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Regulation of spawning behaviour in the female goldfish, Carassius Auratus Stacey, Norman Edward 1977

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THE REGULATION OF SPAWNING BEHAVIOUR IN THE FEMALE GOLDFISH, CARASSIUS AURATUS  by NORMAN EDWARD STACEY B.Sc., University of B r i t i s h Columbia, 1970  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of Zoology We accept this thesis as conforming to the required standard  The University of B r i t i s h Columbia 1977 Norman Edward S t a c e y , 1977  In presenting this thesis in partial  fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for  reference and study.  I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook'Place Vancouver, Canada V6T 1W5  Date  2  7  /  f  ABSTRACT  This study of the regulation of spawning behaviour i n the female goldfish (Carassius auratus) i d e n t i f i e d four endogenous factors believed to play major roles i n spawning behaviour: ( i ) s t i m u l i from ovulated eggs, ( i i ) ovarian steroids, ( i i i ) p i t u i t a r y hormones, and (iv) prostaglandins. Spawning behaviour i s synchronized with ovulation by the stimulus of an intraovarian mass of ovulated eggs.  Normally, female goldfish begin to  spawn on the morning of the day of ovulation and perform as many as several hundred spawning acts over a period of several hours; spawning behaviour ceases when a l l ovulated eggs have been shed.  The duration of spawning be-  haviour was extended i f oviposition (the release of eggs through the ovipore) was prevented by placing a plug i n the ovipore.  Spawning behaviour was ter-  minated when ovulated eggs were removed by hand-stripping and restored when ovulated eggs were injected through the ovipore and into the ovarian lumen. This e f f e c t of eggs on spawning behaviour was not r e s t r i c t e d to the day of ovulation but was seen i n a l l f i s h with ovaries i n any stage of vitellogenesis.  Injection of several substitutes for ovulated eggs induced low levels  of spawning behaviour. Injection of ovulated eggs f a i l e d to induce spawning behaviour i n f e male goldfish with regressed, nonvitellogenic ovaries.  Pretreatment with a  variety of gonadal steroids restored the spawning response to egg i n j e c t i o n i n these i n t a c t , regressed f i s h . Hypophysectomized with ovulated eggs.  f i s h did not perform spawning behaviour when injected Pretreatment of hypophysectomized  f i s h with homogenized  goldfish p i t u i t a r i e s or p a r t i a l l y p u r i f i e d salmon gonadotropin (SG-G100) restored the response to egg i n j e c t i o n .  Aminoglutethimide, an i n h i b i t o r of  steroid synthesis, blocked the effect of SG-G100 on spawning behaviour,  iii s u g g e s t i n g gonadotropin may e x e r t i t s e f f e c t on b e h a v i o u r by s t i m u l a t i n g steroidogenesis.  However, s t e r o i d treatments were t o t a l l y i n e f f e c t i v e i n  r e s t o r i n g the response t o egg i n j e c t i o n i n hypophysectomized Prostaglandin  (PG) appears  response t o o v u l a t e d eggs.  fish.  t o be i n v o l v e d i n m e d i a t i n g the b e h a v i o u r a l  Indomethacin  (IM), an i n h i b i t o r o f PG s y n t h e s i s ,  b l o c k e d the onset o f spawning b e h a v i o u r f o l l o w i n g egg i n j e c t i o n . of PGF„  r e s t o r e d spawning b e h a v i o u r i n e g g - i n j e c t e d , I M - t r e a t e d f i s h ; PGE  was l e s s e f f e c t i v e and PGE^ was w i t h o u t e f f e c t . normal  Injection  I n j e c t i o n of PGF^.induced  spawning b e h a v i o u r i n f i s h which had n o t been i n j e c t e d w i t h o v u l a t e d  eggs, s u g g e s t i n g t h a t o v u l a t e d eggs induce spawning b e h a v i o u r by s t i m u l a t i n g s y n t h e s i s o f PG.  The e f f e c t s o f PG on spawning b e h a v i o u r o f  hypophysectomized  f i s h t r e a t e d w i t h SG^GIOO o r s t e r o i d s p a r a l l e l e d the e f f e c t s o f egg i n j e c t i o n on f i s h r e c e i v i n g s i m i l a r treatments; SG-G100 r e s t o r e d the spawning r e sponse  t o i n j e c t i o n o f PG, w h i l e s t e r o i d treatments were w i t h o u t  effect.  Mechanisms by which o v a r i a n and p i t u i t a r y hormones and p r o s t a g l a n d i n s may i n f l u e n c e spawning b e h a v i o u r a r e d i s c u s s e d and a model of the r e g u l a t i o n of  spawning b e h a v i o u r i s proposed.  In a d d i t i o n , an attempt  v i d e a t h e o r e t i c a l b a s i s f o r comparing female v e r t e b r a t e s .  i s made to p r o -  the r e g u l a t i o n o f s e x u a l b e h a v i o u r i n  iv TABLE OF CONTENTS CHAPTER I - GENERAL INTRODUCTION  Page 1  CHAPTER I I - MATERIALS AND METHODS A. General Maintenance B. Hypophysectomy C. Histology of regressed ovaries D. Behavioural testing procedures E. Description of spawning behaviour  4 5 6 9 13 16  CHAPTER I I I - THE ROLE OF OVULATED EGGS IN THE SPAWNING BEHAVIOUR OF FEMALE GOLDFISH A. Experiment 1. Effect of Removal and Replacement of Ovulated Eggs 1. Introduction 2. Materials and Methods 3. Results and Discussion B. Experiment 2. Induction of Spawning Behaviour by Injection of Egg Substitutes 1. Introduction 2. Methods, Results and Discussion C. Summary of Chapter I I I CHAPTER IV - THE EFFECT OF STEROIDS ON SPAWNING BEHAVIOUR OF FEMALE GOLDFISH A. Experiment 3. Steroid-Induced Spawning Behaviour i n Intact, Regressed Female Goldfish 1. Introduction 2. Materials and Methods 3. Results 4. Discussion B. Experiment 4. Ineffectiveness of Steroids i n Hypophysectomized Female Goldfish 1. Introduction 2. Materials and Methods 3. Results 4. Discussion C. Experiment 5. Ineffectiveness of LongTerm Steroid Treatment i n Hypophysectomized Fish 1. Introduction 2. Materials and Methods 3. Results and Discussion D. Summary of Chapter IV  21 22 22 22 25  29 29 30 34 35  36 36 38 39 44  46 46 46 47 47  48 48 48 49 5  0  V.  Table of Contents (cont'd) CHAPTER V - ROLE OF THE PITUITARY IN THE SPAWNING . BEHAVIOUR OF FEMALE GOLDFISH A. Experiment 6. Effect of Hypophysectomy and P i t u i t a r y Replacement on Spawning Behaviour 1. Introduction 2. Materials and Methods 3. Results 4. Discussion B. Experiment 7. E f f e c t of Salmon Gonadotropin and Aminoglutethimide on Spawning of Hypophysectomized Female Goldfish ...... 1. Introduction 2. Materials and Methods 3. Results ...... 4. Discussion C. Summary of Chapter V CHAPTER VI.  THE ROLE OF "PROSTAGLANDINS IN SPAWNING BEHAVIOUR OF FEMALE GOLDFISH A. Introduction B. Experiment 8. I n h i b i t i o n by Indomethacin of Spawning Induced by Ovulated Eggs 1. Introduction 2. Materials and Methods ...... 