Fish Physiology: Reproduction and Growth Bioluminescence, Pigments, and Poisons. Volume III (3)

FISH PHYSIOLOGY Volume III Reproduction and Growth Biohinescence, Pigments, and Poisons

CONTRIBUTORS J. H. S. BLAXTER RYOZO FUJI1

WILLIAM S. HOAR N. R. LILEY

J. A. C. NICOL M. C. QUIMBY FINDLAY E. RUSSELL KEN WOLF TOKI-0 YAMAMOTO

FISH PHYSIOLOGY Edited by W . S . HOAR DEPARTMENT OF ZOOLOGY UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, CANADA

and

D . J. R A N D A L L DEPARTMENT OF ZOOLOGY UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, CANADA

Volume 111

Reproduction and Growth Bioluminescence, Pigments, and Poisons

(23

Academic Press New York and London

1969

COPYRIGHT@ 1969,

BY

ACADEMIC PRESS,INC.

ALL RIGHTS RESERVED, NO PART OF THIS BOOK MAY BE REPRODUCED I N ANY FORM,

BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W1X 6BA

LIBRARY OF CONGRESS CATALOG CARDNUMBER: 76-84233

PRINTED IN THE UNITED STATES OF AMERICA

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin.

J. H. S. BLAXTER" (177), Department of Zoology, University of Aberdeen, Aberdeen, Scotland R ~ o z oFUJ I I ~( 3071, Department of Biological Sciences, Northwestern University, Evanston, Illinois

WILLIAM S. HOAR ( l ) ,Department of Zoology, University of British Columbia, Vancouver, Canada

N . R. LILEY(73), Department of Zoology, University of British Columbia, Vancouver, Canada J. A. C. NICOL(355), University of Texas, Marine Science Institute at Port Aransas, Port Aransas, Texas

M. C. QUIMBY (253), Bureau of Sport Fisheries and Wildlife, Eastern Fish Disease Labordo y,Kearneysville, West Virginia

FINDLAY E . RUSSELL(401), University of Southern California School of Medicine, Los Angebs, California KEN WOLF(253),Bureau of Sport Fisheries and Wildlife, Eastern Fish Disease Laboratory, Kearneysville, West Virginia Tom-o YAMAMOTO~( 117), Nagoya University, Biological Institute, Fuculty of Science, Chikusa-ku, Nagoya, Japan

* Present address: N.E.R.C. Marine Research Laboratory, Oban, Argyll, Scotland, and University of Stirling, Stirling, Scotland. t Present address: Division of Biology, National Institute of Radiological Sciences, Chiba City, Japan. $ Present address: Biological Laboratory, Meijo University, Yagoto-Urayama, Showa-ku, Nagoya, Japan. V

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PREFACE The topics discussed in the third volume of this treatise are extremely diverse. The first volume was devoted to problems of electrolyte and water balance, excretion, and metabolism; the second included much of the endocrinology of fishes. In contrast, Volume 111 is heterogeneous. The first four chapters are devoted to problems of reproduction, differentiation, and development; the last four are quite unrelated, dealing with tissue culture, physiological color changes, venoms, poisons, and those structures which produce light. Thus, the subjects range from the cellular level in fish genetics, development, and tissue culture to organ physiology and interacting organisms at the social level, with descriptions of many curious devices for protection and communication. Admittedly, this arrangement is one of convenience, for it is manifestly impossible to group all the physiological adaptations of fishes into closely integrated book-sized packages. Although lacking in homogeneity, this volume does emphasize the scope of evolutionary adaptation. Fishes have exploited the gamut of devices associated with sexual reproduction and provide examples of highly successful alternatives to sex; their reproductive behavior is frequently complex, and its various phases are neatly timed and associated with environmental cycles. The fishes are unique among the vertebrates in their ability to create light; the production of venoms and poisons and the use of protective coloration play important roles in the defense and protection of many species. Quite apart from the physiology of fishes, it is evident from the chapters on genetics, development, tissue culture, and chromatophores how important the use of fish tissues can b e in fundamental biological research. This volume brings together many scattered observations as well as the results of recent investigations. We feel confident that the general biologist, the zoologist, and the comparative physiologist, as well as the fish physiologist, will find a wealth of exciting information in it.

W. S. HOAR D. J. RANDALL

August, 1969 vii

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CONTENTS LISTOF CONTRIBUTORS

V

PREFACE

Vii

CONTENTS OF OTHERVOLUMES

xiii

1. Reproduction WillkZTn s. HOUT I. Introduction 11. The Gonads and Their Ducts 111. Viviparity and Gestation IV. The Endocrinology of Reproduction References

1 3 20 40 59

2. Hormones and Reproductive Behavior in Fishes N . R . Liley I. Introduction 11. Gonadal and Thyroid Hormones 111. Pituitary Hormones IV. External Factors and the Endocrine System V. Summary and Discussion VI. Conclusion References

73

75 94 102 104 109 110

3. Sex Differentiation Toki-o Yamamoto I. 11. 111. IV. V.

Introduction: Sexuality in Fishes Hermaphroditism Gonochorism Genetic Basis of Sex Determination Control of Sex Differentiation ix

117 118 127 131 142

CONTENTS

X

VI. Nature of Natural Sex Inducers VII. Differentiation of Secondary Sexual Characters VIII. Summary References

150 153 157 158

4. Development: Eggs and Larvae I. H. S . BlaxCer I. XI. 111. IV. V. VI. VII. VIII. IX. X. XI.

Introduction The Parental Contribution Events in Development Metabolism and Growth Feeding, Digestion, and Starvation Sense Organs Activity and Distribution Mortality, Tolerance, and Optima Meristic Characters Rearing and Farming Conclusions References

178 178 184 191 213 220 221 229 235 238 241 241

5. Fish Cell and Tissue Culture Ken Wolf and M. C. Quimby I. Introduction 11. Physiological Salines

111. IV. V. VI. VII. VIII. IX.

Media Methods Choice of Tissues for Culture Storage and Preservation Fish Cell Lines Shipment of Cell Cultures Needed Developments References

253 260 205 273 286 287 289 294 295 301

6. Chromatophores and Pigments

Ryozo Fujii 1. Introduction 11. Classification and Terminology of Chromatophores

111. Morphology and Chromatophores IV. Chromatophore Pigments V. Physiological Color Changes VI. Morphological Color Changes VII. Other Topics References

307 308 309 313 317 338 341 344

xi

7. Bioluminescence 1. A. C. Nicol I. Introduction 11. 111. IV. V. VI. VII. VIII.

Occurrence Light Organs Biochemistry Regulation of Light Emission Physical Characteristics Significance and Employment of Luminescence Conclusions and Summary References

355 356 357 375 379 384 388 393 394

8. Poisons and Venoms Findlay E . Russell I. Introduction 11. Venomous Fishes 111. Poisonous Fishes References

AUTHOR INDEX SYSTEMATIC INDEX SUBJECTINDEX

401 404 423 440 451 467 477

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CONTENTS OF OTHER VOLUMES Volume I The Body Compartments and the Distribution of Electrolytes W.N . Holmes and Edward M . Donaldson The Kidney Ckueland P . Hickman,

JT.,

and Benjamin F . Trump

Salt Secretion Frank P . Conte The Effects of Salinity on the Eggs and Larvae of Teleosts F . G. T . Holliday Formation of Excretory Products Roy P . Forster and Leon Coldstein Intermediary Metabolism in Fishes P . W . Hochachka Nutrition, Digestion, and Energy Utilization Arthur M. Phillips, I T .

AUTHOR INDEX-SYSTEMATICINDEX-SUBj ~ c rINDEX

Volume I1 The Pituitary Gland: Anatomy and Histophysiology 1. N . Ball and Bridget I . Baker The Neurohypophysis A. M . Perks

Prolactin (Fish Prolactin or Paralactin) and Growth Hormone 1. N . Ball Thyroid Function and Its Control in Fishes Aubrey GOT^ xiii

xiv

CONTENTS OF OTHER VOLUMES

The Endocrine Pancreas August Epple The Adrenocortical Steroids, Adrenocorticotropin and the Corpuscles of Stannius I . Chester Jones,D . K. 0. Chan, I . W. Henderson, and J . N . Ball The Ultimobranchial Glands and Calcium Regulation D. Harold Copp Urophysis and Caudal Neurosecretory System Howard A. Bern AUTHORINDEX-SYSTEMATICINDEX-SUBj ~ c rINDEX

Volume IV Anatomy and Physiology of the Central Nervous System JerakE 3. Bemstein Pineal Organ lames C. Fenurick The Mauthner Neuron

3. Diamond Autonomic Nervous System Graeme Campbell The Circulatory System

D. 3. Randall Acid-Base Balance C. Albers Properties of Fish Hemoglobins Austen Riggs Gas Exchange in Fish D. J. Randall The Regulation of Breathing G. Shelton Air Breathing in Fishes Kiell Iohansen The Swim Bladder as a Hydrostatic Organ J o h n B. Steen

CONTENTS OF OTHER VOLUMES

xv

Hydrostatic Pressure Malcolm S. Gordon Immunology of Fish john E . Cushing AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX Volume V (Tentative)

Vision: Visual Pigments F. W. Munz Vision: Electrophysiology of the Retina Tsuneo Tomita Vision: The Experimental Analysis of Visual Behavior D. Ingb Chemoreception Toshiaki I . Hara Temperature Receptors R. W. Murray

Sound Production and Detection W. N . Tavolga The Labyrinth 0. L0U;emtein Mechanoreceptors: The Lateral Line Organ Receptors Ake Flock Electroreception M . V. L. Bennett Electric Organs M . V. L. Bennett Volume VI (Tentative)

The Effect of Environmental Factors on the Physiology of Fish: An Examination of the Different Categories of Physiological Adaptation F . E. 1. Fry Action of the Environment on Biochemical Systems P . W. Hochachka and G. N . Somero

mi Learning and Memory Paul Rozin and Henry Gleitmun The Ethological Analysis of Fish Behavior G. P. Baerends Locomotion R. Bainbridge Biological Rhythms H. 0. Schwassmann Orientation and Fish Migration A. D. Hasbr Special Techniques D. 1. Randull and W.S . Hoar

CONTENTS OF OTHER VOLUMES

1 REPRODUCTION WlLLlAM S . HOAR

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I. Introduction . . . . . . 11. The Gonads and Their Ducts . A. Embryology and Phylogeny . . . B. The Male . . . . . . . C. The Female . . . . 111. Viviparity and Gestation . . . . A. Evolutionary Considerations . . . , B. Viviparity among the Chondrichthyes . . C. Viviparity among the Teleosts . IV. The Endocrinology of Reproduction . A. The Pituitary Gonadotropins . . . . B. The Gonadal Steroids . . . C. Reproductive Cycles and Their Coordination References . . . . . . . .

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I. INTRODUCTION

The fishes, like all the other vertebrates, reproduce sexually. In the vast majority of species, eggs and spermatozoa are formed in separate individuals (dioecious), and the gametes are expelled into the surrounding water where fertilization takes place immediately and is promptly followed by the development of a new generation. Within this broad pattern there is an amazing array of curious modifications so that the fishes as a group exemplify almost every device known among sexually reproducing animals; indeed, they display some variations which may be unique in the animal kingdom. 1

2

WILLIAM S . HOAR

Although most fishes are dioecious, hermaphroditism does occurparticularly among the cyclostomes and teleosts; in some species it is the normal way of life ( Atz, 1964). Parthenogenesis has not been observed among fishes in nature but has been produced experimentally (Austin and Walton, 1960). Gynogenesis occurs in Poecilia formosa and perhaps also in some populations of Carmsius auratus (Hoar, 1965a). These modes of reproduction are, however, unusual; and most of the 20,000 or more known species of fish have male and female organs in separate individuals. At one extreme, the two sexes are externally indistinguishable. They swim together during the breeding season to discharge their genital products into the water without specialized mating behavior. At the other extreme, marked differences in morphology and coloration are characteristic of the males and females and these play distinctive roles in elaborate presexual behavior, courtship, mating, and parental care. A synchronized spawning without copulation is usual but copulation occurs at all levels in fish phylogeny; it sometimes merely insures close proximity of eggs and sperm discharged into the water but more frequently involves insemination of the females. Although internal fertilization may be followed by the laying of newly fertilized eggs or the release of primitive larvae, viviparity is highly specialized in some elasmobranchs and teleosts with young born in advanced stages of development; in Cymatogaster aggregata the males may even be sexually mature at birth. Sexual activities are normally followed by the prompt fertilization of eggs but sometimes sperm are stored in the female for prolonged periods prior to fertilization; occasionally the fertilized eggs develop only after a period of diapause ( Wourms, 1967); sometimes development occurs but hatching is delayed during adverse environmental conditions ( Harrington, 1959). Breder and Rosen (1966) have summarized these and many other curious specializations in an extended chart; this tabulates information on secondary sex characters, mating, breeding, and parental behavior for each of the many families of fishes. There are many specializations associated with reproduction in fishes but only a few of them are considered in this chapter. Breeding behavior, fertilization, and early development are considered in other chapters. The present account is confined to a discussion of the gonads and their associated ducts, the special physiology of viviparity, and the endocrinology of reproduction. The latter topic has been repeatedly reviewed (Pickford and Atz, 1957; Dodd, 196Oa; Hoar, 1965a, 1966), but the other two aspects of fish reproduction have not been recently summarized (see Hoar, 1957; GBrard, 1958; Bertin, 1958a).

1.

REPRODUCXION

3

11. THE GONADS AND THEIR DUCTS

A. Embryology and Phylogeny

A knowledge of early embryology, as well as adult morphology, is essential to an understanding of several features of the comparative physiology of the reproductive system of fishes. In particular, the two very different patterns of gonadal ontogeny may account for the variable frequency of intersexuality in different groups while comparative studies of spawning, fertilization, and gestation must be based on an understanding of the diversified anatomy of the gonads and their ducts. The gonads of all vertebrates arise in the dorsolateral lining of the peritoneal cavity-one gonad on each side of the dorsal mesentery. Their development is intimately associated with that of the nephric system. In most of the vertebrates each gonad has a double origin, developing from two distinct but closely associated cellular proliferations. The more laterally located cortex or cortical portion arises as an elongated ridge of peritoneal wall and is destined to become an ovary. The medulla or medullary portion which is destined to form the testis arises from a more medial cellular proliferation which also produces the adrenocortical tissue ( interrenal or mesonephric blastema) . Usually, one of these portions grows rapidly while the other fails to develop and the sex of the individual is thus determined at a very early stage. A section through the undifferentiated gonad of a 22-mm dogfish embryo is depicted in Fig. 1.

Fig. 1. Cross section through a 22-mm embryo dogfish, Scyliorhinus canicu~us,to show the origins of the cortex and medulla of the gonad. From Chieffi (1952).

4

WILLIAM S. HOAR

This pattern of gonad differentiation from two different components is characteristic of the elasmobranchs (Chieffi, 1949, 1952, 1967) and all of the tetrapods. In contrast, the gonads of cyclostomes and teleosts develop from single primordia. In cyclostomes and teleosts the entire gonad develops directly in the peritoneal epithelium and corresponds to only the cortex of other vertebrates. There is evidently no contribution from the interrenal (mesonephric) blastema. Atz ( 1964) has summarized the literature and provided an extensive bibliography; D’Ancona (1950, 1956, 1960) did much of the early work on teleosts. It has been suggested that these differences in embryology may account for the more widespread occurrence of intersexuality among the cyclostomes and teleosts. Whether the ontogeny of the gonad is by way of a single or a double primordium, distinctive cells (the primordial germ cells) which are destined to form gametes first appear within or migrate into the cortical portion of the gonad. They can usually be identified as conspicuously larger cells within this modified layer of proliferating mesothelium (germinal epithelium). There is a voluminous literature on the origin of the germ cells with a considerable body of evidence for a widespread development of these cells and a subsequent migration of them into the germinal epithelium. This literature will not be reviewed here; it can be readily reached through standard embryology texts (Nelsen, 1953) and reviews (Everett, 1945; Brambell, 1956; Franchi et al., 1962). From whatever source, these distinctive cells can be identified at an early stage within the thickening layer of germinal epithelium. In genetic male elasmobranchs there is an early migration of germ cells from the cortex, where they first appear, into the medulla (Chieffi, 1949, 1967); in the genetic females there may be a transient migration of some of the germ cells into the medulla but many of them retain their cortical location to form the basis of ovarian differentiation. The origin and segregation of germ cells within the single primordium of the cyclostome and teleost gonad has been frequently described and reviewed (Nelsen, 1953; Brambell, 1956; Franchi et al., 1962). Under normal conditions, genetic factors probably determine whether the developing fish will be a male or a female (Dodd, 1960b); genetic aspects of sex determination are considered in the chapter by Yamamoto, this volume. Earlier workers postulated male and female inductor substances or hormones which controlled the course of development. Witschi (1942, 1950) who carried out the pioneer work on amphibians referred to them as “medullarin” and “corticin,” for male and female, respectively; DAncona (1945, 1950) working with the teleosts called these theoretical substances “androgenin” and “gynogenin,” Reinboth ( 1962) does not

1. REPRODUCTION

5

consider evidence for the existence of these factors to be at all convincing; Atz (1964) agrees with Reinboth's conclusions. Whether or not there are special embryonic hormones concerned with the determination of sex, it is well established that the differentiation of the gonadal primordium can be readily modified with gonadal steroids similar to those found in adults. Androgens stimulate the development of testes, and estrogens promote ovarian differentiation. Findings are consistent and the literature has been frequently reviewed (Dodd, 1960a,b; Atz, 1964; Chieffi, 1967; and the chapter by Yamamoto, this volume). The gonads of vertebrates always originate from biIateral primordia, but many species as adults possess only one reproductive gland. In some of the fishes there is a fusion of the two primordia during development (as in the ovaries of lampreys), while in other cases one of the gonads fails to develop (as in the myxinoid ovary). The literature reveals a range of specializations in all groups of fishes from complete fusions to partial fusions involving only the posterior portion of the gonads or just the gonoducts; sometimes one of the gonads is rudimentary or merely smaller but still present. Franchi et al. (1962) summarize the pertinent literature with specific examples. The comparative anatomist has found some of his most interesting problems in the phylogeny of the gonoducts and their relationships to the mesonephric tubules and ducts. Gonoducts are absent in the cyclostomes. Spermatozoa or ova are discharged from the surface of the gonad directly into the body cavity and then pass through pores into the urinary sinus or urinary duct; they are expelled through the cloaca or urinogenital papilla-depending on the anatomy of the urinogenital opening in the particular sex and species (Dodd, 1960a; Walvig, 1963). Gonoducts are present in all groups of the gnathostomes although they may be secondarily lost in some fishes (for example, in the Greenland shark, Laemurgus borealis, and in the Salmonidae among the Teleostei, Balfour, 1875). Kerr ( 1901) provided the classic description of the origin of the male gonoducts from the mesonephric system (Fig. 2). The sturgeon and garpike are thought to represent a primitive situation where some of the renal tubules throughout the length of the mesonephros have been conscripted into the service of the testis and form vasa efferentia which drain into the mesonephric duct or vas deferens. In the Chondrichthyes (and Amphibia), the testis is thought to have taken over a group of anterior mesonephric tubules which cease to have any relationship with the excretory system; in Lepidosiren, the vasa efferentia are formed from some of the posterior mesonephric tubules. In Polypterus and the Teleostei there is no connection between the mesonephros and the gonad at maturity; and the vas deferens is

6

WILLIAM S. HOAR

Fig. 2. Representative types of urinogenital systems in male fishes. Upper figures, redrawn from Portman (1948) with permission of Dr. A. Portman and B. Schwabe (Basel); lower figures, originals by Margaret Jensen.

quite separate from the ureter or mesonephric duct (Fig. 2 ) . It is generally assumed, however, that the main gonoduct has been derived from the mesonephric duct during phylogeny. More complete descriptions of this system will be found in Goodrich (1930), GCrard ( 1958) and van den Broek (1967).

1. REPRODUCTION

7

In all of the vertebrates, except some of the more specialized fishes, the ova are discharged into the peritoneal cavity and find their way to the outside through oviducts (Mullerian ducts), which pass from open anterior funnels to the cloaca (Fig. 3 ) . In these groups with naked ovaries ( gymnovuriun condition) and open ovarian funnels, the genital ducts are derived as in the male from the mesonephric ducts although

Fig. 3. Representative types of urinogenital systems in female fishes. Upper figures, redrawn from Portman (1948) with permission of Dr. A. Portman and B. Schwabe (Basel); lower figures, originals by Margaret Jensen.

8

WILLIAM S. HOAR

the evidence for this origin is completely lost in the land vertebrates ( Romer, 1955). In the Teleostei, the oviducts are posterior continuations of the ovarian tunic. The embryology of the ovary and its duct varies so that the ovary in some teleosts has a central ovarian cavity continuous with the oviduct while in others the oviducts are para-ovarian ( Goodrich, 1930; GBrard, 1958; Franchi, 1962; van den Broek, 1967); in any case the oviducts are formed by the backward growth of the same peritoneal folds which enclose the ovary during its development ( cystovarian condition). The gonoducts are also continuous with the ovaries in the holostean Lepidosteus, thus providing the exception to the rule that only teleosts fail to discharge their ova into the peritoneal cavity. Some of the teleosts are themselves exceptional in that they do release their eggs directly into the body cavity. In the Salmonidae, Galaxiidae, Hyodontidae, Notopteridae, Osteoglassidae, and the cyprinid Misgurnus, the oviducts degenerate in whole or in part so that the ova pass into the peritoneal cavity and thence through pores or funnels, depending on the degree of degeneration, to the exterior. In the Anguillidae the loss of gonoducts occurs in both males and females ( Goodrich, 1930).

B. The Male 1. THETESTIS AND SPERMATOGENESIS Spermatozoa are formed from the sperm mother cells or spermatogonia through a series of cytological stages collectively referred to as “spermatogenesis.” This process involves a proliferation of spermatogonia through repeated mitotic divisions and growth to form primary spermatocytes; these then undergo reduction division to form secondary spermatocytes; the division of the secondary spermatocytes produces the spennatids which then metamorphose into the motile and potentially functional gametes-spermatozoa, spermia or sperm. This process of spermatid metamorphosis is often called “spermiogenesis,”Details of the cytological changes are similar in all vertebrates as described in standard textbooks of histology and embryology. Physiologists are interested in the factorsboth environmental and hormonal-which trigger waves of spermatogenesis at different seasons and control the essential steps of meiosis (division of primary to secondary spermatocytes) and the metamorphosis of the spermatid with eventual release of mature sperm. In some species -particularly the elasmobranchs and viviparous teleosts-sperm production involves the packaging of sperm into sperm balls or spermatophores which are transferred to the female.

1. REPRODUCTION

9

Spermatogenesis occurs within testicular units which may take the form of small sacs, ampullae, lobules, or tubules; in many groups of fishes these differ radically from the familiar seminiferous tubules of the mammalian testis. In the cyclostome, spermatogenesis occurs within small bladders, follicles, or ampullae. These are separated by a delicate connective tissue; a number of units may be grouped together and bounded by somewhat thicker connective tissue to form lobules (Dodd et al., 1960; Walvig, 1963). Spermatogenesis is almost synchronous throughout the many follicles and just prior to spawning the follicles filled with mature sperm rupture to release their contents into the body cavity. Walvig ( 1963) summarizes the cytological details of spermatogenesis in Myxine. In elasmobranchs, spermatogenesis occurs within a mass of ampullae arranged in a manner which seems to be unique among the vertebrates. The testis of the basking shark, Cetorhinus maximus, carefully described by L. H. Matthews (1950), is divided by connective tissue trabeculae into many lobules each of which corresponds to the entire testis of the dogfish, Scylliorhinus cunicula, as described by Fratini (1953) and Mellinger (1965). The structure of the dogfish testis is shown diagrammatically in Fig. 4. The spermatogenetic units, usually called “ampullae,” are proliferated from a mesoventral area of the testis referred to as the “tubulogenic zone.” Within this zone, nests of cells-somewhat like primary ovarian follicles-arise and proliferate to form small tubules or ampullae which gradually shift toward the dorsal side of the organ while spermatogenesis occurs within them, By the time the ampullae reach the dorsal surface of the testis, the sperm ( a constant number in each ampulla: Stanley, 1962; Mellinger, 1965) are ready for discharge into the efferent ducts which emerge from the testis at this point. At this stage, the Sertoli cells surround the ampullae and are clearly associated with packets of sperm. According to L. H. Matthews (1950), the ampullae shrink after sperm are discharged into these collecting tubules and the Sertoli cells are resorbed. It is of interest that all of the gonocytes within any one ampulla are in the same stage of spermatogenesis and that within the testis, distinct zones are evident from ventral to dorsal surface with all the tubules of a particular zone in a similar stage of development. Thus, in studies of the pituitary regulation of spermatogenesis, Dodd and his colleagues (1960) were readily able to spot a distinct zone of degeneration in the primary spermatocytes when it appeared following hypophysectomy. The cytology of the elasmobranch testis, including spermatogenesis, the development of the Sertoli cells, and the formation of spermatophores, has been detailed by L. H. Matthews (1950) and Fratini ( 1953).

10

WILLIAM S. HOAR

Fig. 4. Structure of the chondrichthyan testis. Redrawn from van den Broek (1967) with the permission of Asher & Co., Amsterdam, and from Fratini (1953) with the permission of Stazione Zoologica di Napoli.

There are now good descriptions of the testicular histology of several species of teleosts. Among the early papers, the following are particularly helpful: C. L. Turner’s description (1919) of the spermary of the perch, Craig-Bennett’s account ( 1931) of the stickleback, S. A. Matthews’

1. REPRODUCTION

11

report (1938) on Fundulus, and Cooper's study (1952) of the crappies. Many other investigations are cited in the bibliographies of these papers and in the reviews by Hoar (1957) and Dodd (1960a). More recent descriptions are available for the minnow Couesius ( Ahsan, 1966a,b), the rockfish Sebastodes ( Moser, 1967a), the sea perch Cymatogaster (Wiebe, 1968b), and the guppy Poecilia (Pandey, 1969a,c). Testes of dBerent species vary in complexity; the brief description which follows is a generalized one. The main sperm duct (vas deferens) arises from the posterior mesodorsal surface of the elongated testis and leads to the urinogenital papilla. It may be traced anteriorly for a variable distance in a connective tissue groove of the testis along with the spermatic blood vessels and nerves. In many teleosts, the paired testes fuse posteriorly and the vasa deferentia are combined into a single sperm duct. Within the body of the testis, the

Fig. 5. Structure of testis of guppy, Poecilia reticulatu. ( A ) Diagrammatic section to show relation of gametogenetic tissue (acini or cysts) to the system of ducts, and ( B ) series of cysts to show differentiation of gametes from spennatogonia ( a t right) to mature spermatophores surrounded by Sertoli cells ( a t left). Sertoli cells become confluent with the epithelia1 cells of the efferent ducts when the spermatophores enter the ducts. Based on Pandey (1969a).

12

WILLIAM S. HOAR

main sperm ducts give rise to smaller ducts (vasa efferentia) which penetrate ventrally and laterally to form a drainage system of variable complexity. In some species these tubules are extremely short (poeciliids, for example), while in others they form an extensive system of seminiferous tubules which can be followed almost to the periphery of the organ (Fundulus, the rockfishes, and the cottids). Testes of the poeciliid type are sometimes referred to as “acinar” (Fig. 5 ) while those with the extensive duct systems are called “tubular.” This difference is one of degree rather than kind. It is to be noted that the seminiferous tubules of the teleost-in contrast to those of the higher vertebrates-lack a permanent germinal epithelium. Whether the testis is acinar or tubular, nests of spermatogonia proliferate from the resting germ cells near the margin of the organ. In the acinar type, these nests of cells or cysts undergo the various stages of maturation as they are displaced toward the sperm ducts into which they eventually discharge their contents (Fig. 5 ) . In the tubular testis, the resting germ cells are particularly evident and packed together at the blind ends of the tubules near the periphery, but many of them migrate or are displaced along the walls of the tubules. In active spermatogenesis, nests of spermatogonia proliferate both from the ends of the tubules and from the resting germ cells along their walls. Thus, at the end of spermiogenesis, the seminiferous tubules are packed with sperm as the masses of gametes from a multitude of matured cysts combine within the tubules. During maturation all of the cells within one of the cysts are in approximately the same stage of development; the degree of synchrony among the many cysts varies in different species.

2. THE ENDOCRINE AND SUPPORTING TISSUES OF

THE

TESTIS

Current histological and histochemical techniques have now resolved a long-standing uncertainty about the tissues responsible for the production of the testicular androgens ( Hoar, 1965,a). Well-vascularized clusters of cells similar to those described by Leydig many years ago in the mammalian testis have been identified between the seminiferous alveoli and tubules of many different fishes. In routine H and E sections, these large cells with spherical or oval nuclei often appear vacuolated because of the removal of lipoidal substances. Their endocrine nature was first postulated from the marked seasonal proliferation which occurs just prior to the breeding season in the stickleback Gasterosteus (Courrier, 1921; Craig-Bennett, 1931); in this same species Gottfried and van Mullem (1967) have recently shown a convincing correlation between the histological development of the tissue and its biochemical

1. REPRODUCI’ION

13

content of androgen. Nests of typical Leydig cells or interstitial tissue have now been identified in the cyclostomes (Chieffi and Botte, 1962; Hardisty et al., 1967), in all groups of elasmobranchs (Marshall, 1960; Chieffi et al., 1961; Chieffi, 1962, 1967), in the lungfishes and the coelacanth Latimeria (Marshall, 1960) as well as the testes of many teleosts ( Gasterosteus Tilapia, Tinca, Solea, Lebistes, Cymatogaster, Oncorhynchus, and others). Staining of the cytoplasmic droplets with sudanophilic coloring agents and the demonstration of steroid dehydrogenases leaves little doubt of their role in steroidogenesis (Marshall, 1960; Follenius and Porte, 1960; Collenot and Ozon, 1964; Delrio et al., 1965; Stanley et al., 1965; Yaron, 1966; Wiebe, 1969a). In some species of teleosts, however, the hormone-producing cells are located in the walls (basement membrane) of the seminiferous tubules. Marshall and Lofts (1956) who first noted this difference in their studies on the pike, Esox lucius, and the char, Salvelinus willughbii, referred to this tissue as the “lobule boundary cells.” The difference between interstitial and lobule boundary cells is largely one of distribution since both tissues arise from the same source and are similar histochemically (reviews by Marshall, 1960; Dodd, 1960a; Hoar, 1965a). It may be of interest to note that the Urodela also possess lobule boundary cells, in contrast to the Anura and all other groups of tetrapods (Marshall, 1960). Sertoli cells (Figs. 4 and 5 ) are also prominent in the testes of all groups of fishes (L. €3. Matthews, 1950; Fratini, 1953; Chieffi and Botte, 1962; Lagios, 1965; Wiebe, 196813, 1969a,b; Pandey, 1969a). The spermatogenetic units-whether cysts, ampullae, or tubules-are bounded by a thin layer of connective tissue (the basement membrane) and contain two types of cells; one of these is the gonocyte giving rise to the several generations of spermatogenetic cells, while the other is the Sertoli or supporting cell believed to play a nutritive role during spermiogenesis. The process whereby spermatids become embedded in the centripetal end of the Sertoli cells to undergo metamorphosis is well described in the higher vertebrates ( Nelsen, 1953; Patten, 1953; Ham, 1965). Cytological details probably vary in the different species, but in all cases the spermatids (perhaps earlier stages in some fishes: Stanley, 1962) become intimately associated with these nurse cells and presumably draw nourishment from them during transformation. The fully developed sperm are attached to the Sertoli cells prior to spermiation; this association can be very nicely seen in fishes such as the sharks, the poeciliids, and the embiotocids which form spermatophores. In these groups, where all of the sperm in a unit mature at the same time, the Sertoli cells form an almost complete layer just inside the basement membrane (Figs. 4 and 5); in many other fishes the situation is similar to that of the higher vertebrates where

14

WILLIAM S. HOAR

Sertoli cells are interspersed at intervals along the basement membrane with the groups of spermatogonia between them. L. H. Matthews (1950) and Fratini (1953) have detailed the cytogenesis of the Sertoli cell system in the basking shark and the dogfish. Pandey (1969a) describes Sertoli cell development in the guppy. In addition to their nutritive role, the Sertoli cells may be concerned with the phagocytosis of unused sperm (Vaupel, 1929; Nelsen, 1953; Lagios, 1965) and involved in hormone production. The presence of hydroxysteroid dehydrogenases has been demonstrated in the Sertoli cells of dogfishes (Collenot and Ozon, 1964; Simpson and Wardle, 1967) and in the surfperch (Wiebe, 1969a). In summary, the cytology of the Sertoli cell suggests three functions : nutritive, phagocytic, and hormonal; in this and in their embryology (L. H. Matthews, 1950; Lofts et al., 1966) they seem very similar to the granulosa cells of the ovarian follicle which are described later.

3. SECRETIONSOF THE SPERMDucrs AND MALE SECONDARY SEX CHARACXXRS Following spermatogenesis, the mature sperm are made ready for discharge or spermiation. The glandular epithelial lining of the sperm duct probably always contributes to the seminal discharge but there seems to be no systematic study of this. However, a wide variation is recognized-at one extreme, the formation of specialized packets of sperm (spermatophores) and, at the other, a mere thinning of the semen through the hydration of the testis and accumulation of fluid in the testicular passages. L. H. Matthews (1950) has detailed the formation of the complex spermatophores of the basking shark, Cetorhinus maximus, where these sperm packets range in size from a few millimeters up to 25 or 30 mm and have a cortex of translucent hyaline material surrounding a central mass of opaque white sperm; the sperm mass may be 10 mm in diameter. The general process, as described by L. H. Matthews, seems to be representative of the elasmobranchs although the structure of the spermatophores varies in digerent species from simple sperm aggregations to the hyaline packets of the basking shark. In the elasmobranchs, sperm released from the ampullae into the efferent canals pass through a mass of coiled glandular tubules (gland of Leydig) which are derived from the anterior nonurinary portion of the mesonephros. Sperm contained in the secretions of the Leydig gland then pass into an expansion of the vas deferens known as the ampulla. As they move through the complex system of septae in this structure they are consolidated and

15

1. REPRODUCXION

receive additional secretions such as the hyaline cortex of the basking shark spermatophore. Borcea’s monograph ( 1906) should be consulted for anatomical details of the gonoducts of elasmobranchs. Spermatophores are also regularly formed in the viviparous teleosts. They have been described in the embiotocids (Lagios, 1965; Wiebe, 1969b) and in the poeciliids ( Phillipi, 1908; Henn, 1916; Pandey, 1969a). In these groups, the aggregations of sperm formed within the seminiferous acini or tubules become arranged with their tails directed centrally and the heads oriented peripherally to form the sperm balls. As these pass through the efferent ducts, they seem to receive a gelatinous secretion which binds them together so that they remain intact during transfer to the female. More complex spermatophores have also been described in teleosts, but there are very few studies of either the cytogenesis or the physiology of fish spermatophores (see review by Bertin, 1958a). Spenniation in the goldfish is typical of the much simpler process involving only a thinning of the semen. Clemens and his associates (1964; Clemens and Grant, 1964, 1965) have described the weight changes in the testis and established a pituitary regulation of the spermiation process. Yamazaki and Donaldson (1968a) have used the spermiation of the goldfish in the bioassay of salmon pituitary gonadotropin. In some teleosts ( Ariidae, Gobeiidae, and Blennoidae ), large structures often referred to as “seminal vesicles” are found as glandular developments from the sperm ducts-occasionally from the testis as in Tachicorystes (von Ihering, 1937). These “seminal vesicles” do not store sperm and are not comparable to the structures of the same name in the higher vertebrates. They provide secretions which are of importance in sperm transfer or other breeding activities. Descriptions of these glands and the many other interesting secondary sex characters of fishes are beyond the scope of this review. Good general accounts with bibliographies have been given by Bertin (1958a) and Breder and Rosen ( 1966). Secretory activities of the sperm ducts, accessory glands, and secondary sex characters show a marked seasonal development and are under the control of the androgenic secretions of the testis (Section IV, C and chapter by Liley, this volume).

C. The Female 1. THEHISTORY OF

THE

OVARIANFOLLICLE

In structure, the fish ovary ranges from an expanded mesentery (mesovarium) which dehisces mature ova from its ventral margin in the hagfish Myxine (Lyngnes, 1936; Walvig, 1963) to a complex hollow

16

WILLIAM S . HOAR

organ in the viviparous teleosts where the gonad produces eggs, stores sperm, serves as a site for fertilization, and provides nourishment for the development of young to an advanced stage. Comprehensive reviews of the earlier literature are available (Hoar, 1955, 1957, 1965a; Dodd, 1960a, Franchi, 1962). The description which follows is a generalized one. Ovarian follicles develop from or in association with the germinal epithelium which covers the surface of the ovary as an extension of the peritoneum ( mesovarium ) . As described earlier, this germinal epithelium also lines the cavity of the hollow teleost (cystovarian ) ovary. The ovary of the basking shark, Cetorhinus maximus-single organ-is evidently exceptional among the elasmobranchs in that the germinal epithelium invaginates to form a series of tubular ramifications within a gonad which is superficially similar to that of the teleosts (L. H. Matthews, 1950). It differs, however, both in its embryology and in its gonoduct. The cavities of the basking shark ovary open into a pocket on the right side of the organ and ova discharged into this pocket pass via the peritoneum into the open end of the Miillerian duct. These hollow ovaries of the teleosts and the basking shark are unique among vertebrates and quite different from the hollow ovaries of some other elasmobranchs and the amphibians, where the lining is not germinal epithelium and where the cavities develop as large lymph spaces within the stroma (medulla) of the gland. The numerous ovarian follicles of the fish ovary are supported by a richly vascular connective tissue stroma which extends into the gland from the somewhat denser connective tissue layer (tunica albuginea) just under the germinal epithelium. The internal lining of the hollow teleost ovary is thrown into a complex series of folds (ovigerous folds) which may almost obliterate the cavity (Fig. 6). Fish eggs are discharged from mature ovarian follicles into the peritoneum or into the cavity of the ovary; it may be significant that the stroma of the ovary is rich in elastic tissue and smooth muscle. At an early stage, the oogonia which arise from primordial sex cells either in or near the germinal epithelium become surrounded by a layer of small epithelial cells to form the ovarian follicle. In cyclostomes and teleosts this follicular epithelium is single-layered while in elasmobranchs and amniotes it is usually composed of several layers (Franchi, 1962). The connective tissue near this nest of cells forms a distinct theca, which in some species assumes a very active role during the later history of the follicle. As the follicle differentiates and the ovum becomes mature, the epithelial cells increase in size and number to form a glandular granulosa while the theca becomes more distinct and may be divided into an interna and an externa. The maturing ovum is separated from the granulosa by a noncellular membrane usually called the “zona pellucida”;

Fig. 6. Structure of the ovary in Cymutogaster. Upper left, section of ovary to show oocytes developing in ovigerous folds: ( A ) nest of oocytes in very early stages of development, ( B ) Class I oocyte, ( C ) Class I11 oocyte, ( D ) Stage I1 atretic oocyte, and ( E ) atretic oocyte almost at Stage 111. Bottom, Cymutogaster before birth to show specidized dorsal fin, Upper left and bottom two diagrams redrawn from C. L. Turner (1938a); other diagrams, courtesy of John P. Wiebe.

17

18

WILLIAM S . HOAR

the terminology of the egg membranes is not always consistent (Nelsen, 1953; Brambell, 1956). An early follicle and an almost mature follicle of Cymatogaster are depicted in Fig. 6. The functions of the follicular epithelium in fishes are still problematic. The granulosa has a recognized responsibility for the deposition of yolk in the developing ovum and for its removal in ova which degenerate before ovulation. Yolk deposition in some elasmobranchs and reptiles apparently takes place through specialized protoplasmic processes which can be seen to penetrate the zona pellucida from particular granulosa cells; nutritive transfer is not microscopically evident in other fishes ( Brambell, 1956; Bertin, 1958a). The phagocytic activities of the granulosa cells have been described repeatedly by histologists since the last century. In all groups of vertebrates, the number of ovarian follicles started on the road to development is greatly in excess of the number of eggs which are eventually produced; some ova are normally resorbed in different stages of development ( Lyngnes, 1936; Busson-Mabillot, 1967; and reviews cited). In addition to its nutritive and phagocytic functions, the granulosa may also be concerned with the elaboration of the ovarian hormones. The fish ovary does not contain interstitial tissue comparable in development and histochemistry to the Leydig cells of the testis; the theca of the follicle, which in some mammals participates in the formation of the corpus luteum (Brambell, 1956; Ham, 1965) and probably secretes progesterone-perhaps also estrogen-never shows a sudanophilia in fishes and is evidently a simple fibroelastic connective tissue. It is evident that the ovarian hormones must be synthesized by the ovum or the granulosa or the corpus luteum (corpus atreticum) which develops from the granulosa. Major interest has centered around the corpora, which in this review are termed “corpora lutea” even though they may not be physiologically equivalent to the corpora lutea of mammals which are known to produce progesterone and are under the control of the pituitary gland. Hisaw and Hisaw (1959) and Chieffi (1962) agree with this terminology. Corpora lutea have now been observed in all groups of fishes. Their histogenesis has been described repeatedly since the beginning of the century and a rich literature is cited in several reviews of fish endocrinology (Brambell, 1956; Ball, 1960; Hoar, 1955, 1965a; Dodd, 1960a). Although there is a remarkable variation in the extent of these proliferations, they regularly appear when yolky ova become atretic (preovulatory corpora lutea or corpora atretica) or during the postovulatory history of the follicle (postovulatory or corpora lutea of ovulation). The two opposing views concerning their physiology have been ably maintained from the studies of elasmobranchs by Hisaw (1959, 1963) who

1.

19

REX’RODUCITON

argues that they are concerned with yolk phagocytosis in the preovulatory follicles or the removal of tissue fragments and blood cells in the postovulatory ones, while Chieffi (1961, 1962, 1967) finds substantial histochemical evidence for steroidogenesis and attributes an endocrine function to them. Their function is just as problematic in other groups of fishes (Ball, 1960). The controversy was detailed recently and will not be repeated here; the conclusion reached at that time (Hoar, 1965a) still seems valid; it is suggested “that estrogen synthesis was one of the responsibilities of the granulosa from the earliest stages of vertebrate phylogeny and that this capacity developed in association with the synthesis of lipid materials present in the yolk; the high estrogen content of yolk in the ova of some species (Gottfried, 1964) may represent their entire reserve of this hormone. With the variety and complexity of reproductive processes and controls found among fishes, it seems entirely reasonable that the same granulosa cells (in some fishes) may have specialized in hormone production to the point where corpora atretica become functional corpora lutea-even though they may synthesize estrogen rather than progesterone as their hormone.” Chieffis studies (1961, 1967) indicate how different the history of the pre and postovulatory bodies may be in closely related groups of fishes.

2. ACCESSORYREPRODUCTIVE STRUCTURES AND PHYSIOLOGY OF THE OVIDUCTS

THE

In contrast to the males, secondary sex characters are inconspicuous in female fishes (Breder and Rosen, 1966). The females are frequently larger, occasionally have specialized ventral fins ( Ariidae) , distinctive ventral surfaces or folds of unknown function on the abdominal wall (Bunocephalidae and Bagridae), tubercles on the head (Osteoglossidae), or an enlarged urinogenital papilla ( Notopteridae, Cyprinidae) . The genital papilla may be only slightly enlarged but in the cyprinid Rhodeus amarus-the European bitterling-it often extends well beyond the caudal fin as a specialized ovipositor (Bretschneider and de Wit, 1947; Shirai, 1964). Studies of the physiology of the oviduct have been almost completely confined to the Chondrichthyes. In this group, the oviduct (Miillerian duct) not only serves as an open tube for the collection of eggs from the abdominal cavity and their transport to the cloaca but also provides the secretions concerned with the formation of horny shelled eggs in the oviparous species and a site for the development of the young in the viviparous forms. In addition, this tube also serves for the reception of sperm and, at least in some species, for sperm storage and the dissolution of the hyaline cortex of the spermatophores (Metten, 1941; L. H.

