposition, unless hydrocortisone persists for a long time in the tissues after isolation. The reactions are slightly better known in the kidney, where diuresis seems to be facilitated by hydrocortisone. On exposure to a hypotonic medium, salts are actively reabsorbed in the proxi- mal tubules (§ 5.311). This would result, as in the mammalian kidney (Fig. 5- 15a, p. 214) in an obligatory endosmosis of water. In so far as the inward diffusion of water failed to keep pace with the transport of the salts, 'Tree water" would remain in the tubules, converting the isosmotic glomerular fihrate into hypotonic urine. The greater the degree of impermeability to water possessed by the kidney tubules, the greater would be the hypotonicity of the urine. There is no clear evidence as to how this impermeability is * Several related 17-OH steroids have the same effect (Part II). 226 METABOLIC HORMONES maintained in frogs, but it is probable that hydrocortisone may act here as in mammals. The removal of such hormones by adrenalectomy in Rana temporaria (but not in R. pipiens) is followed by oedema, or an excessive accumulation of water in the tissues ; but the effect of injecting cortical extracts has not yet been shown (Chester Jones, \9Sla), Intact ia) Intact Isolated skin Fig. 5-20. Increase in rate of water uptake following dehydration or injection of neurohypophysial extract (frog ADH); a. and h. in intact Rana pipiens^ with the cloaca ligatured to prevent loss by excretion; c. in isolated frog's skin (from Sawyer, 1956). Teleostei. Although freshwater teleost fish are in much the same osmotic relation to the environment as Amphibia, the scales usually make the skin more waterproof, and water only enters the tissues through the gut and gills. Diuresis by the kidneys seems therefore to be the main means of water control, and there is some evidence that the cortical cells of the anterior interrenal body (§2.311) may be concerned; but the situation is different in species adapted to different normal environments. In FunduluSy the cortical cells remain inactive when the fish are in sea water, § 5.321 WATER BALANCE 227 which is for them a hypertonic medium (cf. G. Pickford and Atz 1957). The protective action claimed for the thyroid, in allowing fish to migrate into waters of low salinity, has not been fully elucidated (cf. § 5.311) ; but it may be similar to that of thyroxine, in causing water (and salts) to pass from the tissues of mammals to their blood, thereby increasing the possible rate of water diuresis by the kidneys (Fontaine, 1956). The corpuscles of Stannius, derived from the pronephric ducts of teleosts, may also have a diuretic function, as they tend to hypertrophy when the tissues are loaded with water (Rasquin, 1956). Mammalia. When water is plentiful in the tissues, the mam- malian kidney reacts like that of the frog and excretes a hypotonic urine. The volume of urine is considerably less than that of the glomerular filtrate, chiefly because reabsorption of salts by active transport in the proximal tubules results in a correlated reabsorp- tion of at least 4/5ths of the water by obligatory endosmosis, either in the same part or in the thin loop of Henle. Nevertheless, the diffusion rate for water limits the amount of endosmosis, so that, as in the frog, "free water" remains in the tubule. Most of the remaining sodium ions are actively reabsorbed, or exchanged for other cations, and especially for H + , in the distal tubule, which is made relatively impermeable by hydrocortisone. This causes further water to become "free" and to pass on to the collecting ducts. Thence it would be excreted, as it is in the frog, were it not for a "concentrating mechanism", which is apparently independent of hormone control. This is of most significance in reinforcing antidiuresis, and it is referred to in more detail in that connexion (§ 5.322). "Osmotic diuresis" is also unaffected by hormones ; it can occur if the blood is loaded with extra urea, which then passes into the glomerular filtrate and increases the tubular osmotic pressure, so that more water than usual is excreted, instead of being re- absorbed (Mudge, 1954). The action of hydrocortisone, in causing the relative imper- meability of the distal tubules in normal diuresis, has been examined indirectly in "water-loaded rats", i.e. rats which have been given large doses of water by stomach tube. Such rats 228 METABOLIC HORMONES normally show a maximal urine flow, comparable to that of rats with "diabetes insipidus", which follows inactivation, or removal, of the neurohypophysis and therefore of the store of the anti- diuretic hormone (§ 5.322). When adrenalectomized, the water- loaded rats, with or without their neurohypophyses, show less than 1/lOth of the urine flow of either unoperated or merely neurohypophysectomized controls (Chester Jones, 1957^; cf. Table 27). The decrease in diuresis in adrenalectomized rats (Fig. 5-16, p. 216) is greater than could be accounted for by the decrease of the blood pressure to a half, or even the correlated drop to l/6th in G.F.R., the glomerular filtration rate. It may be assumed to be due to the lack of adrenocortical hormones. If rats are loaded with hypertonic saline, instead of water, the urine flow in the controls is halved, as compared with that in the rats with diabetes insipidus. This is mainly because the anti- diuretic hormone, ADH, is secreted in response to increased tissue salinity and higher osmotic pressure of the blood (§ 5.322). Adrenalectomy, by removing the source of the diuretic hormone, reduces urine flow still further, though not so greatly as in the case of water-loading. This is because the higher level of salts in the glomerular filtrate reduces the volume of obligatory endos- mosis. The action appears to be independent of the presence or absence of the neurohypophysis (see Chester Jones, 1957«, for further details). The secretion of hydrocortisone, that causes relative tubule impermeability, is stimulated by adrenocorticotrophin, ACTH (§ 4.231). 5.322 Increase in cell permeability and antidiiiresis leading to dilution of the blood-salts Dehydration can occur both on land and in any hypertonic aquatic environment. The animals' response to this usually includes waterproofing some parts of the body surface to restrict water loss and increasing the permeability of other parts to allow of water uptake or reabsorption. Invertebrates. No example of a hormone that increases tissue permeability to water has so far been found in the reports on § 5.322 WATER BALANCE 229 c3 13 "^ M 5i , ^ > s s .s O O "^ t; n w ,^ t ^ " -"^ ^ 1 3 1 ^ « « S 2^i 9 r;- OS .,1 lO lO LO ^,| ON CO lO T^ .o _H s ^ -^ c S (^' ^ J . o < z a < --H _, r< c« O ON O lO ^o o ^ -il lO ^-1 ^ -11 ro vO ^ HI o ^^ c« ° o fo 1 ^ 1 .S -J ;> C3 O ^ s • o o r^ ON T-. (N r^ ON 2 . ^^ rj- T^ (N O O ^ ON O ^ o^ T-l O ■r-H O '-^ O o o § ^-fl ^ -H ^-H ^ il li < H C O ^ M -^ O Oh iJ ^ S o >. § LO t^ Q < CO -^ m o o o ON CO Tt- O o o O CO CN) lO CO o ^4^ Kg °4^ w ^ "^ ^ a; Th lO t^ to \D '^t w Is 2 VO TH rh .Ln c3 o +2 a =J as c/3 i o z < X ^ ^ a o T3 i^H o ^ ^ ^ £ o -a 11 .5 w u .•O^ = .!« t. 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