3. Results and Discussion C. Experiment 9. Effect of Prostaglandins on Spawning of Indomethacin-Treated Fish 1. Introduction 2. Materials and Methods 3. Results and Discussion 'D. Experiment 10. E f f e c t of Prostaglandins on Spawning Behaviour 1. Introduction 2. Methods, Results and Discussion E. Experiment 11. E f f e c t of Steroids and Salmon Gonadotropin on Prostaglandin-Induced Spawning Behaviour i n Hypophysectomized Fish 1. Introduction 2. Materials and Methods 3. Results 4. Discussion F. Summary of Chapter VI  CHAPTER VII - GENERAL DISCUSSION ' A. Introduction B. The Role of the P i t u i t a r y C. The role of Steroids 1. Introduction 2. Effects of steroids i n i n t a c t female goldfish  5  1  52 52 52 53 56 57 57 58 61 66 67 68 69 74 74 74 77 78 78 78 78 82 82 82 86 86 87 87 88 92 93 94 95 99 99 100  Table of Contents (cont'd) D. E. F. G.  3. Possible mechanisms of steroid action The Role of Ovulated Eggs The Role of Prostaglandins The Regulation of Spawning Behaviour A Comparative Approach to the Study of Female Sexual Behaviour  BIBLIOGRAPHY APPENDIX - Some Aspects of the Histology of Regressed Ovaries - Appendix Figures  vii. LIST OF TABLES Table I  P  a  g  e  Effect of removal and replacement of ovulated eggs on the spawning behaviour of ovulated female goldfish  27  Effect of i n j e c t i o n of egg substitutes on spawning behaviour of female goldfish  32  E f f e c t of steroids bn spawning behaviour of i n t a c t , regressed female goldfish  42  E f f e c t of i n j e c t i o n of goldfish p i t u i t a r y homogenate on the spawning behaviour of hypophysectomized female goldfish.  55  V  Vehicle volumes injected i n Experiment 7  60  VI  Effect of salmon gonadotropin (SG-G100) and aminoglutethimide on the spawning behaviour of hypophysectomized female goldfish  64  E f f e c t of incubation time on latency to the f i r s t spawning act i n recently ovulated female g o l d f i s h . . .  71  Effect of indomethacin on spawning i n response to i n j e c t i o n of ovulated eggs  76  E f f e c t of prostaglandins on the spawning response to i n j e c t i o n of ovulated eggs i n female goldfish treated with indomethacin  80  E f f e c t of prostaglandins on the spawning behaviour of female goldfish without eggs i n the ovarian lumen  ^4  Effect of salmon gonadotropin (SG-GL00) and steroids on prostaglandin-induced spawning behaviour i n hypophysectomized female goldfish  90  II  III  IV  VII VIII  IX  X  XI  viii.  LIST OF FIGURES Figure 1.  Page  Proposed model of the regulation of spawning behaviour i n the female goldfish  118  Early stage of degenerating early yolk v e s i c l e or p r e v i tellogenic oocyte  148  3.  F o l l i c u l a r hypertrophy i n degenerating oocyte  148  4.  Advanced degenerating early yolk v e s i c l e oocyte  148  5.  Advanced degenerating early yolk v e s i c l e oocyte  150  6.  Advanced degenerating previtellogenie oocyte from hypophysectomized f i s h  150  7.  Yolk nucleus i n oocyte of hypophysectomized  150  8.  Nuclear migration stage i n degenerating oocyte  150  9.  Yolk v e s i c l e s i n normal oocyte  152  10. Yolk v e s i c l e s i n normal oocyte  152  11. Yolk v e s i c l e s associated with membrane of presumptive p r e - a t r e t i c oocyte  152  12. Yolk vesicles associated with membrane of presumptive p r e - a t r e t i c oocyte  152  13. Yolk vesicles and presumptive n u c l e o l i i n hypertrophied f o l l i c u l a r layer from hypophysectomized f i s h  154  14. Pre-nuclear breakdown stage oocytes i n ovary of hypophysectomized f i s h  154  15. Degenerating nucleus at early stage of a t r e s i a  154  2.  fish  16. Nucleus with spherical body at periphery of degenerating oocyte  ......  17. Degenerating early yolk v e s i c l e oocyte from hypophysectomized f i s h 18. Advanced degenerating early yolk v e s i c l e oocyte with nucleus and spherical body  15^ 156  .......  156  ix. L i s t of Figures (cont'd) Figure 19. 20. 21. 22.  Page  Nucleoli and spherical body i n cytoplasm of advanced degenerating p r e v i t e l l o g e n i c oocyte  156  Clumped n u c l e o l i i n a t r e t i c oocyte from hypophysectomized f i s h  156  M-l macrophage and presumptive n u c l e o l i i n a t r e t i c oocyte of hypophysectomized f i s h  158  M-2 macrophage i n a t r e t i c oocyte of i n t a c t , regressed fish  158  23.  Macrophage aggregation adjacent to early a t r e t i c oocyte  24.  Macrophages entering early a t r e t i c oocyte of i n t a c t regressed f i s h  158  Advanced degenerating p r e v i t e l l o g e n i c oocytes i n ovary of hypophysectomized f i s h  160  Spaces i n ovary of hypophysectomized f i s h apparently caused by a t r e s i a of p r e v i t e l l o g e n i c oocytes  160  Spaces i n ovary of hypophysectomized f i s h apparently caused by a t r e s i a of p r e v i t e l l o g e n i c oocytes  160  Injected ovulated eggs i n ovary of fish  162  25. 26. 27. 28. 29.  158  hypophysectomized  Macrophage aggregation adjacent to injected ovulated oocyte  162  X.  ACKNOWLEDGEMENTS I am very g r a t e f u l to Dr. N.R. L i l e y , my supervisor, for suggesting the problem, for guidance and support during the i n v e s t i g a t i o n , and for encouragement, constructive c r i t i c i s m , and patience during the preparation of the manuscript.  I would also l i k e to thank my research committee,  Dr. W.S. Hoar, Dr. A.M. Perks, arid Dr; E.M. Donaldson, f o r their h e l p f u l comments and discussion. I am grateful to Dr. D.L. Kramer and Dr. K.H. Khoo for their suggestions and enthusiasm, to Dr. E.M. Donaldson for the generous g i f t of salmon gonadotropin,  to Dr. J . Pike (Upjohn Company) for h i s g i f t of prostaglandins,  and especially to Ms. M.E. Hurlburt. This study was supported by a National Research Council operating grant to Dr. N.R. L i l e y and by a University of B r i t i s h Columbia graduate fellowship to myself.  1  CHAPTER I  GENERAL INTRODUCTION  2 GENERAL INTRODUCTION Research into many aspects of vertebrate reproduction has expanded 'explosively' i n recent years.  However, considering the numbers of e t h o l -  ogists, animal psychologists, neurophysiologists, and biochemists whose c r i t i c a l attentions now  focus on the problems of hormonal control of  female reproductive behaviour i n many species of mammals , the research e f f o r t concerned with these phenomena i n lower vertebrates i s at best modest.  Undoubtedly, this inequitable d i s t r i b u t i o n of s c i e n t i f i c labor  stems from differences i n the degree of relevance to problems of medicine and f e r t i l i t y i n man  and domestic animals.  As greater emphasis i s placed  on the culture of edible f i s h species, greater emphasis also w i l l be placed on the study of reproductive physiology and i t s r e l a t i o n to sexual behaviour i n f i s h . Remarkably l i t t l e information concerning the hormonal regulation of sexual behaviour i n female f i s h i s available (Hoar, 1965;  Liley,  1969)  and much of this pertains to a reproductively specialized teleost, the viviparous guppy ( P d e c l l l a r e t i c u l a t a ) . In this species, the correlation of r e c e p t i v i t y with gonadal state and the results of hypophysectomy, ovariectomy,  and replacement therapies suggest that, as i n higher verte-  brates, ovarian and gonadotropic hormones p a r t i c i p a t e i n the regulation of sexual behaviour  ( L i l e y , 1968,  1969,  1972;  L i l e y and Donaldson,  1969;  L i l e y and Wishlow, 1974). Apart from an early study (Noble and Kumpf, 1936;  discussed i n  L i l e y , 1969) which reported p a r t i a l restoration of sexual behaviour following treatment  of female Hemichromis bimaculatus with an un-  i d e n t i f i e d ovarian extract, replacement therapies (steroid hormones or ovarian extracts) have been i n e f f e c t i v e i n restoring spawning behaviour  3 of ovariectomized oviparous f i s h (Liley, 1969).  