20

WILLIAM S . HOAR

Matthews, 1950). The oviduct of viviparous teleosts is also conccrned with some of these sperm functions, but there are no systematic studies of its physiology. The four regions of the Mullerian duct are shown diagrammatically in Fig. 3; the ostium or funnel, the oviducal or nidamental gland, the connecting isthmus, and the expanded posterior uterus. In some species the ostium is closely applied to an ovarian pocket from which the ova emerge to enter it directly; in other species the eggs are discharged at many points on the surface of the ovary and carried into the funnel by continuously beating cilia which line the abdominal cavity. The ciliation of the abdominal cavity has been described and illustrated by Metten ( 1941). The absence of cilia in males and immature females (Metten, 1941) and changes in the size of the ostium during the breeding season (L. H. Matthews, 1950) suggest a hormonal regulation of these structures; estrogens have been shown to stimulate the development of nidamental glands and other areas of the Mullerian tubes (Thiebold, 1954; Dodd and Goddard, 1961). In all chondrichthyans, the oviducal gland secretes albumen and mucus; it is relatively larger in the oviparous species where it is also responsible for the formation of the shell. Two or three distinct glandular zones may be distinguished. In the oviparous species there is an anterior albumen-secreting area and a posterior shell-secreting zone; an intermediate mucus secreting area may be present, as in the ratfish, Hydrolagus colliei, or absent, as in the ovoviviparous species Rhinobatus granulatus or located caudad as in Raja rhinu (Prasad, 1948). The shellsecreting area of the nidamental gland also serves as a seminal vesicle in the dogfish (Metten, 1941) where sperm are stored to fertilize eggs before or at the time of shell formation, The wall of the uterus is smooth and covered with a 3attened epithelium in the oviparous elasmobranchs where it serves only as a passageway for the eggs. In viviparous species, this portion of the Miillerian duct is variously modified through the formation of villuslike appendages which nourish the developing young. These specializations are considered in the next section.

111. VIVIPARITY AND GESTATION

A. Evolutionary Considerations

If an animal species is to survive, each adult member of the population must on the average produce one reproductively active adult.

1. REPRODUCTION

21

Darwinian evolution rarely operates through one channel, and the fishes as a group have achieved reproductive success in many different ways. At one extreme, eggs and sperm are broadcast in sufficient numbers to balance the unusual pressures of the environment and satisfy the predators while, at another extreme, fertilization is internal and the developing young are housed within the parent's body until ready for an independent existence. The provision of millions of unprotected gametes represents the primitive condition among fishes, Along the road to specialized vviparity many curious and successful devices have evolved to provide a measure of protection during incubation. Parental care must confer great biological advantages in reducing energy demands for production of eggs or by spreading this load over a longer period while the young are developing within the parent. Thus, it is perhaps not surprising that viviparity has evolved independently several times in both the vertebrates and the invertebrates. Between those fishes which represent the primitive situation-scattering millions of unprotected gametes-and the highly specialized viviparous forms, there are numerous species which build nests and exercise parental care (Salmonidae and Gasterosteidae), other species in which incubation takes place either in the buccal (Cichlidae and Bagridae) or branchial chambers ( Amblyopsidae ) , fishes which provide special devices for the attachment of their young to their bodies (cutaneous incubation in the male Kurtus gulliveri or the females of Aspredo cotylephorus and Sobnostomus laciniatus), and the fascinating sea horses and pipefishes (Syngnathidae) in which the males have a ventral marsupial pouch where the young are incubated until ready for an independent existence. Bertin (1958b) has provided a summary with many more examples and illustrations of these interesting and curious devices. Although there is a rich literature on the natural history and functional morphology, there are very few physiological studies beyond those which have shown a strong dependence of secondary sex characters such as the marsupium on the gonadal steroids (Oguro, 1958; Noumura, 1959; Wai and Hoar, 1963). Among the fishes, only elasmobranchs and teleosts have achieved a true viviparity. In each of these groups there is an array of species from the ovoviviparous-where the eggs have sufficient yolk for the nourishment of the young and the female provides only protection-to the truly viviparous species-where the yolk content of the egg is greatly reduced and the developing young establish a connection with the maternal tissues at an early stage to draw nourishment from them and to satisfy the respiratory and excretory demands. The literature on viviparity in fishes has been reviewed several times

22

WILLIAM S . HOAR

(Hoar, 1957; Budker, 1958; Bertin, 1958c; L. H. Matthews, 1955; Amoroso, 1960). The following is a brief summary; since the phenomena are so very different in elasmobranchs and teleosts these two groups will be dealt with separately.

B. Viviparity among the Chondrichthyes The Chondrichthyes produce relatively few eggs which vary greatly in their content of organic matter but are all of the yolky telolecithal type (see Table 8 in Needham, 1942). In the truly oviparous groups (Scyllidae, Heterodontidae, Raiidae, and Chimaeridae) , ova are housed in horny protective shells which provide protection throughout a long period of incubation. In the ovoviviparous and viviparous species, the young develop within the Mullerian duct (uterus) on which they usually depend for nourishment as well as protection. Ranzi (1932, 1934) did much of the pioneer work and his papers should be consulted for details; Needham (1942) gives a valuable English summary and bibliography. Budker (1958) summarizes these important papers in French. 1. INTERNAL FERTILIZATION Fertilization is always internal among the Chondrichthyes. LeighSharpe in a series of classic papers (1920, 1921, 1922, 1924, 1925, 1926) described the modifications of the male pelvic fins which form the copulatory organs or claspers. These posterior extensions of the fins are stiffened not only by the cartilages of the metapterygia (Romer, 1955) but also during copulation by erectiIe tissue. The reIative contributions of cartilage and erectile tissues vary in different species. In all cases, an essential portion of the organ is the clasper groove formed by skin folds, the edges of which overlap to form a scroll-like tube along which the sperm are transported from the cloaca. Another constant feature of this apparatus is the presence of a clasper syphon or gland which contributes to the seminal discharge or provides a pumping mechanism for its release. In the sharks, the syphon takes the form of a blind muscular sac situated just under the skin anterior and lateral to the cloaca1 region; in the skates and rays a clasper gland takes the place of the hollow syphon. LeighSharpe concluded from his anatomical studies that the dilute fluid found in the syphon was mostly seawater and that during copulation the contractions of the muscular syphon wall pumped sperm through the clasper groove in a jet of seawater. It is by no means certain, however, that this is its main function. Botte et d. (1963) and La Marca (1964) among others have reported more recently on the physiology and histochemistry.

1.

REPRODUCTION

23

Mann (1960, 1964; Mann and Lutwak-Mann, 1963) has shown that the syphon of the spiny dogfish, Squalus acanthias, secretes an abundance of 5-hydroxytryptamine (serotonin) ; a pair of syphons in a mature male may contain as much as 20 mg of serotonin or 0.3% (Mann, 1960). The further demonstration of a marked stimulatory effect of serotonin on the isolated uterus of the spiny dogfish is strongly suggestive of a role for the syphon in the transport of sperm after transfer to the female (Mann and Prosser, 1963). At this stage, generalizations are not justified since only traces of serotonin have been found in the syphon sacs of the smooth dogfish, Mustelus canis, and none was demonstrated in the clasper glands of Torpedo and Raja. The clasper glands of skates and rays produce a milky white semiviscous fluid which coagulates on contact with seawater; its function remains speculative and, indeed, there are still many questions concerning the physiological mechanisms of internal fertilization in these fishes. 2. OVOVIVIPARITY The ovoviviparous species are far more numerous than the truly placental or viviparous ones; a true viviparity with yolk sac placenta is confined to certain species of two families of sharks-the Carcharhinidae and the Sphyrnidae, both belonging to the Galeiformes (Tortonese, 1950; Budker, 1958). Actually, the distinction between ovoviviparity and viviparity is a rather artificial one for the physiologist since the maternal contribution of the placental shark to the nourishment of the fetus is an intermediate one in a series ranging from almost zero in the primitive ovoviviparous species to an almost complete dependence in the highly complex ones (Fig. 7 ) . Moreover, in some sharks (Sphyrna tiburo and Mustelus canis) the placenta develops only after several months of an ovoviviparous existence ( Te Winkel, 1950; Schlernitzauer and Gilbert, 1986). These facts are recognized by Budker (1958) who divides the Chondrichthyes into only two major groups-the oviparous and the viviparous-with the latter subdivided into the aplacentals and the placentals. The rectangles to the left of the ordinate in Fig. 7 show the extent to which the newborn fish receives nourishment from its mother while in utero; the rectangles to the right show the content of organic substances in the uterine fluids; the black bars show the relative reductions in size of maternal liver during development. Although the presence of organic material in the uterus is universal, the organic content of the egg may be actually greater by 20-404: than the organic content of the animal at birth (Group I in Fig. 7); in short,

Ia . .

I b Rhinobotur pandumtur)

I

------

I

- - - - - - - - - -tt

Ptsmplotea micruru

?

’------

- - - - - - - - -F,-

Myliobafis bovine

I

Trygon violaceu I

r

I

I

I

I

100

10

1

0.1

0.01

0

I

I

1

1

0

1

1

10 20%

1

orgonic subsfonce

R-

in uterine

milk

I

i

10

100%

1. REPRODUCTION

25

the mother has provided protection but probably does not provide organic nutrients to the fetus. At the other extreme, eggs with very little organic material (only 200 mg in Pteroplatea micrura) depend almost entirely on the maternal secretions for growth; at birth, Pteroplatea contains about 10 g of organic material, an increase of almost 5000% (Needham, 1942). In contrast, the change in organic substance from egg to newborn pup is only 840 and 1050%in two placental speciesCarcharias glaucus and Mustelus laevis, respectively. Selected tables summarizing the biochemical data, largely the work of Ranzi, will be found in Needham (1942) and Amoroso (19csO). A host of research problems await the interested physiologist. It has now been almost 40 years since Ranzi’s classic work on the morphology and biochemistry; most of the endocrinological work does not go beyond correlations between the cytology of the endocrine glands and the cycle of morphological events during gestation. Histologically, the uterine lining varies from a mucus-secreting layer of cuboidal epithelial cells in species which depend on the egg yolk for nourishment (Type Ia in Fig. 7) through forms with moderately folded, serous-secreting linings, as in Torpedo, to those with uterine linings beset with villi or trophonemata of varying lengths and complexities and glandular surfaces which secrete an abundance of fat (8%of the organic substance in Trygon violacea) . Amoroso ( 1960) refers to all these uterine secretions as “uterine milk” and provides a summary of the composition in various species. Budker (1958) describes them as mucous, serous, and lipid. The yolk is mainly digested within the intestine of embryos which depend on it for nourishment. Te Winkel (1943) has described the process in the Squalidae. In Squalus acanthias, she found that the relatively enormous yolk sac established at an early stage in ontogeny decreased rapidly in size while an internaI yolk sac (expansion of the yolk stalk) became relatively larger until the external one remained as only a remnant. Yolk granules are moved by cilia from the external sac through the yolk stalk into the internal sac; then they pass on into the intestine where digestion occurs (Fig. 8). All of these structures, including the Fig. 7. Summary of the maternal-fetal relationships in the elasmobranch fishes (Ranzi, 1934). Rectangles to left of ordinate, extent to which the embryo receives organic substance from mother (negative sign indicates that this does not occur); rectangles to right of ordinate, content of organic substance in uterine fluid (dotted lines show probable values); black bars show degree to which maternal liver is reduced during development as measured by factor R ( Needham, 1942). Sketches of the uterine lining are shown at the right. Based on Ranzi (1934) and Needham (1942).

26

WILLIAM S. HOAR

Fig. 8. Dissection of a 220-mm embryo of Squalus suckleyi.

intestine, are ciliated. Enzymic activity is established in the gut when the embryo is about 60-70 mm long (size at birth about 250-300 mm). A small reserve of yolk still remains in the internal sac at birth. The skates and rays display a variety of specializations from those which are oviparous through a series of curious adaptations in ovovivi-

1.

REPRODUCTION

Fig. 9. Structures concerned with embryonic nutrition in Dasyatis violacea ( =Trygon uiohcea). ( A ) Villi of uterine wall, ( B ) diagram of circulation in uterine villus, and ( C ) and ( D ) cross sections of uterine villi (23 g embryo) taken through the apex ( C ) and base ( D ) of a villus. Bottom series of cells shows progressive stages in development of uterine gland cells with accumulation of fat droplets at right. Redrawn from Ranzi (1934) with the permission of the Stazione Zoologica di Napoli.

28

WILLIAM S. HOAR

parity. In the most highly specialized situations, nutrition by uterine milk or embryotrophe becomes more efficient than placentation in terms of organic material transferred from mother to fetus ( Amoroso, 1960). In the electric ray Torpedo the uterine secretions are taken directly into the digestive tract through the mouth and spiracles; while yolk digestion is going on in the intestine, the uterine secretions are being digested in the stomach, In one of the stingrays Pteroplatea, the highly glandular trophonemata or villi enter through the spiracles of the embryo and extend down into the esophagus to release the secretions directly into the gut. In another stingray (Trygon or Dasyatis) and in the eagleray Myliobatis the villi are shorter but extremely numerous; they produce a secretion rich in fat which is aspirated into the gut mostly through the spiracles (Alcock, 1892; Amoroso, 1960). The structure of the trophonemata of Trygon is shown in Fig. 9. Shann (1923) describes a very different mode of embryonic nutrition in the shark, Lamna cornubica. In this animal, the egg yolk is absorbed early in development and thereafter the developing pups depend almost entirely on the swallowing of immature eggs and degenerating ovarian tissues which pass down the oviduct packaged in a delicate secretion of the shell gland. As L. H, Matthews (1955) notes, some of these ovoviviparous arrangements are not very different from the oviparity found in the Holocephali, where only a small part of the yolk is enclosed in the yolk sac; the remainder breaks up into a thick milky fluid which is first absorbed by the filamentous external gills and later ingested through the mouth. 3. VIVIPARITY

Descriptions of the placentae of the silky shark, C u r c ~ r h i ~ u fa&s formis ( Gilbert and Schlernitzauer, 1966), and the bonnethead shark,

Sphyrna tiburo ( Schlernitzauer and Gilbert, 1966), have recently been added to the literature of true viviparity cited in Amoroso’s review ( 1960). The ova of Mustelus canis, on arriving in the uterus, become isolated into separate uterine compartments. These are formed by the growth of ridges on the uterine wall which eventually fuse so that each embryo is enclosed in a separate chamber (Fig. 10); although uterine compartments are not formed in most of the nonplacental sharks there are at least two exceptions ( Gabus canis and Mustelus vulgaris) cited by Ranzi (1934). Mustelus canis (also Sphyrna tiburo) leads an ovoviviparous existence during this early period and may not establish a placenta for several months. During this time, while nutrients are obtained from the yolk, these embryos develop elaborate circulatory networks in the walls

Fig. 10. Yolk sac placenta of Mustelus cunis ( =be&). ( A ) Diagram of ventral view of right oviduct and uterus to show orientation of egg cases and embryos, ( B ) section through placenta with detailed portion at lower right, and ( C ) transverse section through umbilical cord of 18 g embryo. Upper diagram after Te Winkel (1950); others redrawn from Ranzi (1934) with the permission of the Stazione Zoologica di Napoli.

30

WILLIAM S. HOAR

of their yolk sacs and the yolk sacs themselves become greatly folded. While these developments are taking place in the yolk sac, the uterine mucosa which is initially smooth, loose, edematous, and covered with a columnar or cuboidal epithelium also becomes folded. A placenta is established through the interdigitation of these two series of folds with a thinning of their epithelia to bring the maternal and fetal circulations into close proximity ( Fig. 10). The umbilical cord or stalk which attaches the embryo to the placenta is a modified yolk stalk. The interdigitations characteristic of the placentae of the two fishes just described do not seem to develop in C~rcharhinus( Mahadeven, 1940; Schlernitzauer and Gilbert, 1966). The specialized yolk sac sits on a modified and extremely vascular discoidal patch of the uterus. Detailed descriptions of the umbilical cords will be found in the literature cited. An interesting specialization found in some forms ( Paragaleus, Scoliodon, and Sphyma) is the presence of numerous villi (appendiculae) which appear to be absorptive (see Fig. 1285 in Budker, 1958); these suggest that the dependence on uterine milk was not completely lost in the evolution of viviparity among the sharks. In this and in several other features, it is clearly indicated that the phylogeny of viviparity in these fishes was via a highly specialized ovoviviparity and that the steps involved were not very long ones. Both conditions may be markedly developed within a single genus; Mustelus vulgaris is ovoviviparous while Mustelus canis is viviparous.

C. Viviparity among the Teleosts

Development of the young within the female occurs in only two orders of teleost fishes, the Cyprinodontiformes and the Perciformes. The nine families involved represent only a small proportion of the Teleostei. However, the diversity of mechanisms is as great as that of any other group of vertebrates which range from the oviparous, through several stages of ovoviviparity to highly successful viviparous forms. This, as well as a lack of correlation with particular habitats or geographical regions, argues for a separate evolution on more than one occasion. The viviparous teleosts include such geographically remote groups as the comephorids unique to Lake Baikal in Siberia and the goodeid fishes of the Mexican plateau (C. L. Turner, 1947); both marine and freshwater species are represented. Amoroso’s review (1960) is the most informative of the recent ones and includes citations to the papers of C. L. Turner which are still the richest source of information on these fishes. Amoroso’s summary (1960) is reproduced in Table I and lists the known families of viviparous

31

1. REPRODUCTION

teleosts (see also Breder and Rosen, 1966). It will be noted that internal development in the teleosts always takes place in the ovary; the Mullerian duct, which houses the developing elasmobranch, is absent in the teleosts (Fig. 3 ) . Sometimes the young develop within the ovarian follicle to a very advanced stage before ovulation (Poeciliidae and Anablepidae) ; more frequently, embryogenesis occurs within the cavity of the ovary after fertilization within the follicle. Although one might speculate that the ovarian type of gestation is more primitive, C. L. Turner (1947) believes that with one probable exception (Zoarces) fertilization precedes ovulation in present-day viviparous teleosts. Bertin ( 1 9 5 8 ~ )summarizes the three general situations as follows where “0” represents ovulation, “ F is fertilization, “H” is hatching, and “ P is parturition: Type Zoarces Type Jenynsia Type Gambusia

0-F . . . . . . . . . . . . . . . . .H ...................... P F . . . . . . . .O. ..................... H . . . . . . . . . . P F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0- H-P

Both follicular and ovarian gestation are characterized by ovoviviparous and viviparous types of development. As in the elasmobranchs, the total dry weight of the larva at birth may be less than that of the egg, indicating the complete lack of a maternal contribution to the nourishment of the embryo while in the more specialized viviparous types the egg is endowed with very little yolk and the developing embryo obtains almost all of its nourishment from its parent (Fig. 11). Belonesox be1iza nus

0.018

0.016 0.014

; g 0.012

.,

Aulophallus elongatus

c

-

0.01 ’

g

0.008.

.-

r

0

0.006

0.002

Mollienesia velifera Gornbusia nobilis Sebastes marinus

Fig. 11. Changes in the dry weight (including yolk) of several viviparous fishes during their development. From Scrimshaw (1945).

Table I Summary of Modes of Development in Teleosta with Internal Fertilizationa Order, family, or species Siluridae Trachychoristes Perciformesb Scorpaenidae Sebastodes

Zoarcidae (Zoarcea

Type

Gestation

Fetal nourishment and placental structures

Corpora lutes ?

Oviparous

Ovarian cavity Eggs immediately discharged Yolk in yolk sac and from or gonaduct into water aquatic environment

Oviparous

Follicle zygote Brood remain in ovarian retained for cavity for short period; long period thereafter development oviparous

Yolk in yolk sac and from aquatic environment

Present

Viviparous

Ovarian cavity Retained in ovarian cavity until birth

Very little yolk; ovary furnishes greater part of food; some embryotrophe from dead embryos; no supplementary structures; absorption through skin

Present

Viviparous

Follicle

Secretions; embryotrophe Present from desquamated cells; sperm, dead embryos; absorption through hypertrophied yolk sac. S u p plementary structures for respiration in form of V ~ S cular spatulate processev on fins

viviparus)

Embiotocidae (Cymatogaster aggregatus)

Fertilization

Hatched into ovarian cavity before segmentation mm. pleted. Retained for very long period; embryos born in very advanced state. Males sexually mature a t birth

Cyprinodontes Poeciliidae (Xiphophoriis helleri)

Oviparous

Follicle

Follicular for entire gestation; high degree of superfetsr tion; embryos very immature at birth; gonads in indifferent stage

Yolk provides most of the intraovarian food, not completely absorbed a t birth; “neck” strap forms s u p plementrtry structure for absorption of secretions

Em

I

Poeciliidae (Heferandria formosa)

Viviparous

Follicle

Follicular for entire gestation; high degree of superfetatioii

Ovarian secretions; “neck” strap and follicular pseudoplacen ta

Anablepidae ( A nahleps anahleps)

Viviparous

Follicle

Follicular for most of gestation; short time in ovarian cavity

Ovarian secretions; yolk sac hypertrophied and is principal organ of absorption; enlargement of gut form intestinal pseudoplacenta

Goodeidae Ovoviviparous with Follicle (Ataenohius superimposed tuweri) viviparity (Goodea hilineata) (Zoogonecticus cuitzooensis) (Lermicthys multiradiatus)

Ovarian cavity for entire gestation ; brood in advanced state of development a t birth; high degree of mortality

Ovarian secretions; embryoPresent trophe from stored sperm and dead embryos; Trophotaeniae (fetal) absent in Ataenobius toweri, poorly developed and voluminous in Zoogonecticus cuitzooensis and Lermichthys multiradiatus

Jeny nsiidae

Ovarian cavity for entire gestation; brood in advanced state of develop ment at birth; high degree of mortality

As for Goodeidae but ovarian Present fluids imbibed directly by mouth; trophonemata (maternal) in the form of ovarian flaps constitute a branchial pseudoplacenta

a

b

Ovoviviparous with Follicle superimposed viviparity

From Amoroso (1960). To this list of perciform fishes Breder and Rosen (1966) add certain species of the Clinidae and the Comephoridae.

?

Present

z“

34

WILLIAM S. HOAR

Actually, most of the teleosts which develop within the ovary seem to draw nourishment from the mother and in this sense are not truly ovoviviparous. As indicated in Fig. 11, only Sebastes marinus shows a decline in dry weight between fertilization of the egg and parturition; most of the species show little change in weight indicating that the mother has met the metabolic costs of respiration in the developing tissues so that the total weight of the larva at birth is just balanced by the stored material in the original egg. Only two of the poeciliids (Heterandriu formosa and Aulophallus elongatus) studied by Scrimshaw (1945) are truly viviparous, depending on the mother for both their respiratory needs and the anabolic demands for growth. The weight decrease of 34% in Sebastes marinus is essentially the same as the value 37%reported for the oviparous trout Salmo fario ( Scrimshaw, 1945). 1. INTERNAL FERTILIZATION As in the Chondrichthyes, internal fertilization in the teleosts is not necessarily followed by a development of the young in the female. One of the most complex intromittent organs described in male teleosts (the priapium located on the throat of the Phallostethidae) serves only to fertilize the eggs as they are being laid (Bailey, 1936; Breder and Rosen, 1966). In teleosts, the copulatory organ is usually an enlarged genital papilla or a specialized anal fin. Unlike the Chondrichthyes, the pelvic fins are rarely used directly in the transfer of sperm (Bertin, 1958~);the priapium of the phallostethid fishes is again an exception with skeletal elements derived from pelvic fins (Bailey, 1936). An enlarged genital papilla is sometimes referred to as a “pseudopenis.” Its size bears little relation to viviparity. In the Embiotocidae, a highly successful viviparous group, it appears only seasonally as a modest fleshy papilla; in the Cottidae, an oviparous group, it is often greatly enlarged and may contain erectile tissue (Weisel, 1949). A pseudopenis is found in several other families both oviparous and viviparous (Bertin, 1 9 5 8 ~ ) There . are many strange modifications of the genital papillae of both sexes. In female Orthonopias triacis the protrusible oviduct is smeared with sperm during copulation and then withdrawn to effect fertiIization of the eggs which are laid one at a time ( BoIin, 1941). In Apogon imberbis the urogenital papilla of the female is enlarged and introduced into the male for the reception of sperm (Garnaud, 1950). As noted earlier, the elongated genital papilla of Rhodeus amurus transfers eggs to the gill spaces of a freshwater mussel. These enlarged genital papillae-both male and female-appear as secondary sex characters

1. REPRODUCTION

35

during the breeding season, and their development is probably always regulated by the gonadal steroids. The copulatory organs of the cyprinodonts are specialized anal fins. The fins are only slightly altered in the Goodeidae with a foreshortening of the anterior rays to form a spermatopodium (Langer, 1913; Mohsen, 1961). In contrast, the anal fin of the Poeciliidae, referred to as the “gonopodium,” is profoundly modified through an elongation and specialization of several of its rays; the genital aperture is at its base, but during sexual activity the elongated bony fin rays and the web of tissue connecting these rays interact to form a transitory groove presumably associated with the transfer of sperm bundles to the tip of the gonopodium ( Rosen and Gordon, 1953; Rosen and Bailey, 1963) . The Jenynsiidae and the Anablepidae show a third stage in anal fin specialization to form a large penis containing a permanent tube opening at its tip; at times the organ may be greatly extended; it is a naked structure in the Jenynsiidae but covered with scales in the Anablepidae (Bertin, 1958~). 2. GESTATION WITHIN THE OVARIAN CAVITY

Sebastodes paucispinis (family Scorpaenidae ) undergoes an entirely ovoviviparous development within the ovarian cavity. Moser ( 1967a), who has recently studied this species, found numerous sperm singly or in clumps embedded among the epithelial cells of the maturing follicles or on their outer surfaces but never within the follicles; he believes that fertilization occurs immediately after ovulation and this would place Sebastodes in the “Zoarces type” of Bertin’s classification. Sebastodes embryos lack placentalike connections and depend entirely on their egg yolk for nourishment; they hatch just prior to spawning when the yolk has been largely absorbed and they are about 4-5 mm long; life in the ocean commences as a planktonic larva. Large females may produce broods of as many as two million larvae; the respiratory demands of this large mass of developing tissue must create one of the major problems in maternal physiology. A specialized dual vascular system to the ovary seems to be unique to this group of fishes and is evidently related to these special needs (Moser, 196%). The Embiotocidae show an intermediate situation between the strict ovoviviparity of Sebastodes and the goodeid or jenynsiid fishes which usually form elaborate placentalike connections. Cymtogaster aggregata has been carefully investigated by C. L. Turner (1938a) and Wiebe (1968b,c). Copulation occurs during the spring or early summer and sperm remain dormant in the ovary until fertilization occurs about 6 months later. Eggs, fertilized within the follicle, are shed into the cavity

36

WILLIAM S. HOAR

of the ovary during early segmentation stages and develop there for 10-12 months to an advanced stage (sexual maturity in some males: C. L. Turner, 1938a). The Cymatogaster egg contains relatively little yolk and depends mainly on the secretions of the highly glandular ovigerous folds for nourishment (Fig. 6 ) . By the time the larvae have reached a length of 15 mm, spatulate vascular extensions have developed on the vertical fins (Fig, 6 ) to assist in nutrition and respiration; these are resorbed just before birth. The alimentary canal begins to function later in gestation and long villi located in the gut are evidently concerned with digestion and absorption of foods entering through the mouth (C. L. Turner, 1938a); relationships are also established between the ovarian tissues and the embryonic gills (C. L. Turner, 1952). More elaborate types of placental connections are characteristic of both the Goodeidae (C. L. Turner, 1937a, 1940a) and the Jenynsiidae (C. L. Turner, 1940b). Turner's investigations of the goodeids have revealed a spectrum of species with progressively diminishing yolk stores, a less significant role for the yolk sac and pericardial sac (mentioned below) and an increasing importance of highly vascular rectal trophotaenia of many diverse forms (Fig. 12 and C. L. Turner, 1937a). These are bathed in the copious secretions of the ovarian cavity; the ovarian

Fig. 12. The trophotaeniae of Zoogoneticus quitzeoensis during late gestation with a segment of a longitudinal section through one of the nutritive processes at the lower left. Redrawn from C. L. Turner ( 1933, 1940a).

1. REPRODUCTION

37

secretions are supplemented by degenerating embryos. The brotulid fishes have similar absorptive surfaces (C. L. Turner, 1936). In the jenynsiids there is also an early ovulation following fertilization, an initial dependence on the small supply of yolk, the development of a yolk sac and a pericardial sac. For the major portion of intraovarian development, however, an intimate association is established between the ovarian tissues and the pharyngeal and mouth cavities of the embryo; vascular folds of the ovarian epithelium are in close contact with the gills-an arrangement which Amoroso (1960) refers to as a “potential branchial placenta.” These fishes also imbibe ovarian fluids as a source of nourishment (C. L. Turner, 1940b). In several of the papers cited above, Turner discusses the progressive specialization in ovarian gestation from the strictly ovoviviparous to these several curious placental connections and notes that this trend is evident in a single family-the Goodeidae.

3. FOLLICULAR GESTATION

A prolonged follicular gestation is found only in the Poeciliidae and the Anablepidae. Again there is a series of forms from the ovoviviparous ( Poecilia ) to species with a specialized follicular pseudoplacenta; the complete array is evident within the poeciliids which remain in the follicle for the entire period of gestation (C. L. Turner, 1 9 4 0 ~ ) . The yolk supply is adequate for embryonic nutrition in several of the familiar poeciliids ( guppy, black molly, and swordtail) and respiratory exchanges are effected through an expanded yolk sac which extends over the head as a “neck strap.” Although it is generally assumed that the intimate association of the portal blood system on the yolk sac and the vascular wall of the ovarian follicles serves only for the exchange of gases and nitrogenous wastes, a transfer of nutrients has not been ruled out. In Heterandria formosa the yolk supply is meager and the yolk sac very small; the fetal-maternal exchanges are effected through a greatly expanded and vascular pericardial sac which is wrapped around the anterior part of the embryo; the wall of the follicle is smooth and makes intimate contact with the portal system of the embryo. A further stage of specialization found in this group is an enlarged ventral expansion of the coelomic cavity to form a “belly sac” (only modestly developed in Heterandria) and the formation of finger-like villi on the follicular wall (Fig. 13). Turner applies the term “follicular pseudo-placenta” to this complex of follicular wall, follicular space, and the portal system of the belly sac.

38

WILLIAM S. HOAR

Fig. 13. The embryonic-maternal connections in several poeciliids. ( A ) The portal circulation of the yolk sac of 7.5-mm embryo of Lebistes (=Poecilia) reticuhtus, ( B ) Poeciliopsis embryo showing the portal network of the belly sac (pericardial sac anteriorly and expanded coelom posteriorly), ( C ) low branched villi on the internal surface of the follicle of Poeciliopsis, and ( D ) section of follicle and adjacent Poeciliopsis embryo. Redrawn from C. L. Turner ( 1 9 4 0 ~ ) .

The anablepid fishes show still another stage in the specialization of the follicular pseudoplacenta. Not only is the follicular wall clothed with complex villi but also numerous vascular bulbs appear in the portal circulation of the belly sac (Fig. 14). In addition, there is a spectacular enlargement of the gut in Anabkps (midgut in A. anableps and posterior gut in A. doweri), and this is evidently concerned with the digestion of follicular fluids taken into the alimentary canal (Fig. 14); an involution of this specialized area of the gut occurs before birth. The enlargement of the gut and the presence of vascular bulbs is unique to the Anablepidae (C. L. Turner, 1940d). A multitude of fascinating problems awaits the physiologist who becomes interested in the viviparous fishes. Although the functional morphology has been systematically investigated there are relatively few

1.

REPRODUCTION

39

studies of the physiology. Nutritional demands of elasmobranch embryos were investigated by Ranzi (1934) many years ago and there is a small amount of work on their nitrogen metabolism (Read, 1968); there are also a few studies of the nutrition of developing teleosts (Scrimshaw, 1944, 1945). However, to my knowIedge, there are no carefuI investiga-

Fig. 14. Expanded midgut and maternal-fetal connections in Anableps anableps. ( A ) Dissection of a 21-mm embryo. Redrawn from C. L. Turner (194Od). ( B ) Section of the wall of a follicfe containing a 21-mm embryo. Redrawn from C. L. Turner ( 193813).

40

WILLIAM S. HOAR

tions of the gaseous exchanges, the properties of maternal and fetal blood, or the ultrastructure of the maternal-fetal connections. 4. SUPERFETATION

In some of the poeciliids, several broods of young may be developing simultaneously within the ovary ( superfetation) . C. L. Turner ( 1947) suggests that the condition has evolved through a shortening of the period required for the development and maturation of the ova; a second group of eggs reaches maturity and is fertilized before the previous group completes development. It must also be related to storage of sperm within the ovary. In many fishes the ovarian epithelium assumes a nurse-cell function and immobilized sperm, embedded in its epithelium, remain viable for many months. Superfetation seems to reach a climax in Heterandria formosa where live sperm have been found 10 months after a single contact with the male and as many as nine broods may be developing in the ovary simultaneously with births at intervals of about 10 days (C. L. Turner, 1937b).

IV. THE ENDOCRINOLOGY OF REPRODUCTION

The timing of reproduction, regulation of the associated morphological changes, the mobilization of energy reserves for gonadal development, and intricate breeding behavior are very largely dependent on the glands of internal secretion. Most of the endocrine organs are either directly or indirectly involved since this complexity requires profound metabolic adjustments, associated not only with gonadal development but sometimes also with changes in habitat during the breeding season. Only the pituitary gonadotropic hormones and the gonadal steroids are considered in this chapter. Chapters associated with the other endocrine glands contain pertinent references to their role in reproduction; the endocrinology of reproductive behavior is considered separately in the chapter by Liley, this volume, The literature on fish endocrinology has been reviewed several times during the past decade (Pickford and Atz, 1957; Vivien, 1958; Bern and Nandi, 1964; Hoar, 1966; Matty, 1966), while the reproductive hormones of fishes have been separately considered by Dodd (1960a) and Hoar (1965a). The bibliographies of these reviews are a rich source of the earlier literature; the citations included here have been kept to a minimum.

1. REPRODUCTION

41

A. The Pituitary Gonadotropins 1. PHYLOGENY OF GONADOTROPIC REGULATION-STUDIES IN THE AGNATHA It is now evident that the pituitary gland regulates certain aspects of reproduction in all vertebrate animals. It is equally true that this control is sometimes less precise and embraces fewer elements of reproduction among the fishes and, further, that there may be marked variations among the different groups of fishes. Pickford and Atz (1957) have tabulated the early experiments involving hypophysectomy of fishes. By the midthirties it was evident that gonadal development and the maturation of the sexual products were dependent on the pituitary gland in all gnathostomes. Investigation of the gonadotropic activities of the pituitary in the Agnatha awaited the investigations of J. M. Dodd and his associates in the early sixties. Their studies and investigations of Larsen (1965, 1969) have clearly shown pituitary gonadotropic controls in the cyclostome Lampetra fluuiatilis, one of the representatives of the most primitive living vertebrates. It would seem that the pituitary has dominated vertebrate reproductive processes since the Ordovician, a period of almost 500 million years. Dodds studies, however, went further and revealed a less complete dominance among the Agnatha suggesting a certain phylogeny in the establishment of this physiological regulation. Lampetra fluuiatilis, hypophysectomized in the late autumn and winter, were followed for a period of 5 months and the changes in the gonads carefully described (Dodd et al., 1960; Evennett and Dodd, 1963). At the beginning of the experiment, secondary sex characters were absent, the ovaries contained only small eggs, and the testicular ampullae a high percentage of spermatocytes. During the 5-month period the normal and sham-operated animals matured rapidly, secondary sex characters appeared, the ova increased in size, the testicular ampullae became filled with spermatozoa, and spawning took place in early April. On the contrary, the hypophysectomized animals remained immature in appearance while gonadal development was completely arrested in the females and markedly delayed in the males. Larsen (1965,1969) finds continued relatively slow growth of the ovaries after hypophysectomy but confirms Dodd's findings in the male. The effects on the gonads are particularly interesting. In the gnathostomes, hypophysectomy is followed not only by an arrest of ovarian development but also by a general atresia of the follicles (corpora lutea formation) while spermatogenesis is completely blocked in the testis.

42

WILLIAM S. HOAR

In the lamprey, spermatogenesis, spermiogenesis, and ovarian growth seem to be autonomous processes; they are retarded but not suppressed in the absence of a pituitary. The endocrine tissues of the gonads are evidently pituitary-regulated since the secondary sex characters which are thought to depend on them (Matty, 1966) fail to develop in the operated animals. Dodd et al. (1960) and Larsen’s investigations (1969) of the adult Lampetra fluuiatilis are the only long-term studies of the effects of hypophysectomy in the Agnatha. They have established the indispensable nature of the pituitary for reproduction. The Myxinidae have not yet been critically investigated in this connection (Adam, 1963); these primitive animals should prove particularly interesting and may well increase our understanding of the phylogeny of gonadotropic controls among the vertebrates. On the basis of the lamprey work, it has been suggested that in phylogeny the metabolic controls involving yolk mobilization and gonadal growth preceded those concerned with gametogenesis ( Hoar, 196513). 2. CHEMICAL INHIBITION OF THE ACTIONOF GONADOTROPINS Investigations of the pituitary functions of fishes have sometimes been hampered by the technical dSculties of hypophysectomy. This is true of some of the teleosts which are particularly popular with fish physiologists (Gasterosteus and Cymatogaster, for example); the mouth and opercular openings may be very small or the skull extremely deep dorsoventrally with much vascular tissue between the roof of the mouth and the brain or the fish may be difficult to handle postoperatively. Hoar et at. (1967) and Wiebe (1968a) have used Methallibure as a chemical blocking agent of gonadotropic functions. This agent, prepared by Imperial Chemical Industries ( 33,828), is l~-methylallylthiocarbamoyl-2methylthiocarbamyl hydrazine. It has been carefully tested in the homeotherms and is now finding a use in agricultural practice. We have examined the reactions of four fishes treated with Methallibure either by injection or by addition to the ambient water. It appears to be particularly effective in Cymatogaster aggregata ( Wiebe, 1968a), and Poecilia reticulata (Pandey, 1969d), but much less active in Gasterosteus aculeatus; the work with the goldfish, Carassius auratus, is preliminary. At present it is not known whether the variable response is because of species differences in reaction to the substance or species differences in the physiological actions of the gonadotropins; it is also possible that the effect varies with the environment-sea water vs. fresh water. It is of interest that some species of domestic animals are proving

1. REPRODUCTION

43

more responsive than others; the substance has been particularly effective in regulating the farrowing of pigs. In spite of the variations in response, all species of fish studied show clear evidence of the blocking of gonadotropic action in a manner comparable to that which follows surgical hypophysectomy. Spermatogenesis is either completely suspended or greatly depressed; yolk deposition ceases, and, in the more responsive species, steroidogenesis comes to an end. The side effects of the compound appear to be minimal involving a slight stimulation of the pituitary thyrotrophs (because of a mild thiourea action) and some depression of the somatotrophs (Wiebe, 1967; Leatherland and Pandey, 1969) . In histochemical studies of Foecilia, Leatherland and Pandey (1969) have shown that the most likely locus for the block is in the synthesis of the gonadotropins; there was a sharp and significant decrease in both number and size of these cells and no evidence of suppression of secretory activity in the neurosecretory system of the hypothalamus. Ovine LH was found to partially counteract the inhibitory effects of Methallibure in the gonads of Cymutogastet aggregata ( Wiebe, 1969b). 3. THE PITUITARY-GONADAL RELATIONS IN GNATHOSTOME FISHES

Pituitary-gonadal relations have now been investigated in several elasmobranchs and many teleosts. In general, the findings are consistent; the pituitary regulates both gametogenesis and steroidogenesis. In the hypophysectomized male dogfish, Sc yliorhinus caniculus, Dodd and his associates (1960) found that the effects of hypophysectomy were localized in the transitional zone between spermatogonia and spermatocytes. The zonated structure of the dogfish testis (Fig. 4) was particularly helpful in localizing these effects. It is of considerable interest that the seasonal cycle-presumably regulated by the pituitarycreates an identical picture ( Fig. 15). Spermatocyte production ceases during the springtime or following hypophysectomy, but in both cases spermatocytes already formed continue to differentiate and become mature spermatozoa (Simpson and Wardle, 1967). Dodd has reached the tentative conclusion that the gonadotropin( s ) are essential for the normal transformation which occurs when the spermatogonia (which divide by mitosis) become primary spermatocytes (which divide by meiosis). The testicular changes are well marked in dogfishes 6 weeks after h ypophysectomy. Removal of the ventral lobe of the female dogfish pituitary led to cessation of egg laying within 10 days to be followed somewhat more

44

WILLIAM S. HOAR

Fig. 15. Cycle of spermatogenesis in the dogfish, Squalus acanthius. The stages of gamete development are pictured in the circles representing ampullae. The four columns dividing the testis indicate four periods of the year. The filled circles indicate the position of the degenerate band, and they coincide with the absence of one stage and reduction of the neighboring stages of ampullae. Note the accumulation of evacuated ampullae to a maximum at the mating time ( December-February) followed by a resting phase when breakdown of spermatogonia results in a new degenerate band ( March-May). The rate of development from spermatocyte to spermatozoon is clearly seen using the degenerate band as a marker (7-9 months). 1, Epigonal tissue; 2, ampulla with sperm bundles and no Sertoli bodies; 3, origin of the degenerate band from breakdown of spermatogonia; 4, evacuated ampulla with Sertoli cell nuclei; 5, ampulla with ripe sperm and Sertoli bodies; 6, remains of degenerate band; 7, spermatid metamorphosis; 8, spermatids; 9, spermatocytes; 10, Sertoli cell nucleus; 11, spermatogonia; and 12, germ ridge. From Simpson and Wardle ( 1967 ) .

1. REPRODUCTION

45

slowly by distinct histological changes in the ovaries. Vitellogenesis ceases and all eggs larger than 4 mm become atretic; these changes are evident 3 weeks after hypothysectomy, but it requires about 14 months for the disappearance of all yolk and the reduction of the gonad to the juvenile condition. This atresia which follows hypophysectomy was first noted many years ago (Pickford and Atz, 1957); Hisaw and Hisaw (1959) and Chieffi (1962) have described it in some detail. Dodd and his associates have not observed any changes in the oviducts or secondary sex characters of their hypophysectomized animals. It is assumed but not yet proved that the elasmobranch pituitary like that of the higher vertebrates regulates the steroid-producing endocrine tissues of the gonads. In 1957, Pickford and Atz tabulated 17 species of teleosts and one lungfish known to have been successfully hypophysectomized at that time. This numbcr has increased substantially during the past decade; following are more recent additional species hypophysectomized in studies of pituitary-gonadal relationships: Mollienesia latipinna ( Ball, 1962), Pleuronectes platessa ( Barr, 1963a,b,c), Ophicephalus punctatus ( Belsare, 1965), Couesius plumbeus ( Ahsan, 1966b), Heteropneustes fossilis ( Sundararaj and Nayyar, 1967; Sundararaj and Goswami, 1968), and Poecilia reticulata-both adults and juveniles ( Liley, 1968; Pandey, 1969a,c). Of the species listed earlier by Pickford and Atz, more recent studies of Fundulus heteroclitus (Lofts et al., 1966) and, particularly those of Yamazaki on Carassiw auratus are of special significance in the literature on fish gonadotropins (Yamazaki, 1961, 1962, 1965; Yamazaki and Donaldson, 1968a,b). Wiebe’s work on the pharmacologically hypophysectomized surfperch, Cymutogaster aggregnta, also forms a part of this literature. All investigators agree that the pituitary is required for gonadal maturation; in its absence vitellogenesis is suppressed with atresia of the larger developing oocytes, spermatogenesis is blocked a t the spermatogonia-spermatocyte stage, and steroidogenesis does not occur in the gonadal endocrine tissues. Investigations of the endocrine tissues have been almost entirely confined to the testis. Although these general findings appear to be consistent, there are still many unresolved details. As yet, it is not certain whether gonadotropin is required for ( a ) multiplication of the spermatogonia, ( b ) specifically triggering the reduction divisions, and ( c ) spermiation or ovulation, nor is it certain to what degree the Sertoli cells and other supporting tissues in the gonads are dependent on the pituitary. Barr’s work (1963~)suggests that some of the variations recorded by different workers may have a seasonal basis and depend on the variable stages of gonadal maturation at the time of hypophysectomy. The later stages

46

WILLIAM S. HOAR

of spermatogenesis and spermiogenesis, if well under way, seem to continue with production of mature sperm but spermiation is usually not observed in the absence of the pituitary (Yamazaki and Donaldson, 1968a; Pandey, 1969a) and the mature sperm gradually disappear through phagocytosis or resorption. Spermiation has been reported in the hypophysectomized plaice ( Barr, 1963c) and lake chub ( Ahsan, 1966b); these differences may depend on the stage of sexual maturity at the time of surgery. It has already been noted (Section 11, B, 3 ) that the pituitary normally triggers the secretions which are essential in thinning the seminal fluid prior to its discharge. Pandey’s work (1969a) shows that in the absence of the pituitary spermatophores form from the later spermatocytes, but they rupture and are resorbed without a discharge of the sperm. Pandey’s work ( 1 9 6 9 ~ on ) the juvenile guppy has also emphasized the interplay of both pituitary gonadotropins and androgens in the maintenance of spermatogenesis. The changes in ovarian histology which follow hypophysectomy have been much less frequently examined. However, the findings are consistent with those recorded for the elasmobranchs and indicate that after hypophysectomy a slow but inevitable resorption of ova may be expected; vitellogenesis comes to an end; new ova fail to form; and ovulation and spawning do not occur unless yolk deposition was complete prior to the surgery. Yamazaki (1965) found that relatively few goldfish ovulated even though the operation was performed only a few hours prior to the normal ovulation time. A major component of the literature on fish endocrinology is concerned with the practical matter of regulating ovulation in fish culture operations. The work primarily involves the use of mammalian pituitary or chorionic gonadotropic preparations. Many citations will be found in reviews by Pickford and Atz (1957), Ball (1960), and Dodd (1960a). This topic is also discussed in the chapter by Liley, this volume. The activity of some of the preparations commonly used will be considered in the next section. The endocrine-producing tissues of the gonads seem to be pituitary regulated in all of the vertebrates, and regressive changes may be expected in them after hypophysectomy. Atrophy of the testicular interstitial tissues has now been recorded in several teleost fishes (BuserLahaye, 1953; Lofts et al., 1966; Sundararaj and Nayyar, 1967; Pandey, 1969a) and in lobule boundary cells (Ahsan, 1966b). Regressive changes in the Sertoli cells have also been noted (Pandey, 1969a). The gonadotropic inhibitor Methallibure causes atrophy of both the interstitial cells and the Sertoli cells in the surfperch Cymatogaster; both of these groups of cells are actively concerned with steroidogenesis in this species

1. REPRODUCTION

47

(Wicbe, 1969a). All thcse data are consistent with the concept of a pituitary control of steroidogenesis in the gonadal endocrine tissues. Regressive changes noted in the efferent duct system (Lofts et al., 1966; Pandey, 1969a) are probably secondary to the degeneration of the steroidproducing tissues ( Wiebe, 196813). 4. EFFECTSOF HYPOPHYSECTOMY ON GESTATION Several workers have associated cytological cycles in pituitary secretory activity with the pregnancy cycle of viviparous fishes. Ranzi (1936, 1937) examined the endocrine glands in a part of his extensive studies of selachian viviparity and described pituitary hypertrophy and hyperemia with a decreased acidophilia in pregnant females. Chieffi (1961, 1967) and Della Corte and Chieffi (1961) cite confirmatory recent evidence for elasmobranchs. Likewise, pituitary changes have been associated with the reproductive cycles of viviparous teleosts (Stolk, 1951a). However, in both groups of fishes these pituitary cycles may only regulate the timing of reproduction and the development of the gametes as they do in the oviparous species. From present studies it must be concluded that the pituitary plays little or no part in the maintenance of pregnancy in the elasmobranchs but may be involved in some teleosts (Chester Jones and Ball, 1962; Hoar, 1965a, 1966). However, there is still very little experimental work directed to relatively few species. In the late thirties, Hisaw and Abramowitz (1938, 1939) reported that pregnancy was uninterrupted by hypophysectomy in the viviparous dogfish, Mustelus canis; animals hypophysectomized in early stages of pregnancy showed normal development of young for a period of 3% months (Hisaw, 1959). These seem to be the only experiments of this nature recorded for the elasmobranchs, although Dodd et d. (1960) report that removal of the ventral lobe of the pituitary of Scyliorhinus has no effect on the oviducts which retain their normal secretory capacities. Studies of the effects of hypophysectomy on gestation in the viviparous fishes are likewise preliminary. Ball (1962) found that 12 MoZZienesia latipinna, hypophysectomized during pregnancy, gave birth to normal off spring; the embryos completed their development in the usual time and were as lively and survived as well as the controls. Chambolle (1964)) however, working with another viviparous cyrinodont Gambusia, recorded a highly significant mortality of embryos when females were hypophysectomized during the first days of pregnancy; in part, the mortality was thought to be related to a disturbance in water and electrolyte metabolism-perhaps associated with the loss of the pituitary-adrenal

48

WILLIAM S. HOAR

control, Thus, the evidence from two different species of viviparous cyprinodonts is contradictory but there are further indications from other studies that the pituitary is essential to successful gestation and parturition in some teleosts. Wiebe (1967) found that treatment with the gonadotropin-blocking agent Methallibure leads to an atrophy of the secretory ovigerous epithelium in Cymatogaster. Associated with this, Wiebe noted reduced growth and a 3% mortality of the intraovarian embryos. These results are preliminary and based on only three pregnant females; the effects may also be indirect ones mediated through the gonadal steroids. They are, however, indicative. In addition, there are several reports (reviewed by Chester Jones and Ball, 1962) of premature release of young by pregnant cyprinodonts following injection of mammalian pituitary preparations. It is also pertinent that incubation and parturition of developing young in the male sea horse, Hippocampus hippocampus, is disturbed by hypophysectomy although gonadectomy is without effect (Boisseau, 1964). Finally, there is some suggestive evidence of a role for prolactin in the gestation of certain cyprinodonts and an intimation that the neurohypophysial hormones are active in parturition (Egami and Ishii, 1962; Ishii, 1963). Prolactin is considered in the chapter by Ball, Volume 11, and the neurohypophysial factors in the chapter by Perks, Volume 11. It is obvious that further research is required in this area of endocrinology. It may yet be necessary to modify Hisaw’s conclusions (1959)-based on only one species-that the responsibilities of the fish pituitary end with ovulation and that an involvement in the maintenance of pregnancy developed at a later stage in vertebrate phylogeny,

5. BIOCHEMICALNATUREOF

THE

GONADOTROPIN( s)

Two separate gonadotropins, the follicle-stimulating hormone ( FSH ) and the luteinizing hormone ( L H ) , are physiologically distinct in all tetrapods (Bern and Nandi, 1964; Hoar, 1965a). Although the nature of the fish gonadotropins is still not resolved, the data for a single factor seem to be accumulating steadily. In addition to the physiological Iiterature summarized below, the biochemical evidence for a single protein increases as the preparations (salmon or carp) have been purified more carefully ( Burzawa-GBrard and Fontaine, 1965, 1966; Schmidt et al., 1965; Yamazaki and Donaldson, 1968a; Donaldson et al., 1969). Most of the studies-both physiological and biochemical-have been concerned with teleosts. The excellent discussion by Pickford and Atz (1957) is still valuable. When fish pituitary extracts are tested in tetrapods, both the FSH-

1. REPRODUCl'ION

49

and the LH-like effects are frequently elicited. Witschi (1955) assayed pituitaries of sharks, garpikes and salmon-representatives of the three major groups of jawed fishes. Positive evidence for FSH was obtained by using the vaginal cornification test in the rat and for LH by using the weaver-finch feather reaction. The FSH content was very low; most of the gonadotropic activity appeared to be associated with LH. Ball ( 1960) has summarized these and other investigations-most of which provide suggestive evidence for the presence of the FSH-like effect and strong evidence for the LH action when fish pituitaries are injected into mammals. A series of papers by Otuska (1956a,b,c, 1957) using partially purified salmon pituitaries injected into newts and mice are in agreement with evidence for two gonadotropic proteins with distinct physiological effects. However, not all of this evidence for an action of fish gonadotropin( s ) in higher vertebrates is positive. Burzawa-Gdrard and Fontaine (1965) prepared a carefully purified extract of carp pituitaries which appeared to be a single protein and was entirely inactive in mammals. The histophysiology of the pituitary and the question of two different gonadotrophs is discussed in the chapter by Ball and Baker, Volume I1 (see also van Overbeeke and McBride, 1967). Evidence concerning the nature of fish gonadotropin has also been sought in tests with the pursed mammalian fractions injected into fishes. The preparations of mammalian FSH have consistently given negative results; LH, on the contrary, usually elicits both the gametogenetic and the steroidogenetic actions commonly associated with two different fractions in the higher vertebrates. The earlier literature is summarized in reviews previously cited. Among the more recent studies are those of Sundararaj and Goswami (1966) who report that LH alone, of several pituitary factors tested, would stimulate ovulation in Heteropneustes; Ahsan (1966b) who found a partially purified salmon gonadotropin to have an almost identical physiological action to mammalian LH when injected into hypophysectomized Couesius plumbeus; Fontaine and Chauvel (1961), Fontaine and Gdrard (1963), and Schmidt et al. ( 1965) who have reported strongly positive Galli-Mainini reactions (sperm release in frogs) with partially purified extracts of carp and salmon pituitaries; Ramaswami ( 1962) who has used enzymes which selectively digest or inactivate the gonadotropic factors in mammalian pituitaries (trypsin or pepsin for LH and ptyalin for FSH) and found convincing evidence for the LH-like factor but no support for FSH in Heteropneustes fossilis. Sundararaj and Nayyar's work (1967) with the latter species is confirmatory as is also that of Yamazaki (1965) and Yamamoto and Yamazaki ( 1967) for the acceleration of ovulation in hypophysectomized goldfish. However, it cannot yet be concluded that the fish gonadotropin

50

WILLIAM S. HOAR

is always physiologically similar to mammalian LH. By using the spermiation test in goldfish, Yamazaki and Donaldson (1968a) have obtained strong reactions with a partially purified salmon gonadotropin and with human chorionic gonadotropin (HCG) but no reaction with either FSH or LH. The mammalian chorionic gonadotropins are often used for experimental purposes and in fish culture work because of their ready availability. These factors, produced by the mammalian placenta during later stages of pregnancy, mimic many of the activities of the pituitary gonadotropins although they are definitely known to be different biochemically from the pituitary factors (C. D. Turner, 1966). Two chorionic gonadotropins are in common use: one, prepared from the serum of pregnant mares (PMS), usually has biological properties similar to a combination of FSH and LH; the other, prepared from human pregnancy urine ( HCG), acts more like LH. In mammals, the precise effects depend somewhat on the dosage used (C. D. Turner, 1966). When injected into fish, HCG has often been found to be quite effective (Yamazaki, 1965; Sundararaj and Nayyar, 1967; Yamazaki and Donaldson, 1968a) and this is in line with its LH-like activity. Pregnant mare serum is sometimes active (Ahsan and Hoar, 1963) but has usually been less consistently effective than HCG (reviews by Pickford and Atz, 1957; Dodd, 1960a). Sundararaj and Goswami (1966) report that 100 IU/fish of HCG will induce ovulation in Heteropneustes while 250 IU/fish of PMS are required to elicit the same reaction. In summary the evidence, both biochemical and physiological, now indicates a single proteinaceous gonadotropic factor in the pituitaries of teleost fishes. Although this may sometimes mimic FSH and frequently mimics LH when injected into tetrapods, it is clearly not identical with either of these factors. It more closely resembles LH but has some unique physiological properties and may be expected to differ from its counterpart in the tetrapods and even differ in its effects in different groups of fish. Investigations of some of the more primitive groups of fish should be rewarding. The lactogenic hormone (prolactin, luteotropin) is also gonadotropic in some mammals (the ovary of the rat), but no evidence has yet been presented for a comparable action in the lower vertebrates. The physiology of fish prolactin is considered in the chapter by Ball, Volume 11. B. The Gonadal Steroids

In each of the major groups of fishes, development of the secondary sex characters prior to breeding has been shown to depend on gonadal

1.

REX'RODUCXION

51

steroids (reviews cited and the chapter by Liley, this volume). This broad generalization is now backed by considerable experimental evidence and there seem to be no exceptions to it. Following gonadectomy, secondary sex characters fail to develop or, if seasonal in occurrence, regress. Their differentiation is initiated or stimulated by a wide variety of androgens and estrogens-both natural and synthetic. This may be the only broad generalization possible; in all the later events associated with reproduction ( ovulation, spermiation, spawning, and breeding behavior) the division of regulatory responsibilities between pituitary and gonads appears to be rather variable in different species. The physiologically most active gonadal hormones of higher vertebrates are testosterone from the interstitial cells of the testis, estradiol17p and its derivatives from the ovarian follicle, and progesterone from the corpus luteum. The biogenesis of all these compounds is from acetate via cholesterol, as the parent sterol, to testosterone through intermediate steps involving progesterone; the estrogens are derivatives of testosterone or closely related molecules and form the terminal part of the biosynthetic chain (see Fig. 14 in the chapter by Yamamoto, this volume). Thus, the biologically active gonadal steroids are linked through common pathways and one may expect to find small amounts of any or all of them in tissues which are primarily concerned with the synthesis of a single important hormone. The biogenesis of the adrenocortical steroids is linked to the same chain. These metabolic pathways are very ancient phylogenetically. Progesterone, estradiol-17/3, and some other estrogens have been identified in the ovaries of invertebrates (echinoderms and mollusks) as well as the lower vertebrates ( Botticelli et aE., 1960, 1961; Hisaw, 1963). Estrogens are also well known in plant tissues (Bickoff, 1963). It is not then surprising that both androgens and estrogens of several biochemical sorts have been regularly found in the gonadal tissues and blood of many different fishes. However, the presence of a gonadal steroid is no evidence of its significance as a hormone and the much more difficult task is to identify steroids which are physiologically important in regulating the reproductive activities of fishes. At present, it is by no means certain that all the steroids recognized as physiologically active in higher vertebrates are biologically significant in the fishes. Different groups of fishes seem to react somewhat differently to some of the same steroids (Egami and Arai, 1965). Moreover, it seems certain that there are important biosynthetic pathways of steroidogenesis in fishes which have not been recognized in the higher vertebrates (see below) although the general scheme of biosynthesis is probably similar in both groups (Breuer and Ozon, 1965; Idler and MacNab, 1967). In fishes, as in the higher verte-

52

WILLIAM S. HOAR

brates, some of the synthetic compounds are more active than the naturally occurring ones (Egami and Arai, 1965). The localization, identification, and analysis of synthetic pathways of fish steroids are being investigated in many places using a variety of techniques-physiological, biochemical, and histochemical. The specific histochemical techniques now available for the detection of enzymes involved in steroid biosynthesis ( e.g., 3P-hydroxysteroid dehydrogenase: Wattenberg, 1958; Galil and Deane, 1966) have been particularly useful in localizing the tissue sites of hormone synthesis (Delrio et al., 1965; di Prisco et al., 1965; Bara, 1965; Yaron, 1966; Simpson and Wardle, 1967; Wiebe, 1969a). Bern and Chieffi (1968) have provided a useful and comprehensive bibliography of the steroid hormones of fishes while Nandi (1967) summarizes the comparative endocrinology for the nonmammalian vertebrates. 1. THEANDROGENS The tissues of several different elasmobranchs have now been investigated, and there is good evidence that pathways recognized for biosynthesis of androgens in higher forms are operating in the elasmobranchs and that testosterone is an important end product in the testis. Chieffi and Lupo ( 1961), using chemical methods, investigated the testicular tissues of mature dogfish, Scylliorhinus stellaris, and recorded progesterone (100 pglkg), testosterone (50 pglkg), androstenedione (70 pg/kg), and estradioL17p (20 pglkg). Idler and Truscott (1966) were the first to isolate testosterone from the blood of a male elasmobranch. They found values which were relatively high (7.4 pg/lOO ml in Raja radiata) when compared with human males (about 0.56 pg/lOO ml). Testosterone was also found in the blood of female R . radiata, but the mean values were much lower. Progesterone, androstenedione, androsterone, and other steroids have been isolated from the semen of the dogfish, Squalus acanthias ( Simpson et al., 1963a), and testosterone biosynthesis was demonstrated by incubation techniques in the testis of this fish (Simpson et al., 1964). A number of different androgens have been isolated from the tissues and blood of teleost fishes. The studies of salmon tissues by Idler and his associates (1960, 1961a,b, 1964; Idler and Tsuyuki, 1959; Idler and Truscott, 1963; Idler and MacNab, 1967) are particularly significant. Steroid values have also been recorded for testicular tissues of the teleosts Morone labrax (Chieffi, 1962), Mugil cephulus (Eckstein and Eylath, 1968), and Serranus scriba (di Prisco and Chieffi, 1965); the latter is a hermaphroditic form.

1. REPRODUCTION

53

Idler’s meticulous chemical analyses have included gonadal tissues and blood from both the Atlantic salmon, Salmo salar, and the Pacific salmon-particularly the sockeye, Oncorhynchus nerka. Testosterone has been found in significant amounts together with several other steroids such as 17a-hydroxyprogesterone, recognized in higher forms as a step in testosterone biosynthesis, and androsterone-a product of testosterone synthesis. As might be expected from the known relationships of these substances, androgens have also been found in the tissues of female fish. Testosterone values are extremely variable (ranging up to 17 pg/lOO ml of male blood) ; since tissue values must represent a balance between synthesis and utilization it is always difficult to relate the actual amounts to the physiology. Three particular facets of Idler’s studies require special comment: ( a ) the presence of ll-ketotestosterone, ( b ) the occurrence of conjugated testosterone, and ( c ) the marked variations associated with the life cycle of the salmon. Idler et al. (1960, 1961a,b) were the first to identify ll-ketotestosterone as a natural product and subsequently to show its biological activity as an androgen in stimulating the development of secondary sex characters in salmon and chickens. The isolation of ll-ketotestosterone from both males and females of Atlantic and Pacific salmon suggests that the synthetic pathways of the androgens in these fishes are somewhat different from the usually recognized ones. This steroid is present in amounts up to 17 pg/lOO ml of blood and occurs along with several of the more familiar androgenic compounds including testosterone. Idler and Truscott ( 1963) found that testosterone and l7a-hydroxyprogesterone will serve as precursors for ll-ketotestosterone in the sockeye salmon and, subsequently Idler and MacNab (1967) demonstrated its in vitro synthesis from adrenosterone and testosterone in Atlantic salmon gonads and sperm; they suggest the probable pathways of biosynthesis. Arai and Tamaoki (1967) also studied the in vitro synthesis in Atlantic salmon gonads and sperm from adrenosterone and testosterone; they too suggest probable pathways of biosynthesis. Arai and Tamaoki (1967) have studied the in vitro biosynthesis of ll-ketotestosterone in rainbow trout, Salmo gairdneri. Conjugated testosterone was first reported in fish blood by Grajcer and Idler (1961, 19fX3). In higher vertebrates, the steroids are transported, at least in part, as conjugates with serum proteins and glucuronic acid. Grajcer and Idler obtained the release of testosterone in amounts of 13.7 pg/lOO ml of male sockeye salmon blood after treatment with the enzyme p-glucuronidase. The “free” testosterone in these samples of blood was 1.7 pg/100 ml. The blood of female salmon also contained conjugated as well as “free” testosterone with relatively less of the con-

54

WILLIAM S. HOAR

jugated form (7.6 pg of conjugated to 7.8 p g per 100 ml of free) even though the total amounts were about the same. Since these initial studies of testosterone glucuronoside, Idler and Truscott ( 1966) have reported on conjugated testosterone in the skates, Raia radiata and R. ocellata; these conjugates are probably as common in fish blood as they apparently are in the higher vertebrates (previous references and di Prisco and Chieffi, 1965). Schmidt and Idler (1962) studied the quantitative changes in several plasma steroids ( including 11-ketotestosterone and testosterone) during the migration of the sockeye salmon. Definite differences were recorded and the shift in ratio of 11-ketotestosterone and testosterone may be significant in the physiology and behavior of the spawning migration. Changing levels of androgens have also been reported in females of the ovoviviparous elasmobranch, Torpedo murmrata, during the reproductive cycle (Buonanno et aZ., 1964); there was a moderate increase in androsterone at the end of the gestation period and a marked steady rise in dehydroxyepiandrosterone from pregestation to the midgestation period, These variations may indicate physiological changes in the demands for estrogens rather than imply a role for androgen in the gestating ray. In the stickleback, Gasterosteus aculeatus, where the development of sexual behavior is cyclical and associated with strong agonistic behavior in the males, Gottfried and van Mullem (1967) report that the dominant males have testicular androgen levels which are five to seven times higher than the nondominant individuals; testosterone could not be detected in the testes of the nondominant fish but was present in the dominants. These cyclical and seasonal changes in gonadal steroidogenesis are presumably triggered by variations in the gonadotropic activity of the pituitary; seasonal changes in the latter have been traced in both plaice Pleuronectes (Barr and Hobson, 1964) and the perch Perca (Swift and Pickford, 1965).

2. THEESTROGENS AND PROGESTERONE Estradiol-17P has been found in the ovaries of many fishes at all levels in phylogeny: the lampreys, Petromyzon marinus (Botticelli et al., 1963), dogfishes, Squalus suckbyi (Wotiz et al., 1958, 1960) and Scyliorhinus caniculus (Simpson et al., 1963b), the ray, Torpedo marmorata (Chieffi, 1962; Chieffi and Lupo, 1963), the ratfish, Hydrolugus colliei (Hisaw, 1963), the lungfish, Protopterus annectes ( Dean and Chester Jones, 1959), and many different teleosts (Cedard et al., 1961; Gottfried et al., 1962; Hisaw, 1963; Lupo and Chieffi, 1963). This wide dktributiontogether with its presence in several invertebrate phyla-suggests that it

1. REPRODUCTION

55

will be found in at least small amounts wherever there is active steroid synthesis. Estradiol-17P has also been identified in the blood of elasmobranchs (Simpson et al., 1963b) and teleosts (Cedard et aZ., 1961) and is probably the physiologically most important estrogen. Gottfried ( 1964 ) tabulates estradiol-17,B values for ovarian tissues ranging from a trace to 120 pg/kg, with the most usual amounts varying from 10 to 20 ,ug/kg. Estradiol-l7P is recognized as the parent substance for several other estrogens known to occur widely in the animal world. Particularly frequent in occurrence are estrone (an oxidation product of estradiol-17P) and estriol which is derived from estrone by 16-hydroxylation and reduction at 17. Both estrone and estriol were found in the ovaries of many of the species listed above, but the proportions are quite variable and occasionally one of the compounds is absent (summary by Hoar, 1965a). Again, this is not surprising with a series of related substances in which the active utilization of at least one of them probably varies with the stage of maturity. Cedard et al. (1961) traced seasonal changes in the estrogens of the blood of both male and female Atlantic salmon. The total estrogen content in both sexes showed a five- to sixfold increase at the time of spawning, reaching 5-6 pg/lOO ml of blood. Estrone was found at all seasons; estriol appeared in significant amounts only at spawning; and estradiol was only present in small amounts and seemed to disappear completely in the females at spawning and in the postspawning males. Again, it is difficult to interpret the findings in terms of physiological demands but results such as these emphasize the hazards of conclusions based on a few estimations of the gonadal steroids at only one season. Progesterone has been found in the tissues of all vertebrates and many of the invertebrates (Gottfried, 1964; Botticelli et al., 1960, 1961). It is probably ubiquitous as a link in steroid biogenesis. As yet, however, there is no definitive evidence that progesterone is a physiologically active hormone with distinct endocrine responsibilities. Hisaw ( 1959, 1963) has discussed this problem at length and concludes that the role of progesterone as a hormone was established during the evolution of placentation in the mammals although it probably had its beginning among reptilian ancestors. Chieffi ( 1962, and elsewhere) has convincingly correlated the development of corpora lutea with the stages of gestation in the electric ray Torpedo. He has also demonstrated histochemically the biosynthesis of a variety of steroids-including progesterone-in the corpora lutea ( Chieffi, 1961; di Prisco et aE., 196S), while chemical methods have identified progesterone in Torpedo blood during pregestation (di Prisco et al., 1967) but again the question of its presence as hormone or precursor has )

56

WILLIAM S. HOAR

'O!

oocytes

9 days after parturition

Fig. 16. Variations in the number of oocytes and embryos during the 28-day gestation period of Poeciliu. The number was cdculated as a percentage of the total number of structures present in each ovary per 24 hr. From Stolk (1951b).

not been settled, It appears, however, that active steroidogenesis occurs in the corpora lutea of some fishes; it seems equally evident that this is not the case in all fishes (Lambert and van Oordt, 1965).Further work is required to clarify the endocrinological status of these structures.

C. Reproductive Cycles and Their Coordination Reproduction is almost always a seasonal or cyclical phenomenon. An annual cycle of temperatures and photoperiods characterizes the tem-

1.

57

RFPRODUCTION

perate and frigid zones; the rainy seasons may markedly alter freshwater habitats of the tropics. In these seasonally unstable environments, reproduction is geared to take advantage of seasons which offer the greatest opportunities for survivaI and development of the new generation. Even where conditions are relatively stable and the eggs or young are produced regularly throughout the year, there may still be a cycle of gonadal maturation imposed by the energy demands of maturing a batch of eggs or young (Fig. 16). These cycles of gonadal development frequently alter many aspects of metabolism as well as behavior and reproductive physiology. Cycles of active feeding with storage of fat and long periods of starvation are characteristic of many species (Greene, 1926; Idler and Bitners, 1960, and earlier; Idler and Clemens, 1959; Bentley and Follett, 1965; Tomlinson et d.,1967; Fig. 17). The electrolyte metabolism may change seasonally both in species which inhabit the ocean waters of relativeIy constant salinity (Woodhead, 1968) and in the euryhaline and anadromous forms (see chapters by Holmes and Donaldson, and Hickman and Trump, Volume I ) . The basic cycle is probably the one imposed by the seasonal nature of reproduction, and these regular changes in metabolism are secondary to it. The endocrine system forms the major link between the environment and the organs concerned with reproduction. Changing environmental

80-

c

60-

0

\

\

/

Feeding

\\

/

/ \

/

Y L?

40

-

-

20

Gonad development

'.J

L z L

n "

( 1

I

I

I

I

'

Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jui Aug

Fig. 17. Relation between feeding cycle (percent sample with food in stomach) and reproductive cycle (gonad development, percent fish with ripening gonads; spawning, percent ripe fish) of the haddock, Melanogrummus aeglifinus. Data from Homans and Vladykov ( 1954 ) .

58

WILLIAM S . HOAR

conditions, operating through the sensory system and specific centers in the brain, trigger neurosecretions which in turn regulate the activities of the pituitary gland. The pituitary hormones have direct effects on gametogenesis, metabolism, and behavior; these hormones also regulate the development of the gonadal endocrine tissues. Gonadal hormones take over some of these pituitary responsibilities and carry on the coordination of events in the production of gametes, sexual behavior, fertilization, and sometimes parental care. There are, in fact, several cycles within the gonads (Figs. 16 and 18), but many of the details of regulation at the cellular level remain to be unraveled. Studies of the cyclical changes in the structure of the gonads and endocrine organs, as well as variations in actual secretion of hormones (Barr and Hobson, 1964; Swift and Pickford, 1965; Gottfried and van Mullem, 19671, form one of the most voluminous components of the literature concerned with reproduction. No attempt will be made to summarize it here; the reader is referred to the many existing reviews (L. H. Matthews and Marshall, 1956; Pickford and Atz, 1957; Hoar, 1959, 1965a)b; see chapter by Liley, this volume). Although the pattern of. regulation via the peripheral sense organs, the neural centers, pituitary, and gonads is general throughout the vertebrates, the details vary considerably even in closely related species. A definite breeding season is absent in the female of the spotted dogfish, Scyliorhinus caniculus, and in the male spermatogenesis is continuous with a regular progression in the ampullae from spermatogonia to sperm (Fig. 4 ) ; in comparison there is a limited breeding period of about 2 months in the spiny dogfish, Squulus acanthias, and during this period Female Oocytes

Yo1 k formation

.15

Maturation Male Spermatogenesis Sex characters

10

5

Interstitial ti s u e OC

Fig. 18. Seasonal cycle of reproduction in Gasterosteus. Ordinate, months; curve, temperature in "C; horizontal black bar, spawning period; horizontal arrows, periods when more than 50%of samples showed characters listed on the left. Data from Craig-Bennett (1931); reproduced from Hoar ( 1955) by permission of the Cambridge University Press.

59

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there is a maximum accumulation of semen in the efferent ducts which is followed by a pause in sperm production (Fig. 15).This suspension of sperm production is the result of a cessation in the proliferation of spermatogonia several months earlier ( Simpson and Wardle, 1967). Since a band of degenerating ampullae appears in the testis of the spotted dogfish following hypophysectomy (Dodd et ul., 1960), there seems little doubt that these cyclical changes in the sperm production of the spiny dogfish are regulated by the pituitary gonadotropins. Similar examples could be drawn from many different groups of fishes. Occasionally, the details of regulation may even be somewhat different in the males and females (Wiebe, 1968b,c, 1969b). There are diurnal as well as seasonal cycles. In the cyprinodont, Oryzius lutipes, there is a daily cycle of ovulation (between 1 AM and 5 A M ) , mating behavior ( 4 AM to 7 A M ) , and the laying of fertilized eggs which follows soon after mating. Ovulation and oviposition are independent phenomena; the former depends on the temperature and the light cycle while the latter depends on contact stimuli associated with the sexual embrace (Egami, 1959a,b; Egami and Nambu, 1961). In another cyprinodont, RivuZus murmorutus, there is an internal self-fertilization which is also timed by the daily light cycle. Rivulus murmorutus is hermaphroditic with functional ovotestes. The timing of events within these organs is so correlated that there is a peak frequency in ovulation; fertilization occurs at dawn with a peak in oviposition at noon. Harrington (1963) also found evidence of seasonal changes in the cycles in accordance with light conditions. The modifications in physiological controls are fully as numerous and diverse as those of an anatomical nature. In a sense, the reproductive system of an animal is independent of the other organ systems and can, perhaps, respond to evolutionary pressures more freely; although reproduction is indispensable to the survival of the species, it is not a matter of life or death for the individual. Perhaps, for this reason, the adaptive variations in anatomy and physiology are particularly diverse and curious in the biology of reproduction. At any rate they emphasize the opportunism, the compromise, and the adaptiveness of Darwinian evolution. ACKNOWLEDGMENT

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2 HORMONES AND REPRODUCTIVE BEHAVIOR IN FISHES N . R. LZLEY I. Introduction . . . . . . . . . . 11. Gonadal and Thyroid Hormones A. Gonadal Hormones in Male Fish . . . . . B. Gonadal Hormones in Female Fish . . . . C. Nonspecific Effects of Gonadal and Thyroid Hormones D. Thyroid Hormones and Migratory Behavior . . 111. Pituitary Hormones . . . . . . . . A. Prespawning Behavior . . . . . . . B. Spawning Behavior . . . . . . . C. Prolactin and Parental Behavior . . . . . IV. External Factors and the Endocrine System . . . V. Summary and Discussion . . . . . . . VI. Conclusion . . . . . . . . . . References . . . . . . . . . . .

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73 75 76 88 90 93 94 94 97 100 102 104 109 110

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The significance of hormones in the regulation of reproduction presumably derives from the fact that in most animal species breeding occurs during restricted periods of the life history. The endocrine system provides a relatively slow acting link between the external environment and the internal state. The result is that reproductive behavior is nicely synchronized with the maturation of the gonads and the environmental conditions appropriate to breeding. The term “reproductive behavior” as used here encompasses a diverse range of activities involved in a propagative function. It includes sexual, parental, and nestbuilding behaviors. Agonistic and migratory behavior are also considered “reproductive” insofar as they are essential preliminaries to, or are involved in, reproduction. 73

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In comparison with studies involving birds and mammals the investigation of the hormonal regulation of reproductive behavior in fish is at a relatively primitive stage. Consequently it is frequently necessary to draw upon avian and mammalian examples to illustrate some of the principal features of the relationship between hormones and behavior. It is well established that in all classes of vertebrates the pituitary gonadotropins and gonadal steroids are the hormones most directly involved in the regulation of reproductive behavior. There is a number of ways in which the CNS-pituitary-gonad axis may regulate reproductive behavior; pituitary hormones may act directly on behavioral control mechanisms; pituitary hormones may stimulate the secretion of gonadal hormones which in turn regulate behavioral activity; both pituitary and gonadal hormones may act synergistically to regulate behavior. In addition, these hormones may in%uence behavior indirectly by their role in the development of secondary sexual characteristics. In its turn the CNS-pituitary-gonad axis is influenced by internal and external factors acting through the sense organs and brain. Studies involving birds and mammals, particularly those of Lehrman (1965) and Hinde (1965) with ring doves and canaries, respectively, have emphasized that the integration and coordination of the components of reproductive behavior depend upon a continuous interplay of external and internal factors; not only do hormones affect behavior but also behavior affects the endocrine state. Although the hormones of the gonads and anterior pituitary seem to play the major role in controlling reproductive processes, there is some evidence that neurohypophysial and thyroid hormones are also directly involved in the regulation of certain components of reproductive behavior in fish. Other hormones may affect reproductive behavior indirectly by their effects on general metabolism and growth and will not be considered here. Little is known of the details of the mechanisms by which hormones exert their effects upon behavior. This aspect will be considered in Section V. However, it is relevant to point out that hormone-induced changes in behavior differ widely in the time span involved, suggesting that the same or different hormones may act in a number of different ways. In general, hormones serve as relatively slow acting chemical links between the environment and an effector system. Thus there may be long-term seasonal changes over weeks or months, as in the annual cycles of very many animals. Changes may take place over hours or days, e.g., the switch from sexual to parental phases in birds (Hinde, 1965; Lehrman, 1965). In addition, there are some situations in which it appears as if hormones may be involved in short-term changes over periods of minutes

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or seconds, e.g., the spawning of the female in response to courtship in the medaka, Oryzias Zatipes (Egami and Nambu, 196l), or the postcopulatory “after reaction” in the rabbit ( Kawakami and Sawyer, 1959). There is a danger of assuming that all reproductive behavior must be regulated by hormones. It is possible that only certain components of the behavior repertoire are under hormonal control, while other activities which are usually associated with reproduction may be causally linked to hormone-regulated activities without being themselves under the influence of the hormone state. Thus, there may be situations comparable to the causation of maternal responsiveness in rodents, reviewed by Richards ( 1967). In rodents, initiation of maternal responsiveness may depend on the endocrine state associated with parturition, while its maintenance is dependent on a nonendocrine mechanism stimulated by the presence of pups, Alternatively, certain behavior patterns associated with reproduction may occur outside the breeding season in other functional contexts. For example, agonistic behavior may be associated with reproduction, but it may also occur at times and places unrelated to breeding. Such activities are unlikely to be under the same hormonal control as seasonally changing behavior and may not be regulated by hormones at all. This emphasizes the need for care in the use of terms included under the general heading of reproductive behavior. Clearly, it would be misleading to refer to a behavior pattern obviously associated with reproduction during the breeding season as “reproductive” when it occurs out of the season in a different functional context. Beach (1967) makes a similar point with respect to studies of mammalian behavior. Lastly it is important to be aware of the possibility of a process in fish analogous to Beach‘s “corticalization of function” in the control of sexual activities in mammals ( 1964). Although hormones may be essential to the development of coordinated reproductive behavior, these activities may become less dependent on gonadal function after maturation and experience of breeding.

11. GONADAL AND THYROID HORMONES

It is well established that fish possess a CNS-pituitary-gonad axis similar in general pattern to that established for other classes of vertebrates (see chapter by Hoar, this volume). Furthermore, a wide range of steroids has been identified from male and female fish (see reviews by Gottfried, 1964; Hoar, 1965a; and chapter by Hoar). Surprisingly, there

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have been few unequivocal demonstrations for any fish that reproductive behavior is in fact induced or regulated by gonadal steroids, Many workers have demonstrated effects of exogenous steroids on sex determination and the development of secondary sexual characteristics in fish, but in most cases there is little mention of the effects of these treatments on reproductive behavior (see chapter by Yamamoto, this volume).

A. Gonadal Hormones in Male Fish The three-spined stickleback, Gasterosteus aculeatus, has been the subject of more intensive ethological and behavior-oriented endocrine investigations than any other species of fish and therefore provides a particularly suitable starting point. Reproductive behavior and the annual cycle are described by van Iersel (1953) and Baggerman (1957). There are two distinct races of the stickleback: the leiurus form which spends its entire life cycle in freshwater, the trachurus form which breeds in freshwater in spring and summer but migrates to the sea in the fall where it remains until a return migration to freshwater in the spring. There are a number of consistent morphological and ecological differences between the two races. Hagen (1967) found that the two races are almost completely reproductively isolated and questions their inclusion in a single species. In spring the males, which were previously associated in loose bisexual schools, become spaced out and acquire territories which they defend against other males. At the same time males acquire a distinctive appearance: red on throat and belly, dark back, and a blue iris. Internally, the kidney tubules begin to secrete mucus which is used in gluing materials used in nestbuilding. From this stage the male may go through one or more reproductive cycles lasting 20-30 days. Each cycle involves a series of overlapping phases or subcycles: nestbuilding, sexual, and parental (van Iersel, 1953; Baggerman, 1968; van Mullem, 1967). Throughout these cycles the male defends his territory against males and other intruders. Baggerman (1966) also traced cyclical changes in displacement fanning and frequency of comfort movements. Castration of the male in full breeding condition brings about a reversion to the nonbreeding condition-loss of nuptial coloration, a reduction in kidney tubuIes and cessation of sexual and nestbuilding behavior ( Ikeda, 1933; Baggerman, 1957; Hoar, 1962a,b). However, Hoar (1962a,b) working with the leiurus form noted that agonistic behavior persisted in fish castrated shortly before nestbuilding had commenced and held under long photoperiod (16L:SD). In contrast, agonistic be-

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havior decreased along with other reproductive behavior in males placed under short photoperiod (8L:l6D) after castration. Baggerman ( 1966), working with the truchurus form, carried out a more detailed study of the effects of castration at different stages of the reproductive cycle. Castration of animals in full breeding condition, 9-19 days after the onset of breeding, resulted in a decrease in the frequency of swimming movements and a reduction in agonistic, sexual, nestbuilding, and displacement fanning behaviors to the level exhibited by intact animals after the end of the breeding period. In this study the onset of breeding is taken as the day a male built its first nest of the season in the presence of a male separated by a glass partition. Males castrated about 1 week before they were expected to build their first nest showed a decline in swimming activity and sexual behavior to normal postbreeding levels; nestbuilding and displacement fanning disappeared completely. In contrast, agonistic behavior increased to just below the level achieved by males in breeding condition and remained high for at least 3-4 weeks. This finding supports the suggestion by Hoar (1962a,b) that there is a change in the internal causation of agonistic behavior. Hoar (1965b) and Baggerman (1966) hypothesize that in the period before the onset of breeding the level of aggressive behavior is regulated by an increasing level of pituitary gonadotropic hormone. Gradually the mechanism underlying this behavior becomes less sensitive to gonadotropin and increasingly under the control of gonadal hormone ( s ) , Recent work by Wootton (1968) suggests that it is important to maintain the distinction between the leiurus and trachurus forms of the three-spined stickleback. Working with leiurus, Wootton finds that males castrated in the prenestbuilding stage and held under long photoperiod show a high level of aggressive behavior when presented stimulus males or models in a test situation. Fish castrated after they have built nests also maintain a level of aggressive behavior, not less than that of prebreeding fish but lower than control fish with nests present. This result contrasts with Baggerman’s finding (1966) that in trachurus castration of fish which have built nests results in a marked reduction in aggressive behavior. In spite of important differences in testing procedures, which make direct comparison difficult, it may be tentatively suggested that the two findings reflect real differences in the endocrine mechanisms underlying the reproductive cycle which presumably are related differences in the biology of the two forms. These findings concerning the control of agonistic behavior raise the semantic difficulties referred to on p. 75. Each worker has used different testing procedures which tend to emphasize different aspects of

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the agonistic response: Hoar (1962a,b) tested small groups of males and females in a situation in which few males succeeded in acquiring territories and building nests. Baggerman ( 1966) and Wootton ( 1968), using different procedures, tested isolated males which in many cases were able to build nests. Quite clearly, it is important to distinguish between agonistic behavior involved in defence of a nest and territory, and agonistic behavior without reference to a territory. In the latter case agonistic behavior may provide a mechanism of general dispersion and perhaps simply represent the reaction of a fish to an intruder within its “individual distance” (Hediger, 1950). Defense of a territory is undoubtedly an important component of reproductive behavior, whereas defense of “individual distance” is a much more general phenomenon and cannot be considered as exclusively reproductive in function. Therefore, although the agonistic motor patterns involved in both contexts are similar, it is not surpising that the underlying causal mechanisms appear to be somewhat different. It is evident from the preceding paragraph that in the stickleback investigations, and also in other similar studies, a final evaluation of the role of endocrine mechanisms in the control of agonistic behavior requires a considerable understanding of the functional significance of the behavior in different situations. This will in turn influence the choice of behavioral testing procedures. The acquisition of nuptial coloration and the onset of breeding behavior has been correlated with evidence of increased steroidogenesis. Gottfried and van Mullem ( 1967) identified testosterone, androstenedione, dehydroepiandrosterone, and progesterone in the testes of male sticklebacks. They estimated that “dominant” males, which had acquired territories, had 5 7 times the steroid level of fish caught at the same time but which had not been allowed to pass into full breeding condition. Replacement therapy has confirmed the role of androgens in inducing secondary sexual characters and reproductive behavior in the stickleback (Hoar, 1962a,b; Wai and Hoar, 1963). Methyltesterone treatment stimulates kidney tubule development and male nuptial coloration in gonadectomized adult males and females as well as intact juveniles of both sexes. However, juvenile fish failed to perform any nestbuilding behavior, and only 5 out of 86 gonadectomized females held under long photoperiod built nests. In these five fish most of the nestbuilding movements were present but occurred sporadically and resulted in poorly constructed nests, Masculinized females never showed sexual behavior toward introduced females. In the case of castrated males treated with methyltestosterone, Hoar (1962a) found that in those maintained under long photoperiod (16L:BD) 87.5% built nests, whereas only 57% of

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males under short photoperiod (8L: 16D) built nests and in addition took more than twice as long to commence building. The apparent refractoriness of androgen-treated fish under short photoperiod leads Hoar to suggest that although reproductive behavior requires the gonadal hormone its full expression occurs only when gonadotropic activity of the pituitary is maintained at a high level by long photoperiod. Smith and Hoar (1967) investigated the endocrine involvement in displacement and parental fanning behavior. They found that low doses of methyltestosterone restored only the earlier stages of reproductive behavior in castrate males, i.e., digging, collecting and gluing. Pushing and displacement fanning appear only after treatment with higher doses of androgen. Castration early in the parental phase resulted in a decline in fanning even though live eggs were present in the nest. Smith and Hoar conclude that displacement and parental fanning are regulated by a testis hormone. Baggerman (1966) makes the important point that in interpreting the effects of gonadectomy and hormone treatment it may be difficult to distinguish the direct from the indirect effects of a hormone. It appears that sexual, nestbuilding, and parental behaviors are correlated with a high level of gonadal hormone, suggesting that the hormone is playing a causal role. The same could be true of displacement fanning, changes in swimming activities, and frequency of comfort movements. Alternatively, these latter activities may occur as a result of the arousal and perhaps interaction of the mechanisms underlying sexual, agonistic, and parental behavior. Baggerman (1957, 1959) considered the possibility that gonadal hormones might be involved in the prespawning migration into freshwater of the trachurus form of the stickleback. Using salinity preference as a measure of migration disposition, Baggerman found that gonadal maturity coincides with a change in preference from saltwater to freshwater, whereas gonad regression was correlated with a change in preference from freshwater to saltwater, But, since similar changes in salinity preference can be induced in gonadectomized fish by manipulation of the photoperiod and temperature, it is clear that gonadal hormones are not directly involved. Baggerman (1962) did, however, find the testosterone treatment resulted in an increase in locomotory activity of a type associated with migrating fish (further discussion in Section 11, C). Poeciliids have been used in numerous studies in which attention has centered on the effects of steroid hormones on morphological secondary sexual characters and on sex determination (see Pickford and Atz, 1957, and chapter by Yamamoto, this volume, for full reviews). In a few cases the effects of these treatments on behavior are mentioned.