Though the results of  many studies acknowledge the 'close temporal relationship between ovulation and the spawning act' i n female f i s h ( L i l e y , 1969), the possible significance of this sequence i n the regulation of spawning behaviour . seems generally to have been overlooked.  However, the observation that  removal of ovulated eggs terminated the spawning behaviour of female goldfish led Yamazaki (1965) to hypothesize that 'ripe eggs i n the ovarian lumen stimulate the spawning behaviour of females v i a some pathway'. Yamazaki's results further suggested that a role for ovulated eggs i n spawning behaviour could explain why the technique of ovariectomy and steroid replacement therapy had yielded negative results i n e a r l i e r studies of egg-laying f i s h ( L i l e y , 1969) . In this thesis I have tested and found much support for Yamazaki's hypothesis concerning the role of ovulated eggs i n spawning behaviour. In subsequent experiments, evidence i s presented which indicates that p i t u i t a r y and ovarian hormones influence the tendency of female goldfish to perform spawning behaviour when ovulated eggs are i n the ovarian lumen. The mechanism by which eggs evoke spawning behaviour has been examined and i t i s suggested that protaglandins are involved.  Emphasis has been  placed on correlating sexual r e c e p t i v i t y with ovarian histology i n both treated and untreated f i s h .  In summarizing  the results of this study,  comparative aspects of hormonal control of reproductive behaviour are emphasized  and a basis for comparing reproductive behaviour i n female  vertebrates i s proposed.  CHAPTER I I  MATERIALS AND METHODS  General Maintenance A l l f i s h used i n this study were purchased from Hartz Mountain Pet Supplies Limited (Richmond, B.C.).  To ensure an adequate supply  of female g o l d f i s h , most shipments of f i s h were sexed at the warehouse; the remainder were shipped unsexed to the lab at the University of B r i t i s h Columbia. used.  Common, comet, and intermediate types were  Colors ranged from deep orange to s i l v e r and included many  variations i n mottled patterns, black tipped and white tipped etc.  fins,  Individuals with the w i l d , o l i v e coloration were not used. The  f i s h weighed from 15 to 60 g but the majority were i n the range of 20 to 30 g. To 'regulate' the reproductive state of both males and females, f i s h were maintained under two environmental regimes; cold water, long photoperiod (1.2 C; 16L:8D) and warm water, long photoperiod (20 C; 16L:8D).  As reported by Yamamoto et a l (1966), goldfish kept  at 14 C or less develop to a prespawning nor spermiate.  stage but neither ovulate  When raised to 20 C, f i s h i n this condition ovulate  or spermiate within several days. Usually, f i s h newly acquired from the warehouse (where they had been kept i n running, dechlorinated, cold tapwater) were placed i n large stock tanks of various sizes supplied with running dechlorinated tapwater thermostatically regulated at 10-13 C and illuminated f o r 16 hours per day withh20 - 40 watt fluorescent l i g h t s .  In some cases,  cold water stocks were kept outdoors i n six-feet diameter fibreglass tanks under natural photoperiod but with some continuous low-level illumination. as 5 C.  These tanks were not heated and sometimes were as cold  To avoid introduction of disease, new shipments of cold  water stock f i s h were kept separate from established stocks f o r at least several months.  Cold water stock tanks contained no vegetation  6 or loose substrate.  In some cases, cold water stocks were separated  by sex. Warm water stocks were kept 3 - 6  per 40 1 tank i n a f i s h room  with a i r temperature control (water temperature 20 - 22 C) on a 16L:8D photoperiod.  Each tank was supplied with 1" - 2" of quartz  sand as bottom substrate, a f l o a t i n g mat of water s p r i t e (Ceratopterus t h a l i c t r o i d e s ) , and an a i r stone.  F i l t r a t i o n was used  at the outset but quickly abandoned: outside box f i l t e r s because of the maintenance involved, subgravel f i l t e r s because of the acid water conditions produced when used i n conjunction with Ceratopterus.  A  rapid f a l l i n pH was by f a r the greatest cause of mortality i n the warm water stock f i s h .  In f i s h exposed to this condition, skin (and  presumably g i l l ) mucous apparently i s denatured and becomes opaque; moribund individuals transferred to neutral water nearly always r e covered within a few hours.  Fortunately, an e a s i l y observable i n -  dicator, an increase i n water c l a r i t y , always preceded the development of c r i t i c a l l y low pH by at least a day.  Entrapment of much suspended  detritus i n the fine roots of Ceratopterus aids greatly i n maintenance of water c l a r i t y , although this was l i k e l y of more importance to the experimenter than to the subjects. A l l stocks were fed frozen brine shrimp ad libitum at least every other day.  In addition, cold water stocks were fed Clark's  New Age Trout Feed (Moore-Clark Company, Salt Lake C i t y , Utah) on an i r r e g u l a r basis. B. Hypophysectomy Hypophysectomized f i s h were used i n a number of experiments r e ported i n this thesis.  M o r t a l i t i e s d i r e c t l y due to the operation were  low; however, disease and a r e l a t i v e l y high incidence of incomplete  7 removal of the p i t u i t a r y usually resulted i n less than half the  operated  f i s h being used for experiments. Hypophysectomy was communication).  performed as demonstrated by Yamazaki (personal  F i s h which had been kept at 20 C f o r at least several  days were c h i l l e d for 30 to 60 minutes i n ice water to which had been added a small amount of t r i c a i n e methanesulphonate (MS-222,  0.01  %) .  Fish used d i r e c t l y from cold water stock tanks often f a i l e d to remain anaesthetized for the duration of the operation; addition of more MS-222 i n these cases usually resulted i n higher Anaesthetized  mortality.  f i s h were wrapped i n moist paper towelling, positioned  belly-up on a grooved wet gular membrane on the l e f t  sponge, and covered with crushed i c e . side was  slit  The  and a r e t r a c t o r inserted  between the second and t h i r d g i l l arch to expose the roof of the buccal cavity.  The dorsal buccal epithelium was  then cut and folded back and  the underlying parasphenoid bone d r i l l e d away with a dental burr. With p r a c t i c e , the l o c a t i o n of the p i t u i t a r y could be accurately determined by reference to conspicuous nerves on the surface of the bone; these nerves were cut i n a l l hypophysectomies and sham operations.  When  the p i t u i t a r y could be seen through the bone, probe and forceps were used to expose the p i t u i t a r y which then was  removed by a s p i r a t i o n .  cut edges of the buccal epithelium were repositioned but not  The  sutured.  Following removal of the p i t u i t a r y , f i s h were placed i n 40 1 aquaria f i l l e d with 25% sea-water (7?>/'pp) t 5 C and allowed to warm a  slowly to room temperature.  Each tank had a subgravel f i l t e r and a i r -  stone but, i n order to maintain s a l i n i t y and avoid pH problems, no plants were added. ation.  No food was  given u n t i l 3 or 4 days after the oper-  A l l f i s h were kept i n the d i l u t e seawater throughout post-  operative recovery, ovarian regression, hormone treatment, and havioural testing.  be-  8 A disease (or diseases) which v i r t u a l l y every hypophysectomized f i s h i n my lab contracted was not i d e n t i f i e d .  However, i t developed  with a readily i d e n t i f i a b l e set of symptoms and could be cured e a s i l y with low mortality provided treatment was begun early.  The f i r s t  indi-  cations of i n f e c t i o n were a lowering of the dorsal f i n , folding of the paired f i n s , s l i g h t loss of balance, and accelerated swimming near the surface.  Within a day, areas of the body surface became reddened,  apparently by enlargement and rupture of the blood vessels i n the skin, and the i n i t i a l symptoms were more pronounced. the addition of 0.5  to 0.75  Treatment consists of  g K^C^O^ to a 40 1 tank.  Fish  undergoing  treatment do not feed, apparently an e f f e c t of the drug, as healthy treated f i s h behave s i m i l a r l y .  Several days following the disappear-  ance of the reddening and the resumption  of normal swimming, 50% of the  aquarium water i s replaced with 25% sea-water, and several days l a t e r the water i s changed completely. Preliminary tests showed that f i s h which had been completely hypophysectomized  for more than one month (1) l o s t a l l body color, becoming  a pale white, (2) had highly regressed ovaries, and (3) could not be induced to spawn i n standard testing procedures. P r i o r to assignment to experimental groups, prospective test females could be rejected for incomplete hypophysectomy on the basis of body coloration.  This method i s e f f e c t i v e but not foolproof, pre-  sumably as pigmentation and reproduction are regulated by d i f f e r e n t populations of p i t u i t a r y c e l l s which may be removed separately i n an i n complete ablation.  Following behavioural testing, heads were checked  for p i t u i t a r y remnants under a dissecting microsope; heads were not examined h i s t o l o g i c a l l y due to the time involved and a lack of confidence i n the technique.  Various aspects of ovarian histology were the f i n a l  9 c r i t e r i a for accepting or rejecting behavioural data from f i s h judged on the basis of body coloration to be completely hypophysectomized.  This  procedure i s discussed i n the following section. C. Histology of Regressed Ovaries i n Intact and Hypophysectomized Fish In interpreting the effects of various hormonal manipulations  on  the spawning behaviour of female g o l d f i s h , I have placed considerable emphasis on the ovarian histology of treated f i s h .  The basic assump-  tions involved i n this approach derive from the results of a number of studies of female teleost reproductive endocrinology which indicate that p i t u i t a r y hormones stimulate growth of ovarian f o l l i c l e s and position of yolk.  de-  Correlations observed i n the present study between  the state of ovarian development and sexual r e c e p t i v i t y have been used to develop h i s t o l o g i c a l c r i t e r i a by which spawning behaviour data were accepted or discarded.  The application of this procedure to various  types of experiment i s discussed below.  A more detailed account of  some aspects of the histology of regressed ovaries of intact and hypophysectomized f i s h i s given i n the Appendix. In previous studies, the growth of goldfish oocytes has been divided into two basic stages. (Yamazaki, 1961), a f i r s t growth phase i n which the oogonia develop into primary yolkless oocytes about 150y ,in diameter, and a second growth phase characterized by the f o r mation of two types of cytoplasmic inclusions, yolk v e s i c l e s granules) and yolk granules  (proteinaceous yolk).  appear i n oocytes about 150\L i n diameter.  (cortical  Yolk vesicles f i r s t  The yolk granule stage,  which does not begin u n t i l the oocyte i s about 300u. i n diameter, i s divided into primary, secondary, and t e r t i a r y stages depending on the cytoplasmic pattern of granule d i s t r i b u t i o n . Following hypophysectomy of female goldfish, yolk-laden or second growth phase oocytes become a t r e t i c (Yamazaki, 1961,  1965).  This  10 s e n s i t i v i t y of the yolky oocytes i s related to stage of development. Oocytes i n late yolk v e s i c l e , primary and secondary yolk granule stages are the most sensitive and begin to degenerate within a few days of p i t u i t a r y removal. stage oocytes.  Next to degenerate are the t e r t i a r y yolk  Least sensitive are the early yolk v e s i c l e stage  oocytes which often are present and apparently healthy several months after the operation.  There i s some evidence from this study that  d i s t i n c t changes i n the d i s t r i b u t i o n of the yolk v e s i c l e s precedes a t r e s i a i n these oocytes. ate  Eventually, a l l oocytes with yolk degener-  i n the absence of the p i t u i t a r y , no new yolk formation occurs,  and the ovary remains i n a regressed condition composed of various stages of f i r s t growth phase oocytes (Yamazaki, 1965).  Published  accounts suggest that i n g o l d f i s h , as i n other teleosts, p r e v i t e l l o genic oocytes are independent of the p i t u i t a r y , at least to the extent that they do not degenerate following hypophysectomy.  How-  ever, i n the present study a t r e s i a of p r e v i t e l l o g e n i c oocytes was observed many times i n inteact and i n hypophysectomized  f i s h and their  presence served as a valuable indicator of advanced ovarian regression. Details of p r e v i t e l l o g e n i c a t r e s i a are discussed i n the Appendix. Treatment  of hypophysectomized  f i s h with gonadotropin prepar-  ations induces growth of oocytes and deposition of yolk vesicles and yolk granules (Yamazaki, 1965; Yamazaki and Donaldson, 1968). I t appears that the mechanism by which the p i t u i t a r y stimulates formation of yolk granules i n f i s h i s similar to that i n other vertebrates (Chester Jones et a l . , 1972; G a l l i e n , 1975). At the l e v e l of the ovarian f o l l i c l e , gonadotropin stimulates both the production of estrogens (which induce the formation and mobilization of hepatic yolk proteins) and the uptake of yolk proteins from the blood  11 (Campbell and Idler, 1976; Emmersen and Petersen, 1976).  