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Cohen (1946) and Tavolga (1949) found that treatment with pregnenolone induced male characters in maturing, genetically female platyfish, Xiphophorus ( =Platypoecilus ) muculatus. These sex-reversed “males” showed male courtship patterns and attempted to copulate but, according to Tavolga ( 1949), courted less vigorously than genetic males under the same treatment. In contrast, genetic males treated with estradiol benzoate are said to have behaved like females and were pursued by normal males (Cohen, 1946). Hildemann (1954) was able to induce male courtship behavior in female platyfish by treatment with methyltestosterone. Noble and Borne (1940) report without giving details that females of X. helbri treated with testosterone propionate rose in the pecking order until a reversal in sexual behavior occurred. As the sword and gonopodium developed, the sex-reversed “male” made attempts to copulate. Treatment of females with estradiol dipropionate failed to induce any change in social rank. These workers found that gonadectomized males and females maintained rank position for several months. They concluded that male sex hormone brought about a rise in rank of the female only by reversal in sex. Laskowski (1954) treated both immature and adult genetic females of Xiphophorus ( =Platypoecilus ) variatus with testosterone. The females performed what is referred to as phase 1 courtship which is characterized by a lateral display. Even after several weeks of treatment during which gonopodia developed in the young fish, the genetic females failed to perform the zigzag dance (phase 2) and the mating attempt (phase 3). The question arises as to whether phase 1 behavior should be regarded as a courtship movement. Males perform similar activities in establishing dominance hierarchies. Laskowski ( 1954) points out that in a community of untreated females a definite hierarchy is set up. Females introduced into such a community show considerable phase 1 type of interaction with other females over a period of several hours until dominance relations become established. Androgen-treated adult females differed from untreated fish chiefly in that phase 1 behavior persisted for several days. Evidently the movements which initiate a courtship sequence are similar to those involved in agonistic behavior, and perhaps sex recognition depends upon the response of the female to an initial threat by the male. Thus, it appears that phase 1 activities should be regarded as agonistic behavior common to both male and female. Under normal circumstances agonistic behavior occurs more frequently among males than among females. Androgen treatment of genetic females increases the

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frequency of agonistic behavior and in this respect has a masculinizing effect but is relatively ineffective in inducing movements which are functionally restricted to the sexual context. Gonadectomy of the male platyfish, Xiphophorus muculatus, resulted in a decrease in frequency or duration of copulation thrusts, swings, and sidling, whereas pecks, backs, and approaches remained the same (Chizinsky and Aronson in Aronson, 1959). An interpretation suggested by the previous discussion of Laskowski’s work is that those activities which persist after castration are also components of agonistic behavior common to both sexes. Clemens et al. (1966) treated guppies with testosterone from birth to 60 days. After treatment they found a very marked increase in the proportion of males-in some cases the males outnumbered females 9: 1. However, when paired with females only 14%of these males, including sex-reversed genetic females, sired young, even though in many cases they showed full male coloration and yielded viable sperm on stripping. Clemens et at. suggest that the failure to breed resulted mainly from a behavioral deficiency which was not simply the result of a lack of endogenous androgen. A possibility, suggested by the present author’s work with the guppy (Liley, 1966, p. 160), is that males failed to respond to females because of the prolonged isolation from responsive females. Working with the medaka, Oryzias latipes, Okada and Yamashita (1944) confirmed by castration and testosterone treatment that the male secondary sexual characteristics are under hormonal control. Testosterone treatment of females or implantation of a testis results in masculinization and the performance of male behavior including pursuit of normal females, Yamamoto (1962, and chapter, this volume) has confirmed that complete functional sex reversal may be achieved with androgen treatment of genetic female medakas. Unfortunately, there has been no detailed camparison of the behavior of normal males and sex-reversed genetic females. Tavolga (1955) found that castration of male gobies, Bathygobius soporator, abolished aggressive behavior toward an introduced male. Instead the operated males failed to discriminate between males and females, and between gravid and nongravid females, and courted all equally. Spawning behavior of castrated males with gravid females appeared to be normal. The male brooded the infertile eggs which resulted from the spawning. Tavolga (1956) also investigated the stimulus situation involved in prespawning behavior of goby males. Intact males readily distinguished gravid females from nongravid females and males. However, if stimulus fish were presented in glass containers males failed to discriminate and courted all females and nonaggressive males equally. This suggests that

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normally the males recognize the gravid female on the basis of chemical cues. Tavolga (1956) demonstrated that a substance present in the ovary of gravid females, and presumably released through the genital pore, will elicit a vigorous courtship response in a male, even in the absence of a stimulus fish. Tavolga (1956) suggests that in the male goby a gonadal hormone affects the sensitivity of the olfactory organs perhaps by making the male differentially sensitive to chemical factors released by the gravid female. Castration may lower the sensitivity threshold and permit chemicals other than the ovarian fluid to stimulate courtship behavior. Cichlids and anabantids have been the subjects of many intensive ethological studies ( e.g., Baerends and Baerends-van Roon, 1950; Forselius, 1957; see also chapter by Baerends, Volume VI). A few species have been investigated from the point of view of the endocrine mechanisms involved in their complex reproductive processes. Noble and Kumpf (1936) reported that males of Hemichrornis bimaculatus performed typical courtship, fertilization, and brooding movements for 202 days after castration. The males developed nuptial coloration and genital tubes at each spawning. After a normal spawning both parents are inhibited from eating their young and perform brooding behavior which consists of collecting and guarding the young. Noble et at. (1938) found that brooding in response to donated young could be induced by treating nonbrooding fish, both male and female, with a variety of hormones. Positive responses were only obtained with fish which had had previous experience of brooding and/or spawning. Thirteen fish, including four castrates, began to brood normally after treatment with corpus luteum extracts ( source not given: presumably rich in progesterone). Proluton and prolactin were also highly effective in inducing brooding. Anterior pituitary extract, fresh fish pituitary, thyroxin, desiccated thyroid, and testosterone were also effective in a smaller proportion of fish treated. Nearly half the fish given control phenol injection also began brooding. These results are difficult to interpret but serve as a useful warning of ( a ) the role of experience in determining the outcome of hormone therapy, and ( b ) the possibility that a wide variety of agents may produce the same behavioral effects: In this case it seems likely that some or all the agents were exerting their effects indirectly, perhaps by stimulating pituitary secretory activity. Aronson (1951) found that castration of male Tilapia macrocephala resulted in a reduction of the genital tubes and loss of the yellow coloration of the operculum. Castrated males continued to dig nests as frequently as intact males. The effects of castration were reversed by testosterone treatment (Levy and Aronson, 1955). Previously, Aronson

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and Holz-Tucker (1947) had shown that testosterone treatment of ovariectomized and intact females resulted in the growth of the genital papilla and the acquisition of male coloration on the operculum. Working with another species, Tilapiu mossambica, Clemens and Inslee ( 1968) obtained functional sex reversal in genetic females by treating them with methyltestosterone for the first 69 days of life. The sex-reversed fish exhibited male mating coloration and nestbuilding behavior when placed with ripe females. Aronson ( 1959) compared the reproductive behavior of three males of Aequidens latifrom before and after gonadectomy. Up to 6 weeks after castration all elements in the mating pattern were still present, most of these showing little change in frequency of occurrence or duration from their own preoperative levels. There was an increase in nest passing and several other items of behavior after castration. The increase was particularly marked for the period 1 day after spawning. On the other hand, there was a noticeable decline in nestdigging after castration. Noble and Kumpf report (1936), without giving details, that gonadectomized males of the anabantid, Betta splendens, perform courtship movements, whereas gonadectomized females do not. These authors also found (1937) that a small number of ovariectomized B. splendens females developed a testis and in such cases acquired male coloration and behavior. Also working with anabantids, Forselius (1957) reports that sterile Colisa hbiosa x lalia hybrids acquired male secondary sex characteristics and exhibited “migratory” and reproductive behavior. Histological examination of a large number of the hybrids failed to reveal any trace of gonadal tissue. However, evidence that androgens are involved in the control of male characteristics is provided by the finding (Forselius, 1957) that testosterone propionate treatment of females of C. lalia induced male coloration and, in some cases, nestbuilding and defense, Johns et al. (1969) found that some males of the blue gourami, Trichogaster trichopterus, performed reproductive behavior after castration. Of a total of 16 males castrated, 11 failed to build bubble nests or show any sexual behavior. The other five built nests and “spawned within 7 days of being paired with females. Apart from a relatively long delay between introduction of females and actual spawning, the reproductive behavior of these five did not differ from that of control fish. Castrate males which had spawned (but not fertilized the eggs) readily accepted fertile eggs and performed parental behavior for several days. Fertile eggs given to nonspawning castrates were eaten. Castrate males placed with intact or other castrate males performed agonistic behavior until a dominance relationship was established. Agonistic behavior of castrates

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did not obviously differ qualitatively or quantitatively from that of intact males. The dorsal fin of the male gourami is longer and more pointed than that of the female. The dorsal fin of castrated males became shorter and rounded, more like that of the female. Significantly, however, the dorsal fins of the castrates which spawned were more like those of intact males than were the fins of nonspawning castrates. Castrated males treated with methyltestosterone developed the long pointed dorsal fin and in all cases spawned when paired with ripe females. These results indicate that sexual behavior and the characteristic male dorsal fin are under gonadal hormone control. Of the five castrates which succeeded in spawning before testosterone treatment only one showed identifiable regenerating testis. The similarity in behavior and length of dorsal fin in the testosteronetreated fish and those which spawned after castration suggests that in the latter there was either undetected testicular tissue or an extragonadal source of androgenic steroid. Johns et al. (1969) conclude that their results emphasize the need for considerable caution in the interpretation of castration experiments and demonstrate the importance of independent checks on the presence of androgen before concluding that reproductive behavior is not dependent on the direct action of testicular hormone. Machemer and Fiedler ( 1965) investigated the hormonal involvement in nestbuilding of another anabantid, the paradise fish, Macropodus opercularis. Two out of eight intact males given only methyltestosterone showed an increase in building tendency but, apparently because of inadequate mucus production, building was incomplete. Testosterone in combination with prolactin ( which increases mucus production ) resulted in nestbuilding in three males and two females. Machemer and Fiedler do not appear to have run a control series, and it is important to note that, in at least some species of anabantid, isolated males begin nestbuilding apparently spontaneously ( Forselius, 1957; own observations of Betta splendens and Trichogaster trichopterus) . Greenberg (1947) and Hale (1956) note that in the green sunfish, Lepomis cyanellus, immature and mature fish of both sexes perform agonistic behavior, although defense of a nest is limited to sexually mature males. There was no decrease in agonistic behavior after gonadectomy in a small number of males and females placed in a test situation (Hale, 1956). Smith (1967) found that castrated males of Lepomis megalotis and L. gibbows maintained high levels of aggressive behavior but failed to build nests. Treatment with testosterone restored nestbuilding but, in the case of L . megalotis placed in large experimental pools, the males did not cluster together to the same extent as sham-operated fish.

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Wiebe (1967) demonstrated that in the male of the viviparous seaperch, Cymatogaster aggreegata, the occurrence of full reproductive behavior coincides with maximum gonadal development and histochemically demonstrated steroidogenesis. The gonads of fish in winter condition (October) matured in fish placed under long photoperiod ( 16L:8D) and high temperature (20.C). Such fish also acquired the secondary sexual structures and performed sexual behavior, including chasing, heading-off the female, courtship dance, lateral quiver, and mating attempt. Castrated fish under the same conditions did not acquire the sexual structures. However, they still performed some of the elements of sexual behavior: chasing and mating attempt, but without the darkening characteristic of full reproductive behavior. Castrated males treated with methyltestosterone acquired the secondary sexual characteristics and showed an increase in sexual behavior, although the level of sexual behavior did not reach that of intact males under the same environmental conditions. Significantly, chasing and mating attempts which persisted after castration occur in intact fish with reduced vigor and frequency throughout the year. Thus, it appears that certain components of the sexual behavior occur only at times at which insemination is known to occur. These activities disappear after gonadectomy and reappear as a result of testosterone therapy. In contrast, chasing and mating attempt appear to be to some extent independent of gonadal control and for much of the year are not obviously associated with a reproductive function. Surprisingly little is known of the role of gonadal hormones in the reproductive behavior of salmonids. Jones and King ( 1952) castrated four adult males of Salmo salar. In three castrated males treated with testosterone propionate sexual behavior was partially restored: males followed females and one male quivered. The one castrate which was not treated with androgen showed no interest at all in the females. In all species of fish studied, male secondary sexual characteristics and at least some components of reproductive behavior are under gonadal hormone control. In a number of cases it has been claimed that complete reproductive behavior persists in castrated fish. The most striking example of this is provided by Tavolga’s study (1955) of Bathygobius soporator. Others involve cichlids and anabantids. However, Johns e t al. (1969) have reason to suggest that in the gourami the persistence of reproductive behavior may result from the presence of unidentified regenerated testis or the existence of an extragonadal source of androgen. In addition to the above there are several examples of situations in which behavior usually classified as “reproductive” persists after castration. Most of these examples apply to behavior involved in a nonreproductive context out of the breeding season and thus, not surpris-

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ingly, these activities turn out to be more or less independent of gonadal control. This applies particularly to agonistic behavior which occurs in very many species in both reproductive and nonreproductive situations and frequently plays a prominent role in the preliminary encounters between the two sexes.

B. Gonadal Hormones in Female Fish The hormonal regulation of female behavior has received far less attention than that of the male. This is at least partly because the female is in most cases a more passive partner. Even where nestbuilding and parental care occur, in many species it is the male which is responsible. Thus the behavior of the female is usually difficult to detect or quantify, with the result that it may be difficult to assess the effects of gonadectomy or hormone treatment. Numerous investigators have applied estrogen treatment to male and female poeciliids. Most of this work has been reviewed by Pickford and Atz (1957) and need not be detailed here. Indeed most investigations have been concerned with the effects of estrogens on the gonads and morphological structures. In general, estrogens suppress the testis and male characteristics in developing genetic males. When treatment is withdrawn the testis and male gonopodium and coloration develop. None of these investigations have given any clear indication that estrogenlike hormones are involved in the regulation of female behavior. Ovariectomy of poeciliid females has been performed in a small number of investigations. Noble and Kumpf (1936) and Ball (1960) found that females of Xiphophorous helleri and Poecilia ( =Mollinesia) sp., respectively, remained sexually attractive to males after ovariectomy. However, there is no indication that an attempt was made in either of these studies to assess female responsiveness to male courtship. It is well known that poeciliid males will direct persistent and vigorous courtship toward unresponsive females, Liley (1966) has described the sexual response of the female guppy, Poecilia reticulata, and finds that nonvirgin females go through a cycle of receptivity which can be correlated with the cycle of brood production. Females respond most readily and are more likely to accept copulation in the few days following the birth of a brood of young. Virgin females are more persistently receptive until fertilization of the eggs occurs. Ovariectomy of virgin females results in an initial decline in receptivity over a period of 5-6 days. However, nonvirgin females tested several

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weeks after gonadectomy still occasionally responded to male courtship. It was concluded that sexual behavior may still occur in the absence of gonads, but that in intact fish the ovary exerts a regulatory and perhaps stimulating effect upon a more direct pituitary or neural control. The importance of the gonadal contribution is emphasized by the finding ( Liley and Donaldson, 1969) that treatment of hypophysectomized females with the gonadotropin fraction of salmon pituitary extract brought about a restoration of sexual responsiveness only after the gonad recovered from its regressed state resulting from hypophysectomy. In the medaka, Oqzias Eatipes, Okada and Yamashita (1944) found that ovariectomy of females and estriol treatment of adult males had no effect on secondary sexual characteristics. However, Yamamoto (1962), and Yamamoto and Matsuda (1963) have established that functional sex reversal of genetic males may be induced by treatment of young with estrogens. Sex-reversed genetic males are fully functional as females and breed with true males. Unfortunately, there has not been a detailed behavioral comparison of sex-reversed genetic males with normally developing females. Little is known of the role of gonadal hormones in the sexual response of the medaka. Egami (1955) found that treatment of females with estradiol benzoate brought about a reduction in the occurrence of oviposition. This result may have been caused by the suppression of pituitary gonadotropin secretion and the consequent regression of the ovaries. Wai and Hoar in an unpublished study (1968) compared the effects of gonadectomy on the aggressive behavior of male and female threespined sticklebacks maintained under long photoperiod. In one series of experiments involving the leiurus form, aggressive behavior was measured by placing two fish of the same sex in a tank and scoring the number of attacks during a series of 5 min observations over a period of several weeks. The results, Table I, confirm earlier findings (Hoar, 1962a,b) that prenestbuilding agonistic behavior of males is hardly affected by castration. Intact females attacked each other at about onethird of the rate shown by intact males. Ovariectomized females performed twice as many attacks as intact females. These results suggest that ovarian secretions normally suppress aggressive behavior to some extent. Wai and Hoar noted that most of the intact females which showed high levels of aggressive behavior were immature and therefore, presumably, estrogen levels were low. The fact that gonadectomized females were less aggressive than gonadectomized males suggests that there are genetic sex differences in the levels of aggression, or that the presence of gonadal hormones earlier had some persistent effects. Forselius (1957)

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Table I Mean Number of Attacks per 5 min in Pairs of Intact and Gonadectomized Male and Female SticklebacksoJ' Treatment

No. of pairs of fish

Mean No. of attacks/5 min

Intact male Gonadectomized male Intact female Gonadectomixed female

45 48 48 44

33 28 11 21

Unpublished data from Wai and Hoar (1968). Means are for two or three 5-min observations per week in the fourth and fifth weeks after gonadectomy in four experimental series.

found evidence that estrone injections resulted in a marked reduction in aggressive behavior in maIes of Colisa l a b . In contrast to their findings with castrated males, Noble and Kumpf (1936) found that ovariectomy of females of Hemichromis bimuculutus resulted in total loss of reproductive behavior. Injection of ovarian extract (source not stated) restored most of the sexual behavior. As noted earlier, Noble et al. (1938) found that brooding behavior could be induced in experienced females by treatment with a variety of gonadal and pituitary hormones. Of these agents corpus luteum extract proved to be one of the most effective. Ovariectomy of Tilupia macrocephula brought about a reduction of the genital tube and the reappearance of a silvery immature condition of the operculum ( Aronson and Holz-Tucker, 1947). Estradiol treatment of ovariectomized fish induced the growth of the genital tube but had no effect on the silvery operculum. Aronson found (1951) that the majority of intact females built nests prior to spawning. Ovariectomy resulted in a reduction in nestbuilding to about the level of intact or castrate males, that is, nests were build in about 11%of the pairings. Aronson notes (1957) that in Tilupiu mucrocephala completion of ovulation is marked by the occurrence of nest-passing, an activity closely associated with spawning. The close temporal relationship between ovulation and spawning, which has been noted by several workers, will be considered further in Section 111, B. Noble and Kumpf (1936) mentioned briefly their finding that Bettu splendens females no longer performed sexual behavior after ovariectomy. This contrasts with the results of castration of males. Johns et al. (1969) considered the effects of ovariectomy in the blue gourami, Trichogaster trichopterus. Normally the female initiates the spawning sequence by approaching and butting a dark, nestbuilding male. None of

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the eight ovariectomized females made sexual responses when exposed to males in reproductive condition. Two ovariectomized females treated with estrone also failed to respond. In addition, ovariectomized females were evidently less attractive to males than were intact females: Males directed significantly fewer contacts with their long pelvic rays toward ovariectomized females. There is histological and behavioral evidence (Scharrer et al., 1947) that the pelvic rays are chemosensitive. Thus the effect of ovariectomy on female “attractiveness” suggests that the ovary is responsible directly or indirectly for the elaboration of a specific chemical detected at the female body surface by the male. Such a secretion might provide the means by which males discriminate between males and females and between gravid and nongravid females. It should be noted however that Picciolo (1964) was unable to find behavioral evidence that male anabantids responded to chemical stimuli during reproductive behavior, although he did find that amputation or cauterization of the pelvic fins reduced reproductive success. Ovarian involvement in the production of a chemical attractant has been well established by Tavolga (1956) in his work with Bathygobius soporator. The chemical has not been identified but is evidently only produced in effective quantities in gravid fish ready to spawn. Amouriq (1964, 1965a,b) has concluded that a substance produced in the ovaries of female guppies which elicits increased male activity, is in fact an estrogen (discussed in Section 11, C ) , Ball (1960) reviewed much of the work concerning the hormonal control of ovipositor growth in the bitterling, Rlaodeus amarus. Early work had suggested that ovipositor growth is stimulated by ovarian hormones. However, Bretschneider and de Wit (1947) and others found that ovipositor growth could be induced by a wide variety of chemical and physical stimuli. Ball suggests that although there is no reason to suppose the normal ovipositor growth is not under ovarian steroid control, in many experimental situations it was mainly a response to internal changes involved in a stress reaction. Shirai (1962, 1964) finds a clear correlation between ovipositor growth and ovarian condition in the Japanese bitterling, R. ocellatus. He notes (1964) that there is a period of slow steady growth in the prebreeding season and a periodic fluctuation in length during the breeding season. Shirai (1964) speculates that there may be two hormone factors involved: an estrogenlike hormone, which stimulates the long-term growth, and another factor involved in the short-term cyclical changes during the breeding season. In this last phase fish show maximum length of ovipositor at the time that ripe eggs are extruded into the ovarian lumen, and it is at this time that oviposition occurs. If there

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is no mussel present, maturation, ovulation, and ovipositor growth are all inhibited. The clear correlation between ovipositor growth, ovulation, and oviposition suggests that gonadal hormones are in some way involved in the regulation of the spawning act. Shirai (1962) makes an alternative suggestion that a physical stimulus caused by the extrusion of mature eggs into the lumen brings ovipositor growth under nervous control. Apart from an obvious correlation between ovarian maturation and spawning readiness, there is no direct evidence that gonadal hormones are involved in the regulation of spawning behavior in female fish. There are relatively few reports of the experimental use of steroid estrogen, none of these has revealed behavioral responses to hormone treatment. Sundararaj and Goswami ( 1966) treated sexually mature but unovulated female catfish, Heteropneustes fossilis, with estradiol benzoate, testosterone propionate, and progesterone 3 days after hypophysectomy. These steroids failed to induce ovulation or spawning behavior. In contrast, three corticosteroids induced ovulation and, in some cases, oviposition in the absence of males. Thus, it appears that corticosteroids can act as ovulating agents and proved almost as effective in this regard as mammalian luteinizing hormone ( LH) . Furthermore, hypophysectomy of the experimental fish ruled out the possibility that the steroids exerted their effect by an action involving the pituitary. Sundararaj and Goswami ( 1966) direct attention to investigations which have shown that in the Pacific salmon the 17-hydroxycorticosterone titer in the blood progressively increases in fish as they migrate from the sea to spawning grounds. They speculate that perhaps the corticosteroids are directly involved in ovulation and spawning in fish. Changes in corticosteroid levels in migrating and spawning fish have usually been interpreted, e.g., Chester Jones and Phillips (1960) and Fagerlund (1967), as a response to activity and stress. However, considering that treatment with gonadal steroids has been so conspicuously unsuccessful in inducing reproductive behavior in female fish it seems worthwhile considering the possibility that corticosteroids may be in some way involved. C. Nonspecific Effects of Gonadal and Thyroid Hormones So far this review has been concerned with hormones which regulate the performance of behavior directly involved in reproduction. There are a number of indications that gonadal and thyroid hormones may have more general effects on behavioral activity. Stanley and Tescher (1931) reported a considerable increase (400%) in the locomotory activity of

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goldfish fed on ground mammalian testicular substance. This effect became marked within hours of feeding and continued for at least 24 hr. Hoar et al. (1955) found that goldfish immersed in solutions of thyroxine, testosterone, or stilbestrol showed greater locomotory activity and were more responsive to electrical stimulation than untreated fish. Forselius reports (1957), without giving details, “An increase of appetitive behavior and general locomotory activity” in the anabantid, Colisa lalia, after injection of thyroxine, testosterone, and chorionic gonadotropin. Thyroxine treatment resulted in increased locomotory activity and jumping in guppies, Poecilia reticutata ( Sage, 1968). Hoar et al. (1952) showed that chum salmon fry treated with methyltestosterone and synthetic thyroxine became more active and showed less marked schooling than control or thiourea-treated fry. Coho and sockeye salmon yearlings treated with thyroxine, testosterone, or stilbestrol, and yearling sockeye treated with estrogens, showed a decrease in the time required to make a standard response to flowing water (Hoar et al., 1955). Van Iersel ( 1953) found that male three-spined sticklebacks (trachums form) treated in midwinter with testosterone propionate began “fluttering” (persistent swimming against the glass walls of the aquarium) which did not occur in control fish at that time. Iersel regards this fluttering as an expression of migratory behavior. Both androgen and thyroid stimulating hormone ( TSH ) treatments resulted in significant increases in swimming movements and bouts of fluttering in castrated male sticklebacks ( Baggerman, 1962). Baggerman suggests that TSH produced this effect indirectly by stimulating the secretion of thyroid hormones. A different mechanism of hormone action is suggested by Amouriq (1964, 1965a)b) who found that male guppies, Poecilia ( =Lebistes) reticulata, placed in water which had previously held females, exhibited a marked increase in their locomotory activity; water which had previously held males was ineffective. Amouriq added extracts of skin, intestine, and ovary of females to the aquarium water and found that only the latter induced a significant increase in male activity. An estrogen, hexestrol dipropionate, when added to the water, resulted in a marked increase in male activity. More recently Amouriq ( 1967) has established that the range of concentrations of the steroid affecting locomotory activity is quite narrow: 0.025 mg/ml evokes hyperactivity; 0.05 mg/ml results in hypoactivity; concentrations below the first and above the second have no effect, Amouriq (196%) concludes that an ovarian hormone induces hyperactivity in males and maintains female attractiveness to males. Thus, he is suggesting that the hormone act as a pheromone in addition, presumably, to its endocrine role in the female.

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Earlier studies, Breder and Coates ( 1935), Clark and Aronson ( 1951) , Baerends et al. ( 1955), and Liley (1966), as well as Amouriq (1965a), have emphasized the role of visual stimuli in guppy courtship. Although not tested experimentally there has been little evidence to indicate that chemical stimuli are involved. On the basis of data provided it is difficult to appreciate the functional significance of the response described by Amouriq. The response was only obtained using water from a 2liter container which had held as many as 10 females for 24 hr ( Amouriq, 1965a). Maximum locomotory effects occurred 5 hr after the start of the addition of ovarian extract (Amouriq, 1965b). The effects of hexestrol remained slight for 6-7 hr from the start of treatment after which there was a marked increase in male activity. This suggests that the hormone may not be acting directly as an external stimulus but is being taken up through the skin and gills of the male and acting on the central nervous system or sense organs, perhaps increasing the susceptibility of the fish to external stimuli and thereby bringing about an increase in locomotory activity. Because of the delay in obtaining a locomotory response, the relatively high density of females required to obtain effective concentrations, the mobility of both male and female, and the lack of obvious contact behavior in courtship (compared with a species such as Poecilia uiuipara, see Liley, 1966) it is difficult to see how the chemical released by the female could play an important role as a sexual attractant or even by simply increasing the searching behavior of the male. It is of course possible that more refined behavioral measures may reveal femaleoriented male responses at lower concentrations and with less delay. This work with several species of fish indicates that hormones can affect behavior by bringing about an increase in general activity. However, it is not clear whether the hormones influence activity by an effect on general metabolism or whether they act directly on neural mechanisms underlying the behavioral responses. Evidence that steroid and thyroid hormones may directly affect CNS and sense organ states is provided by a number of electrophysiological studies. Several investigations involving goldfish have revealed that various steroids and thyroid hormones exert differential facilitatory and inhibitory effects on the excitability of the olfactory bulb and regions of the fore and midbrains (Oshima and Gorbman, 1966a,b, 1968; Hara, 1967; Hara et al., 1965, 1966). Godet and DupC (1965) found that in Protopterus an active thyroid or thyroxine treatment facilitates responsiveness to olfactory stimuli at the level of the forebrain. Although little is known of the behavioral significance of most of the above neurophysiological findings, they appear to indicate that steroid

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and thyroid hormones influence the responsiveness of the CNS to external stimuli. It seems reasonable to suppose that hormone-induced changes in responsiveness to general or specific external stimuli are in part responsible for increased locomotory activity. Such changes in activity and responsiveness may be important components in migratory and other reproductive behavior in fishes.

D. Thyroid Hormones and Migratory Behavior The previous section surveyed a number of investigations in which it was found that treatment with steroid and thyroid hormones resulted in an increase in locomotory activity. In most examples this “activity” was not obviously linked with any specific behavioral system. In a few cases it was suggested that the activity induced by the hormone treatment was reminiscent of the behavior of migrating fish held in captivity ( van Iersel, 1953; Forselius, 1957; Baggerman, 1962). Baggerman (1957) showed that in the stickleback the gonads are not essential to the onset of migration disposition (as measured by salinity preference). Baggerman ( 1962) mentions other investigations which have indicated that gonadal maturation and regression are not factors underlying migration. However, Baggerman ( 1962) suggests that gonadal hormones are involved in timing and perhaps augmenting the action of the thyroid hormone which, she believes, plays an important causative role in the onset of migration in certain anadromous fish. Baggerman (1957, 1959) tested the effects of thyroxine, TSH, and various thyroid blocking agents on the salinity preference of the truchurus form of the three-spined stickleback. The results suggest that an increase in the level of circulating thyroid hormone induces a preference for freshwater. This agrees with the fact that the stickleback migrates to freshwater in spring when the thyroid gland shows signs of heightened activity. Low levels of thyroid hormone, as is believed to occur in the fall, result in a preference for saltwater. As mentioned previously, Baggerman (1962) also found that both thyroid hormone ,and androgen treatments stimulate a form of locomotory activity characteristic of migrating fish. Baggerman (1962) concludes that an increase in thyroid activity brought about by long photoperiod induces two changes which are known to be associated with migration. Baggerman (1960, 1963) carried out similar experiments with juvenile Pacific salmon, Oncorhynchus, and found an increased preference for saltwater associated with the downstream migration. The saltwater preference is in some species correlated with heightened thyroid activity.

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Juvenile coho saImon treated with TSH showed a change from freshwater to saltwater preference ( Baggerman, 1963). Thiourea treatment induced a freshwater preference in coho which initially showed a preference for saltwater ( Baggerman, 1960). Similarly, underyearling pink salmon treated with thyroid blocking agents showed a change from saltwater to freshwater preference ( Baggerman, 1963). Although the changes in salinity preference induced by thyroid hormone are directly opposite in sticklebacks and salmonids, Baggerman (1962) argues that the thyroid gland is directly involved in the causation of migration of these species. On the other hand, Hoar ( 1 9 6 5 ~ )reviews the available information regarding the role of the thyroid and concludes that the data suggest a generalized function of thyroxine in metabolism and electrolyte balance rather than a specific causative role associated with migration. Hoar suggests that the onset of migration may depend upon general growth and maturation with associated, but not regulating, hormone changes.

111. PITUITARY HORMONES

A. Prespawning Behavior Reviews by Pickford and Atz (1957), Dodd (1960), and Hoar (1965a, 1966; and chapter by Hoar, this volume) survey a considerable body of evidence which indicates that at least one pituitary hormone regulates gonadal development and hormone secretion and thereby exerts an indirect control over reproductive activities. Indeed, treatment of intact fish with fish or mammal pituitary material has proved a highly effective approach to the breeding of commercially important freshwater fishes. Pickford and Atz (1957) provide a thorough review of these procedures. Ramaswami and Lakshman ( 1958), Sneed and Clemens (1959), Chaudhuri (1960), Sneed and Dupree ( 1961), Das and Khan (1962), Clemens and Sneed (1962), Stevens (1966), and Sundararaj and Goswami (1966) have provided additional information on techniques and the effectiveness of various substances of mammalian and piscine origins. In addition to their gonadotropic role there is reason to believe that in a number of cases pituitary hormones may have a more direct influence on behavior, perhaps by acting on the CNS directly. Much of the evidence for such a pituitary control is of an indirect nature. Usually the first indication that the pituitary may have a direct effect on behavior arises from experiments in which it is found that reproductive behavior

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persists after gonadectomy. Of course it does not necessarily follow in such cases that the pituitary is directly involved; other endocrine glands may be important, for example, the thyroid in stickleback migration, or there may be no endocrine involvement at all. There have been relatively few attempts to investigate direct pituitary effects by hypophysectomy and replacement therapy. The work of Baggerman (1966) and Hoar (1962a,b) leaves little doubt that in the stickleback gonadal development and hormone secretion (at least in the male) are under pituitary gonadotropic control. But, in addition to the indirect effects of the pituitary on reproductive behavior, both of these workers have hypothesized that prenestbuilding aggressive behavior is directly controlled by the pituitary. Baggerman (1966) found that castration of males prior to nestbuilding did not affect aggressive behavior, whereas castration after the onset of breeding resulted in a marked decline in aggressive behavior. Hoar's evidence (1962a,b) for a direct pituitary action is based upon a comparison of castrated sticklebacks under regimes of long and short photoperiod. The long photoperiods are assumed to produce a high level of pituitary gonadotropin, while the output of these pituitary factors is greatly depressed or eliminated under short photoperiods. Males castrated prior to nestbuilding show a high level of aggression if they are maintained under long photoperiod. Castrates held under short photoperiod are much less aggressive. In experiments designed to measure the effects of a series of mammalian pituitary hormones on aggressive behavior only treatment with LH consistently produced an increase in aggressive behavior (Hoar, 1962a). Ahsan and Hoar (1963) also found LH to be the most effective mammalian gonadotropic hormone in eliciting gonadal development in immature fish. There are indications that in the stickleback androgens and pituitary hormones act synergistically to maintain full reproductive behavior. Hoar ( 1962a,b) found that methyltestosterone treatment was less successful in inducing nestbuilding in castrate males kept under short photoperiod than in males under long photoperiod. These findings suggest that although the behavior of the nestbuilding phase requires the gonadal hormone its full expression only occurs when the gonadotropic activity of the pituitary is high. Smith (1967) found that in males of two species of Lepomis aggressive behavior remained high after Castration and was not affected by androgen treatment. Aggressive behavior remained high under short photoperiod provided the temperature remained high ( WOC), but it declined at low temperatures ( 13°C). Treatment with human chorionic gonadotropin, although effective in stimulating nestbuilding, did not

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affect aggressive behavior. Smith suggests that in these species aggression, although inhibited by low temperature, seems more closely related to social conditions than hormonal state. Wiebe (1967) was able to relate the reproductive cycle of Cymatogaster aggregata to the seasonal light and temperature cycle, indicating that pituitary gonadotropins are involved in stimulating gonad development and hormone secretion. Treatment with methallibure (ICI 33,828), a substance shown to have antigonadotropic activity in a number of fish species (Hoar et al., 1967; Wiebe, 1968; Pandey and Leatherland, 1969), effectively blocks gonadal development and results in the complete elimination of reproductive behavior including chasing and mating attempt. As these two activities persist after castration Wiebe concludes that they are normally under direct pituitary control. Burger ( 1941) hypophysectomized males of Fundulus heteroclitus and noted that the later stages of spermatogenesis came to a halt. Implantation of Fundulus pituitary material, five pituitaries at each treatment, at 3 5 day intervals, resulted in vigorous sexual behavior. By the tenth day the fish were in a “frenzy of display.” As the treatment also resulted in the recrudescence of the testes, it is not known whether the behavioral effects were the results of a direct action of the pituitary material on the CNS or a result of the stimulation of gonadal secretory activity. Tavolga ( 1955) hypophysectomized males of Bathygobius soporator and found that courtship and agonistic behavior were eliminated. This result is evidently not simply an effect of a reduction in gonadal hormone secretion since castration does not eliminate courtship behavior. Hypophysectomy of virgin female guppies, Poecilia reticulata, results in an immediate and complete decline in sexual responsiveness (Liley, 1968). Because this effect contrasts with the relatively slow decline in sexual behavior following gonadectomy, and because of the reappearance of sexual responses several weeks after gonadectomy, Liley proposed that there is a pituitary factor directly involved in the control of receptivity in the female. On the other hand, the decline in receptivity after gonadectomy and the cyclical changes in responsiveness of nonvirgin fish suggest that the ovary is also producing a hormone which regulates, or acts synergistically with, the more direct pituitary mechanism. Liley and Donaldson (1969) treated female guppies with a partially purified gonadotropin fraction of salmon pituitary material. Several of a group of hypophysectomized, ovariectomized fish exhibited a low level of sexual responsiveness. Hypophysectomized females with their ovaries intact showed a marked increase in sexual responsiveness after several days of hormone treatment. This increase coincided with the growth of

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the ovaries which had previously regressed. These results appear to support the suggestion that in the female guppy sexual responsiveness is not completely dependent upon any one hormone, but under normal circumstances the gonadotropic and ovarian hormones act synergistically-the absence of one hormone may be partly compensated for by the other. B. Spawning Behavior

It is convenient to give separate consideration to the role of hormones involved in the actual spawning act involved in the release of gametes as opposed to the more lengthy prespawning procedures. There is some evidence that the two phases may be under separate controls. In general, the distinction between prespawning and spawning behavior is more clearly defined in males than in females. In females there may be little in the way of overt prespawning behavior before a female in “ripe” condition responds to male courtship and proceeds rapidly to actual spawning. Pituitary hormone treatment procedures described by Pickford and Atz ( 1957) and others cited (Section 111, A ) have proved highly effective in the induction of spawning in fish which normally prove difficult to breed under laboratory or artificial pond conditions. Because females generally prove more refractory than males under such conditions most of the investigations involving pituitary treatment have involved females. Pituitary hormone therapy has been used to accelerate maturity in fish in nonreproductive condition (e.g., Combs and Burrows, 1959; Ahsan and Hoar, 1963). More often these techniques have been applied to fish already mature to induce or accelerate ovulation (release of ova into the ovarian lumen) and perhaps actual spawning. In most studies reported treated fish are stripped by hand and the eggs fertilized artificially. However, if stripping is not carried out but the females are placed with mature males, spawning occurs within a few hours of ovulation. Fontenele (1955) reports that in Brazilian fish culture practices a hormone-treated female is separated by a partition from a male until readiness to spawn is indicated by a state of accentuated agitation. The partition is then removed and the fish allowed to spawn. Spawning usually occurs within 24 hr of the first of a series of injections of fish pituitary material. This last report brings to the fore the close temporal relationship between ovulation and the spawning act already referred to in Section 11, B. It appears that spawning only occurs after ovulation of mature eggs. The apparently close dependence of spawning on ovulation gives rise to

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a number of suggestions as to the endocrine or neural involvement in the control of spawning: the gonadotropic hormone responsible for ovulation may also act directly on the CNS to induce a state of sexual receptivity; the gonadotropin may induce a marked increase in gonadal hormone which in turn affects behavior; the release of ova into the ovarian cavity may also involve a sudden release of gonadal hormones from the follicles; the physical presence of ova in the ovarian cavity may have a direct neural effect on the CNS, this effect may be accentuated by gonadal hydration which takes place at ovulation (Clemens and Grant, 1964); and, lastly, the endocrine events involved in ovulation, or behavioral events which follow, may stimulate the secretion of a neurohypophysial hormone which in turn induces actual spawning. There is some evidence that in male fish readiness to spawn is associated with seminal thinning (Clemens and Grant, 1964, 1965). This increase in fluidity of the semen appears to be the same as the process referred to as “spermiation” by Yamazaki and Donaldson (1968a,b). Both pairs of workers have demonstrated for a number of species of fish that spermiation (or gonadal hydration) occurs in response to treatment with fish and mammalian gonadotropin preparations. More recently, Yamazaki and Donaldson (1968~)have found that androgen alone will induce spermiation in hypophysectomized goldfish. However, it is evident from work discussed in Section 11, A that spawning behavior may occur, or be induced by androgen treatment, in castrated fish. Thus, spawning in males is not dependent upon the presence of intact gonads as appears to be the case in females. At present there is little convincing evidence for or against the above suggestions regarding the significance of ovulation in the timing of oviposition. Yamazaki (1965) has gone further than most workers in an attempt to determine the factors involved in the regulation of spawning. Under natural spawning conditions the male goldfish courts the female for 1 or 2 days before ovulation and spawning occur. Evidently male courtship is important in the induction of ovulation. Spawning will not occur in females from which ovulated eggs have been stripped, suggesting that the presence of ova is an important factor. On the other hand, females hypophysectomized after ovulation may oviposit normally about 3 hr after the operation. A small number of hypophysectomized females in which ovulation had been induced by hormone treatment 1 4 days after hypophysectomy spawned normally when paired with males. Yamazaki (1962) also found that male goldfish would spawn several hours, or in some cases, the day after hypophysectomy. Yamazaki (1965) concludes that, although ovulation is induced by pituitary gonadotropic hormone, actual spawning is not under direct pituitary control. However,

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he recognizes the possibility that in his experiments residual pituitary hormone may remain in effective quantities for some time after hypophysectomy. In addition he draws attention to the possibility that neurohypophysial hormones or their precursors may be released directly from the hypothalamus of hypophysectomized fish and could be involved in the regulation of spawning behavior. There have been a number of suggestions that neurohypophysial hormones are directly involved in the causation of spawning behavior. Wilhelmi et al. (1955) found that purified fish and mammalian neurohypophysial preparations and synthetic oxytocin would induce the spawning reflex in Fundulus heteroclitus. The response usually appears within 10 min after injection and may persist for 2030 min. This behavior occurs in hypophysectomized or castrated fish of either sex. However, there is no coordination of the response when males and females are injected and placed together. It is suggested that this response is mediated by direct excitation of a nervous center and that sex hormones play no part in mediating this phase of the sexual behavior pattern. Egami ( 1959) injected mammalian neurohypophysial substances into Oryzias Zutipes and observed spawning movements in both males and females. These responses occurred within 10-60 min of treatment. Females isolated from males prior to treatment oviposited even though there was no male present. Egami and Nambu (1961) consider these findings in relation to the normal breeding activity of Oryzias. During the breeding season the female lays eggs almost every morning. Maturation of oocytes and ovulation may take place in a female which is isolated from a male, but oviposition only occurs naturally after a period of male courtship. Egami and Nambu suggest that stimuli arising from male courtship induce the secretion of neurohypophysial hormone which, when it reaches a certain level, causes oviposition to occur. Females of Gambusia afinis performed behavior resembling a spawning reflex and released their embryos shortly after treatment with oxytocin, or homogenates of frog neurointermediate lobe or rat neural lobe (Ishii, 1963). Egami and Ishii ( 1962) mention unpublished work by Shirai in which it was found that injection of neurohypophysial substances will induce oviposition in ovulated female bitterling, Rhodeus ocelhtus. These authors also refer to reports, without giving details, that loach, salmonids, and some other species fail to exhibit any definite response to the injection of a neurohypophysial extract. Blum (1968) injected reserpine into immature angelfish, PterophyZlum scaZure. One to three hours after the injection the fish darkened; 6-8 hr after injection several of the fish performed typical spawning movements. Blum suggests that reserpine may exert its effect by stimulating the release of melano-

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phore stimulating hormone, responsible for the darkening, and a neurohypophysial hormone, responsible for the induction of the spawning response. Pickford and Atz (1957) review earlier work in which it had been suggested that pituitary secretions pass directly to the brain and induce the final stages of maturation and spawning activity. Gerbilskii (1938, cited in Pickford and Atz, 1957) named this secretion the “spawning hormone.” Support for the existence of such a hormone was derived from the findings by Gerbilskii and others that intracranial injections of pituitary substances were more effective in inducing final maturation than intraperitoneal or intramuscular injections. However, Pickford and Atz (1957) point out that other Russian workers have not confirmed these results. Vivien (1941) also noted that intracranial implantation of pituitary gland into female Gobius paganellus were far more effective in inducing spawning than similar amounts injected into the body cavity. It is impossible at this stage to draw any firm conclusions regarding the mechanism by which spawning is regulated in fish. In general, gonadotropin-induced ovulation must occur before spawning can proceed-it may turn out that this is not an essential precursor to spawning behavior. Several workers have suggested that spawning is directly evoked by a neurohypophysial hormone secreted in response to appropriate sexual and environmental stimuli. However, it has not been possible to confirm this in several species of fish tested. Also, the interpretation of the effects of neurohypophysial preparations has been questioned on the grounds that the doses required to elicit an effect are extremely high (Dodd et al., 1968). C. Prolactin and Parental Behavior

Several studies have implicated a prolactinlike hormone in the regulation of parental behavior in fish, Noble et al. (1938) found that injections of prolactin, as well as several other hormone preparations, induced parental behavior in both males and females of Hemichromis bimculutus (discussed in Section 11, A ) . Fiedler ( 1962) reports that males of the wrasse, Crenilabrus ocellatus, treated with prolactin would perform parental fanning even though no nest was present. Similarly, prolactin treatment of Symphysodon aequifasciata resulted in a number of changes associated with the parental phase of the reproductive cycle (Blum and Fiedler, 1964). At the lower dose leveIs tested there was a marked increase in the number of mucus-secreting cells at the. body surface. The secretions of these cells normally provide supplementary nutrient for the

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young. At high doses the fish began to perform parental-type fanning directed to a particular place in the aquarium. Agonistic behavior in response to a test fish was also affected: At intermediate doses there was a decrease in fighting behavior, at higher doses fighting increased. Blum and Fiedler (1965) examined the effects of pituitary hormone treatments on several species of cichlid including Pterophyllum scalare, Aequidens latifrons, Cichlasoma seuerum, and Astronotus ocelhtus. In all cases prolactin treatment resulted in an increase in mucus cell production. This effect was less marked than in Symphysodon which is the only species in which the mucus serves as a nutrient for the young. Some behavioral effects of prolactin treatment were noted. In the case of Pterophyllum a low dose of hormone induced a parental type of fanning directed toward a fixed point in space; at higher doses fanning decreased. Agonistic activity in response to a stimulus animal was depressed by prolactin treatment in Pterophyllum and Aequidens but unaffected in the other species. Prolactin tended to make fish very calm and to depress the feeding response. Blum and Fiedler regard this latter effect as a component of parental behavior which prevents eggs or young being eaten by the parents. They also note that hormone-induced parental responses occurred in fish which had had no previous experience of breeding. Blum ( 1966) tested progesterone and several adenohypophysial hormones on Pterophyllum and Symphysodon. Only prolactin treatment resulted in the appearance of parental behavior, although both somatotropin and thyrotropin elicited an increase in mucus cell production. Luteinizing and follicle-stimulating hormones resulted in an increase in aggressive behavior. Blum (1966) concludes that prolactin is the only hormone involved in the control of parental behavior in cichlids. Prolactin treatment resulted in an increase in mucus cell production in the paradise fish, Macropodus operculuris ( Machemer and Fiedler, 1965). This effect was more pronounced in males than in females which do not normally build bubble nests or show parental care. However, prolactin treatment alone did not produce an increase in nestbuilding even though the fish produced more mucus. Chorionic gonadotropin alone, or in combination with prolactin, increased nestbuilding behavior. Similarly, methyltestosterone combined with prolactin treatment resulted in extensive nestbuilding in both males and females. Machemer and Fiedler (1965) conclude that for full nestbuilding activity two hormones are necessary: Androgen increases building activity, but this effect is only expressed fully in the presence of a prolactin-induced increase in mucus production. Smith and Hoar (1967) studied the effects of prolactin treatment on parental and displacement fanning behavior in the stickleback. They

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conclude that there is no evidence of a pituitary regulated control of fanning behavior in the stickleback. In fact, injections of prolactin appeared to depress fanning, although in no case was the difference between the treatment groups statistically significant. Smith and Hoar suggest that this depression effect may have resulted from a feedback which reduced the output of natural gonadal androgen. Blum (1966) also obtained evidence that prolactin treatment suppresses gonadotropin production in cichlids. It is difficult to draw any definite conclusions regarding the role of prolactin in reproductive behavior in fish. It is important to separate two types of effect. First, in a number of fish tested prolactin treatment results in an increase in mucus production (Egami and Ishii, 1962; Blum, 1966; Blum and Fiedler, 1964, 1965; Machemer and Fiedler, 1965; Leatherland and Lam, 1969; see review in chapter by Ball, Volume 11). However, this effect on epithelial tissues is evidently not universal among fish (Egami and Ishii, 1962; Bern, 1967). Second, evidence suggests that prolactin, or rather a prolactinlike hormone, is involved in the regulation of parental behavior. The situation is complicated by the fact that untiI now only mammalian prolactin has been used in treatment of fish. Thus differences among various species of fish might result in part from differences in specificity in responsiveness to tetrapod hormone. Evidence reviewed by Bern (1967) indicates that fish “prolactin” is only partly related to the mammalian hormone in terms of its capacity to produce typical prolactin effects in birds and mammals.

IV. EXTERNAL FACTORS AND THE ENDOCRINE SYSTEM

The role of photoperiod, temperature, and other physical factors in the regulation of seasonal changes in reproductive behavior has been reviewed by Aronson ( 1965), Hoar (1965a), and in the chapter by Schwassman, Volume VI. In general, physical stimuli serve as proximate factors which act through the brain-pituitary-gonad axis to ensure that the fish are ready to carry out the reproductive processes at a time and place most favorable to the production and survival of eggs and young. The synchronization achieved in this manner is relatively crude. A “fine adjustment” is provided by the animals in their responses to the physical environment and by their behavioral interaction with conspecifics. Little is known of the internal mechanisms by which the final syn-

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chronization is achieved. It is possible that there is no endocrine involvement other than that the sex hormones induce a state of responsiveness to certain classes of behavioral stimuli. On the other hand, it seems more likely that, apart from immediate behavioral responses, the performance of reproductive behavior may induce endocrine changes in the performing and responding animals, providing the appropriate conditions for the later phases of reproductive activity. In other words, it seems reasonable to look for a situation in fish comparable to that described most clearly in the canary ( Hinde, 1965) and the ring dove (Lehrman, 1965). Not only do hormones affect behavior, but behavior in turn affects the endocrine state. The interaction of these relationships results in the integration of behavior with other physiological conditions and the smooth progression through successive stages of the reproductive cycle. Relatively little attention has been directed to the details of the breeding requirements of fish, The difficulties encountered by aquarists and commercial fish culturists in obtaining spawning in gravid fish indicates that for the final act certain key stimuli are necessary. Aronson (1965) points out that it is common aquarium practice to lower the temperature of the water to induce cyprinids and characins to breed in captivity. Chaudhuri (1960) remarks that in several species of Indian cyprinid spawning only occurs on overcast days and appears to be inhibited by direct sunlight. Yamazaki (1965) points out that goldfish will only spawn in the presence of green plants. Bitterling require the presence of a freshwater mussel before they will show any reproductive behavior (Shirai, 1962). Courtship plays an important role in the final synchronization of the sexes. It seems likely, although there is virtually no experimental evidence, that courtship affects the endocrine state of one or both partners. Johns et al. (1969) found that 2-3 days of exposure to a nestbuilding male is necessary before a female gourami will spawn. This suggests that the exposure to the male affects the final maturation and preparation of the endocrine state before mating. Egami and Nambu (1961) maintain that in Oyxias male courtship stimulates neurohypophysial activity which in turn is responsible for the induction of oviposition behavior in the female. Exposure to other individuals of the same sex may also be important. Aronson (1951) notes that females of Tilapia macrocephalu maintained in visual isolation from conspecifics could be spawned, on average, three times a year. Females able to see other fish, males or females, went through seven or eight spawnings a year. Aronson (1965) refers to work involving salmon, herring, whitefish, and minnows which suggests that social facilitation may occur in the synchronization of breeding.

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Van Mullem (1967) provides evidence of a considerable degree of synchronization in the reproduction of the stickleback in natural populations. Van den Assem (1967) found that in the three-spined stickleback, males in winter condition came into breeding more readily if paired, one on each side of a glass partition, than did solitary males. The presence of vegetation also had a stimulating effect upon nestbuilding. On the other hand, the presence of males in breeding condition may have an inhibitory effect upon those without nests. The mechanism of these inhibitory and stimulatory effects is unknown but could involve the endocrine system. In conclusion, although much is known regarding the proximate environmental factors involved in the long-term control of breeding, little is known regarding the short-term synchronization of sexual partners and the regulation of the successive subcycles which constitute reproductive behavior in many species of fish. The little evidence available suggests that we may find that endocrine mechanisms are involved and that, as in other vertebrates, we will find that not only do hormones affect behavior but in turn behavior affects the endocrine system.

V. SUMMARY AND DISCUSSION

The preceding survey of the literature reveals a considerable diversity in the hormonal regulation of reproductive behavior, even though relatively few species have been studied, It proves virtually impossible to draw any general conclusion about the role of any one hormone or the control of any one class of behavior. It is well established that in male fish morphological secondary sex characters are dependent on gonadal hormones. In several species examined certain components of reproductive behavior are evidently under gonadal control, but there are others in which much or all reproductive behavior persists after castration-with the implication that this behavior is independent of gonadal hormones. The effects of ovariectomy have been studied in only a few species, usually without the careful behavioral examination necessary to detect subtle changes resulting from the operation. Ovariectomy abolishes the sexual response in those species examined. In the guppy sexual behavior reappears some time after ovariectomy (Liley, 1968). There has been no unequivocal demonstration that administration of estrogen or progesterone will induce reproductive behavior in fishes. On the basis of salinity preference tests, thyroid hormone has been

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implicated in the migratory behavior of the stickleback (Baggerman, 1957, 1959). However, no consistent picture emerges in studies involving other anadromous fishes, and there has been no exhaustive study of the effects of other factors on the migratory behavior in sticklebacks. A similar lack of uniformity emerges in studies of the pituitary hormones. The pituitary is inevitably involved in the regulation of reproductive behavior by its gonadotropic control of gonad activity. In addition, there have been a number of suggestions that gonadotropic hormones act directly upon behavioral control mechanisms or many act synergistically with gonadal hormones (Hoar, 1962a,b; Liley, 1968). Neurohypophysial hormones appear to play a role in the control of spawning in certain species of fish (Wilhelmi et al., 1955; Egami and Ishii, 1962). Although these findings have not been widely confirmed they do raise the possibility that the act of spawning may be under different control to those activities which precede it. Prolactin is said to induce parental behavior in several species of cichlid and a wrasse (Fiedler, 1962; Blum and Fiedler, 1964, 1965). There has been no widespread confirmation of a relationship between prolactin and parental behavior: One attempt with the stickleback (Smith and Hoar, 1967) resulted in negative evidence. There are several possible explanations for the apparent lack of uniformity in the behavioral results of experiments involving surgical and hormone treatments. First, the apparent diversity may be spurious, arising out of the experimental procedures themselves. Second, fish may show varying degrees of cephalization in the control of reproductive behavior, analogous to Beach‘s “corticalization of function” in mammals ( 1964). Third, the diversity in endocrine mechanisms may be real, arising from a considerable flexibility in the responsibilities of the hormones. These three possibilities will be considered in turn. Experimental procedures. Most experiments involving hormone treatment have used synthetic hormones or naturally occurring hormones from other species of fish or mammal, It would not be surprising to find species or group differences in responsiveness to hormones from such sources. Although there is no absolute biological specificity, important phylogenetic differences have been recognized in the protein and peptide hormones of the pituitary (Pickford and Atz, 1957; Dodd et al., 1966; Bern, 1967). On the other hand, the similarity of most of the steroids identified so far in fish (Gottfried, 1964; see chapter by Hoar, this volume) to those of mammals suggests that phylogenetic specificity is not too serious a problem in applying steroid therapy. But, in the case of steroids a different problem is raised by the apparent similarity of the biosynthetic pathways involved in the production of estrogens, proges-

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terone, androgens, and corticosteroids (see Fig. 23.5 in Hoar, 1966). This gives rise to the difficulties in the interpretation of the results of hormone therapy or identification discussed by Hoar (1966, p. 699, and chapter given in this volume), e.g., an exogenous hormone may be metabolized to another rather different form which is in turn responsible for the observed physiological effects. Furthermore, there is the possibility that another steroid secreting gland may produce sufficient estrogen or androgen to act on reproductive processes-a situation which occurs under some circumstances in mammals (Julesz, 1967). Aronson (1959) stressed the danger that even very small fragments of gonadal tissue may regenerate and provide sufficient hormone to induce reproductive behavior. Thus, undetected regeneration may have been responsible for the persistence of reproductive behavior in some of the gonadectomy experiments reported earlier. Lastly, work with fish has so far been characterized by the small number of replications within any one study, or a lack of independent replications by different workers investigating the same species. The exception here is work with the stickleback. Significantly, Baggerman (1966) and Wootton ( 1968), studying the stickleback, obtained rather different results which could result from differences in the races used or the testing procedures. Cephalization of the control of reproductive behavior. If we assume that the surgical operations were completely successful then a striking feature of many gonadectomy experiments is the persistence of reproductive behavior after the operation. This has been interpreted as an indication that the regulation of this behavior is not dependent on gonadal hormones. At first sight these findings appear to be similar to the results of gonadectomy experiments with several species of mammal. Beach (1947, 1964) has pointed out that in certain groups of mammals, notably the carnivores, ungulates and primates, we find a tendency toward a “corticalization of function” in the control of sexual activities; that is, although hormones may be essential to the development of reproductive behavior, these activities become less dependent on gonadal function after maturity and experience of breeding. In males, in particular, sexual arousal and performance appear to depend to a considerable extent upon learning and experience. Is there a process in fish analogous to the “corticalization of function” in mammals? Aronson (1959) considers this question and concludes that although the teleost forebrain differs in important respects from that of a mammal “we must recognize the possibility that, incorporated within the complex teleostean forebrain, there may be a mechanism equivalent to that indicated in higher mammals, whereby some elements of the

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reproductive pattern have been released from the functional control of the gonadal hormones.” It is important to recognize that independence of gonadal hormones does not necessarily imply a cephalization of function in the sense outlined by Beach. In particular, Beach (1964) stresses the role of learning and experience in arousal and performance of sexual behavior. Little attention has been given to the role of experience in studies of hormones and behavior in fish. One of the few cases in which experiential factors have been considered is the investigation of Noble et al. (1938). They found that several of their treatments would induce parental behavior in Hemichromis, but this only occurred in fish with previous breeding experience. Although in most gonadectomy experiments there is little direct evidence available, it seems unlikely that experiential factors could have played an important role in the persistence of reproductive behavior after gonadectomy. Furthermore, it is important to stress that in several cases the behavior which persists after gonadectomy is not exclusively reproductive in function. This applies particularly to agonistic behavior which, while it is prominent in reproduction, is also involved in the maintenance of individual distance or dispersal of young and adults not in breeding condition. Therefore, it is not surprising to discover that agonistic behavior as such is more or less independent of gonadal hormone. In a number of cases there are indications that such activities are more directly controlled by the pituitary. But, one might go further and question the existence of any endocrine control at all. Smith (1967) has suggested that in Lepomis agonistic behavior depends more on social conditions rather than on hormonal state. Flexibility in hormone responsibilities. It may be unrealistic to expect uniformity in the endocrine regulation of behavior in such a diverse group as the teleosts. Endocrine evolution has evidently involved the evolution of the uses to which hormones are put as well as the hormones themselves. Even among closely related species we may expect to find considerable flexibility in the responsibilities of certain hormones. Hoar (196513) has pointed out that in the anadromous form of the stickleback there appears to be a shift in hormone responsibilities within the annual cycle. Long photoperiods stimulate the secretory activity of the anterior pituitary; its hormones first induce the physiology associated with the change in tonicity of the environment and the migratory and presexual phases of behavior. Three factors, luteinizing, lactogenic, and thyroidstimulating hormones, are known to be involved. At the same time, the pituitary-presumably the luteinizing hormone-activates the interstitial tissue of the gonads which produce the gonadal steroids; these dominate the sexual phases, gradually taking over complete control of behavior

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during the parental phase. In other words, there is a shift in the endocrine control of reproductive events during the cycle. It seems reasonable to assume that the same type of shift or flexibility in endocrine responsibility which appears to occur within the life cycle of a single species will also provide the basis for variability in control mechanisms of different species or major groups. The three possible sources of diversity in the results of endocrinebehavior investigations in fish are not mutually exclusive. However, at present few species have been studied with sufficient thoroughness to exclude one or more of the various “explanations.” The manner in which hormones exert their effects on behavior in fish is poorly understood. Studies involving other vertebrate classes have suggested a number of possible mechanisms (Lashley, 1938, cited by Hinde, 1966): ( 1 ) The hormone stimulates the growth of new nervous connections ( 2 ) The hormone acts by inducing specific changes in various organs, and these mediate sensory impulses which influence the central nervous system ( 3 ) The hormone increases the excitability of specific sensory-motor mechanisms in the central nervous system (4) The hormone increases the general excitability of the organism It is possible to point to what seem to be examples of each of the mechanisms listed above. Much of the work with poeciliids and Oryzias suggests that gonadal hormones are involved in sex determination and, it would appear, in the development of the neural mechanisms appropriate to each sex (see chapter by Yamamoto, this volume). In a few cases we have evidence that hormones induce changes in peripheral structures and thereby affect the animals’ sensitivity to certain stimuli. Tavolga ( 1955) believes that castration of Bathygobius soporator results in a change in olfactory sensitivity. Electrophysiological studies involving goldfish (Section 11, C ) reveal that hormones may affect the sensitivity of the olfactory organ. It seems likely that in many cases hormones exert their effects on the excitability of specific sensory-motor mechanisms in the CNS. However, the only direct evidence that hormones affect central mechanisms in the fish brain comes from electrophysiological studies of the goldfish. Hara ( 1967), Hara et al. ( 1965, 1968), and Oshima and Gorbman ( 1966a) recorded from the fore- and midbrain of the goldfish and report changes in electrical activity and responsiveness to stimulation, brought about by treatment with steroids. The behavioral significance of these findings is not known. Lastly, several investigators have shown that hormones may result

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in changes in locomotory activity. Such changes in behavior may be in part the indirect result of metabolic effects of the hormones concerned. But, in addition, it is suggested that the increased activity reflects changes in general susceptibility to external stimuli. In either case these changes in activity may play an important role in behavior leading up to and including reproduction. In addition to their direct effects on the nervous system, hormones evidently influence reproductive behavior by their role in the development of secondary sexual characteristics which serve as signals or accessories during the breeding process. The most obvious of these are morphological structures and coloration. There is some inconclusive evidence that pheromones are present and under gonadal control in Bathygobius soporator ( Tavolga, 1956), Trichogaster trichopterus (Johns et al., 1969), and possibly in the guppy (Amouriq, 1964). A different form of indirect hormone effect is suggested by Baggerman (1966), who points out that in the stickleback displacement fanning and perhaps changes in activities such as swimming or comfort movements may arise as a result of the interaction of other motivational systems which are in their turn under hormonal control. Clearly these categories of mechanisms of hormone action are not mutually exclusive; the same hormone may act in one or all of the possible ways.

VI. CONCLUSION

The preceding review reveals an apparent diversity and lack of agreement in studies of the role of hormones and behavior in fish. This is perhaps an inevitable outcome of the relatively primitive stage of development of this field of investigation. There is considerable need for more investigations using basic endocrine procedures of gland removal and replacement therapy, with the addition of the more refined techniques of hormone implantation and electrical recording. Such studies must incorporate a thorough analysis and understanding of the normal behavior of the species under investigation. Not only do we require more "in depth" studies but also it is essential to consider a wider variety of species. Comparison of different species and a comparison of fish with other vertebrates will almost certainly provide insight into the significance of the differences between taxa and perhaps shed further light on the evolution of endocrine mechanisms. Undoubtedly the investigation of hormones and behavior in fish can

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gain great impetus from the ideas and experimental procedures which have emerged from the more sophisticated studies of birds and mammals. However, it is well to bear in mind the warning implicit in Bern’s comments (1967) that “there is an elementary and unjustifiable tendency to view the fishes, in view of their collective ancestral position, as much like their landliving descendents only more primitive.” As Bern points out the fishes reveal a broader range of variation and a longer history of adaption than do the landliving vertebrates. In other words, while benefiting from the more advanced state of our knowledge of other vertebrates, it is important to be prepared to find basic differences in the details of the mechanisms which have evolved. REFERENCES Ahsan, S. N., and Hoar, W. S. (1963). Some effects of gonadotropic hormones on the three-spined stickleback, Gasterosteus aculeatus. Can. J . ZOOZ.41, 1045-1053. Amouriq, L. (1964). L’activitk et le phbnomkne social chez Lebistes reticulatus ( Poeciliidae, Cyprinodontiformes ) . Compt. Rend. 259, 2701-2702. Amouriq, L. (1965a). L’activit6 et le phknomkne social chez Lebistes reticulatus (Poeciliidae, Cyprinodontiformes). Ann. Sci. Nat. Zool. Biol. Animale [12] 7 , 151-172. Amouriq, L. (1965b). Origine de la substance dynamogene 6mise par Lebistes reticulatus femelle ( Poisson Poeciliidae, Cyprinodontiformes) . Compt. Rend. 260, 2334-2335. Amouriq, L. (1967). L’optimum de sensibilit6 de Lebistes reticulatus (Poisson Poeciliidae, Cyprinodontiformes ) a l’hormone synthbtique femelle. Rev. Comp. Animal 3, 57-60. Aronson, L. R. ( 1951). Factors influencing the spawning frequency in the female cichlid fish, Tilupia mucrocephala. Am. Museum Novitates 14.84, 1-26. Aronson, L. R. (1957). Reproductive and parental behavior. In “The Physiology of Fishes” (M. E. Brown, ed.), Vol. 2, Chapter 3, pp. 271-304. Academic Press, New York. Aronson, L. R. ( 1959). Hormones and reproductive behavior: Some phylogenetic considerations. In “Comparative Endocrinology” (A. Gorbman, ed. ), pp. 98-120. Wiley, New York. Aronson, L. R. ( 1965). Environmental stimuli altering the physiological condition of the individual among lower vertebrates. In “Sex and Behavior” ( F . A. Beach, ed.), pp. 290318. Wiley, New York. Aronson, L. R., and Holz-Tucker, M. (1947). Morphological effects of castration and treatment with gonadal hormones on the female cichlid fish, Tilapia macrocephulu. Anat. Record €@, Suppl., 572. Baerends, G. P., and Baerends-van Roon, J. M. (1950). An introduction to the study of the ethology of cichlid fishes. Behaviour Suppl. 1, 1-243. Baerends, G. P., Brouwer, R., and Waterbolk, H. Tj. (1955). Ethological studies on Lebistes reticulatus (Peters). I. An analysis of the male courtship pattern. Behaviour 8, 249-334. Baggerman, B. (1957). An experimental study on the timing of breeding and migration in the three-spined stickleback (Gasterosteus acukatus L,). Arch. Neerl. Zool. 12, 105417.

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Baggerman, B. (1959). The role of external factors and hormones in migration of sticklebacks and juvenile salmon. In “Comparative Endocrinology” (A. Gorbman, ed.), pp. 24-37. Wiley, New York. Baggerman, B. (1960). Factors in the diadromous migrations of fish. Symp. ZOO^. SOC. London 1, 33-60. Baggerman, B. (1962). Some endocrine aspects of fish migration. Gen. Comp. Endocrinol. Suppl. 1, 188-205. Baggerman, B. (1963). The effect of TSH and antithyroid substances on salinity preference and thyroid activity in juvenile pacific salmon. Can. J. Zool. 41, 307419. Baggerman, B. ( 1966). On the endocrine control of reproductive behaviour in the male three-spined stickleback (Gasterosteus acukatus L.). Symp. SOC. Exptl. Biol. 20, 427-456. Ball, J. N. (1960). Reproduction in female bony fishes. Symp. Zool. SOC. London 1, 105-135. Beach, F. A. (1947). A review of physiological and psychological studies of sexual behavior in mammals. Physiol. Reu. 27, 240407. Beach, F. A. ( 1964). Biological bases for reproductive behavior. In “Social Behavior and Organization among Vertebrates” (W. Etkin, ed.), pp. 117-142. Univ. of Chicago Press, Chicago, Illinois. Beach, F. A. ( 1967). Cerebral and hormonal control of reflexive mechanisms involved in copulatory behavior. Physiol. Reo. 47, 289-316. Bern, H. A. (1967). Hormones and endocrine glands of fishes. Science 158, 455-462. Blum, V. (1966). Zur hormonalen Steuerung der Brutpflege einiger Cichliden. Zool. Jahrb., Abt. Allgem. Zool. Physiol. Tiere 72, 264290. Blum, V. ( 1968). Die Auslosung des Laichreflexes durch Reserpin bei dem sudamerikanischen Buntbarsch Pterophyllum scalare. Z . Vergleich. Physiol. 60, 79-81. Blum, V., and Fiedler, K. (1964). Der Einfluss von Prolactin auf das Brutpflegerverhalten von S ymphysodon aequifasciata arelrodi L. P. Schultz ( Cichlidae, Teleostei). Naturwissenschaften 51, 149. Blum, V., and Fiedler, K. (1965). Hormonal control of reproductive behavior in some cichlid fish. Gen. Comp. EndocrinoZ. 5, 186-196. Breder, C. M., and Coates, C. W. (1935). Sex recognition in the guppy, Lebistes reticulatus. Zoologica 19, 187-207. Bretschneider, L. H., and d e Wit, J. J. D. (1947). “Sexual Endocrinology of Nonmammalian Vertebrates.” Elsevier, Amsterdam. Burger, J. W. (1941). Some experiments on the effects of hypophysedomy and pituitary implantations on the male Fundulzls heteroclitus. Biol. BUZZ. 80, 31-36. Chaudhuri, H. (1960). Experiments on induced spawning of Indian Carps with pituitary injections. Indian J. Fisheries 7 , 2048. Chester Jones, I., and Phillips, J. G. (1960). Adrenocorticosteroids in fish. Symp. Zool. SOC. London 1, 1 7 3 2 . Clark, E., and Aronson, L. R. (1951). Sexual behavior in the guppy, Lebistes reticulatus (Peters). Zoologica 36, 49-66. Clemens, H. P., and Grant, F. B. (1964). Gonadal hydration of carp (Cyprinus carpio) and goldfish (Carassius auratus) after injections of pituitary extracts. Zoologica 49, 193-210. Clemens, H. P., and Grant, F. B. (1965). The seminal thinning response of carp (Cyprinus carpio) and rainbow trout (Salmo gairdnerii) after injections of pituitary extracts. Copeia, 1965, No. 2, 174-177.

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Clemens, H. P., and Inslee, T. (1968). The production of unisexual broods by Tilupia mossambica sex-reversed with methyl testosterone. Trans. Am. Fisheries SOC. $7, 18-21. Clemens, H. P., and Sneed, K. E. (1962). Bioassay and use of pituitary materials to spawn warm-water fishes. U.S.Fish Wildlife Serv. Bur. Sport Fisheries Wildlife Res. Rept. 61, 1 3 0 . Clemens, H. P., McDermitt, C., and Inslee, T. (1966). The effects of feeding methyl testosterone to guppies for 60 days after birth. Copeia, 1966, No. 2, 280-2234. Cohen, H. (1946). Effects of sex hormones on the development of the platyfish, Platypoecilus mucukztus. Zoologica 31, 121-128. Combs, B. D., and Burrows, R. E. (1959). Effects of injected gonadotrophins on maturation and spawning of blueback salmon. Progressive Fish Culturist 21, 165-168. Das, S. M., and Khan, H. A. (1962). The pituitary and pisciculture in India with an account of the pituitary of some Indian fishes and a review of techniques and literature on the subject. Ichthyologica 1, 43-58. Dodd, J. M. (1960). Gonadal and gonadotrophic hormone in lower vertebrates. In “Marshall’s Physiology of Reproduction” (A. S. Parkes, ed. ), 3rd ed., Vol. 1, Part 2, Chapter 11, pp. 417-582. Longmans, Green, New York. Dodd, J. M., Perks, A. M., and Dodd, M. H. I. (1966). Physiological functions of neurohypophysial hormones in submammalian vertebrates. In “The Pituitary ButterGland” (G. W. Hams and B. T. Donovan, eds.), Vol. 3, pp. 57-23. worth, London and Washington, D.C. Egami, N. (1955). Effect of estrogen administration on oviposition of the fish, Olyzius kztipes. Endocrinol. Japon. 11, 89-98. Egami, N. (1959). Preliminary note on the induction of the spawning reflex and oviposition in Oryzius latipes by the administration of neurohypophysial substances. Annotattones Zool. Japon. 32, 13-17. Egami, N., and Ishii, S. (1962). Hypophysial control of reproductive functions in teleost fishes. Gen. Comp. Endocrinol. Suppl. 1, 248-253. Egami, N., and Nambu, M. (1961). Factors initiating mating behavior and oviposition in the fish, Oryzius lutipes. J . Fac. Sci., Univ. Tokyo, Sect. IV 9, 263-278. Fagerlund, U. H. M. (1967). Plasma cortisol concentration in relation to stress in adult sockeye salmon during the freshwater stage of their life cycle. Gen. Comp. Endocrinol. 8, 197-207. Fiedler, K. (1962). Die Wirkung von Prolactin auf das Verhalten das Lippfisches Crenilabrus ocellatus ( Forskil ). Zool. Jahrb., Abt. Allgem. Zool. Physiol. Tiere 69, 6-20. Fontenele, 0. ( 1955). Injecting pituitary (hypophysial ) hormones into fish to induce spawning. Progressive Fish Culturist 17, 71-75. Forselius, S. (1957). Studies of anabantid fishes. Zool. Bids. Uppsak 32, 97-597. Godet, R., and Dup-5, M. ( 1965). Quelques aspects die relations neuroendocriniennes chez Protoptesus annectens (Poisson dipneuste ). Arch. Anat. Microscop. Morphol. Exptl. 54, 319-330. Cottfried, H. ( 1964). The occurrence and biological significance of steroids in lower vertebrates. A review. Steroids 3, 219-242. Gottfried, H., and van Mullem, P. J. (1967). On the histology of the interstitium and the occurrence of steroids in the stickleback (Casterosteus aculeatus L.) testis. Acta Endocrinol. 56, 1-15.

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maphroditism, protandrous and protogynous hermaphroditism, to gonochorism. At the outset, it is necessary to clarify the concept of sex. The male and female can best be defined as sperm and egg producers, respectively. This definition, although self-evident, is needed to correct statements that appear frequently in literature, with respect to spontaneous sex reversal in gonochorists (Sections IV, B and C). The term “bisexuality” used to denote hermaphroditism is likely to lead to a misunderstanding. This term should be used to describe gonochorist. The term “intersex” (Goldschmidt, 1915, 1927, 1931) is used in the present review to denote either sporadically appearing or experimentally produced hermaphroditic individuals of a species in which all or nearly all individuals are gonochoristic. Whether hermaphroditism is the more primitive condition from which bisexuality or gonochorism may have arisen or a specialization derived from the more usual vertebrate gonochorism is a matter for debate. The solution of this intriguing problem will require a great deal more information in the future. Nevertheless, fishes provide excellent material to approach the problems of sex differentiation and of evolution of sex among animals. In this study, stress is laid on sex differentiation as a process rather than examining sex phenotypes as they appear in adults. For the latter approach, Gordon’s review (1957) may be consulted.

11. HERMAPHRODITISM

Unlike other vertebrates, a number of teleost fishes are hermaphrodites. Atz (1964), among others, defined the types of hermaphroditism. An individual is hermaphroditic if it bears recognizable male and female tissues. If all, or nearly all, individuals possess both ovarian and testicular tissues, that species is hermaphroditic. Synchronous ( balanced) hermaphrodities are those in which the male and female sex cells ripen at the same time, regardless whether or not self-fertilization is possible. In consecutive ( metagonous) hermaphrodites there are two types: protogynous hermaphrodites that function first as females and then transform into males and protandrous hermaphrodites that transform from males into females. Atz lists 13 families of teleosts, belonging to five orders, that include species of these types. The transformation may be accomplished in several ways, depending upon the arrangement of sexual tissues (Reinboth, 1962, 1967; Smith, 1985, cf. Fig, 3).

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Johns, L. S., Liley, N. R., and Seghers, B. H. (1969). The effects of gonadectomy on the reproductive behaviour of the blue gourami, Trichogaster trichopterus. In preparation. Jones, J. W., and King, G. M. (1952). The spawning of the male salmon parr (Salmo salar Linn. juv.). PTOC.Zool. SOC. London 122, 615-619. Julesz, M. (1967). Recent results concerning the clinicopathology of the function of the pituitary, adrenal cortex and gonad. In “Symposium on Reproduction” ( K . Lissak, ed.), pp. 125-148. Akad. Kiado, Budapest. Kawakami, M., and Sawyer, C. H. (1959). Induction of behavioral and electroencephalographic changes in the rabbit by hormone administration or brain stimulation. Endocrinology 65, 631-643. Laskowski, W. ( 1954). Einige Verhaltensstudien an Phtypoecilus vaht us. Biol. Zentr. 73, 429-438. Leatherland, J. F., and Lam, T. J. (1969). Effect of prolactin on the density of mucus cells on the gill filaments of the marine form (trachurus) of the threespine stickleback, Gasterosteus aculeatus L. Can. J . Zool. In press. Lehrman, D. S. ( 1965). Interaction between internal and external environments in the regulation of the reproductive cycle of the ring dove. In “Sex and Behavior” (F. A. Beach, ed.), pp. 355-380. Wiley, New York. Levy, M., and Aronson, L. R. (1955). Morphological effects of castration and hormone administration in the male cichlid fish Tilapia macrocephah. Anat. Record 122, 450-451. Liley, N. R. (1966). Ethological isolating mechanisms in four sympatric species of poeciliid fishes. Behauiour Suppl. 13, 1-197. Liley, N. R. (1968). The endocrine control of reproductive behaviour in the female guppy. Poecilia reticulata Peters. Animal Behauiour 16, 318-331. Liley, N. R., and Donaldson, E. M. (1969). The effects of fish pituitary material on the behaviour of hypophysectomized female guppies, Poecilia reticulata Peters. Can. J . Zool. 47, 569573. Machemer, L., and Fiedler, L. (1965). Zur hormonalen Steurung des Schaumnestbaues beim Paradiesfisch, Macropodus operculuris L. ( Anabantidae, Teleostei ) . Naturwissenschaften 52, 648-649. Noble, G. K., and Borne, R. (1940). The effect of sex hormones on the social hierarchy of Xiphophorus helleri. Anat. Record 78, Suppl., 147. Noble, G. K., and Kumpf, K. F. (1936). The sexual behavior and secondary sex characters of gonadectomized fish, Anat. Record 67, Suppl., 113. Noble, G. K., and Kumpf, K. F. (1937). Sex reversal in the fighting fish, Betta splendens. Anat. Record 70, Suppl., 97. Noble, G. K., Kumpf, K. F., and Billings, V. N. (1938). The induction of brooding behavior in the jewel fish. Endocrinology 23, 353-359. Okada, Y. K., and Yamashita, H. (1944). Experimental investigation of the manifestation of secondary sexual characters in fish, using the medaka, Oryzias latipes, as material. J. Fac. Sci., Univ. Tokyo, Sect. IV 6, 383437. Oshima, K., and Gorbman, A. (1966a). OIfactory responses in the forebrain of goldfish and their modification by thyroxine treatment. Gen. Comp. Endocrinol. 7 , 39-09. Oshima, K., and Gorbman, A. (1966b). Influence of thyroxine and steroid hormones on spontaneous and evoked unitary activity in the olfactory bulb of goldfish. Gen. Comp. Endocrinol. 7, 482-491. Oshima, K., and Gorbman, A. (1968). Modification by sex hormones of the spon-

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taneous and evoked bulbar electrical activity in goldfish. J . Endocrinol. 40, 409-420. Pandey, S., and Leatherland, J. F. ( 1989). Effects of a dithiocarbamoylhydrazine derivative on the histology of the thyroid gland and adenohypophysis of the male guppy, Poecilia reticuluta Peters. In preparation. Picciolo, A. R. (1964). Sexual and nest discrimination in anabantid fishes of the genera Colisa and Trichogaster. Ecol. Monographs 34, 53-77. Pickford, G . E., and Atz, J. W. (1957). “The Physiology of the Pituitary Gland of Fishes.” N.Y. Zool. Society, New York. Raniaswami, L. S., and Lakshman, A. B. (1958). Spawning catfish with mammalian hormones. Nature 182, 122-123. Richards, M. P. M. (1967). Maternal behaviour in rodents and lagomorphs. In “Advances in Reproductive Physiology” (A. McLaren, ed.), Vol. 2, pp. 5 g 1 1 0 . Academic Press, New York. Sage, M. (1968). Respiratory and behavioral responses of Poecilia to treatment with thyroxine and thiourea. Gen. Comp. Endocrinol. 10, 304309. Scharrer, E.,Smith, S. W., and Palay, S. L. (1947). Chemical sense and taste in the fishes Prionotus and Trichogaster. 1. Comp. Neurol. 86, 183-198. Shirai, K. (1962). Correlation between the growth of the ovipositor and ovarian conditions in the bitterling, Rhodeus ocellatus. Bull. Fac. Fisheries, Hokkaido Univ. 13, 137-151. Shirai, K. ( 1964). Histological study on the ovipositor of the rose bitterling, Rhodeus ocellatus. Bull. Fac. Fisheries, Hokkaido Univ. 14, 193-197. Smith, R. J. F. (1967). Endocrine control of prespawning behaviour in two species of sunfish, Lepomis megalotis and L. gibbosus. Ph.D. thesis, Lawson Library, University of Western Ontario, London, Ontario. Smith, R. J. F., and Hoar, W. S. (1967). The effects of prolactin and testosterone on the parental behaviour of the male stickleback Gasterosteus muleatus. Animal Behaviour 15, 342452. Sneed, K. E., and Clemens, H. P. (1959). The use of human chorionic gonadotrophin to spawn warm-water fishes. Progressioe Fish Culturist 21, 117-120. Sneed, K. E., and Dupree, H. K. (1961). The effect of thyroid stimulating hormone combined with gonadotropic hormones on the ovulation of goldfish and green sunfish. Progressive Fish Culturist 23, 179-182. Stanley, L. L., and Tescher, G . L. (1931). Activity of goldfish on testicular substance diet. Endocrinology 15, 55-56. Stevens, R. E. (1966). Hormone induced spawning of striped bass for reservoir stocking. Progressive Fish Culturist 28, 19-28. Sundararaj, B. I., and Goswami, S. V. (1966). Effects of mammalian hypophysial hormones, placental gonadotrophins, gonadal hormones, and adrenal corticosteroids on ovulation and spawning in hypophysectomized catfish, Heteropneustes fossilis (Bloch). 1. Ezpptl. Zool. 161, 287-296. Tavolga, M. C. (1949). Differential effects of estradiol, estradiol benzoate and pregnenolone on Platypoecilus maculatus. Zoologica 34, 215-237. Tavolga, W. N. (1955). Effects of gonadectomy and hypophysectomy on prespawning behavior in males of the gobiid fish Bathygobius soporator. Physiol. Zool. 28, 218-233. Tavolga, W. N. (1956). Visual, chemical and sound stimuli as cues in the sex discriminatory behavior of the gobiid fish Bath ygobius soporator. Zoologica 41, 49-64.