Except for  a study by Khoo (1974) i n which e s t r a d i o l , estrone, and e s t r i o l were reported to induce yolk v e s i c l e formation i n hypophysectomized  female  goldfish, there i s no information concerning the e f f e c t of steroids on :formation of this type of yolk. Under the experimental conditions of this study, spawning behaviour could be induced without hormonal pretreatment i n i n t a c t f i s h with ovaries i n any stage of v i t e l l o g e n e s i s :  f i s h i n which the ovaries  contained only f i r s t growth phase oocytes were not receptive.  For this  reason, spawning behaviour data from a l l f i s h with ovaries containing any yolk are discarded from the results of experiments designed to test the a b i l i t y of exogenous hormones to induce r e c e p t i v i t y i n i n t a c t , fish.  In this study, ovaries are described as nonvitellogenic i f the  oocytes contain neither yolk vesicles nor yolk granules. In experiments involving hypophysectomized  f i s h , the problem of  distinguishing acceptable behavioural data was more d i f f i c u l t .  Basic-  a l l y , data from f i s h judged on the basis of body coloration to be comp l e t e l y hypophysectomized  were considered acceptable i f there was  h i s t o l o g i c a l evidence of previous and ongoing a t r e s i a . treatment of hypophysectomized  However, as  f i s h either with p i t u i t a r y homogenates  or with gonadotropin preparations arrested a t r e s i a and induced yolk formation, completeness  of hypophysectomy i n these cases was  determined  on the basis of body coloration alone. Unlike the s i t u a t i o n i n intact f i s h , the decision to accept or reject behavioural data was based not only on the presence or absence of yolk v e s i c l e s , which may p e r s i s t for months after hypophysectomy, but also on the presence or absence of degenerating p r e v i t e l l o g e n i c and v i t e l l o g e n i c oocytes.  This was complicated by the fact that ovarian  histology i n hypophysectomized  f i s h i s influenced by the state of ovarian  12 development at the time of the operation.  For example, ovaries from  two females sampled one month after complete hypophysectomy may show very d i f f e r e n t patterns of degeneration.  The ovaries of a female  spontaneously regressed at the time of hypophysectomy w i l l show no signs of second growth a t r e s i a but l i k e l y w i l l contain various stages of degenerating p r e v i t e l l o g e n i c oocytes.  In contrast, a female hypo-  physectomized when the ovaries were i n the early stages of yolk granule formation w i l l have extensive second growth phase a t r e s i a , but normal yolk v e s i c l e stage oocytes w i l l probably be present and the degeneration of p r e v i t e l l o g e n i c oocytes w i l l not have commenced. In general, information from previous studies (Yamazaki, 1961, Khoo, 1974)  1965;  on the sequence of events and time course of a t r e s i a  following hypophysectomy has aided i n distinguishing ongoing but incomplete ovarian regression from: finding (Khoo, 1974) hypophysectomized  incomplete hypophysectomy.  However, the  that estrogens induce yolk v e s i c l e formation i n  female goldfish suggested that i n  f i s h receiving steroid treatment, completeness  hypophysectomized  of p i t u i t a r y  removal  could not be accurately determined on the basis of ovarian histology. Ih the present study, the results of Experiment 5, equivalent i n design to that carried out by Khoo, appeared to show that e s t r a d i o l and drotestosterone induced formation of yolk v e s i c l e s .  -dihy-  However, as de-  generation of yolk v e s i c l e stage oocytes may not be complete by the sixth week after hypophysectomy (Yamazaki, 1965; personal observation) and start-of-treatment controls were not included i n Khoo's nor i n the present study, i t i s equally reasonable that these steroids may the degeneration of early yolk v e s i c l e stage oocytes.  simply i n h i b i t In f a c t , Khoo's  statement that i n some oocytes the 'induced' v e s i c l e s were scattered randomly i n the cytoplasm, suggests that these oocytes were i n the  13 early stages of a t r e s i a (see Appendix).  In Experiment 11 of the  present study, where steroid treatment was not begun u n t i l the t h i r d or fourth month after hypophysectomy, at which time degeneration of early yolk v e s i c l e stage oocytes i s complete, the ovaries of f i s h receiving e s t r a d i o l showed no evidence of yolk v e s i c l e formation. D. Behavioural Testing Procedures A l l behavioural tests were carried out i n 60 1 aquaria supplied with an undergravel f i l t e r , 2" of quartz sand, and a f l o a t i n g layer of Ceratopterus.  Development of low pH from the use of undergravel  f i l t e r s was not a problem as these tanks were set up for only short periods. A l l experiments involved the induction and maintenance of sexual a c t i v i t y i n male goldfish and most experiments also involved the i n duction of ovulation i n females.  Both techniques are simple and  dependable. For the induction of ovulation, gravid female goldfish which had a distended abdomen (preferably soft and asymetrical) and expanded ovipore were selected from the cold water stock tanks about noon on day 0 and warmed to approximately 20 C over a period of several hours. Females treated i n this way were never observed to ovulate on day 1, while a highly variable proportion (presumably dependent on the state of ovarian maturity) ovulated spontaneously the morning of day 2. Intraperitoneal (i.p.) i n j e c t i o n of approximately 3 IU /g HCG(0.6% NaCl vehicle) on the afternoon of day 1 induced ovulation by 0600 h on day 2 i n v i r t u a l l y a l l cases and this technique was used routinely (for a more thorough discussion of the timing of events i n goldfish ovulation, see Stacey and Pandey ['1975]). In g o l d f i s h , the paired ovaries are enclosed dorsally by ovisacs  14 which, are separate a n t e r i o r l y and which j o i n at the posterior of the ovaries to form a short oviduct.  Following ovulation, many released  oocytes move dorsally between the ovarian lamellae and f i l l the o v i duct and ovisacs.  The remainder of the ovulated eggs remain within  the ovary u n t i l those i n the ovisacs and oviduct are gradually released through the ovipore during o v i p o s i t i o n . by applying s l i g h t pressure from the  Ovulation i s detected  to the abdomen to release a stream of eggs  ovipore.  The technique for inducing sexual a c t i v i t y in.male goldfish i s s i m i l a r to that for the induction of ovulation i n females. males were usually separated kept under standard  Stock  from females at the time of purchase and  cold water stock conditions.  