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van den Assem, J. ( 1967). Territory in the three-spined stickleback (Gasterosteus aculeatus L. ) , An experimental study in intraspecific competition. Behaoiour Suppl. 16, 1-164. van Iersel, J. J. A. (1953). An analysis of the parental behavior of the male threespined stickleback ( Gasterosteus aculeatus L. ). Behauiour Suppl. 3, 1-159. van Mullen, P. J. (1967). On synchronization in the reproduction of the stickleback ( Gasterosteus aculeatus L. forma leiura Cuv. ) . Arch. N e e d Zool. 27, 258-274. Vivien, J. H. (1941). Contribution a I'6tude de la physiologie hypophysaire dans ses relations avec l'appareil ghital, la thyroide et les corps suprarknaux chez les poisons sblacians et t616ostkns Scylliorhinus canicula et Gobius paganellus. Bull. Bid. France Belg. 75, 257-309. Wai, E. H., and Hoar, W. S. (1963). The secondary sex characters and reproductive behaviour of gonadectomized sticklebacks treated with methyl testosterone. Can. J. Zool. 41, 611-628. Wai, E. H., and Hoar, W. S. (lSe8). Unpublished study. Wiebe, J. P. (1967). The reproductive physiology of the viviparous seaperch Cymtogaster aggregata Gibbons. Ph.D. thesis, University of British Columbia, Vancouver, Canada. Wiebe, J. P. (1968). Inhibition of pituitary gonadotropic activity in the viviparous seaperch Cymatoguster aggregata Gibbons by a dithiocarbamoylhydrazine derivative (I.C.I. 33,828). Can. J. Zool. 46, 751-758. Wilhelmi, A. E., Pickford, G. E., and Sawyer, W. H. (1955). Initiation of the spawning reflex response in Fundulus by the administration of fish and mammalian neurohypophysial preparations and synthetic oxytocin. Endocrinology 57, 243-252. Wootton, R. J. (1968). A comparison of measures of aggression in the male threespined stickleback. Ph.D. thesis, University of British Columbia, Vancouver, Canada. Yamamoto, T. (1962). Hormonic factors affecting gonadal sex differentiation in fish. Gen. Comp. Endocrinol. Suppl. 1, 341355. Yamamoto, T., and Matsuda, N. (1963). Effects of estradiol, stilbestrol and some alkyl-carbonyl androstanes upon sex differentiation in the medaka, Oryzias kztipes. Cen. Comp. Endocrinol. 3, 101-110. Yamazaki, F. (1962). Effects of hypophysectomy on the ovulation, oviposition and sexual behavior in the goldfish, Carassius auratus. Bull. Fac. Fisheries, Hokkaido Unio. 13, 3946. Yamazaki, F. (1965).Endocrinological studies on the reproduction of the female goldfish Carussius uuratus L., with special reference to the function of the pituitary gland. Mem. Fac. Fisheries, Hokkaido Uniu. 13, 1-64. Yamazaki, F., and Donaldson, E. M. ( 1968a). The spermiation of goldfish (Carassius auratus) as a bioassay for salmon ( Oncorhynchus tshawytscha) gonadotropin. Gen. Comp. Endocrinol. 10, 383-391. Yamazaki, F., and Donaldson, E. M. (1968b). The effects of partially purified salmon pituitary gonadotropin on spermatogenesis, vitellogenesis, and ovulation in hypophysectomized goldfish ( Carussius auratus) , Gen. Comp. Endocrinol. 11, 292-299. Yamazaki, F., and Donaldson, E. M. ( 1 9 6 8 ~ )Personal . communication.

3 SEX DIFFERENTIATION TOKI-0 YAMAMOTO It thus becomes of great interest to discaver the mechanism by which sex is determined. and to find whether by any means we can bring it under our control . Julian Huxley ( 1938) I . Introduction: Sexuality in Fishes . . . . . . . I1. Hermaphroditism . . . . . . . . . . A . Synchronous Hermaphroditism B . Consecutive Hermaphroditism . . . . . . . I11. Gonochorism . . . . . . . . . . . A . Undifferentiated Gonochorists . . . . . . . B. Differentiated Gonochorists . . . . . . . C . All-Female Species . . . . . . . . . IV . Genetic Basis of Sex Determination . . . . . . A . XX-XY and WZ(Y)-ZZ(YY) Types . . . . . . B. Polygenic Sex Determination and So-called . . . . . . . . Genetic Sex Reversal . C. “Spontaneous Sex Reversal” in the Swordtail . . . . V. Control of Sex Differentiation . . . . . . . . A . Surgical Operation . . . . . . . . . B . Modification of Sex Differentiation by Sex Hormones . . C. Complete (Functional) Reversal of Sex Differentiation . . VI . Nature of Natural Sex Inducers . . . . . . . A . Steroid versus Nonsteroid Theories . . . . . . B . Detection of Steroids and Relevant Enzymes in Fish Gonads . . . VII . Differentiation of Secondary Sexual Characters . VIII . Summary . . . . . . . . . . . . References . . . . . . . . . . . . .

. . . . . . .

117 118 119 120 127 127 129 129 131 132 134 139 1 4 142 142 144 150 150 153 153 157 158

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I INTRODUCTION: SEXUALITY IN FISHES

Members belonging to the class Pisces exemplify an almost complete range of the various types of sexuality from synchronous her117

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maphroditism, protandrous and protogynous hermaphroditism, to gonochorism. At the outset, it is necessary to clarify the concept of sex. The male and female can best be defined as sperm and egg producers, respectively. This definition, although self-evident, is needed to correct statements that appear frequently in literature, with respect to spontaneous sex reversal in gonochorists (Sections IV, B and C). The term “bisexuality” used to denote hermaphroditism is likely to lead to a misunderstanding. This term should be used to describe gonochorist. The term “intersex” (Goldschmidt, 1915, 1927, 1931) is used in the present review to denote either sporadically appearing or experimentally produced hermaphroditic individuals of a species in which all or nearly all individuals are gonochoristic. Whether hermaphroditism is the more primitive condition from which bisexuality or gonochorism may have arisen or a specialization derived from the more usual vertebrate gonochorism is a matter for debate. The solution of this intriguing problem will require a great deal more information in the future. Nevertheless, fishes provide excellent material to approach the problems of sex differentiation and of evolution of sex among animals. In this study, stress is laid on sex differentiation as a process rather than examining sex phenotypes as they appear in adults. For the latter approach, Gordon’s review (1957) may be consulted.

11. HERMAPHRODITISM

Unlike other vertebrates, a number of teleost fishes are hermaphrodites. Atz (1964), among others, defined the types of hermaphroditism. An individual is hermaphroditic if it bears recognizable male and female tissues. If all, or nearly all, individuals possess both ovarian and testicular tissues, that species is hermaphroditic. Synchronous ( balanced) hermaphrodities are those in which the male and female sex cells ripen at the same time, regardless whether or not self-fertilization is possible. In consecutive ( metagonous) hermaphrodites there are two types: protogynous hermaphrodites that function first as females and then transform into males and protandrous hermaphrodites that transform from males into females. Atz lists 13 families of teleosts, belonging to five orders, that include species of these types. The transformation may be accomplished in several ways, depending upon the arrangement of sexual tissues (Reinboth, 1962, 1967; Smith, 1985, cf. Fig, 3).

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A, Synchronous Hermaphroditism Dufoss6 (1854, 1856) found that Serranus scriba (Serranidae) is a synchronous, functional hermaphrodite. This was confirmed by van Oordt ( 1929). D’Ancona’s intensive studies ( 1949a,c,d, 1950) revealed that other Mediterranean serranids such as S. cabrilla and Hepatus hepatus belong to this category. The gonad of these fishes is separated into the ovarian and testicular areas (Fig. 1).D’Ancona ( 1949a,c,d, 1950) suggested the possibility of self-fertilization. Clark (1959, 1965) in S . subligerius, a Florida serranid proved that self-fertilization and development are possible. In captivity, an isolated individual can emit sperm and fertilize its own eggs. In both nature and in the aquaria containing two or more fish, the fish may form spawning pairs. Immediately after one fish in the female phase spawns, the partner fertilizes the eggs. At this moment, the color pattern (vertical stripes) in the female phase changes to that of the male phase. Then, the first fish reverses its sexual role and acts as a male. Of serranid fishes from Bermuda studied by Smith (1959), four species (one belonging to the genus Hypoplectrus and three of the genus

Fig. 1. Transverse section of the gonad of the synchronous hermaphrodite, Serranus scribn (Serranidae). Upper portion is the ovary and lower region the testis. After D’Ancona ( 1950).

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Prinodus) are synchronous hermaphrodites while at least nine species are protogynous hermaphrodites (see Section 11, B, 2). Harrington (1961, 1965) demonstrated that most Aiuulus marmomtus, an oviparous cyprinodont, are genuine hermaphrodites capable of internal self-fertilization. It is remarkable that over 10 uniparental generations have been propagated and each fish has been kept in lifelong isolation ab ouo. Tissue grafts between the parent and its offspring and among siblings were histocompatible, thus providing tight evidence of self-fertilization. According to Harrington ( 1967), low temperature ( 18"20°C)tends to transform the hermaphrodites to males. The order Perciformes includes four families ( Serranidae, Sparidae, Centracanthidae, and Labridae) in which either synchronous or consecutive hermaphroditism occurs normally. In all these families, however, there are some gonochoristic species. Mead (1960) and Gibbs (1960, cited in Atz, 1904) discovered that several fishes of four families belonging to the order Myctophiformes (also called Iniomi) are hermaphroditic (cf. Atz, 1964), Some of Meads fish gave evidence of being synchronous hermaphrodites. However, at least five of the remaining dozen families in the order are gonochoristic.

B. Consecutive Hermaphroditism 1. PROTANDROUS HERMAPHRODITES Since the time of Syrski (1876) and Brock (1879), it has been known that the Mediterranean bream, Sparus auratus, is a protandrous hermaphrodite. Pasquali (1941) and D'Ancona (1941) described the precise process of gonadal differentiation from male to female phases. They demonstrated that its gonad consists of both testicular and ovarian areas from a very young stage. In smaller fish the lateroventral testicular region predominates over the ovarian zone, and in larger fish the reverse is true (Fig. 2). Similar patterns have been reported by Syrski ( 1876), McLeod (lSSl), Hoeck (1891a,b), Williamson (1910), and van Oordt (1929). The latter author found in Sargus (Diplodus) annularis that there are some individuals in which the sexes are separated, Le Gall (1929) found that Pagellus centrodontus is a protandrous hermaphrodite. D'Ancona (1949a-d, 1950, 1956) showed that some sparids such as Sparus auratus, Sargus (Diplodus) sargus, and Pagellus mormyrus are protandrous hermaphrodites (see Fig. 2), while others such as S. annuluris, S. uulgaris, Puntazzo ( C h a r m ) puntazzo, Boops boops, Obludu

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Fig. 2. Transverse sections of the gonad of the protandrous hermaphrodite, Sparus auratus (Sparidae). ( A ) Male phase, ( B ) transitory phase, and ( C ) female phase.

Here Ov stands for ovary and Tes for testis. After D’Ancona ( 1950).

mlanulu, and Dentex dentex are rudimentary hermaphrodites. Pagellus a c a m is protandrous ( Reinboth, 1962). The classification of Japanese sparids is notoriously unsatisfactory. Here, the writer follows Dr. Abe’s recent personal communication ( 1967). Kinoshita (1936) reported “sex reversal” from male to female in Acanthopagrus schlegeli (syn. Sparus longispinis, Mylio mucrocephalus). He ( 1939) also reported sex reversal in A. ( Sparus, Mylio) latus and Sparus sarba (Sparus arks, Rabdosargus sarba). He remarked that not all individuals transform from male to female, i.e., some males retain maleness even when they become large. Okada’s histological study (1952b) indicates that an ambisexual organization is present in the early stages, so that the situation is similar to the reports of DAncona and others. In the flat-head fish, Inegocia (Cociella) crocodila (Platycephalidae), Aoyama et al. (1963) examined a large number of individuals ( >lOOO) caught by trawls in the East China Sea and the Yellow Sea. Small individuals had testes and medium-sized fish hermaphroditic gonads with functional testes, whereas large individuals had ovaries. Another flat-head fish, Inegocia (Suggrundus) merderuoort, is also a protandrous hermaphrodite (Aoyama and Kitajima, 1966). Okada (1966) postulated that this form repeats the hermaphroditic state. In Gonostomu gracile ( Gonostomatidae ) , a deep-sea luminescent fish, Kawaguchi and Marumo (1967) found that individuals less than 7 cm are mostly males and those more than 9 cm are invariably females. Sex

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succession takes place in the medium-sized fish (7-9 cm), and hermaphrodites are frequently found in the specimens of 6 7 cm. 2. PROTOGYNOUS HERMAPHRODITES

Certain fishes belonging to the Sparidae, i.e., Pagellus erythrinus ( DAncona, 1950; Larrafieta, 1953, 1964) and Spondyliosomu (Cantharus) canthurus ( D’Ancona, 1950, 1956; Reinboth, 1962) are protogynous hermaphrodites. Among Japanese sparids, Dentex ( Taius) tumifrons seems to be protogynous. Inversion of sex is brought about by development of the testicular region and regression of the ovarian part. In Dentex ( Taius) tumifrons ( now Sparidae), Aoyama ( 1955) found fishes with hermaphroditic gonads. He concluded that some of the females change into males. Of serranids from Bermuda four species were synchronous hermaphrodites, as already stated, while at least nine species belonging to the genera Epinephelus, Mycteroperca, Alphes, Petrometopon, and Cephalopholis were protogynous hermaphrodites ( Smith, 1959). Smith pointed out that there are three patterns of hermaphroditism in serranids: the Serranus, Rypticus, and Epinephelus type (Fig. 3 ) . The patterns are essentially similar to those hermaphrodites reported by Reinboth (1967; cf. Fig. 3 ) . Epinephelus and its allies have gonads in which the male tissue is present throughout the germinal epithelium lining the central lumen of the gonad. This male tissue becomes functional only after the female tissue has ceased to function. The genus Rypticus shows an intermediate type of gonad in which scanty male tissue is present in the lower part but is also found intermixed with the female tissue. The protogynous Chelidoperca hirundinacea is also of this type ( Reinboth, 1967). The eastern Pacific Paralabrax chthratus seems to be secondarily gonochoristic. Here, we can make an inference on the process in evolution of gonochorism from hermaphroditism. Figure 3 illustrates the three types. Kuroda (1931) postulated that larger and red Sacura margaritacea (Serranidae) and smaller and yellow S. pulcher are males and females, respectively, of the same species. He found some intermediate individuals. The protogynous hermaphroditism in this species was demonstrated by Reinboth ( 1963) and Okada ( 1965a,b). Protogynous sex reversal in this species seems to occur by degeneration of ovarian tissue after spawning. The Atlantic sea bass, Centropristes striatus ( Serranidae), is also a protogynous hermaphrodite ( Lavenda, 1949; Reinboth, 1965). In Centracanthidae ( Maenidae ) , three species Spicara smuris, S . chryselis, and S . m e n u , of the genus Spicara (Maena) are known to occur in the

Fig. 3. Gonadal development and phylogenetic relations in serranid fishes, Redrawn after Reinboth (1987).

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Mediterranean. Zei (1949) has postulated that the two species S . smuris and S . chryselis are protogynous hermaphrodites. Lozano Cab0 (1951, 1953) confirmed this in S. smaris and concluded that S. smuris and S . akedo are two sexes of the same species. Lepori (1960) also conchded that S . maena and S . chryselis are protogynous species. Reinboth (1962) performed an elaborate study on S. maenu. For a long time the greenish and reddish Japanese wrasses (Labridae) have been regarded as different species; the former was called Julis poecilopterus and the latter I. pyrrhogrammu. Jordan and Snyder (1902) suggested that the two forms might be sex variants of the same species, Halichoeres poecilopterus. Y. Kinoshita ( 1934,1935,1936) has verified this experimentally. He observed that the blue wrasse rapidly loses its secondary sex characters after castration, while the red wrasse is not affected by ovariotomy. Transplantation of a testis into the red wrasse transforms it into a blue wrasse. However, transplantation of an ovary into the blue wrasse causes no change in coloration of the recipients. Kinoshita found testis-ova in some individuals and postulated that this fish may be protogynous. Okada (1962) corroborated this. He (1964b) performed experiments on the effects of androgen and estrogen on sex reversal. The Mediterranean labrid fishes Cork giofredi and C . julis have long been regarded as separate species. The two types differ in both size and color (Fig. 4 ) . Smaller individuals (C. giofredi) are usually females and larger ones (C. julis) are males. Some medium-sized fish show intermediate color and have testes (Bacci and Razzauti, 1957, 1958). The two types are the same species, C. julis, a protogynous hermaphrodite. An interesting fact is that a few large fish with C. giofredi coIor are nevertheless maIes when their gonads are examined. Because each individual is at all times either a male or female, this labrid is termed a “false gonochorist.” The two species of labrids in the waters of the Mediterranean around Livomo, Labrus turdus and L. merula, are also protogynous species Sordi, 1962). In both species, all individuals less than 27 cm have ovaries. In the largest fishes of L. turdus, all individuals have testes. In L. merula, the largest individuals have a balanced sex ratio. This seems to indicate that while 50%of the fish change from female to male, 504: never change but remain females. In both Coris julis and a Caribbean labrid, Thalassomu bifasciatum, Roede (1965) observed 70% 0 o 30%d d in the young and 100%d d in adults. Reinboth (1962) pointed out the existence of two types of morphologically distinct males in labrids (Coris julis and Haliochoeres poecilopterus). The primary male looks like a female but remains a male through-

+

3. SEX

DIFFERENTIATION

Fig. 4. Change of color and sex phase in the labrid, Cork iulis. After Bacci, 1966: “Sex Determination,” Fig. 7.1. Reprinted with permission from the author and Pergamon Press.

out its life, and the secondary male that changes to a male from the female. However, according to Vandini (196!5), there are no primary males that retain the C . giofredi color throughout their life cycle, viz., even the C . gwfredi males eventually develop C. iulis color. In the striped wrasse, Labrus ossifagus, there are some individuals with a red and others with a blue pattern. It was believed that the former is the female and the latter the male. Lonneberg and Gustafson (1937) reported that females greatly outnumber males in red fish and the reverse is true in blue-striped fish. They also found intersexual individuals changing from femaIes to males. This seems to indicate that this form is protogynous and most of these fish, if not all, exhibit sex succession. Liu (1944) discovered that small individuals of the synbranchoid eel, Monopterus albus ( M . jauanensis), are females and the large ones males and offered a good case for protogynous hermaphroditism (cf. also Bullough, 1947). Liem (1963, 1985) confirmed this using nearly 1000 fish, and

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concluded that every individual starts its reproductive cycle as a functional female and then becomes a functional male. The sexuality of this fish has the following sequence: juvenile hermaphrodite + functional female + intersex + functional male. In consecutive hermaphrodites such as Sparidae and Serranidae, either protandrous or protogynous, there is a basic feature in common. The juvenile gonad has an ambisexual organization. From the very beginning of differentiation of the gonad, ovarian and testicular rudiments are present in every fish, although the topographical arrangement of the male and female tissues and change of the dominant tissue in time differ from species to species (cf. Figs. 3 and 5 ) . A question arises as to whether all individuals in a consecutive hermaphroditic species inverse sex in sequence. In observations made on natural populations, occasional individuals are found smaller or larger than is usual for this sex. Smith (1959) noted unusually large females in some protogynous serranids, and Reinboth (1965) reported unexpectedly small males of Centropristes striatus, also a protogynous species. In Dentex dentex only some individuals undergo a transitional hermaphroditic stage ( Lissia Frau, 1964). According to Larrafieta ( 1964) about 5% of the fish in a population of Pagellus erythrinus are males throughout their life, while 45%transform from female to male and 50% never transform but remain females. Rijavec and Zuvanovic (1965) obtained comparable results in the same species. Smith (1967) proposed a

Fig. 5. Schematic transverse sections of early developmental stages ( A + E ) of the gonad in Sparidae, where ov stands for ovogonia; sp, spermatogonia; gc, gonocoelom; ef, efferent ducts; lc, lacunae; and bv, blood vessels. Redrawn from D’Ancona ( 1950).

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theory of hermaphroditism in which he postulated that sex reversal takes place in different individuals at different sizes and ages viz., sexual succession is a prolonged continuous process for the population. “During the first time interval following sexual maturity, only a part of the population changes sex, and during each time period a fraction of the remaining individuals transform.” He interpreted Larraiieta’s results as indicating that 5%of the individuals change before sexual maturity and that the life span is such that half the females do not live to change. Researches on consecutive hermaphrodites have been mostly based on observations on natural populations. There are only a few experimental or physiological strides and these are not satisfactorily documented. Hence, we know little about the cause of sex reversal. It seems that sex reversal may be caused by sex hormone imbalances. In the female phase the female hormone might dominate the male hormone and in the male phase the reverse might be true.

111. GONOCHORISM

As Witschi (1914a,b, 19.30)has pointed out for amphibians so also in gonochoristic fishes there are “undifferentiated and “differentiated species. In the former, the indifferent gonad first develops into an ovarylike gonad and then about one-half of individuals become males and the other half females. In the latter, the indifferent gonad directly differentiates into either a testis or an ovary. In both types, sex differentiation seems to be brought about by male- and female-inducing substances (Section VI, A ) . It is natural that undifferentiated species are more unstable than differentiated ones in sexuality. Unfortunately, however, the two types have been studied embryologically in only a few fishes. In the absence of embryological evidence, it is as yet hazardous to correlate the two modes with the occurrence of sporadic intersexes which represent a remnant of the embryological condition manifested late in some adults. However, a few species in which undifferentiated and differentiated conditions are known provide evidence that spontaneous intersexes among gonochorists occur mostly in the “undifferentiated” species while in the “differentiated ones the occurrence of intersex is rarely or never seen. A. Undifferentiated Gonochorists The lampreys and hagfishes ( Agnatha ) are undifferentiated species. Sexuality of the brook lamprey, Lurnpetra lumottei (Entosphenus

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wildmi), has been studied by Lubosch (1903) and more extensively by Okkelberg (1921). Ammocoete larvae up to 3.5 cm possess indifferent gonads. After this stage, some of the larval gonads contain both male and female germ cells. Of 15 adult males (sperm producers) undeveloped ova were found in the testis of seven. In ammocoete larvae of L. planeri and L. fluuiatilis, differentiation of the gonads is mostly completed at 5-6 cm in length. Hardisty (1960) recorded the sex ratio in animals longer than 6 cm. In L. planeri ammocoetes there is a small but significant excess of females. There are a number of transitional individuals with disintegrating oocytes and developing stroma, which may eventually become males. Among hagfish, Atlantic species, Myxine glutinosa, seems to be undifferentiated. Conel's ( 1917) and Schreiner's (1904, 1955) reports seem to indicate that this species is an intermediate type between hermaphroditism and gonochorism, in which juvenile hermaphrodites are common. The Japanese hagfish, Eptatretus burgeri, is also an undifferentiated gonochorist. Dean (cited by Conel, 1931) found only one male with an ovotestis out of 569 fish. Turning to the class Pisces, there are many undifferentiated gonochorists which normally develop into either male or females. However, sporadic intersexes are known to occur in these fishes. It is beyond the scope of the present chapter to cite a vast number of instances; Freund (1923) and Atz ( 1964) have listed numerous cases. Grassi (1919) in the eel, Anguilla anguilla, and Mrgid (1923, 1930) in the rainbow trout, Salmo gairdneri irideus, have demonstrated that these species are undifferentiated. In the latter, accidental intersexes have been reported not infrequently. In Gadidae, sporadic intersexes have also been frequently reported. In the herring, Clupea harengus, there are several types of the intersexual gonad (Gabler, 1930; Rudolf, 1931). In the minnow, Phoxinus laeuis, Bullough (1940) found 10 fish with intersexual gonads. In all cases the ovarian portion was suppressed while the testicular region was normal. He thought that these intersexes represented transitory stages changing from female to male. The effects of an androgen and an estrogen supported this postulation. However, a complete sex reversal was not achieved with these hormones. These are three species of the paradise fishes: Macropodus operculark,M . chinensis, and M . concolor; all three are undifferentiated species. Whereas the male index ( o o J o o + d d ) of M. opercularis and M . chinensis is 50$, that of M . concolor varies from 68% to 91%;thus there is a preponderance of males (Schwier, 1939). The presence of multiple sex factors in M . concolor is apparent. While the offspring from M . opercularis X M . chinensis are fertile, males from M . concolor X M . chinensis are sterile.

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The live-bearing cyprinodont Poecilia ( formerly Lebistes) reticulata is an undifferentiated gonochorist ( Goodrich et al., 1934; Dildine, 1936). Blacher (1926a) and Winge (1927) found a few aged intersexes with an XX constitution. Eggert’s report (1931, 1933) on intersexes of the mudskipper, Periophthalmus vulgaris ( Gobiidae) , seems to suggest that there are two geographical races in the same species. In the fish on the south coast of Java, the two sexes differentiate independently from the start, whereas on the north coast the testes of young have basically ovarian structure indicating that the male pass through the female phase. However, number and sizes of specimens examined by him were very limited.

B. Differentiated Gonochorists Only a few fishes have been shown to be differentiated gonochorists. Sexuality of differentiated species is fairly stable among fishes. Wolf ( 1931) demonstrated that the “domesticated platyfish, Xiphophows ( formerly Pktypoecilus ) maculatus, is a differentiated species. Bellamy and Queal (1951) stated that not a single sporadic intersex has ever been found among 50,000 specimens. The situation is the same in the medaka, OTYZ~US latipes (Cyprinotontidae ), No spontaneous, true intersex has ever been found among more than thirty thousand fish studied during 40 years. Yamamoto (1953) showed that this species is a differentiated gonochorist. In this connection, Oka’s report (1931b) on the occurrence of oviform cells in three males of this species may be mentioned. His finding was based on fish which had been left to starve for 3 months. It is unfortunate and even strange that the title of this paper included the words “accidental hermaphroditism” since Oka carefully observed the cytological difference between these enlarged cells and genuine ovocytes, calling them “pseudoovocytes.” It is probable that these cells are enlarged proto- or spermatogonia prior to degeneration, As to the so-called testis-ova in this species, induced by various agents, comments are given in Sections V, C and VI, A.

C. All-Female Species The viviparous toothcarp, Poecilia ( Mollienisia) formosa, which is thought to be a form of hybrid origin, inhabits northeastern Mexico and southwestern Texas. The “all-female’’ phenomenon in this form was first found by C. L. Hubbs and Hubbs (1932, 1946). In natural habitats,

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it propagates itself by mating with sympatric, related bisexual species, either P . latipinnu or P . sphenops. However, paternal characters are not transmitted to off spring which are solely females. Subsequently, this fact has been confirmed by Meyer (1938), C. Hubbs et al. (1959), Haskins et al. (lWO), C . Hubbs (1964), Kallman (1962, 1964a,b). C. L. Hubbs and Hubbs, and Kallman concluded that the monosexuality is the result of gynogenesis, the mate providing a stimulus to activate development of the ovum without syngamy. Nevertheless, there is strong evidence that the species is diploid (cf. C. Hubbs et al., 1959; Kallman, 1962). In terms of DNA levels, P. formosu nucleus is the same as in the diploid spaces P. sphenops (Rasch et al., 1965). C . Hubbs et al. (1959) discovered a single wild male in the Brownville population, and Haskins et al. (1960) reported a single male in their laboratory stock. It proved fertile in mating to P . formosu siring all females. The appearance of extremely rare males is still a matter for debate. In this connection, Kallman’s study (1964b) deserves attention. He found ammg thousands of off spring from gynogenetic P . formo~amated with males of P . vituttu or P . sphenops 14 exceptional fish of which 12 exhibited paternal patterns and possessed morphologically intermediate features while the remaining two had mosaic patterns with some areas patriclinous and others matriclinous. This shows that in rare cases syngamy occurs or a single chromosome from the male nucleus, governing pattern formation, may become accidentally incorporated into some or all cells of developing embryos giving rise to mosaic patterns. Gynogenesis has been reported in certain natural populations of the crucian carp, called the “silver goldfish,” Carrussius auratus gibelio, which produce all-female progeny (Lieder, 1955, among others). He regarded this phenomenon as a natural parthenogenesis. Artificial insemination revealed that a gynogenetic stimulus to the ova can be provided by the common goldfish, C. aurutus uuratus, or by the carp, Cyprinus carpio. Spurway ( 1953) claimed the occurrence of “spontaneous parthenogenesis” in two anomalous guppy females. Later ( Spurway, 1957) this postulation was withdrawn and she assumed that the phenomenon results from the self-fertility of “functional” intersexes. Subsequent to Spurway’s study, Stolk ( 1958) reported pathological gynogenesis observed in the guppy and the swordtail. The ovary of each female was infested by a phycomycete fungus, Ichthyophorus hoferi, which apparently was responsible for the pathological activation of the ova. More than 64 broods were produced by these fishes; these without exception were daughters. Stolk criticized Spurway’s finding by saying that her fish might also be infected by the parasite. The mode of reproduction of the two unisexual “species” or strains of the genus Poeciliopsis, viviparous toothcarps of northwestern Mexico,

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131

is unique to vertebrates (Miller and Schultz, 1959; Schultz, 1961, 1966). At first, it was considered that each of the two undescribed species had both bisexual and unisexual strains. Bisexual strains were tentatively referred to as C and F and unisexual ones as Cx and Fx, respectively. Miller (1960) identified the C and Cx as P . lucida, but F and Fx are still undescribed. Later, Schultz (1966) showed that monosexual Cx is a form different from the bisexual P . lucidu but closely allied to it. In nature, Cx propagates itself by mating with P . lucida. Hybridization experiments between Cx and males of two bisexual species P . lucidu or P . latidens provided evidence that genetic factors of both parent combine to form the F1 offspring. However, the entire male genome appears to be eliminated during ovogenesis, probably at meiosis. Hence, ova have no paternal genome in each generation. Besides Cx, Schultz ( 1967) further reported two additional all-female stocks, Cy and Cz, which were previously thought to be Cx. Poecilia Zucida provides sperm for these monosexual forms. While Cx and Cz are diploid expressing characteristics of both parents in the F,, Cy is a triploid and in mating with males of various bisexual species, e.g., P . latidens, produces all-female, triploid offspring by gynogenesis devoid of paternal characters. Thus, the mode of reproduction of Cy is quite different from that of Cx and Cz in which paternal chromosomes of the F, generation are not transmitted through the ova; only those characteristics of the female germ line pass to the next generation. In gynogenetic diploid forms, the diploid complement might be maintained by suppression of one of the meiotic divisions, reentry of the second polar body, or suppression of the first mitotic cleavage. The means by which the triploid Cy undergoes meiosis and produces fertile triploid eggs is obscure. At this point, it may be mentioned that Rasch et al. (1965) mated the gynogenetic, diploid Poecilia formosa to P. vittata and obtained offspring which were triploids as judged by their nuclear DNA. However, these triploids were sterile. IV. GENETIC BASIS OF SEX DETERMINATION The genetic study of sex is important not merely because sex is instinctively our major interest, but it lies at the root of Mendelian heredity itself and is the major factor in evolution. It provides such admirable material for the study of gene interaction, of phenogenetics, that is, of developmental physiology. Abbreviated from H. J. Muller (1932)

The classic sex factor studies use notations such as FF = ? ,FM = d , heterogametic species, and F M = 0 , MM = d, where M > F in

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where F > M in o heterogametic forms, which are oversimplified and are not generally found in nature, if the F and M symbolize single sex determiners (genes), Hartmann ( 1951, 1956) has proposed the formulas ( 8 G ) F F = o and (A@)MF = d , where the M and F represent the sex determiners (realizers), the AG stands for ambisexual potencies and the symbols # and $ represent the inhibiting effects exerted by the combinations of sex realizers. The supposed existence of the double system is also a formalization and has been a matter for debate since experiments on the localization of the AG complex have not been fruitful. Kosswig (1964) is also an opponent to these formulas.

A. XX-XYand WZ(Y ) -ZZ( YY) Types At this point, it may be appropriate to comment on the designation of sex chromosomes. The symbols in male heterogametic forms, that is, XX for female and XY (or XO) for male, are in universal agreement. Fishes which were found to have this type are listed in Table I. Among fishes, those which have this type are more numerous than those with female heterogamety. On the other hand, in organisms with female heterogamety, some Table I Fishes with Male Heterogamety (XX 0 , XY 3 ) Based Mostly on Genetic Evidence Species Oryzias latipes Poecilia" reticulata Poeciliab nigrofasciata Xiphophorus variatus X . ziphidium X . maculatusc X . couchianus X . milleri X . montszumae cortezi Betta splendens Mogrunda obsculad COttllS polluxd Carassius auratus'

Family

Author

Year

Cyprinodontidae Poeciliidae Poeciliidae Poeciliidae Poeciliidae Poeciliidae Poeciliidae Poeciliidae Poeciliidae Anabantidae Gobiidae Cottidae Cyprinidae

Aida Winge Breider Kosswig Kosswig Gordon Gordon and Smith Kallman Kosswig Kaiser and Schmidt Nogusa Nogusa Yamamoto and Kajishima

1921 1922 1935 1935 1935 1946 1938 1965 1959 1951 1955 1957 1969

Formerly Lebistes. Formerly Limia. Mexican populations. d Cytologically established. Cultivated goldfish.

a b 0

(I

3.

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SEX DIFFERENTIATION

Table I1 Symbols of Sex Chromosomes in the ? Heterogametic Platyfish, Xiphophorus maculatzcs" ~

0

3

Author

Year

Stock

XY

wz

xx zz

1922 1951 1926

Domesticated Domesticated Domesticated

XY WY WY WY

YY YY YY YY

Bellamy Bellamy and Queal Gordon, Kosswig, Breider, Bellamy Castle Gordon Gordon Kallman

1936 1947 1950 1965

Domesticated Domesticated Belize river Rio Hondo

a

From several authors, but chiefly Gordon (1952)

authors use the formula WZ( OZ) P -ZZd, while others, including outstanding geneticists, use the formula XY (or XO) ?-XXd. The latter denotation is not only perplexing but also is based on the misleading assumption that the Z = X and W = Y, which is in contradiction to the experimental facts revealed in fishes. This notation becomes seriously confusing when matings are made between male heterogametic and female heterogametic species or races. In Table 11, symbols used for the female heterogametic platyfish by several authors are listed. As a result of the studies that follow, the symbol XY P -XX$ should be eliminated. In connection with the Bellamy's experiment (1936) of the interspecific matings between P heterogametic Xiphophorus maculatus and heterogametic X . variatus, Castle (1936) suggested use of the terms XY for P and YY for in the 9 heterogametic species, where the Y is equivalent to the Z and the X to the W. In fact, Gordon (1946a,b, 1947a) proved experimentally in xiphophorin fishes that the Z = Y. However, since there was no evidence that W = X, he suggested that the formula WZ 0 -ZZd might have been better written as WY Q -YY d. German authors continue to use the WZ-ZZ system, whereas American investigators, particularly Kallman (1965b), stress that it is best to eliminate the symbol Z. The W of ? heterogametic forms is considered to have strong F gene(s), and the Y of a" heterogametic forms is considered to have strong M gene(s). In this connection, it may be noted that in the interspecific mating between o heterogametic X . maculatus ( WZ-ZZ) and 8 heterogametic X. variatus (XX-XY), Kosswig (1935a) stated that the Z of the former behaved like the Y of the latter, although at that time he considered that Z = X and W = Y. Heterosomal sex-determining mechanisms have often been called

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“monogenic.” This term is inadequate at least in fishes since sex is “determined indirectly by the totality of sex genes in heterosomes and autosomes. Polygenic sex determination with or without heterosomes will be discussed later (Section IV, B). Female heterogamety in the “domesticated” platyfish, Xiphophorus (Platypoecilus) muculatus, has been established by Bellamy ( 1922, 1928), Gordon (1927), Fraser and Gordon (1929), and Kosswig (1933). The dual mechanism of sex determination operating in the same species has been found by Gordon (1946a,b,c, 1947a, 1950, 1951a) who showed that, while the Mexican wild populations are male heterogametic, those originally from British Honduras are female heterogametic. The “domesticated” varieties are considered to have derived from British Honduras. Kallman (1965a,b) extended Gordon’s findings by studying the genetic behavior of populations from a vast area of Guatemala, intermediate between Mexico and British Honduras. The populations in this area contain both male and female heterogametic types even in a single pond. They interbreed with each other with the result that WX females were also collected. In view of evolution of the WY-YY and XX-XY types, this study is most interesting. TiEapia mossambica (Cichlidae) is considered to be a superspecies or group of closely related forms. Crosses of Malayan females to African males result in progeny consisting only of males, or nearly so, whereas the reciprocal crosses produce offspring in a sex ratio of 1o :3d (Hickling, 1960). Whether the Malayan form has the XX-XY and the African form the WZ(Y)-ZZ(YY) system, analogous to the dual system in the platyfish, X. maculatus, has not yet been clarified.

B. Polygenic Sex Determination and So-called Genetic Sex Reversal To grasp the real situation of sex determination, we must adopt the broader view originally advanced by Bridges (1922, 1925) for the fruitfly and elaborated by Winge (1934) for fish viz. that “a given property, the sex included, depends upon all the chromosomes, some of which pull in one direction and others in the other direction, some strongIy and others faintly or not demonstrably at all” (Bridges, 1939). In fishes in which the homogamety/ heterogamety is established, exceptional XXd d or XY o o and WZ(Y) d d appear occasionally, although no exceptional ZZ(YY) o o are reported. These exceptions have been misleadingly called genetic “sex reversals” even by distinguished geneticists. This is based on the concept that only sex chromosomes are carriers of the sex determiners. Only a few geneticists-of whom Winge

3.

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(1934) is the most celebrated-have grasped the intrinsic nature of sex differentiation. He has never referred to such exceptions as sex reversals because his theory is based on multiple sex factors with superior sex genes in the allosomes. The genetic evidence indicates that the guppy, Poecilia ( Lebistes ) reticulata, has sex chromosomes, XX for p and XY for d’ (Winge, 1922b). However, minor or polygenic F and M genes are distributed throughout the autosomes. The Y has superior (epistatic) M gene(s) and the X is supposed to possess epistatic F gene(s) (Winge, 1934; Winge and Ditlevsen, 1947, 1948). In the majority of individuals, autosomal sex genes are more or less in balance. Consequently, sex in most individuals is determined by the heterosomal combination. However, by fortuitous combinations of autosomes or recombinations a few exceptions may appear in which E M > EF in spite of an AAXX constitution. In Mutatis mutandis, exceptional XY females are considered to be individuals in which the totality of sex genes becomes SF > EM. These exceptions cannot be regarded as genetic “sex reversals” since they have a genetic basis to develop either into males or females. Aida’s breeding results ( 1930) in the medaka, Oryzias ( Apbcheilus) Zatipes, can be interpreted by this postulation (Winge, 1930). Since polygenic sex genes are numerous, by selective breedings of XX- d d , the sex ratio of offspring can be varied. Aida (1936) established an XX-XX strain of the medaka and adopted a theory of polygenic sex differentiation which is somewhat different from that of Winge. He suggested that XX males may be the result of a lowering of the femaledetermining potency of the X chromosome. It is unfortunate that he referred to XX males as “sex reversals.” In our d-rR strain of the medaka, where normally X’X‘ are females and XrYR males, about 0.5% of the progeny are exceptions (0.2%crossovers and 0.3%of X‘X‘ d plus XrXR o , Yamamoto, 1959a, 1984a,b). These rare X’X‘d d and X‘YR 9 o are regarded as exceptions in which autosomal M genes and F genes, respectively, over-accumulate in such a way that the sum of autosomal sex genes outbalances the superior heterosomal sex factors. As pointed out earlier (Yamamoto, 1963), the number of possible autosomal combinations is 2‘O, an astronomical number. Even if only some of autosomes are assumed to be sufficiently different in respect to M and F genes (or modifiers), the rest being more or less in even balance, the number of combinations would be enormous. Figure 6 is a graphical diagram illustrating the concept of the polyfactorial sex determination with epistatic sex genes in sex chromosomes, as illustrated in the guppy, the platyfish, and the medaka. For the sake of simplicity, it is taken for granted that the ratio of EM of AA:xF of AA of a popula-

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TOKI-0 YAMAMOTO

c

.

I F> I M

33

I

I M>IF

$8

I

3

Fig. 6. A diagram of polyfactorial “sex determination” with epistatic sex genes in sex chromosomes.

tion (where A stands for a set of autosomes) has a center (mean or mode) around which the individuals are fluctuating, thus forming a normal distribution curve (broken line). In an AAXY constitution the AA curve is shifted in the male direction (right) because of epistatic M gene( s ) in the Y, and in the AAXX constitution the curve is pulled to the female direction (left), perhaps because of the presence of epistatic F gene( s ) in the X or absence of the Y. Exceptional XX males and XY females encountered in normal breeding may be regarded as individuals which fall in the two extreme regions of the AA curve (stippled and oblique hatched), i.e., the z M greatly overweighs the Z F of AA or vice versa. By inbreeding and systematic selections, the AA curve may be shifted in either direction. In fact, the concept of polygenic sex factors has been substantiated by selective breeding. Thus, Aida (1936) obtained a strain of the medaka, in which both female and male have the XX constitution. In Aida’s stock, exceptional XX males were rare at first; by continued selection for XX males a breed was established in which XX males outnumbered XX females. Formerly, Kosswig ( 1932, 1933, 1935a,b) and Breider (1935a) have asserted that sex of the platyfish is determined genotypically, while that of the swordtail is determined phenotypically. Kosswig ( 1939a,b, 1954) has maintained that the X in d” heterogametic species and the Z in ? heterogametic forms are devoid of sex factors and that ? determiners in the former and d determiners in the latter are present in autosomes. Later, Kosswig and dktay (1955) modified their earlier views on mechanisms of sex determinations in xiphophorin fishes (X. mculatus, X. uariutus, X . xiphidium, and X . helleri). In this article, they maintained

3.

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that in d heterogametic (XX ? -XYd ) fish the X contains the F gene which is weaker than the M gene of the Y, and thai the W has a strong F gene which is stronger than the M gene of the Z in P heterogametic fish. The x of X. helleri has a strong masculinizing potency, Kosswig and his colleagues (Breider, 1942, 1949; Oktay, 1959, Anders and Anders, 1963; Peters, 1964; Dzwillo and Zander, 1967) eventually adopted the concept of polygenic sex determination (cf. Kosswig, 1964). This being so, exceptions are either prospective males or females from the very start of development in spite of their opposite sex chromosome formulas. Oktay (1959) and Anders and Anders (1963) correctly attributed the occurrence of exceptional XX males and XY females, appearing in the Mexican platyfish, to the effects of autosomal sex genes. MacIntre (1961), working on the same animal, reported a not infrequent occurrence of phenotypic females with the XY constitution. When mated to normal XY males, “sex-reversed females gave rise to XX daughters and XY and YY sons. It seems that autosomal F genes must have been accumulated in these strains by several generations of inbreeding. In fishes with entirely polygenic sex determination, sex chromosomes are as yet not differentiated. They are characterized by diverse sex ratios. In crossing them with a species with a homogameticIheterogametic system, sex chromosomes of the latter usually become epistatic. The swordtail, X. helleri, is the most famous gonochorist with a polygenic system. Kosswig (1932, 1935b), Breider and Kosswig (1937), and Gordon (1948) symbolized a pair of chromosomes which are counterparts of sex chromosomes as xx or X’XJ in both sexes. Sengiin (1941) reported that the Wx class of F, (Mh) from the interspecific matings between X . muculutus (WZ) and X. helleri ( x x ) and of backcross ( M h h ) to X. heZZeri are all 0 Q , while Xx class of F, (Mh) of the Mexican X . muculatus (XX) Q x X. helleri d (xx) comprised both sexes. This means that the W is stronger than the X and Xx-d d are the result of X. helleri-polygenic M genes, Oktay (1963) performed a hybridization between the swordtail, X . helleri, and d heterogametic platyfish, X . xiphidium. The F, ( Hx) from X. helleri P ( x x ) x X . xiphidium d (XY) were comprised of both 0 0 (xX) and d d ( x Y ) . In the backcross (Hxh) cross, the F, (Xh) from XX Q x xxd consisted of xx 0 and x X d d.This can be interpreted as indicating that the totality of sex genes in X . helleri 0 is XF > EM, while that of X . helleri d is XM > EF. There are three species of the genus Poecilia (Linzia) ( Poeciliidae) in the Caribbean Islands, P. nigrofasciata, P. caudofasciata, and P. uittata. Of these, P. nigrofasciata possesses the XX-XY mechanism while the

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TOKI-0 YAMAMOTO

other two species show polygenic sex determination (Breider, 1935b, 1936b; Breider and Kosswig, 1937). The F, of XX-P. nigrofasciatu o x xx-P. cuudofasciutu d are all females (Xx). In the reciprocal cross, xx-P. caudofasciata o x XY-P. nigrofasciata 8,the F, are in a ratio of 19 ( xX):1 g (xY). “he Y of P. nigrofasciata contains a strong M factor. In Section 111, A, Schwier’s studies (1939) on the genus Macropodus ( Anabantidae) are cited. Of three species, M . opercularis, M . chinensis, and M . concolor, M . opercularis and M . chinensis seem to have a homogametic/heterogametic mechanism, because the sex ratio of the latter two species is 19 :l d . In the absence of any sex-linked character the heterogametic sex cannot be decided. In M . concolor, males outnumber females. Zander (1965) interpreted this fact as indicating that the X chromosome might be absent in this form, viz., sex formulas, being xx o -xY d type. Some males are XY and the others are xx males produced by autosomal, polygenic EM > zF genes. Females are considered to be invariably xx constitution with autosomal Z F > EM genes. In short, sex determination in gonochorists is polyfactorial with or without epistatic sex gene( s ) in sex chromosomes, in both cases, a zygote in which xM > xF differentiates into a male and that in which Z F > ZM develops into a female. In the experiments of Bellamy and Queal (1951) using the o heterogametic platyfish, X . muculatus, a type of exceptional male appeared, which sired offspring in a ratio of 3 :Id as if it had the genotype WZ(Y). About one-third of the females bred as though they were WW and produced only daughters. These exceptions were regarded as “sex reversals.” If such were the case, we would expect to find some intersexes at the transitional stage. Confronted with the fact that not a single intersex has ever been found among 50,000 fishes handled in the past 28 years, the authors postulated that early sex inversion might have taken place. However, Wolf (1931) demonstrated that this species is a differentiated one. Hence, no juvenile hermaphroditism can occur. In the platyfish, X . muculatus, with opposing sex-determining mechanisms, one XX-XY the other WY(Z)-YY(ZZ), exceptional XX or WY males and XY females appear occasionally. Besides Bellamy and Queal, Gordon (1946b,c, 1947a, 1951a), MacIntre ( 19sl) and Kallman ( 1965b) regarded these as “sex reversals,” notwithstanding the lastmentioned author’s statement that sexuality of this fish is stable and that although more than 100,000 platyfish have been examined during the last 25 years, not a single female has ever changed into a male (fish with a testis). Consequently he defined a sex-reversed fish as one that is func-

3.