before they are required for behavioural  Two  or three days  testing, males which have well  developed tubercles on the opercula or on the leading rays of the pectoral f i n s are injected i . p . with 3 IU/g HCG over several hours.  and then warmed to 20 C  Males i n prespawning condition spermiate on the  day a f t e r being warmed and injected and usually display s u f f i c i e n t sexual a c t i v i t y the following day.  Most males remain highly active for  at least a week;  injections or exposure to spawning  supplementary HCG  females usually extend this period. In e a r l i e r experiments, only one female at a time was each observation  tank as i t was  tested i n  f e l t that the spawning behaviour of  one  female might influence the behaviour of another and that, as males given a choice of females usually show marked preference  for certain  individuals,unequal stimulation of the females would r e s u l t . tions-confirmed neither of these suspicions.  Observa-  To increase the number of  females which could be observed at one time, and i n some cases to reduce the  amount of courtship directed toward each female, as many as  5 females were tested simultaneously  i n a 60 1 tank.  15 The i n i t i a l method of behavioural testing was i n the observation tank shortly before testing was  to place the female to begin.  Obser-  vation commenced with the introduction of an active male which had previously been kept i n an all-male tank.  It soon became evident  that the sexual a c t i v i t y of males was much greater i f the female was introduced to the male's tank and i f more than one male was  present.  Thus, i n l a t e r experiments, from 3 to 5 active males were kept i n each observation tank from the time they had been warmed and injected with HCG.  The general l e v e l ,of sexual a c t i v i t y i n each tank was  assessed  on  the morning of testing by placing an ovulated or gravid female into each observation tank and waiting u n t i l chasing had commenced. where male a c t i v i t y was  In tanks  only moderate, the 3 most active males were  used for courting test females and the remaining males were removed f o r the duration of the test period.  Only two males were used when male  a c t i v i t y was high (the usual case), and only one male when the a c t i v i t y was  extremely high.  In a small aquarium, excessive chasing and butting  can k i l l a female goldfish which i s unable to escape. Behavioural testing sessions were usually of 3 h duration. responding  females commenced spawning within the f i r s t hour of testing  and stopped spawning by the end of the t h i r d hour. extending  Most  It was  the test period would increase the incidence of  (see experiment 2).  Spawning a c t i v i t y was  f e l t that egg-binding  recorded on grid charts as  numbers of spawning acts i n each 5 minute i n t e r v a l of the test period. Following most tests, females were k i l l e d by over-anaesthetization i n MS-222, and the ovaries fixed i n Bouins F l u i d , embedded i n p a r a f f i n , sectioned and stained with Mallory connective tissue s t a i n .  16 E. D e s c r i p t i o n o f Spawning Behaviour When an o v u l a t e d female and a spermiated male g o l d f i s h are p l a c e d t o g e t h e r i n an aquarium p r o v i d e d w i t h green v e g e t a t i o n , the u s u a l course of events i s as f o l l o w s . contact  The male approaches  the female and may  ( e s p e c i a l l y i n the r e g i o n o f the o v i p o r e ) o r may  approach s e v e r a l times b e f o r e making c o n t a c t .  make  t u r n away and  U s u a l l y the female  does  not swim q u i c k l y a t t h i s stage and o f t e n appears to p r e s e n t the o v i p o r e r e g i o n to the male.  T h i s o c c u r s as a t u r n i n g away from the male,  sometimes w i t h the t a i l h i g h e r than the head. fails  to i n v e s t i g a t e a r e c e p t i v e female, i t may  female w i l l  E s p e c i a l l y when the male be observed t h a t the  approach the male, sometimes making c o n t a c t , but u s u a l l y swim-  ming p a s t the head o f the male. .-Normally, males t a k e - t h e i n i t i a t i v e i n i n v e s t i g a t i n g the female, and the female appears to p l a y a p a s s i v e  role.  The male soon b e g i n s to b u t t the female (pushing the female w i t h h i s snout) i n the r e g i o n of the b e l l y , o v i p o r e and c a u d a l peduncle i f the female i s swimming away, and i n v i r t u a l l y any p a r t of the body i f the female remains s t a t i o n a r y .  A chase d e v e l o p s , the female swimming  at a moderate speed, the male e i t h e r swimming v e r y c l o s e b e h i n d , o r swimming b e s i d e and m a i n t a i n i n g c o n t a c t w i t h the female.  I f green  v e g e t a t i o n i s not p r e s e n t , the female does not perform o v i p o s i t i o n b e h a v i o u r though some eggs may  be dropped, a p p a r e n t l y i n a d v e r t e n t l y  due to the p r e s s u r e o f the male. The female i n i t i a t e s spawning b e h a v i o u r by p e r f o r m i n g a  'rise'.  In t h i s , she swims up toward f l o a t i n g v e g e t a t i o n , pushes:.the snout i n (the head may vegetation.  o r may  not be c o v e r e d ) , and o c c a s i o n a l l y mouthes the  T y p i c a l l y , r i s i n g i s f o l l o w e d by a spawning  act(ovi-  p o s i t i o n ,),. The female e n t e r s the v e g e t a t i o n and t u r n s on h e r s i d e , as the male p a r a l l e l s  t h i s motion from s l i g h t l y behind and below.  The  pair  17 (female above, male below) then quickly swims up, breaking the water surface, and then down, leaving the vegetation.  It i s during this rapid,  synchronized r i s i n g and arching, i n which the male appears to attempt to push the female out of the water,  that the eggs and sperm are released.  Whether the female exercises any control over egg release during the spawning act i s not known; however, even s l i g h t pressure against the b e l l y such as occurs during prespawning chasing i s sometimes s u f f i c i e n t to release some eggs.  Netting ovulated females i n v a r i a b l y causes egg  release. Following emergence from the vegetation, the female may swimming at a moderate speed, followed closely by the male.  resume More often,  the female w i l l return to the vegetation and complete one or several spawning acts i n quick succession.  This 'clumping' of spawning acts  interspersed with periods of chasing and butting agrees with Yamazaki's (1965) description. Rising does not always lead to completion  of a spawning act.  Most  females which have not begun to spawn w i l l perform a number of r i s e s before completing  the f i r s t spawning act, after which the r a t i o of rises  to spawning acts decreases.  Occasionally, the male i s responsible for  the f a i l u r e of r i s i n g to lead to spawning, either because the speed of his approach s t a r t l e s the female, or because of incorrect orientation to the female.  