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139

tionally one sex, but genotypically the other. However, if we take autosoma1 sex genes with various potencies into consideration, it is likely that it is genotypically also one sex and not the other. If we accept these exceptions as real sex reversals, we are confronted with an array of paradoxical facts: (1)Both the medaka and the platyfish are sexually stable among fishes; ( 2 ) not a single sporadic intersex has ever been found, however, so-called sex reversals have been reported occasionally; and ( 3 ) no artificial induction of reversal in sex differentiation by any means, including sex hormones, has ever been accomplished in the platyfish, despite the occurrence of “spontaneous sex reversals.” These puzzling facts can be clarified if we accept that they are genotypic males ( z M > Z F ) and genotypic females ( Z F > Z M ) , respectively. On the basis of the concept of multiple autosomal sex genes or modifiers these exceptions are not sex reversals at all. Breider (1942) described a WZd of 9 heterogametic X . maculatus. In mating this exception with a normal WZ P , the offspring were in a ratio of 3 o (1WW P , 2 WZ o ) : l d (ZZ). Breider did not detect the WW female. Bellamy and Queal (1951) detected the genotype by progeny tests. To sum up, in strictly “differentiated gonochorists such as the platyfish and the medaka the occurrence of spontaneous sex reversals is impossible.

C. “Spontaneous Sex Reversal” in the Swordtail A male and female swordtail are illustrated in Fig. 7. Amateur breeders have repeatedly claimed instances of spontaneous sex reversal in some of their fishes, particularly in aged swordtail X . helleri. Biologists have also reported the same phenomenon ( Harms, 1926a,b; Friess, 1933). Hild (1940) regarded sex reversal in this form as a regular phenomenon. To accept a fish as a true functional sex reversal, it is necessary to ascertain that (1) the fish first functioned as a female and ( 2 ) later it turned out to be a functional male. Some reports on the matter have not ascertained these facts. Popoff (1929) and Sacks (1955) doubted that a true functional sex reversal occurred. Pathological masculinization in two guppy females was recorded by Wurmbach ( 1951). The fish developed a gonopodium. Histological study revealed that they were infected by a fungus, Zchthyophorus hoferi, which caused degeneration of peripheral ovocytes, followed by appearance of spermatogonia derived from ovarian-cavity epithelium. He

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TOKI-0 YAMAMOTO

Fig. 7. ( A ) Male and ( B ) female of the swordtail, Xiphorphorus helleri. Redrawn by Dr. G. Eguchi after Gordon (1956).

pointed out that Fries’ “rest bodies” in the masculinized gonad of the swordtail are not remnants of degenerated ovocytes but are cysts of the parasite. Peters (1964) found that in X. helleri from Honduras there are two types of males: the normal or rapidly growing males ( d F ) and the slowly developing males ( d s ) which reach larger sizes and differentiate sexually more slowly. With this background, his criticism on reported sex-reversal merits special attention. Among some hundreds of females which he observed during several years, not a single sex reversal has ever been found, although he obtained some arrhenoid advanced-aged females (Fig. 8A ) . The outer appearance of d s looks like females in the immature stage, and male secondary sex characters manifest themselves only later (Fig. 8B). It is natural that slowly developing d s can fertilize ova. Peters considered that alleged sex reversals might not be males but arrhenoid females caused by hormonal upsets in old age, and unripe d s are likely to be taken as females. Philippi (1908) described arrhenoid females with male sex characters in Glarichthyes (Poeciliidae). The most famous cases are in Essenberg’s report (1926) on spon-

3.

SEX DIFFERENTIATION

141

taneous, functional sex reversal in the swordtail. He claimed that two fish (B,, and G ) , after producing broods, turned out to be males and sired a few offspring in mating with “virgin females.” This article has been cited in zoology textbooks without qualification. However, Gordon (1956, 1957), a celebrated poeciliid geneticist, expressed doubt as to the validity of Essenberg’s conclusions. He pointed out that a functional swordtail male must have not only a complete testis but a functional gonopodium (modified anal fin) as well as its suspensorial skeleton. If the fish in question were first female and then turned out to be male, the female-type anal fin and suspensoria must have transformed into a

Fig. 8. ( A ) An arrhenoid 0 (above) and a rapidly growing 8 I’. ( B ) A slowly $S (above) and a rapidIy developing $ F (below) of the swordtail, Xiphophorw helleri. Drawn by Dr. G . Eguchi from photos by Peters (1964). growing

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TOKI-0 YAMAMOTO

perfect gonopodium. For him, it is inconceivable that such a drastic change should have occurred. Incomplete male secondary sexual characters engendered by hormonal upsets appear in aged swordtails. He questioned the virginity of the females used as mates of alleged sex reversals. It is well known that sperm from a single copulation can sire up to eight broods in the subsequent 8 months. Gordon (1956) states that “unconditional evidence is required before the reality of spontaneous, complete and functional sex reversal in a fish can be accepted.” V. CONTROL OF SEX DIFFERENTIATION Science unfolds and controls nature. Toki-o Yaniamoto

A. Surgical Operation Surgical castrations of female Siamese fighting fish, Betta splendens, have in rare instances produced fish in which the regenerated gonad was a functional testis. These experiments were first performed independently in the United States (Noble and Kumpf, 1937) and later in Germany (Kaiser and Schmidt, 1951). The American authors obtained only seven reversals out of 150 spayings, of which only three proved to be fertile, siring both sexes in mating with females. The German authors obtained three reversals, of which only one was fertile and fathered solely a total of 109 daughters. The latter experiment proved that the male is heterogametic.

B. Modification of Sex Differentiation by Sex Hormones In the rainbow trout, Salmo gairdneri irideus, Padoa (1937, 1939a) reported that injection of a follicular hormone produced ova in the testes, while testosterone induced testicular tissue in the ovaries. Ashby (1952, 1956, 1959) reported the paradoxical similarity of action of estradiol and testosterone in the brown trout, Salmo trutta; both hormones retarded gonadal development and produced hypertrophy of the somatopleure. There was no evidence of sexual inversion. The result might be ascribed to the fact that he started hormone administration in alevins in which gonadal sex differentiation has already been established. A number of attempts have been made in viviparous toothcarps to modify sex differentiation by administration of sex hormones, especially androgens, starting with newly born broods or adults. However, andro-

3.

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143

gens mainly affect the secondary sexual characters and the action on the gonad has been either incomplete, pathological, or negative. In the guppy, Poecilia reticulatn, the effects of sex hormones have been studied by Witschi and Crown (1937), Berkowitz (1937, 1938, 1941), Rkgnier ( 1938), Eversole (1939, 1941), Gallien (1946, 1948), Hildeman (1954), Miyamori (1961), and Querner (1956). Berkowitz caused the production of ovo-testis by administering estrogens to young male guppies. He reported that the optimal dosage resulted in an almost complete reversal of testis to ovary. The same effect was observed by Querner. Miyamori produced ovo-testis by administration of androgen to young females. In the top minnow, Gambusia holbrookii, which shows “transitional intersexuality,” Lepori ( 1942a,b, 1948) produced testis-ova in females by administration of an androgen and in males by an estrogen. Comparable results were obtained in G. afinis by Okada (1944). In the swordtail, X . helleri, Baldwin and Goldin (1940) reported that androgen administration to young females not only simulated the male secondary characters such as formation of a caudal sword and the gonopod but also induced masculinization of the gonad after degeneration of ovocytes. Querner (1956) showed that androgens produce ovo-testis in genetic females. Contrary to the cases in amphibians, corticoids have no effect on sex differentiation. Vivian ( 1952a) reported that 11 incompletely hypophysectomized female swordtails developed an involuted ovary with so-called “rest bodies” and claimed that two or three of them showed partial masculinization. The fact that these rest bodies are not degenerated ovocytes but are cysts of a parasitic mycomycete has already been mentioned (Section 111, C ) . This pathological change might be caused by infection of Ichthyophorus hoferi. Failure to obtain complete reversal of sex differentiation in these studies may be because administration of hormones was started after the onset of gonadal sex differentiation. As stated before, although the guppy and swordtail are “undifferentiated” species, gonadal sex differentiation has already been established when broods are born (Essenberg, 1923, in the swordtail; Goodrich et al., 1934; Dildine, 1936, in the guppy). Dzwillo’s success (1962) in obtaining complete reversal in sex differentiation in the guppy will be discussed later. In the oviparous toothcarp, Oryzias htipes, Okada (194313, 1949, 1952a) claimed that the formation of testis-ova in adult males can be induced by administration of either estrogens or androgens. Treated males had gonads containing large oviform cells which look like ovocytes but which are not surrounded by follicle cells. The nature of these largesized cells will be discussed later (Section VI, A ) . Okada (1964b) obtained a true testis-ova by estrogen administration starting in juveniles.

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In this case, ovocytes are genuine since they are accompanied by follicle cells.

C. Complete (Functional) Reversal of Sex Differentiation In the swordtail (undifferentiated species), Dantschakoff ( 1941) claimed to have induced males by the administration of androgens to females. There have been some reservations regarding these observations (cf. Gordon, 193-8, 1957, and Peters, 1964, in Section IV, D). If, however, this is true, it is possible that the induction was accomplished because this “undifferentiated species is very labile in sexuality. As stated before (Section III, A ) this species has no sex chromosomes. By treatment with an androgen, female germ cells including ovocytes might degenerate and protogonia might differentiate ,into male germ cells. In the differentiated species such as the platyfish and the medaka, where neither spontaneous sex reversal nor even sporadic intersexes occurs, induction of complete reversal may be impossible if hormone treatment starts after the onset of gonadal sex differentiation. The rank of stability in sexuality seems to be platyfish 5 medaka > guppy > swordtail. No sex reversals have ever been successful by administration of sex hormones in the platies (Cohen, 1946; Tavolga, 1949; Laskowski, 1953) because heterologous sex hormones have inevitably been administered after birth when the gonadal sex differentiation has already been established. The medaka, Oryzias latipes (Fig. 9 ) , an oviparous toothcarp, is a strict gonochorist. Genetically, the sex-determining mechanism norma1 to this fish was established as XX for female and XY for male (Aida, 1921). Embryologically, it is a differentiated species ( Yamamoto, 1953). Using our genetically analyzed strain (d-rR) of the medaka, in which white is female X‘X‘ and orange-red is male X7YR,where R stands for xanthic pigmentation, functional reversal in sex differentiation has been accomplished in both directions by heterologous sex hormones (Yamamoto, 1953, 1958). Thus, it has become possible to control sex differentiation ad libitum either from genetic males (XY) to functional females or from genetic females (XX) to males. In other words, we are able to inverse the prospective fate of the fish, genetically destined to develop into one sex and direct it to differentiate into the other. Success in reversing sex differentiation in both directions in the medaka rendered it possible to mate estrone-induced XY females with niethyltestosteroneinduced XX males (Yamamoto, 1961, Fig. 10). In contrast to viviparous poeciliids, the newly hatched fry ( g 4 . 8 mm) has an indifferent gonad (gonad primordium) and gonadal sex differentiation takes place between the 6 and 11 mm stages. To accomplish

3. SEX

145

DIFFERENTIATION

Fig. 9. ( A ) Female and ( B ) male of the medaka, Oryzias latipes.

complete reversal in sex differentiation in any differentiated gonochorists, certain conditions should be fulfilled. First, a heterologous sex hormone should be administered starting with the stage of indifferent gonads and passing through the stage of sex differentiation. In our experiments, heterologous hormones were administered by the oral route to newly hatched fry and continued until they reached 12 mm or longer. Therer?

7-?

Fig. 10. A diagram illustrating the progeny of estrone-induced X'YR female mated with methyltestosterone-induced X'X' male (from Fig. 4, Yamamoto, 1961).

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TOKI-0 YAMAMOTO

Table 111 GDaoDosage Levels of Estrogens Acting as Gynotermone (Gynogenin) to Induce 50% X Y Females in the Medaka, Oryzias latipes Estrogens

GD5o ( r d g diet)

Hexesterol Euves tinn

0.4 0.8

Ethynyl estradiol

1.7

Estradiol-17b Stilbestrol Estrone Estriol 11

5.8 7.5 20 130

Authors Yamamoto and Iwamatsu (unpublished data) Yamamoto, Hishida, and Uwa (unpublished data) Yamamoto, Noma, and Tsuzuki (unpublished data) Yamamoto and Matsuda (1963) Yamamoto and Matsuda (1963) Yamamoto (1959b) Yamamoto (1965)

p,p’-Dicarboethoxyoxy-trans-a#-diethylstilbene.

after, they were raised on a normal diet until they reached maturity. Second, adequate dosage levels of hormones should be used; these differ in potency with different sex hormones (Tables I11 and IV). Estrogen-induced XTYRfemales (egg producers) were first obtained by Table IV AD50 Dosage Levels of Androgens Acting as Androtermone (Androgenin) to Induce 50% X X Males in the Medaka, Oryzias Zatipes Androgens

19-Nor-ethynyltestosterone

1.0

Fluoxymesterone (Halotestin)

1.2

17-Ethynyltestosterone (Pregneninolone, ethisterone) Methylandrostenediol

3.4

Methyltestosterone Androstenedione Testosterone propionate Androsterone Dehydroepiandrosterone

7.8 15 500 560 580 >3200

Yamamoto, Hishida, and Takeuchi (unpublished data) Yamamoto and Hishida (unpublished data) Yamamoto and Uwa (unpublished data) Yamamoto and Onitake (unpublished data) Yamamoto (1958) Yamamoto (1968) Yamamoto et al. (1968) Yamamoto et al. (1968) Yamamoto and Oikawa (unpublished data)

3.

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SEX DIFFERENTIATION

estrone and stilbestrol ( Yamamoto, 1953, 1959a). The induced X'YR females in matings with normal X7YR males produced offspring in a ratio of 1o : 2 g instead of 1o : 3 g ,indicating that viable YRYR males are rare. However, two YRYR males which sired all-male progeny were actually detected among 57 ( X'YR YRYR)males singly tested by X'X' females (Yamamoto, 1955, 1959a). By using other mating systems (cf. Table V ) , it happened that YRYr males produced by mating estrogeninduced X'YR females with X'Y' males were all viable (Fig. 11). The Y'Y' males yielded by mating induced XRF or X'Y' females with X'Y' males also all survived. By administration of estrone in two consecutive generations, it was possible to invert sex differentiation in YY zygotes (Yamamoto, 1963, 1967). Although it does not relate directly to the main theme of this section, reference should be made to the intriguing problem of viability of YY zygotes. This problem has been fully discussed elsewhere (Yamamoto, 1964a,b). In short, the regular YR chromosome contains an inert section ( - ) which in duplex condition ( YR,-YR*-) renders

+

Table V Production of YrY' (White) Males and Estroneinduced V V Females in the Medaka, Orvrias Zutiveso*b r(X'Xr) 9 0 (12) x R(XRY') $ 3 (12) Normal diet 10 R ( X R F ) 9 9 (44) r(XrY.) $ 3 (40) PI T(X'X') 9 9 (12) x R(XRY') $ 3 (12) Estrone diet I I, ER(XRXr) 9 0 (38) Er(X'Y.) 9 9 (24) Er(X'Y7) 3 8 ( 2 ) ~ Po Er(X'Yr)d 9 9 (24) X R(XRYr) $ 3 (12) Normal diet R(XRX') 9 9 (11) R(XRYr) 3 3 (6) r(XrYr YrYr) 30' (17) 11, .....__...._....__ Detected 5 + 4 Po E r ( P Y r I d 9 9 (24) X R(XRY.) 8 3 (12) Estrone diet I ER(XRXr XRYr) 9 9 (28) Er(X'Yv YrYr) 9 9 (13) IL ..-..-............. ...........-..... Detected 3 + 5 3 + 3 P1

I

I

+

+

+

From Yamamoto (1967). Subscripts c and e denote control and experimental, and I and I1 are the first and the second generations. Numbers enclosed in parentheses represent numbers of parents and offspring obtained. Animals designated by broken lines were submitted to testcrosses. Free figures are numbers of fish detected by testcrosses. Sex chromosome formulas following r or R are presumed sex genotypes. E denotes estronized fish; r, white, and R, orange-red. c Nonreversals. d In the original paper (1967) this formula was misprinted as XrXr. a

b

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TOKI-0 YAMAMOTO

r 99

R db ,:;:

:~r~yj;?$:;"':~ r = White

Estrone diet

r ??

R ?9

R= Orange-red r dd

Sex reversal

Sex reversal

Sex reversal

Fig. 11. ( A ) A diagram of production of Y'Y" males feeding to offspring from estrone-induced X'Y' females males, ( B ) Induction of YRY' females by estrone feeding induced X'Y' females mated to normal X'Y" males (from 1963a )

.

Sex reversal

(control of B ) by normal mated with normal X'Y' to offspring from estroneFigs. 1 and 2, Yamamoto,

the zygote nonviable, while both regular X' and XR possess the viability section (+). Rare survivaI YRYR zygotes are experimentalIy verified to have the YRi-YcR,+constitution, the ( + )of which is known to have been derived from X'*+by crossing over. This being so, it is quite rational that the Y' is Y',+ in constitution and YRYr is YR,-Yrv+.It is hardly surprising that practically all Y'Y' zygotes (Y",+Y'>+)are viable (Fig. 12). Using white females (X'x') and orange-red (XRYr) males as parents in the first generation, estrone-induced YRY and YrYr females were produced by administration of estrone in two consecutive generations (Yamamoto, 1963, Fig. 11; 1967, TabIe VI).

3.

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SEX DIFFERENTIATION

AA XX [ZF >ZM]--

Gyminductor

-

Indifferent gonad +Ovary

(0

-

Andr6AA X Y b M >ZF]-inductor (Androterrnone)

Indifferent '\ gonad --Testcs(d)

Fig. 12. A scheme of normal sex differentiation (solid lines) and reversal in sex differentiation b y means of heterologous sex inductors (from Yamamoto, 1962).

Returning to the main theme, adequate dosage levels are very different for various estrogens (Fig. 13). The symbol GD,, was suggested as the designation of the dosage level at which there are 50%induced XY females (Yamamoto and Matsuda, 1963). The writer and his co-workers have performed experiments to estimate GD,, values of both natural and synthetic estrogens (Table IV) . Reversal in sex differentiation in the opposite direction, viz., induction of X'X' males by methyltestosterone, has also been accomplished (Yamamoto, 1958); these males in mating with normal X'X' females sired allfemale progenies. Androsterone and testosterone propionate have also a male-inducing action (Yamamoto et al., 1968). The symbol AD,, may be used as a notation of dosage level at which there are 50% induced XX males. In Table X, AD,, values of various androgens are listed. In the guppy, an undifferentiated species, all attempts to control sex by hormone administrations after birth when the gonadal sex differentiation is already established (cf. Goodrich et al., 1934) failed until Dzwillo (1962) succeeded in getting functional XX males by androgen administration to gravid females containing embryos having indifferent gonads. Clemens (1965) was able to obtain androgen-induced XX males in Tilapia mossambica by administration starting with newly hatched fry having indifferent gonads. In the goldfish, Carassius auratus, Yamamoto and Kajishima (1969) obtained functional reversals in both directions by administering heterologous sex hormones for 2 months to newly hatched fry with indifferent gonads. By testcrosses of the hormone-treated fish, Table V1 Terminology of Sex Inducers Female inducer

Male inducer

Author

Cortexin (corticin) Gynogenin Gynotermone

Medullarin Androgenin Androterrnone

D' Ancona

Witschi Hartmann

Beginning 1931 1949b 1951

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TOKI-0 YAMAMOTO

1

2

s

10

50

20

DOSAGE

100

1,EVE;L

200

wo

IN 9 g / g

loon

2000

DIET

Fig. 13. Potencies of estradiol, estrone, and estriol in inducing XY females (from Fig. 2, Yamamoto, 1965b).

evidence for male heterogamety ( X U ) in the goldfish was first obtained. In differentiated species, neither spontaneous intersex nor sporadic sex reversals can appear. Nevertheless, induction of complete reversal in sex differentiation can be achieved if the following necessary condition is fulfilled. Sexogens (estrogens and androgens) at suitable dosage levels should be administered consistently, beginning with the stage of the indifferent gonad and passing through the stage of sex differentiation. In summary, by finding the correct dose of a suitable hormone for treating embryos or larvae and by starting medication at the indifferent gonadal stage, it is possible to convert an otherwise potential female into one that functions as a male or vice versa. This principle will hold true not only for “undifferentiated” species but also for “differentiated” ones. VI. NATURE OF NATURAL SEX INDUCERS

A. Steroid versus Nonsteroid Theories

Witschi (1914, 1929) postulated the inducer theory of sex differentiation in amphibians. Although the sex genes are effective sex determiners, their action appears to be mediated by the sex-gene-controlled sex inducers as far as the vertebrate is concerned. Terminology of sex inducers differs among authors (Table VI) . The exact chain of events which leads from sex genes to sex differ-

3.

SEX DIFFERENTIATION

151

entiation has not yet been clarified but is outlined in Fig. 12 (Yamamoto, 1962). The important point is that sex genes are not direct sex determiners, but it is the sex-gene-controlled inducers that determine sex differentiation. There are pros and cons for the steroid theory of sex inducers. In amphibians, Witschi (1942, 1950, 1955, 1957) and his followers are “cons” and postulated a protein nature of the sex inducers without evidence. Chieffi (1965) also stated that “the embryonic sex inducers, . . . , are in all probability different from sex hormones of the adults.” The postulation is based inter alia on (1) the so-called paradoxical feminization of the gonad by high doses of androgens, ( 2 ) the nonspecificity of sex hormones as sex inducers, viz., non-sex-hormonic corticoids are believed to also have the potency of sex inducers, and ( 3 ) the ineffectiveness of androgens on some WZ amphibians. These statements are based on the sex ratio scored by a limited number of gonads of young, the sex genotype of which is unknown. Neither a single case of androgen-induced functional nor a corticoid-induced sex reversal has ever been produced. Furthermore, criteria for ovocytes are questionable. Experiments on the adult male medaka show that not only estrogens but androgens, corticoids, and any noxious treatments such as heat treatment and starvation result in the formation of large-sized cells in the testis (testis-ova), without characteristics of ovocytes such as the presence of follicle cells (cf. Egami, 1955a-e, 1956a,b). These oviform cells, which actually look like auxocytes, may be regarded as degenerating protogonia or spermatogonia enlarged prior to deterioration (Yamamoto, 1958). As to point ( 3 ) above we should not rely on negative results. Reported experiments have been made by immersion in hormone. It may well be that androgen immersion cannot simulate the natural condition surrounding the protogonia and induce them to differentiate into male germ cells. Inducers are thought to be densely produced only by cells surrounding the protogonia. In the medaka the following occurs: (1) Estrogens act as female inducers and androgens function as male inducers; no paradoxical phenomenon are engendered. ( 2 ) Sex hormones act specifically as sex inducers; non-sex-hormonic corticoids have no effect. ( 3) Effective dosage levels of estrone and methyltestosterone are so low that it may be possible for juvenile fish to elaborate a small amount of sexogens ( Yamamoto, 1959b, Hishida, 1965). (4)By administration of radioactive te~tosterone-l6-~T propionate and diethylstilbestrol-( m~noethyl-l-’~C), Hishida (1962, 1W) showed that these are selectively incorporated into the juvenile gonad. Point ( 4 ) seems to indicate that the sex-differentiating gonad requires more sex hormones than other organs. On the basis of

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TOKI-0 YAMAMOTO

these results, we are inclined to the sex steroid theory of natural inducers. The current view of pathways of biosynthesis of sexogens in adults is illustrated in Fig. 14 based on Meyers (1955a,b), Solomon et al. (1956), and others. Whether or not the pathways are valid for cells surrounding the indifferent gonad is not known. Of these naturally occurring steroids, pregnenolone, progesterone, and 17a-hydroxyprogesterone are found to be ineffective (Yamamoto and Matsuda, 1963; Yamamoto, 1968) while androstenedione and other sex steroids are effective ( Tables I11 and IV).

-

&o

/

no Cholesterol

Pregnenolone

Progesterone

Androstenedione

110-Hydroxyprogesterone

19- Hydroxy testosterone

Androsterone

19-Hydroxyandrostenedione

Estriol

Eetradiol-170

Eetrone

Testosterone

-

HO

Fig. 14. A diagram of the current view of biosynthesis of key sex steroids (based on Meyers, 1955a; Solomon et aZ., 1956; and others).

3. SEX

DIFFERENTIATION

153

B. Detection of Steroids and Relevant Enzymes in Fish Gonads First of all, it is not known whether all the sex hormones of adult fishes are identical with those of mammals. The identity of some hormones has been established by Chester Jones and Phillips (1960), Chieffi and Lupo ( 1962, 1963), Lupo and Chieffi (1963), and Wotiz et al. (1960). Research on the detection of steroids in the larval gonad at the onset of sex differentiation by classic chemical methods is difficult because of the paucity of available material. Therefore, almost all approaches depend on histochemical and enzymic methods. In the elasmobranch Scylwrhinus canicuhs Chieffi (1955) found a few sudanophilic granules in the gonadal medulla before sex differentiation. After the onset of sex differentiation, there appear many sudanophilic as well as Schultzpositive granules. The identification of enzymes involved in biosynthesis of sex steroids is another approach. Techniques have been developed for the histochemical demonstration of three relevant enzymes: A5-3P-dehydroxysteroid dehydrogenase ( A6-3p-HSDH), A5-3P-ketoisomerase ( A53P-KIM ) and 17P-hydroxysteroid dehydrogenase ( 17P-HSDH) which is DPN and TPN dependent. Together with A5-3p-KIM,A5-3p-HSDHis involved in the pathway from pregnenolone to progesterone, one of the first steps of biosynthesis of sex steroids (cf. Fig. 14). Chieffi and coworkers (1961-1963) were unable to detect A6-3p-HSDH in the embryonic gonads of the elasmobranchs, Scyliorhinus caniculas, S. stellaris, and Torpedo murmorata. The enzyme, however, was detected in the interrenal tissue of S . stellaris and T. mumorada. Bara (1966) showed the presence of 3P-HSDH in the testis of Fundubs heteroclitus. Many questions remain unanswered. It is not known, for instance, where the supposed sex inducers are produced and in what way they influence the indifferent gonia ( protogonia ) to develop into either ovocytes or spermatocytes. Finally, the chemical nature of natural sex inducers still remains obscure. Research along these lines should be fruitful.

VII. DIFFERENTIATION OF SECONDARY SEXUAL CHARACTERS

Our primary concern is with gonadal or primary sex differentiation. References to the manifestation of secondary sexual characters are so numerous that only a brief account is presented here. Within the last two decades some excellent reviews have been pub-

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TOKI-0 YAMAMOTO

lished (Hoar et al., 1951; Hoar, 1955, 1957a,b; Dodd, 1955). Pickford and Atz (1957) presented a monograph on the piscine pituitary gland and its role in reproduction. It is convenient to divide secondary sex characters into two categories: (1) temporary characteristics which normally appear only during the breeding season such as nuptial coloration, pearl organs, and the ovipositor of bitterlings; ( 2 ) permanent organs developed fully at the onset of sexual maturity such as the gonopodium of viviparous cyprinodonts and the papillar processes on the male anal fin and the urinogenital papilla in the female medaka, 0yzias. It is also convenient to define male- and female-positive secondary sex characters (Yamamoto and Suzuki, 1955). The former is a character that is either specific to the male or is more developed in the male than in the female. The latter is the reverse. The mechanism by which these characters manifest themselves may only be elucidated by experiments. Most of secondary sex characters of fishes are male-positive. Kop3 (1918, 1928) was the first to demonstrate in the minnow Phorinus laevis that the nupital coloration is dependent on the testicular hormone. This has been confirmed in the sticklebacks, Gasterosteus pungitius and G. muleatus, by van Oordt (1923, 1924), van Oordt and van der Mass ( 1927), Bock ( 1928), Craig-Bennett ( 1931), and Ikeda ( 1933); in the bitterlings by Tozawa ( 1929), Wunder ( 1931), and Glaser and Haempel (1932); and in the guppy by Blacher (1926b) and Samokhvalova (1933). Similar results were obtained in the medaka (Niwa, 1965a)b). Tozawa (1923) proved that the pearl organs of the goldfish, Carassius auratus, are controlled by testicular hormone. While castration results in the disappearance of these male-positive characters, ovariotomy causes no effect. This means that the absence of these in the female is not because of the inhibitory action of the ovary. On the contrary, in the ganoid, Amia calva, Zahl and Davis (1932) showed that the gray-black pattern (male-positive) is absent in the female because of an inhibitory actiton of the ovary; ovariotomy produced this pattern. The male swordtail and the platyfish have complicated suspensorial skeletons including three hemal spines in the caudal vertebrae; these are necessary for proper functioning of the gonopodium. In the female, the three hemal spines are absent providing an undivided space required by the gravid female for the brood of embryos. Vivian (1952b) reported that spayed swordtails develop the three hemal spines as in the male, indicating that these are absent in the female by an inhibiting action of the ovary. Rubin and Gordon (1953) showed in the platyfish that .westradio1 benzoate induced the dissolution of one or two of these hemal spines in the male and methyltestosterone induced

3.

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155

fusion of basal bony elements (mesonost and baseost) in the female, which are normally separated. The secondary sex characters of the medaka, Oryzias latipes, have been described by Oka (1931a). Nagata ( 1934, 1936) demonstrated that castration results in a regression of both the shape and papillar processes of the male anal fin. Transplantation of a testis into the sprayed fish produces an almost perfect replica of the male characters. Administration of an androgen to females induces the male-type anal fin (Okada and Yamashita, 1944a). Other sexual differences such as teeth, bones, and body shape were reported by Egami (1956b, 1959b) and Egami and Ishii ( 1956). In the topminnow, Gambusia holbrookii, Dulzetto (1933) noted the correlation between testicular development and the gonopod formation. A host of experiments on gonopod formation has been performed by Turner (1942a,b, 1947, 1960) and Okada and Yamashita (1944b) in the topminnow G . ufinis, by Grobstein (1940a,b, 1942a,b, 1948) in the platyfish X . muculatus, by Hopper (1949) and Hildemann (1954) in the guppy, and by Taylor (1948) in the guppy-swordtail hybrid. Some of the earlier studies of hormone-induced characters, aimed at inducing modifications of sex differentiation by administration of steroids in poeciliids, have already been discussed (Section V, B ) . Vivian ( 1950) reported that X-ray irradiation in the females induced formation of a malelike anal fin ( incomplete gonopod). At this point, it may be remarked that not all fins are sensitive to androgens. For example, androgens have a stimulating action on the anal and dorsal fins of the medaka but exert no effect on the caudal fin. We will refer to the pelvic fin later. In the gobiid fish, Pterogobius zonoleucus, Egami ( 1 9 5 9 ~ )stated that while the dorsal fin is elongated by administration of an androgen, other fins are not affected. Another malepositive character is the large tooth in the male medaka. Large teeth can be produced in the female by administration of methyltestosterone (Egami, 1957; Takeuchi, 1967). Turner’s extensive researches ( 1960) demonstrated that regional parts in the anal fin of Gambusia have a differential sensitivity to androgens. The 3-4-5 ray complex is the most sensitive. He among others suggested that the enzyme pattern may be different in each area of the fin. Only a few female-positive secondary sex characters are present in teleosts. The urinogenital papilla (UGP) and the pelvic fin of the medaka and the ovipositor (elongated UGP) of the bitterlings are examples. Although the UGP is stimulated by both estrogen and androgen, it is far more sensitive to the former than the latter (Yamamoto

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and Suzuki, 1955). It appears that normally the UGP of the female is produced by an estrogen from the ovary. The UGP of the Indian catfish, Heteropneustes fossils, is also femalepositive. Kar and Ghosh (1950) reported that injections of either an estrogen or an androgen in females resulted in drastic hypoplasia of the ovaries, oviduct, and the UGP. They attributed this seemingly curious effect of the estrogen to the paucity of gonadotropin through suppression of its action by a large amount of exogenous hormone and concluded that the UGP is under the control of the ovarian hormone. The pelvic fin of the female medaka is longer than that of the male (Oka, 1931a). Niwa (196513) showed that this is because of an inhibitory action of the testicular hormone and cannot be attributed to a stimulating action of the ovarian hormone. In the Japanese bitterling Acheilognathus indermedia Tozawa ( 1929), showed that ovariotomy inhibits ovipositor lengthening. Research on the ovipositor formation in the European species, Rhodeus umurus, has provided a very confusing and puzzling problem. Fleishmann and Kann (1932) and Ehrhardt and Kuhn (1933) found that estrogen induces this reaction in the nonbreeding season. In 1935, American and German authors proclaimed that this response can be used for the diagnosis of pregnancy. It has been reported that urine from both men and pregnant women usually gives a positive reaction, while that from nonpregnant women may or may not cause this reaction. A male hormone in the urine was believed to be responsible. Banes et al. (1936) reported, however, that crude extracts of adrenal cortex gave a positive response while androsterone was negative. On the other hand, Kleiner et al. (1937) obtained a positive response by administration of androgens. De Wit (1939) confirmed this and further stated that progesterone and deoxycorticosterone (DOCA) are far more active than an androgen. Bretschneider and de Wit (1940-1941, 1947) and de Wit (1940, 1941, 1955) developed a quantitative method and studied this phenomenon. However, a survey of literature reveals that not only the abovementioned substances but also adrenaline and anesthetics have been reported to be active in lengthening the ovipositer. De Groot and de Wit (1949) reported that alcohol, heat shock, and strong light also causes ovipositor growth. This would seem to indicate that stresses in the sense of Selye also cause the reaction. Furthemore, the presence of mussels and/or males and an exhalent current from mussels have the influence on the cyclic ovipositor growth (de Wit, 1955). It appears that normally environmental factors exert their effects on the pituitary gland through intermediacy of the nervous activity evoked by sense organs. Further work must be done before these questions can be adequately answered.

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VIII. SUMMARY

The need to clarify our thinking about sexuality in fishes is emphasized. First, it is unsound to consider that sexuality in all members of the class Pisces is labile, There are in fact many sex types: synchronous, protandrous, and protogynous hermaphrodites, as well as gonochorists. Among gonochorists, there are “undifferentiated and “differentiated species in the sense of Witschi. The latter are more stable than the former in sexuality. Evidently, there are graded levels in sexuality among fishes. Second, sex is a phenotypic expression. In gonochorists, it is important to clarify the concept of sex to eliminate confusion. A male is a sperm producer and a female an egg producer. This definition, although selfevident, is particularly important in avoiding controversy in the problem of sex reversal. By definition, sex genotypes of zygotes before sex differentiation such as AAXX and AAXY are usually presumptive or prospective females and males, respectively. Although combinations of sex genes are determined at the time of fertilization, sex is actually determined by sex-gene-controlled sex inducers at a certain critical period of development, that is, sex genes act only indirectly upon sex differentiation. Their action is directly mediated by sex inducers. Sex genes are present not only in the sex chromosomes but also distributed over a great number of autosomes. In view of multiple (polygenic) sex factors, exceptional AAXX males or AAXY females and WZ( Y ) males are not “genetic sex reversals.” These exceptions appear when the totality of sex genes become ZM > XF by fortuitous combinations of autosomes in spite of AAXX constitution and zF >XM despite AAXY constitution. This concept may also be applied to exceptional WZ(Y) males in female heterogametic species. They have the genetic basis for either male or female. A diagram illustrates this concept (Fig. 6). In general, sex differentiation is induced by sex inducers controlled by sex genes with or without (in species without sex chromosomes) sex chromosomes. Sex chromosomes contain epistatic (superior) sex genes. Reports on spontaneous sex reversals in gonochorists have been criticized. Such cases seldom, if ever occur in fishes. Most cases are masculinized females (with ovary) brought about by hormonal upsets that accompany advanced age. Artificial control of sex differentiation may be one of the key projects in biology. One approach is to override the AAXX (in which XF > XM) or AAXY (in which XM > zF ) constitution by artificial means. This

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has been successfully accomplished in both directions in the medaka and the goldfish by administration of heterologous sex hormones, i.e., estrogens to XY zygotes and androgens to XX zygotes. In the medaka, it is possible to invert sex differentiation of even YY zygotes into females by administration of estrone in two consecutive generations. Reversal in one direction, from the presumptive female to the functional male, was achieved in the guppy ( Dzwillo, 1962) and Tilapiu (Clemens, 1965). It is of paramount importance that heterologous sex steroid administration must start at the stage of the indifferent gonad and continue through the stage of gonadal sex differentiation and that a suitable dosage should be used. GD,, of estrogens and AD,, of androgens which indicate relative potencies in inducing XY females and XX females in the medaka, respectively, are listed. The chemical nature of the endogenous sex inducers are discussed. There are pros and cons of the steroid theory of the natural sex inducers. Because of ( 1 ) the specificity of sex steroids as exogenous sex inducers, ( 2 ) the very low effective dosage of natural sex steroids, and ( 3 ) the selective incorporation of sex steroids into the differentiating gonad, it is likely that endogenous sex inducers are allied to or identical with sex steroids. A brief review on the manifestation of secondary sexual characters was given. REFERENCES Abe, T. ( 1967). Personal communication. Aida, T. (1921). On the inheritance of color in a fresh-water fish Aplocheilus Zatipes Temminck and Schlegel, with special reference to the sex-linked inheritance. Genetics 6, 554-573. Aida, T. (1930). Further genetical studies of Aplocheilus Zutipes. Genetics 15, 1-16. Aida, T. ( 1936). Sex reversal in Aplocheilus Zatipes and a new explanation of sex differentiation. Genetics 21, 138.153. Anders, A., and Anders, F. ( 1963). Genetisch bedingte XX- und XY-0 und XYund YY- 8 beim wilden Platvpoecilus maculutw aus Mexico. Z. Verefbungslehre 94, 1-18. Aoyama, T. (1955). O n the hermaphroditism in the yellow sea bream, Taius tumifrom. Japan. I . Ichthyol. 4, 119-129 (in Japanese with English &sun&). Aoyama, T.,and Kitajima, T. (1966). Sex reversal in the flat-head fish, Suggrundus meerderuoort ( Bleeker ) . Oral communication. Aoyama, T., Kitajima, T., and Mizue, K. (1963). Study of the sex reversal of Inegochi, Cociella crocodila (Tisesius). Bull. Seikai Regional Fisheries Res. Lab. 29, 11-33. Ashby, K. R. (1952). Sviluppo del sistema riproduttivo di Salmo truttu L. in condizioni normali e sotto l'influenza di ormoni steroidi. Rio. Biol. (Perugia) 44,3-19. Ashby, K. R. (1956). The effect of steroid hormones on the brown trout (Salmo trutta L.) during the period of gonadal differentiation. J. Embryol. Erptl. Morphol. 5, 225449.

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1. Introduction I1. The Parental Contribution . . . A . Conditions for Incubation . . . B Fecundity and Egg Size . . . 111. Events in Development . . . . A. Fertilization . . . . . . B . Incubation (Fertilization to Hatching) C Hatching . . . . . . D The Larva E . Metamorphosis . . . . . F.Timing . . . . . . . IV. Metabolism and Growth . . . . A . Rate of Development . . . . B . Yolk Utilization . . . . . C. Viviparity D . Biochemical Aspects . . . . E Respiration . . . . . . F. Growth . . . . . . . G. Endocrines. Growth and Metamorphosis V. Feeding, Digestion, and Starvation . . VI . Sense Organs . . . . . . A. Vision . . . . . . . B. Neuromast Organs . . . . VII . Activity and Distribution . . . . A . Phototaxis and Activity . . . B. Vertical Distribution . . . . C . Buoyancy and Pressure . . . D. Locomotion and Schooling . . . E . Searching Ability . . . . . VIII . Mortality. Tolerance. and Optima . . IX . Meristic Characters . . . . . X . Rearing and Farming . . . . A Techniques . . . . . . B. Sensory Deprivation . . . . XI . Conclusions . . . . . . Acknowledgments . . . . . . References . . . . . . . . 177

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I. INTRODUCTION

Fish eggs and larvae provide a relatively untapped source of biological material, increased by the recent improvements in techniques for rearing marine species. Apart from their intrinsic interest, experimentally based information on these early stages is required for further progress in the advancing fields of fish culture and fisheries research. General textbooks on ichthyology such as those of Lagler et al. (196Z), Nikolsky (1963), Norman (1963), and Marshall (1965) and on reproduction in fish by Breder and Rosen (1966) provide both general and some detailed information. Identification of eggs and larvae, apart from specialist papers, is possible from publications of Ehrenbaum ( 1909), DAncona et al. (1931-1933), through the current series of plankton sheets issued by the International Council for the Exploration of the Sea, and with the help of the extensive bibliographies by Dean (1916) and Mansueti ( 1954). Most species of fish pass through a larval stage before assuming the adult form at metamorphosis. Sometimes the newly hatched fish is called a “prolarva” (or alevin in salmonids) until the yolk is resorbed, and then a “postlarva” (or fry), The term “larva” is used here for all stages to metamorphosis in marine fish, although alevin and fry may be used when referring to salmonids or other freshwater groups.

11. THE PARENTAL CONTRIBUTION

Apart from the more obvious genetical effects on differentiation, rate of development, body form, size, and behavior, the parents, and especially the female, have an important influence on the viability of the offspring both on a species and individual level, in terms of ( a ) the conditions for incubation, ( b ) fecundity, and ( c ) egg size. A. Conditions for Incubation Differences of spawning season and time and of spawning sites and substrate mean that incubation can take place in a great variety of conditions which influence the early development and physiology of the off spring.