In the cases where r i s i n g does not lead to spawning, the  female either swims quickly away from the vegetation or remains motionless i n the vegetation for as long as several minutes, not  responding  to the actions of the male. Though female goldfish w i l l sometimes spawn oh submerged vegetation, and a minority of i n d i v i d u a l s seem to prefer this s i t e (B.Partridge, personal communication), the majority of ovipositions occur on f l o a t i n g  18 vegetation both i n the laboratory and under natural conditions (Innes, 1949) . Due  to the problem of distinguishing a spawning act on submerged  vegetation from an attempt by the female to avoid the male, and because only f l o a t i n g vegetation was provided i n warm water stock tanks, only f l o a t i n g vegetation was Spawning may spawning acts.  used i n experimental  situations.  continue for several hours and involve several hundred  Generally, termination of spawning i s coincident with the  shedding of a l l ovulated eggs, though on occasion small numbers of eggs have been removed from females which had ceased spawning behaviour (Yamazaki, 1965; personal observation).  Though this was observed  on only a  few occasions and was not investigated further, i t appeared that females which had finished spawning ( i . e . had shed a l l ovulated eggs) i n h i b i t e d the male's chasing and butting by increasing the swimming speed and becoming very e f f e c t i v e l y evasive for a short period of time. Observations by myself and by others (Innes, 1949) }  under more natural  conditions, suggest that the courtship of male goldfish i s a highly competitive, vigorous, and lengthy event.  In experimental situations where .3 or .4 males  were placed with an equivalent number of females i n 60 1 aquaria, one male invariably i n i t i a t e d courtship.  Usually, this quickly stimulated courtship  of the same female by one or more of the remaining males. p e t i t i v e stimulation of courtship between males was  In f a c t , the com-  so r e l i a b l e , that adding  a  new,^active male to an experimental tank to stimulate courtship i n inactive resident males became standard experimental procedure. (Partridge, L i l e y , and Stacey, 1976)  There i s evidence  that male goldfish are attracted to and  stimulated to court female goldfish by a pheromone, probably of ovarian origin. This description of events i n a normal spawning s i t u a t i o n has ignored the question of whether v a r i a b i l i t y i n the stimulus provided by the male i s a s i g n i f i c a n t factor i n the responsiveness of the female.  This problem was  not  19 investigated experimentally as several observations indicated that provided the male i s s u f f i c i e n t l y aroused to complete the spawning act, the i n t e n s i t y of male courtship has no obvious effect on the spawning behaviour of the female. For example, on many occasions i n which receptive females (either natura l l y ovulated or with experimentally induced receptivity) had been placed with r e l a t i v e l y inactive males which had not begun to court, the female was seen to r i s e (a behaviour which usually e l i c i t s r i s i n g even i n inactive males) and, after allowing the male to p o s i t i o n , perform a normal spawning act without any of the usual preliminary investigation, butting, or chasing on the part of the male.  This i s not evidence that prespawning  courtship  has no stimulating e f f e c t on the female, but i t does demonstrate that i t i s not prerequisite.  Furthermore, i n the multi-male, multi-female testing pro-  cedure used i n many of my experiments, the males invariably showed a preference for certain of the females.  While most i n i t i a l l y  'unattractive' females  became and remained a t t r a c t i v e after they had begun to spawn, the usual s i t uation was that certain females consistently e l i c i t e d more courtship throughout the test, regardless of their spawning behaviour.  In extreme cases, one  or more females which never spawned throughout a test received almost constant courtship, while other spawning females were attended only at the times they entered the f l o a t i n g vegetation. These observations have been interpreted as i n d i c a t i n g that the occurrence and rate of spawning are functions of the physiology of the female goldfish.  Obviously, the presence of the male i s required at the time of  the oviposition behaviour, but provided the male i s sexually active enough to respond to the stimulus of a r i s i n g female and complete the spawning act, the i n t e n s i t y of the male's other courtship a c t i v i t i e s do not a f f e c t the spawning behaviour of the female.  This view i s at odds with that of Yamazaki (1965)  20 who  suggests, without providing data, that prespawning courtship or chasing  by males stimulates both ovulation and o v i p o s i t i o n . An important problem i n the recording of female goldfish spawning behaviour i s the choice of an appropriate parameter with which to measure sexual r e c e p t i v i t y (the tendency to perform spawning behaviour).  The apparent lack  of female prespawning behaviours leave only two obvious indices of r e c e p t i v i t y , r i s i n g behaviour and the spawning act. Though r i s i n g behaviour might at f i r s t appear to be an acceptable measure of sexual r e c e p t i v i t y , i t has several serious drawbacks.  The most important  i s that behaviour similar to r i s i n g occurs commonly i n a nonsexual both males and females.  context i n  Second, though each spawning act i s necessarily pre-  ceded by a r i s e , the tendency f o r r i s i n g to be disrupted p r i o r to the comp l e t i o n of a spawning act usually results i n the performance of many more r i s e s than spawning acts.  This i s p a r t i c u l a r l y evident when spawning be-  haviour i s beginning or i s proceding at a low frequency. (page 17), aspects of male spawning behaviour may  As noted previously  a l t e r the r e l a t i o n between  the numbers of r i s e s and spawning acts. The spawning act i s a d i s t i n c t i v e , stereotyped, and e a s i l y quantified behaviour which i s r e s t r i c t e d to a sexual context; sexual r e c e p t i v i t y used i n this study.  i t i s the only measure of  The term r e c e p t i v i t y i s used s t r i c t l y  i n a behavioural-senseuandniimplies nothing' ahoutuunderlyingrphysiological mechanisms.  CHAPTER III  THE ROLE OF OVULATED EGGS IN THE SPAWNING BEHAVIOUR OF FEMALE GOLDFISH  22 A.  Experiment 1.  Effect of Removal and Replacement of Ovulated Eggs on the Spawning Behaviour of Ovulated Goldfish  1.  Introduction That ovulated eggs within the ovary are involved i n the induction  of spawning behaviour i n female goldfish was suggested by Yamazaki (1965) whose observations on the effects of egg removal led him to speculate that 'ripe eggs i n the ovarian lumen stimulate the spawning behaviour of females v i a some pathway'. In the present study, preliminary examination of the role of ovulated  eggs i n stimulating spawning behaviour u t i l i z e d several simple tech-  niques.  Yamazaki's results were repeated.  