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1. EGGSSINGLE, WITH No PARENTAL CARE ( a ) Buoyant, planktonic-most marine fish, e.g., gadids, clupeids, flathh, and deep-sea fish. ( b ) Nonbuoyant, loose or attached to substrate-a common freshwater characteristic, e.g., cyprinids, pike Esox, or in littoral species, e.g., blenny Bbnnius, bullheads Cottus, sand eels Ammodytes; also found in some marine species, e.g., herring Clupea harengus, capelin Mallotus uillosus, cathh Anarhichas, and American flounder Pseudopburonectes americunus. Tendrils for attachment are found in many oviparous elasmobranchs, in the hagfish Myxine, smelt Osmerus, saury Scomberesox, and flying fish Exocoetus. ( c ) Nonbuoyant, buried in sand or gravel-many salmonids, grunion Leuresthes tenuis, and lamprey Petromyzon; in peat or mud Aphyosemion and Cynolebias where the eggs undergo diapause during the dry season. 2. EGGSSINGLE,SPECIAL ENVIRONMENTS The bitterling Rhodeus amarus lays eggs in the gills of the freshwater mussel and the lumpsucker Cureproctus under the carapace of the Kamchatka crab. 3. EGGSSINGLE,WITH PARENTAL CARE

( a ) No nest, but eggs protected-found in many littoral forms, e.g., the bullheads Cottidae, blennies Blenniidae and gobies Gobiidae. ( b ) Nests, often with parental protection and ventilation-also found in littoral species, e.g., blenny Ictalurus, sticklebacks Gasterosteus, and in other freshwater species such as sunfish Centrarcidae, bowfin Amia, lungfish Protopterus and Lepidosiren, and in the Cichlidae. Bubble nests giving good aeration are found in tropical or swamp species, e.g., Siamese fighting fish Betta splendens. ( c ) Parents carrying eggs-sea horses Hippocampus and pipefish Syngnathus have brood pouches and the sheat fish Platystacus a specially modified area of “spongy” skin. Marine catfish Ariidae, cardinal fish Apogonidae and Tilapia are mouth brooders, and Tachysurms incubates the eggs intestinally. ( d ) Ovovipiparity and viviparity (see Section IV, C)-elasmobranchs include picked dogfish Acanthius, smooth hound Mustelus vulgaris, electric ray Torpedo, stingray Trygon, and the nurse hound Mustelus laevis. Teleosts include redfish Sebastes, Heterandria, Anubleps, poeciliids such as Xiphophorus and half beaks Hemirhumpw.

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Angler fish Lophius and yellow perch Perca flavescens have massed but unprotected eggs; in the lumpsucker Cyclopterus and butterfish Blennius pholb the eggs are protected by the male.

B. Fecundity and Egg Size In higher latitudes the spawning season is often short and the eggs are liberated over a brief period of perhaps hours (clupeids) or over periods of some days, probably at certain times of the day or night (flatfish and gadids). Where the seasons are less marked spawning may occur over a much longer period or be intermittent throughout the year, especially where the time between generations is only a matter of weeks or months. Fecundity may be considered as the number of eggs produced in one year by a female although this may be very difficult to determine where spawning is protracted. Some examples of fecundity, egg size, and length at hatching are given in Table I. In general, fecundity is high where the eggs are liberated into open marine waters; it is lower in freshwater species and where there is parental care. There is also a strong tendency for fecundity and egg size to be inversely related. Apart from enormous interspecific differences, there are also considerable variations of fecundity within a species. Many authors have found that fecundity increases with length, weight, or age (see Fig. 1)) the relationship usually being of the form F = aLb, where F is fecundity, L is length, and a and b are constants. Year-to-year differences resulting almost certainly from environmental effects are also well established. For instance, sea temperature was correlated by Rounsefell (1957) with the fecundity of pink salmon, Oncorhynchus gorbuscha, higher temperatures apparently resulting in lower fecundities. (Here the effect of temperature on growth is a complication.) Bagenal (1966) reported density-dependent factors operating in Scottish flatfish, high densities being correlated with low fecundity. Anokhina (1960) found that fecundity in Baltic herring could be related to feeding conditions, high fat content of the female being reIated to high fecundity. Experimental studies by D. P. Scott (1961) indicated in rainbow trout, Salmo gairdneri, that an insufficient diet caused a reduction in egg number: in the guppy Lebistes fewer offspring were also produced when the females were kept on short rations (Hester, 1963). Extensions of this type of work are badly needed. Differences within a species resulting from latitude, area, race, or

Table I Fecundity (Eggs/Female/Yemr), Egg Diameter, and Length a t Hatching Species Molva molva Gadus morhua Melanogrammus aegleJinus Pleuronectes platessa Solea solea Swmber swmbrus Clupea harengus Clupeonella delicatula Salmo salar Osmerus eperlanus Acipenser sturio Cyprinus carpio Aeanthurus triostegus Oryzias latipes Scyliorhinus caniculus Lebistes reticulatush Zoarces viviparusa Sebastes viviparusb Mustelus mustelusb Squa1u.s amnthiasa a

a

Common name Ling Cod Haddock Plaice

Sole Mackerel Herring Kilka Salmon Smelt Sturgeon Carp Convict surgeon fish Medaka Spotted dogfish GUPPY Blenny Redfish Smooth hound Spur dogfish

Fecundity

20-30 X 10' 20-90 X 106 12 X 103-30 X 16 X 103-35 X 15 x 104 35-45 x 104 50 x 102-20 x 10-60x 103 103-104 50 X lO"50 X 80 X lo4-24X 18-53 X lo4 40 x 1 0 3 ~ 20-40" 2-20 10-50" 20-300 12-30 x 103 10-30 2-7

Number per spawning, which may be repeated often in one year. Viviparous or ovoviviparous.

Diameter (mm)

lo6 lo4

104

1.0-1.1 1.1-1.6 1.2-1.7 1.7-2.2 1.0-1.5 1.0-1.4 0.9-1.7 1.0 5.0-6.0

lo3

lo6

0.9 ?

0.9-1.6 0.7 1 .O-1.3 65.0(length) -

24-32

Length at hatching (mm)

3.0-3.5 4.0 4.O-5.0 6.0-7.0 3.2-3.7 3.0-4.0 5.0-8.0 1.3-1.8 15.0-25.0 4.0-6.0 9.0 4.8-6.2

1.7 4.5-5.0 100.0 6.0-10.0 35.0-40.0 5.0-8.0 250.0 240-310

182

J. H. S . BLAXTER

I06

-

5 1 0

0

x 0" 2 .-

-U C

3 U

u

' Lenqth,

loq scale (crn)

Fig. 1. The relationship between fecundity and length within a species. 1. Acipenser stellatus (Nikolsky, 1963); 2. Salmo salar (Pope et al., 1961); 3. Cyprinw carpio (Nikolsky, 1963); 4 . Pleuronectes platessa (Clyde) (Bagenal, 1966); 5. Clupea harengus (northern North Sea) ( Baxter, 1959); 6. Mekznogrammus aeglefinus (see Parrish, 1956); 7. Osmerus eperlanus ( Lillelund, 1961); 8. Salvelinus fontinalis (Vladykov, 1956); 9. Sardinops caerulea ( MacGregor, 1957); and 10. Sebastes marinus (Faroe Island) (Raitt and Hall, 1967).

season are no doubt interconnected. Considerable differences of this type were reported for plaice, PZeuronectes platessu ( Bagenal, 19fH), for herring (Baxter, 1959; Kandler and Dutt, 1958), for species of Oncorhynchus (Rounsefell, 1957), and for Sulmo sulur (Pope et ul., 1961). An interesting characteristic of certain species is a difFerence in fecundity of the left and right ovary. The left gonad contains more eggs in Oncorhynchus (Rounsefell, 1957), Salmo (Pope et al., 1961), and smelt Osmerus ( Lillelund, 1961) . The significance of this is not clear. Egg size varies at the interspecific level (see Table I ) , with larger eggs being especially associated with freshwater species like the sal-

4. DEVELOPMENT:

183

EGGS AND LARVAE

monids or where fecundity is very low, as in many elasmobranchs. At the intraspecific level, differences of egg size as a result of area or river were noted by Rounsefell (1957) in Oncorliynclzus, Salmo, Cristivomer, and Salvelinus species. In Salmo salar (Pope et al., 1961) there are differences in egg diameter related to length, fecundity, and river. In Tilapia egg weight may increase 2-4 times or even more depending on the size of the female (Peters, 1963); in the flounder, Platichthys flesus, large females, or females from low salinities, have larger eggs (Solemdal, 1967). Larger eggs are also found in larger females of the spur dogfish Acantliias ( Templeman, 1944). There are differences in average dry weight of the order of four times among the various races of herring (Hempel and Blaxter, 1967) and small differences between very young first spawners and repeat spawners. In two darter Etheostoma species with long spawning seasons, the egg diameter tends to be greater in the cooler winter months (Hubbs et al., 1968). The connection between egg size and fecundity in related species may be correlated with the conditions for incubation. For example, Tilapia tholloni, a substrate brooder, has 5003000 eggs depending on length. On the other hand, T . mossambica and T . macrocephala, which are mouth brooders, have less than 500 eggs that are considerably greater in weight (Peters, 1963). Garnaud (quoted by Smith, 1957) reported two species of Apogon, one, A. imberbis, with an egg diameter of 0.5 mm and fecundity of 22,000, and the other, A. conspersus, with an egg diameter of 4.5 mm and fecundity of 150. The relationship between egg size and egg weight within a species is shown for the different races of herring in Fig. 2. The winter-spring spawners have a low fecundity and large eggs,

I .

\

'.._ --

I

/

y'.

! 0.15

' F ' M ' A ' M ' J'J ' A ' S ' O ' N ' D

Spawning month

Fig. 2. Fecundity and average egg dry weight in different races of herring (data from Baxter, 1959; Hempel and Blaxter, 1967; Parrish and Saville, 1965; Kandler and Dutt, 1958).

184

J. H.

S.

BLAXTER

an adaptation to poor food for the young, but a low predator population. In summer-autumn conditions fecundity is high and egg size low, presumably an adaptation to good food supplies and many predators. Intraspecific differences in fecundity and egg size deserve further study in terms of a link between ecological conditions and the physiology of maturation of the ovary. A general pattern seems to emerge of marine fish with many, small buoyant eggs, a short incubation period, and vulnerable larvae. Freshwater fish have larger demersal eggs, a long incubation period and larger, less vulnerable larvae, while the littoral forms exhibit protective devices to prevent losses in this particularly difficult environment. The initial conditions of development determined by the genotype of the parent and reproductive behavior must have a considerable influence on the viability of the young and its physiology, in that environmental conditions such as temperature affect the speed of development, salinity presents problems of osmoregulation, and oxygen must be obtained for respiration. The egg is susceptible and yet cannot make defensive responses to mechanical shock, drift by current, toxins, light, and predators. The larva should live in conditions where food can be obtained and protective behavioral devices can be practiced.

111. EVENTS IN DEVELOPMENT

A. Fertilization The physiology of fertilization in fish, with special reference to the extensive Japanese work, has been fully reviewed by Yamamoto (1961) from which much of the present account is taken. There is some evidence for the action of gamones in Lampetra and in teleosts; these activate the sperm and serve as chemical attractants toward the egg, while other gamones are known to paralyze or agglutinate sperm. In the bitterling species Acheilogwthus and Rhodeus sperm aggregation and activity have been noted in the micropyle region of the chorion. The chorion or egg case is relatively tough with a funnel-shaped micropyle at the animal pole. Within the chorion a plasma or vitelline membrane [also called a pellicle or surface gel layer (Trinkaus, 1951)] surrounds the yolk and cytoplasm (ovoplasm) of the egg. Fertilization, which requires the presence of small concentrations of Ca or Mg ions, is normally monospermic in teleosts, the micropyle being too narrow to

4.

DEVELOPMENT: EGGS AND LARVAE

185

allow more than one sperm to pass at a time. The ovoplasm and chorion separate as the egg is activated by the sperm and a plug forms in the micropyle, further sperm being rejected. Where polyspermy occurs, as in some elasmobranchs, only one sperm fuses with the egg nucleus, the rest probably being resorbed and used as nutrient. Removal of the chorion permits polyspenny in teleost eggs; it seems that polyspermy is usually prevented by rapid changes at the micropyle, rather than over the egg cortex. In salmonids water activation (not to be confused with activation by sperm) takes place (e.g., see Prescott, 1955). When the egg is released into hypotonic solutions like river water the vitelline membrane becomes opaque and there are changes in its permeability. If sperm are not immediately avaiIable these changes may also affect f ertilizability. Following fertilization, the prominent alveoli in the egg cortex of salmonids, acipenserids, and lampreys disappear. In the medaka, Oryxius Zutipes, these alveoli break down progressively from the animal pole. The separation of the cortex from the chorion leads to the appearance of the perivitelline space. The chorion is permeable to water and small molecules, but larger molecules of a colloidal nature are retained in the perivitelline fluid. In Oryzias and Lampetru these colloids maintain an osmotically based tension within the chorion. It seems likely that the colloid is derived from polysaccharide material in the cortical alveoli so that the formation of the perivitelline space is partly owing to a decrease in volume of the ovoplasm, as the alveoli release colloid, and partly due to an osmotic distension of the chorion. According to Ginsburg ( 19Sl), polyspermy is blocked in sturgeon and trout eggs by the discharge of the cortical alveoli in the micropylar region. Chemicals, such as urethane, which cause polyspermy apparently retard the secretions of the alveoli, while removal of the perivitelline fluid in trout eggs allows the penetration of many sperm. The chorion also hardens (see Section VIII) thus protecting the embryo in the early, more vulnerable stages. qrobably the inner layer of glycoprotein is mainly responsible for this and it has been suggested that the alveolar colloid, Ca ions, phospholipids, or hardening enzymes also play a part. In salmonids, Zotin (1958) reported hardening of the chorion because of an enzyme in the perivitelline fluid; Ca ions affect the enzyme rather than the chorion itself. Ohtsuka (1960) considered that a phospholipid was liberated from the cortex (not from the alveoli) in Oryxias eggs. In Fundulus the chorion hardened with oxidizing agents but not with reducing agents. The soft chorion appeared to be impregnated with protein containing SH groups. Hardening resulted from oxidation of SH to SS groups by means of aldehydes produced from

186

J. H. S. BLAXTER

polysaccharides with a-glycol groups. Zotin distinguished between the initial enzyme action and subsequent hardening processes which last much longer and where the enzyme is no longer functioning. Thus the initial enzyme reaction is blocked when Ca ions are bound by citrate or oxalate or by the use of NaCl or other chlorides. Later hardening is not susceptible to many of these factors. The eggs of Oryzias, Gasterosteus, and Lampetra can be activated by pricking. Other activating agents are surface-active chemicals and lipid solvents (perhaps emulsifying the cytoplasm at the animal pole) and thermal shock, electric fields, ultraviolet light, and high frequency vibrations. This type of artificial parthenogenesis usually leads to irregular cleavage, but stringent precautions are needed to prevent contamination with sperm in such experiments. Of interest is the ability of eggs and sperm to retain their fertilizability after leaving the parent. According to Yamamoto (1961) fish eggs lose this capacity after a very short time, but this can be increased if they are retained in isotonic Ringer's solution. While this is true of some freshwater eggs, presumably as a result of water activation, in seawater the capacity for fertilization is retained for much longer-certainly for hours in the herring. Nikolsky (1963) states that sperm motility is short lived where spawning takes place in fast-flowing water, for example, 1015 sec in Oncorhynchus. In slower flows, sturgeon sperm is motile for 230-290 sec, and in the sea herring sperm may be motile for hours or days. Observations of short-lived activity are difficult to make; thus, some of these figures must be considered approximate. Storage of gametes is a useful technique in fish farming to allow controlled fertilizations in the laboratory and to obviate the need for transporting eggs in the susceptible pregastrulation stages. Salmonid gametes are best stored dry below 5°C (e.g., Barrett, 1951, Withler and Morley, 1968); those of the herring may be held in buffered egg-yolk diluents, but are also best kept dry at about 4°C (Blaxter, 1955). While this permits storage for, at most, a few days long-term techniques are also possible. Herring sperm, but not eggs, were kept for some months in a diluent consisting of 12.5% glycerol in 3% salt solution (diluted seawater) at -79°C and crosses made successfully between spring and autumn spawning races (Blaxter, 1953, 1955; Hempel and Blaxter, 1961). Sneed and Clemens (1956) also succeeded in holding out-of-season carp sperm immotile for 30 days at 3'5°C in frog Ringer. Carp sperm could also be frozen and stored at -73°C in isotonic Ringer containing &12%glycerol with some survival when thawed after 60 hr of storage. More recently, Truscott et al. (1968) have shown that salmon sperm can be stored for 1-2 months at temperatures of -3" to -4.5"C using diluents such as 5%

4.

DEVELOPMENT: EGGS AND LARVAE

187

ethylene glycol or 5% dimethyl sulfoxide, retaining 7040%fertility. Horton et al. (1967) obtained alevins from salmon eggs fertilized with sperm frozen in liquid nitrogen with dimethyl sulfoxide as a protecting agent, but the fertility rate was low. Hoyle and Idler (1968) have also obtained fertile salmon sperm after storage in liquid nitrogen using ethylene glycol with added lactose or serine. Slow freezing produced the best results. Mounib et al. (1968) succeeded in storing cod sperm for up to 60 days using 17-24% glycerol and temperatures of -79” and -196°C. Initial experiments suggest that faster cooling rates gave the better results with cod sperm.

B. Incubation ( Fertilization to Hatching ) The progress of cleavage, formation of layers, and morphogenesis have been described in a number of standard textbooks such as Rudnick ( 1955), Waddington ( 1956) and Smith ( 1957), with Oppenheimer (1947) and Devillers (1961) stressing structural changes from the viewpoint of experimental embryology. More detailed information is limited mainly to freshwater species like the trout Salmo trutta, killifish Fundulus, medaka Oryzias, goldfish Carassius, and to the dogfish. New (1966) gives information on the problems of culturing Fundutus, Oryzias, and Salmo eggs for the purpose of experimental embryology. Most fish eggs are round, although in the anchovy Engraulis and bitterling Rhodeus they are ovoid, and in certain gobies pear-shaped. Most species have telolecithal eggs with yolk more concentrated at the vegetative pole; some marine species have oil globules of varying size and number (see Simpson, 1956). Before fertilization the cytoplasm may be mixed with or separate from the yolk. The extent to which polarity exists at this stage has not been described in many species. After fertilization (as the cortical alveoli release colloid, the perivitelline space develops and the chorion hardens) cytoplasm migrates to the future blastodermal region, most of it arriving by the first cleavage. The remaining cytoplasm forms a “halo” or periblast under the blastoderm (see Fig. 3 ) . In lampreys cleavage is holoblastic but with the formation of microand macro-meres. In hagfish, elasmobranchs, and teleosts it is meroblastic. Other groups such as bowfin Amia, gar Lepidosteus, and sturgeon Acipenser have intermediate features. With some variation, the meroblastic group possess a blastodermal cap of cells at the animal pole after the initial stages of cleavage. In Fundulus the surface gel layer overlying the blastoderm, being sticky on its inner surface, serves to hold the outer blastomeres together (Trinkaus, 1951). Usually cleavage is not complete,

188

J. H. S. BLAXI’ER

Fig. 3. ( A ) Transverse section of early blastula showing adhesion of blastomeres to the surface gel layer and attachment of blastoderm to the periblast at the periphery only (Fundulus, after Trinkaus, 1951 ). ( B ) Sagittal section of later blastula showing gastrulation; epiboly shown by arrows. ( C ) and ( D ) Surface view of eggs in later stages of gastrulation. Key: b, blastoderm; bp, blastopore; dl, dorsal lip of blastopore; gr, germ ring; n, notochord; np, neural plate; p, periblast; sgc, subgerminal cavity; sgl, surface gel layer; and y, yolk.

and in the deeper layers the periblast becomes syncytial and is involved in mobilizing the yolk reserves. There are substantial cohesive forces between the developing blastomeres and the surrounding periblast which are important in the subsequent morphogenetic movements. The blastoderm now commences to thin and overgrow the yolk (epiboly) and at the same time invaginate at its periphery (Fig. 3 ) . The periblast seems closely connected with this spreading tendency of the blastoderm, which at the junction may be thickened to form a germ ring. The syncytial periblast seems to have the property of autonomous spreading, and it is likely that cell proliferation in the blastoderm is relatively unimportant. Devillers (1961) suggests that the periblast acts as an intermediary between two “non-wettable” components-the blastoderm and yolk. As epiboly proceeds the blastopore contracts as does the surface gel layer over the yolk. In Fundulus this layer probably solates and passes inward (Trinkaus, 1951). The form of the developing germ then seems to be controlled by a balance of the forces of adhesion of the deeper blastomeres with each other and the syncytium, of tension in the surface (yolk) gel layer and of contractility of the periblast. The embryonic axis is laid down by a process of convergence and concentration in relation to the dorsal lip of the blastopore, the quantity of yolk present having some influence on the time at which this event occurs. Presumptive areas have been mapped in the early gastrulae of

4.

DEVELOPMENT: EGGS AND LARVAE

189

some species and show considerable variation. In earlier stages the eggs seem to be of the regulatory type. In FunduZus the 2-cell and 4-cell stage can withstand a loss of half the number of blastomeres. The embryos of Carussius can be divided at the 8-cell stage, each part sometimes giving a normal embryo. Up to the 1Bcell stage two embryos can be fused resulting either in twinning or an oversized single embryo. Removal of the yolk from the blastoderm before a critical stage is reached ( 8 cell stage in Carassius, 32 cell stage in Fundulus, blastula in Salrrw) brings development to a halt. Incomplete removal of the yolk before the critical stage may, however, not prevent further development. It is likely that organizer substances rather than nutrient material diffusing from the yolk are more important. At later stages even the embryonic shield of Fundulus may be isolated and cultured to a fairly advanced stage, with the development of primitive axial organs, ears and eyes, and even with cardiac contractions and independent movement ( Oppenheimer, 1964 ) .

C. Hatching The time to hatching is both a specifically and environmentally controlled character with temperature and oxygen supply exerting a considerable effect. Hatching results from a softening of the chorion (see Fig. 15) because of enzymic or other chemical substances which are secreted from ectodermal glands usually on the anterior surface or from endodermal glands in the pharynx. In the sturgeon Acipenser the latter are innervated by the palatine nerve (Ignat’eva, quoted by Deuchar, 1965). The activity of the larvae, which may be enhanced by increase of temperature or light intensity or by reduction of oxygen tension, assists in breaking through the chorion. The biochemical aspects of hatching are dealt with by Hayes ( 1949), Smith ( 1957), and Deuchar ( 1965). The enzymes have apparently been identified in a number of species, but certainly in salmonids Hayes’ work shows there is doubt about their mode of action. The chorion, which resists digestion by trypsin and pepsin, appears to be of “pseudo-keratin.” Kaighn (1964) measured the amino acid and carbohydrate components in Fundubs. Cystine comprised only l%,compared with 12%in keratin. It is therefore unlikely that disulfide links play a role in stabilizing chorionic protein as they probably do keratin. The hatching enzyme works best under alkaline conditions, pH 7.2-9.6 and temperatures of 14”-20°C having been reported as optima. Very little hydrolysis takes place and Hayes speculates that the enzyme may be a reducing agent which liquefies the chorion. In Oyzias the enzyme is probably a tryptase. In Ftmdulus, Kaighn (1964) obtained purified chorionase and concluded

190

J. H. S. BLAXTER

that digestion of the chorion was mainly a proteolytic process, although he could not duplicate its action with other proteases. Whatever the mode of action of the enzyme, it is likely that a considerable part of the nutrient material in the chorion can be utilized by the embryo via the perivitelline fluid and the losses at hatching may not be too serious ( Smith, 1957).

D. The Larva At hatching the larva is usually transparent with some pigment spots of unknown function. Notochord and myotomes are clear with usually little development of cartilage or ossification in the skeleton. A full complement of fins is rarely present, but a primordial fin fold is well developed in the sagittal plane. The mouth and jaws may not yet have appeared, and the gut is a straight tube. Although the heart functions for a considerable period before hatching, the blood is colorless in the majority of species and the circulation and respiratory systems poorly developed, The yolk sac is relatively enormous with, presumably, hydrodynamic disadvantages. Pigmentation of the eyes is very variable, but where the eye is not functioning at hatching it very soon develops. The kidney is usually pronephric with very few glomeruli. Very little is known about the endocrine glands, gonads, and other organs of the body cavity at such an early stage. As the yolk is resorbed, the mouth begins to function, the gut and the eyes develop further, and the larva becomes fitted for transfer to sources of external food. One of the earlier systems to develop is that responsible for locomotion and support, the primordial fin being fairly soon replaced by median fins and the skeleton laid down. This is one of the better known aspects of later development because it is a system less easily damaged in such delicate organisms and because of the importance of meristic characters (see Section IX) in racial studies of fish. Branchial replaces cutaneous respiration as the gill arches and filaments appear. The swim bladder may or may not be present during the larval phase. It is possible that this and the eyes, which are potentially dangerous in making the transparent larva visible, are silvered in such a way as to render them inconspicuous.

E. Metamorphosis A clear change or metamorphosis from the larval to adult form is to be found in many species. In others there may be a number of less

4.

DEVELOPMENT: EGGS AND LARVAE

191

marked metamorphoses, e.g., in salmonids and eels. The most obvious signs are the laying down of scales and other pigmentation and often the first appearance of hemoglobin in the circulation. The swim bladder and lateral line may also develop first at this stage. In flatfish there is rotation of the optic region of the skull and the change in the normal orientation of the body so that they eventually come to lie on one side. There are often concomitant changes in distribution and behavior such as schooling. Barrington ( 1961) gives detailed consideration to the physiological changes associated with metamorphosis in salmonids, eels, and the lamprey, especially from the aspect of thyroid activity and osmoregulatory functions. The time to reach metamorphosis may be a matter of days in tropical species, a few weeks or months in the majority of fish from temperate latitudes, or periods of years in the sturgeon Acipenser and eel Anguilla. It is controlled not only genetically but also by temperature and food supply, which may affect the rate of growth, and possibly by social (hierarchical) factors as well.

F. Timing To give some idea of the timing of the events just described, examples of the approximate duration of different stages under natural conditions are given in Table 11. The modification of these times experimentally or by fluctuations in environmental conditions is discussed in the succeeding pages.

IV. METABOLISM AND GROWTH

A. Rate of Development Obvious specific differences in time to hatching may be masked by variations in ambient temperature, which is one of the most potent influences on rate of development. Detailed observations of temperature effects during development are scarce. Fluchter and Rosenthal ( 1965), however, showed between 3.5" and 9°C a doubling of heart rate in the embryos of the blue whiting, Micromesistius poutassou, and more rapid embryonic movements. The effect of temperature on time to hatching, a commonly used criterion, is shown in Fig. 4. Low temperatures retard hatching (see reviews by Battle, 1930; Hayes, 1949; Kinne and

192

J , H. S. BLAXTER

Table I1 Duration of Events in the Development of Some Species Weeks from fertilization to Species

Hatch

First feeding

Metamorphosis

11-13 0.8-1.5 1.3-2.0 Scomber scombrus (mackerel) 4-5 0.75 0.25 Roccus saxatilis (striped bass) 8-10 2.0-4.0 2.5-5.0 Osmerus eperlanus (smelt) ? 0.15 0.7 Acanthurus triostegus (convict surgeon fish) 16-18 2.5 3.5 Clupea harengus (Clyde herring) 10-12 4.0 2.5 Pleuronectes platessa (plaice) 1.5-2 .o 2 . 0 - 2 . 4 Not clear cut Oryrias latipes (medaka)

Temp. range ("C) 9-15

Main author Sette (1943)

17

Mansueti (1958)

4-14

Lillelund (1961)

26

Randall (1961)

7-10

Blaxter and Hempel (1963) Ryland (1966)

7-11

Salmo salar (salmon)

20-22

26-28

Gradual

1-7

Squalus acanthias (spur dogfish) Scyliorhinus caniculus (spotted dogfish)

ca. 104

ca. 104

< 104

4-12

New (1966); T. Iwai, personal communication D. H. A. Marr, personal communication Templeman

24-32

28-36

28-36

4-12

Amoroso (1960)

20-25

(1944)

Kinne, 1962), and at a theoretical low temperature (the biological zero) the incubation period will be infinite. The product of incubation time ( D ) and temperature ( T)-day-degrees-was originally thought to be constant, i.e.,

TD = k (1) This was modified to use the temperature, not from zero, but from the biological zero ( T o ) ,i.e., ( T - T,)D = k (2) There has been increasing criticism of the concept, for example, by Kinne and Kinne (1962) and Garside (1966), on various grounds. Plots of 1/D against T are curvilinear over wide ranges of temperature, simple linearity only applying over a narrow range. This invalidates the formulas given above. Furthermore, there may be inflections even of the curvilinear relationship at extreme temperatures. The biological zero, which

4.

DEVELOPMENT: EGGS AND LARVAE

193

Fig. 4. Time from fertilization to hatching at different temperatures. 1. Saluelinus fontinulk (see Hayes, 1949); 2. Pleuronectes platessa (see Simpson, 1956); 3. Gadus mucrocephulus (Forrester and Alderdice, 1966); 4. Sardinops caerulea ( Lasker, 1964); 5. Clupea harengus (Blaxter and Hempel, 1963); 6. Coregonus clupeaformis (Price, 1940; Braum, 1964); 7. Osmerus eperlunus (Lillelund, 1961); 8. Roccus saxatih (Mansueti, 1958); 9. Cyprinodon macularius (Kinne and Kinne, 1962); 10. Salmo gairdneri (irideus) (Garside, 1966); 11. Salmo trutta (fario) (Gray, 1928b); 12. Enchelyopus cimbrius (Battle, 1930); and 13. Scomber scombrus (Sette, 1943).

is usually given between 0" and -2°C and most often around -1.5"C, may be below the freezing point of water or the body fluids themselves. In addition, abnormalities may occur at less extreme temperatures which are not necessarily lethal in the strict sense. Improvements in describing the mathematical relation between D and T arise in later work. For example, Blaxter (1956) used the equation

(T - To)(D - Do) = k (3) for development of Clupea harengus, where Do is the theoretical time to hatching at infinite temperature, not in itself a very satisfactory additional constant. Lasker (1964), working with the eggs of Sardirwps caerulea, used the equation

D = aTb (4) where a and b are constants, and Braum (1964), using the eggs of whitefish Coregonus and pike Esox lucius, D

=

D, $- 1.26T=-T

(5)

194

J. H. S. BLAXTER

where D, is the minimum possible incubation time at the maximum permissible temperature T,. The van't Hoff values over different temperature ranges reflect the nonlinearity of plots of log D against T (see Fig. 4), the values being higher at lower temperatures. Thus in EncheEyopus cimbrius the Qlo varies between 6.5 and 1.5 over the temperature range 5"-23"C (Battle, 1930) and between 6.5 and 2.0 over the range 3"-18°C in herring (Blaxter, 1956). The value of this type of theoretical consideration may lie in establishing criteria for optimum conditions of development. Thus the optima may be where van't Hoff values lie between certain limits. Certainly the day-degree concept is useful as an approximation for predicting events in normal hatchery practice. Other environmental factors influence the rate of development. Low salinities may accelerate or retard the time to hatching (see Kinne and Kinne, 1962; Forrester and Alderdice, 1966), while oxygen lack has a retarding effect on development, especially at higher temperatures ( see Garside, 1966). Laale and McCallion (1968) found that the development of the zebra fish, Brachydanio rerio, could be arrested before gastrulation by the use of the supernatant of homogenates produced from other zebra fish embryos. This arrest, which could be reversed, appeared to be an effect at the cellular level, the nuclei of the arrested embryos all being in interphase.

B. Yolk Utilization The efficiency with which yolk is transformed to body tissue and the effect of the environment on utilization is important in that larger larvae may be expected to be stronger, better swimmers, less susceptible to damage, and less liable to predation. Efficiency at any time may be expressed as a percentage: dry weight increment of body dry weight decrement of yolk

x

100

More often efficiency is measured from fertilization to final yolk resorption (or to maximum weight attained on the yolk reserves). This is gross efficiency, i.e., dry weight of final body dry weight of original yolk or from fertilization to intermediate stages as

x

100

4. DEVELOPMENT:

195

EGGS AND LARVAE

dry weight of body dry weight of original yolk - dry weight of remaining yolk

x

100

or more precisely dry weight of body

dry weight of body

+ dry weight of yolk used for maintenance x 100

The difficulties of measuring efficiency by dry weight lie in the need for taking samples of an egg population at different stages with the accompanying problems of initial differences in egg weight. Utilization of material from the chorion or losses of excretory products are also difficult to allow for, as are the possibilities of uptake of organic matter from the environment (fitter’s theory, see Section V ) . Another serious problem when comparing, for example, environmental effects such as temperature on efficiency is the question of making dry weight measurements at “equivalent” stages (D. H. A. Marr, 1966). Both hatching and maximum weight (attained on the yolk) can be questioned for staging; hatching at different temperatures can result in larvae of quite different appearance, while full yolk utilization is often not complete when maximum weight is reached, some yolk remaining in the yolk sac (see Fig. 5 ) . Furthermore, the disappearance of the yolk sac is no certain indication that all the yolk has been used as it may be present in storage spaces within the larval body, for example, in the subdermal spaces of cod and plaice larvae (Shelbourne, 1956). D. H. A. Marr ( 1966) adopted the ratio dry weight of body X 100 dry weight of body remaining yolk

+

(as percentage)

as a criterion for equivalent staging, comparing in S . salar the efficiency Herring (a-9”C)

10

30

50

Salmon (IO’C)

1 1 0

130

I50

I70

I90

Days after fertilization

Fig. 5. The relative proportions of yolk ( Y ), embryo ( E ) , and chorion ( C ) during the development of a small egg (herring: Blaxter and Hempel, 1963) and a large egg (salmon: D. H. A. Marr, 1966). The vertical dashed line represents hatching. Note the dihrence in the scale of the ordinates.

196

J. H. S. BLAXTER

of development at different temperatures between the 15% and 80%stages. Ryland and Nichols (1967) used the ratio rate of growth in length rate of yolk disappearance

x loo

for equivalent staging when comparing the efficiency of development at different temperatures during the yolk sac stage of P. platessa. The use of maximum weight as a stage for making comparisons still remains, however, a useful criterion and one with immediate meaning when deciding on the optimum conditions for hatchery practice. Calculations of efficiency by various methods are given in Table 111. Efficiency over the whole process of yolk utilization is mainly between 40% and 70%although clearly cumulative efficiency must decrease as growth proceeds and the maintenance requirements increase ( Gray, 1928a). Experiments with temperature ( see Fig. 6 ) indicate certain optima for maximum efficiency. Other influences on efficiency are the original egg weight at the intraspecific level in C. hurengus (Blaxter and Hempel, 1966), and light conditions, contour of the substrate, and turnover of water in the photonegative alevin of s. salar living within the interstices of the spawning redd (D. H. A. Marr, 1965, 1967). Here highest efficiency is achieved under dark conditions, on a grooved substrate, with a rapid turnover of water. A word of caution is required where larval feeding may occur well

\

\

3

0

5

1 0

15

20

TemflC)

Fig. 6. Efficiency of development (see text and Table 111), at different temperatures. 1. Salmo truttu (furio)-yolk sac period (see D. H. A. Marr, 1988); 2. Sdmo sulur-alevin stage (D. H. A. Marr, 1986); 3. S. sah-early alevin (Hayes and Pelluet, 1945); 4. Pleuronectes plutessu-yolk sac larva (Ryland and Nichols, 1987); 5. Clupea hurengus-small eggs, yolk sac period; 6. Clupea harengus-large eggs, yolk sac period (Blaxter and Hempel, 1968).

Table III Efficiency of Yolk Utilization Species Salmo trutta (trout) Salmo trutta (trout)

Method Dry weights Dry weights

Stage Fertiluation to 50-80 days Fertilization to max. weight

Hatching to 10 days after Calorific values Fertilization to max. weight Special dry weight % 15430% (yolk index (see text) sac stage)

Temp. ("C)

Efficiency

10

63

Gray (1926)

15

56

Gray (1928%)

0-16

Author

( %)

Salmo salar (salmon) Salmo salar (salmon) Salmo salar (salmon)

Dry weights

Salmo gairdneri (rainbow trout)

Dry weight and metabolic criteria

Fertilization to max. weight

10

60

Smith (1957)

Fundulus kteroclitus (killifish) Silurus glanis (sheat fish)

Wet weights

19.4-21.4

62

C. G. Scott and Kellicott (1916)

(?)Wet weights

Fertilization to max. weight During yolk sac stage

?

66

Ivlev, quoted by Lasker (1962)

Sardinops caerulea (California sardine)

Calorific values and respiration

Fertilization to yolk resorption

14

79

Lasker (1962)

Clupea harengus (herring)

Dry weights

Pleuronectes platessa (plaice)

Rate of growth in length + rate of yolk disappearance

Fertilization to hatch Fertilization to max. weight During yolk sac stage

42-59

10

41

7.G14.3

64-70

t312 8-12 2.6-9.8

40-80 5M.5 35-58

Hayes and Pelluet (1945) (Fig. 6) Hayes (1949)

D. H. A. Marr (1966) (Fig. 6)

Blaxter and Hempel (1966) (Fig. 6)

Ryland and Nichols (1967) (Fig. 6) Y

w

-a

198

J. H. S. BLAXTER

before final yolk resorption. High efficiency may result from low activity, a high proportion of yolk being used for growth; if this is reflected in low feeding activity it could be a very undesirable trait.

C. Viviparity Amoroso (1960), who gives a comprehensive review of this subject, points out the rather indistinct barrier between ovoviviparity where the young develop within the female only on their yolk reserves, and viviparity, where the nutrient requirements are obtained from the mother. In the first place, there are a wide variety of methods of obtaining these nutrients: by absorption through simple external surfaces, by swallowing, or by “placental” connections. Perhaps all these may be considered as viviparous. In the second place, initial development may be ovoviviparous (on the yolk supply) with viviparity superimposed later. This is likely to be the case in the very early stages of all viviparous fish, but in the smooth dogfish, Mustelus laevis, and goodeid teleosts, for example, there is a change from one to the other rather later. Details of the functional morphology of viviparity are dealt with in the chapter by Hoar, this volume. The changes of weight found during the development of various species ( Amoroso, 1960) can be very striking. Some of the ovoviviparous ones, where maternal nutrients are scarce, show decreases of organic matter between the fertilized egg and final embryo, for example, of 23434% in Torpedo spp. This gives an efficiency of 6674%which puts this species very much in the same category as oviparous forms (Section IV, B ) although it is rarely certain what nutrient is obtained from the mother. The long gestation period of 4 6 months with this order of efficiency suggests some nutrients are being absorbed from the oviduct. In other “ovoviviparous” species there may be gains in organic matter, e.g., of 35%% in Mustelus uulgaris and 16288 in Trygon. In Mustelus laeuis, sometimes more strictly called viviparous, the gain is 1064%,although Te Winkel (1963) reported a fall in weight of organic matter early in the development of a close relative, M. canis.

D, Biochemical Aspects Much work has been done in the past on the larger salmonid eggs which can provide a greater bulk for analysis (see Hayes, 1949; Smith, 1957, 1958) or on whole ovaries (Lasker, 1962). Some more recent work

4. DEVELOPMENT:

EGGS AND LARVAE

199

which includes the use of isotopes, chromatography, and histochemistry is mentioned by Deuchar (1965) and Williams (1967).

1. WATERRELATIONS The swelling of eggs, with the formation of the perivitelline space as a result of water uptake, is a general phenomenon signifying fertilization. The chorion at fertilization is permeable to water and also to urea, glucose, salts, and certain dyes. It seems likely that the colloidal materia1 liberated from the cortical alveoli cannot escape through the chorion and creates an osmotic pressure which draws in water. This effect can be inhibited by high osmotic pressure in the outside medium, The chorion then hardens, a process taking a matter of a few hours (see Fig. 15) and the osmotic forces become matched by the resistance of the chorion. In hypertonic solutions the eggs of salmonids and of Mullus barbatus lose water only from the perivitelline space and not from the embryo (see Zotin, 1965). Some loss does, however, occur in acipenserids. The use of D,O on water-activated and early fertilized eggs of Oncorhynchus tshawytscha seems to confirm the view that only the perivitelline space is penetrable by water (Prescott, 1955). Subsequent use of 3H20,?,NNaCl, and Na13*Ion the water-activated eggs of Salmo gairdneri has shown a definite but limited permeability of the vitelline membrane to anions, cations, and water (Kalman, 1959). Unfortunately, this was not done on fertilized eggs. Recently, however, Potts and Rudy (1969) have confirmed with 3 H , 0 that the vitelline membrane of fertilized eggs of S. salar has a high permeability before laying and during water hardening. Subsequently permeability is low until the eyed stage. The use of 24Na(Rudy and Potts, 1969) showed that sodium exchange is confined initially to the perivitelline fluid but accumulation within the embryo occurs during the eyed stage. Terner (1968) reported that during the eyed stage of Sabno gairdneri external substrates such as I4C-labeled pyruvate and acetate were apparently taken up and metabolized as judged by the presence of 14C0, in the respiratory CO,. Wedemeyer (1968) also found that s5Zn was taken up by developing eggs of coho salmon, 0. kisutch. Almost all was bound to the chorion, but 26%was found in the perivitelline fluid, 2%in the yolk, and 1% in the embryo. Mounib and Eisan (1969) found that both "C-labeled pyruvate and glyoxylate could be utilized in the form of an exogenous substrate by salmon ( S . salar) eggs. Lactate was produced and any carbon atom in these compounds could be incorporated by the eggs into organic acids, lipids, nucleic acids or proteins. The formation of 14C-aminoacids indicated the presence of an active transaminase system.

200

J. H. S. BLAXTER

The use of freezing point measurements on the yolk of herring and plaice eggs (Holliday and Jones, 1965, 1967) gives an alternative picture. In the herring, which has a deniersal egg, the yolk is not regulated osmotically until after gastrulation when it is covered by cells. However, in the pelagic floating egg of the plaice, regulation occurs from fertilization, indicating the ability of the vitelline membrane to osmoregulate. This seems necessary from the buoyancy aspect. If Gray's (1926) measurements of wet and dry weights of the embryo and yolk of trout are generally true of salmonids, then the yolk as a high density nutrient (41%dry weight) requires considerable quantities of water for transformation to the relatively watery embryo (16% dry weight). Obtaining this water may be a problem if the permeability of the vitelline membrane remains limited throughout much of development. Smith (1957) suggests that growth may be retarded until hatching occurs and water becomes more readily available. It is likely that highly desiccated yolk is only feasible in the demersal egg; in Sardinops caerulea the water content of the larval yolk is about 91%(Lasker, 1962).

2. CHEMICAL COMPOSITION The chemical composition of the eggs of two species are given in Table IV. Further data are given by Phillips and Dumas ( 1959), who showed in particular that there was no difference in the constituents of different sized eggs of Salmo trutta. There is considerable difficulty in deciding on the sequence of utilization of various materials for energy production. Analysis of the change in chemical components may be unreliable where substances like carbohydrates are being synthesized. Heat production and respiratory quotients are difficult to determine accurately in small eggs, and COz Table IV Analyses of Eggs" Percent of wet weight Dry weight, Species

(%)

Protein

Total fat

Oil

PhosCarbopholipid hydrate

S . gairdneri (irideus) (rainbow troot) Sardinops caerzileab (California sardine)

33.8

20.2

-

3.6

3.8

0.2

1.3

29.3

21.0

3.8

-

3.2