Spawning ceased i f a l l ovula-  ted eggs were removed by hand-stripping (gently squeezing the area between the pectorals and the ovipore between moistened f i n g e r s ) .  As stripping  only a portion of the eggs did not terminate spawning, i t was concluded that the e f f e c t of complete egg removal was not due to disturbance or i n jury, but rather to the absence of ovulated eggs, as Yamazaki had suggested. It was found that the use of an 'oviduct plug' to prevent the release of ovulated eggs greatly extended the duration of spawning i n ovulated fish.  Whereas the normal duration of spawning i s less than two hours,  'plugged' females would spawn throughout the day of ovulation and sometimes continue the following day. The following experiment was carried out to determine whether placement of ovulated eggs i n the ovarian lumen would restore rather than simply o  prolong spawning. 2.  Materials and Methods' Females which had ovulated spontaneously ( i n response to warming but  23 without HCG  injection) the morning of the test day were taken from holding  tanks and placed singly i n 60 1 observation tanks. introduced into each tank a f t e r 20 to 30 minutes.  A spermiated  male was  The latency to the  first  spawning act and the number of spawning acts i n the following 20 minutes provided a record of the normal spawning a c t i v i t y of each f i s h . following this i n i t i a l 20 minute spawning period, each f i s h was  Immediately removed,  anaesthetized i n MS-222, and the eggs removed and stored i n egg-injection syringes.  For egg removal, females were dried gently with paper towelling  and hand-held such that the ovipore was  over (and the anal f i n outside) a  small p l a s t i c cup of about 5 ml capacity.  It i s e s s e n t i a l to use p l a s t i c  apparatus when handling ovulated eggs, as eggs w i l l eventually adhere to glass.  Furthermore, eggs must not be allowed to come into contact with  water as this causes adhesion and hardening.  Eggs are released into the  cup. by gentle pressure against the b e l l y and drawn up into a p l a s t i c 1 ml ;  syringe f i t t e d with bevelled PE tubing. Preliminary tests had shown that a single sequence of s t r i p p i n g r e moved only a portion of the ovulated eggs even i f considerable pressure exerted on the b e l l y ; when stripping was  was  repeated i n 10 or 15 minutes,  additional eggs could usually be expressed with only s l i g h t pressure.  It  i s believed that i t i s the eggs i n the oviduct and i n the posterior portions of the ovisacs which are removed during each stripping.  Following  removal of these accessible eggs, additional ovulated eggs move dorsally along the channels between the ovarian lamellae and into the ovisacs and oviduct, from which they can be removed by further s t r i p p i n g . Following egg removal the f i s h were revived and returned to the observation tanks where the behaviour of male and female was  recorded for one  24 hour.  During the f i r s t 30 minutes, the females were removed at 10 minute  intervals and squeezed to remove remaining ovulated eggs; for the second 30 minutes, the pairs were l e f t undisturbed. observation period was  The purpose of this one hour  to ensure that egg removal was  spawning had terminated.  complete and that  In preliminary tests i t had been found that low  l e v e l s of spawning a c t i v i t y occurred i f even a small number of ovulated eggs remained i n the oviduct. Following the observation period, the females were removed from the observation tanks, anaesthetized i n MS-222, and given one of the following treatments: Group I - a no-treatment handling control i n which each f i s h  was  simply revived and returned to the observation tank, Group II - an ovipore plug control i n which each f i s h was  fitted  with an ovipore plug, revived, and returned to the observation tank with the male, Group I I I - an egg-injection treatment  i n which each female was  injected with i t s own ovulated eggs, f i t t e d with an ovipore plug, revived, and returned to the spawning tank with the male. Oviduct plugs consisted of PE tubing (various diameters) with a f i r e polished glass plug i n the proximal end and recurved barbs cut i n the d i s t a l end ( t o t a l length 4-8 mm).  In inserting the plugs, i t was  often help-  f u l to f i r s t insert a fine glass probe into the oviduct and stretch the anterior margin (the junction with the anus) forward. duces the chance of placing the plug i n the rectum.  This precaution reNo attempt was made  to replace a l l the eggs removed from each Group III female as preliminary tests had shown that this usually resulted i n the rupture of the oviduct  25 and f a i l u r e to induce spawning behaviour.  Each female was injected with  approximately 0.025 ml eggs per gram body weight. Though the time between treatment of each f i s h and i t s return to the observation tank was not constant (as more than one f i s h was time), e f f o r t was made to minimize this i n t e r v a l .  treated at a  Thus, of the 33 f i s h  tested, a l l but one were returned to the observation tanks within 10 minutes of treatment.  Time of the f i r s t spawning act and a l l subsequent spawn-  ing a c t i v i t i e s were recorded for one hour, 3.  Results and Discussion There were no differences between the three treatment groups i n the  latency to the f i r s t pretreatment spawning act following i n i t i a l introduction to the male, nor i n the number of spawning acts i n the 20 minute pretreatment period (Table I ) . Removal of ovulated eggs was highly e f f e c t i v e i n terminating spawning behaviour; only one of 11 control f i s h (Group I) spawned during the posttreatment period.  The i n j e c t i o n of ovulated eggs  (Group III) restored spawning behaviour.  This i s obvious whether the number  of f i s h responding or the spawning rate of responding f i s h i s considered. Presence of an ovipore plug may have contributed to the spawning behaviour of Group III f i s h , as more f i s h spawned following treatment with an ovipore plug (Group II) than following the control treatment (Group I ) ; however, the posttreatment responses of Group I and Group II were not s i g n i f i c a n t l y - d i f f e r e n t (Mann-Whitney U-test). A number of informal tests conducted concurrently with the present experiment showed that the a b i l i t y of egg i n j e c t i o n to induce spawning behavior i s not r e s t r i c t e d to the day of ovulation.  Injection of eggs from  26  TABLE I EFFECT OF REMOVAL AND REPLACEMENT OF OVULATED EGGS ON THE SPAWNING BEHAVIOUR OF OVULATED FEMALE GOLDFISH  27 Pre-Treatment Spawning Group  I  Treatment  Latency (min)  No Treatment  6 6 5 8 6 7 50 200 6 41 4  (n=ll)  mean = 14.4 II  Ovipore Plug  19 15 20 2 3 10 4 5 15 2 2  (n=ll)  mean = 18.8 III  (n=ll)  No. Spawning Acts/20 min.  34 60 21 17 25 18 41 14 56 31 33 mean =31.8 44 20 26 55 38 54 40 25 38 40 56  Post-Treatment Spawning No. Spawning Acts/20 min. 0 0 0 0 0 0 7 0 0 0 0  0 0 0 0 0 0 17 0 0 0 0  (1/11) 0 0 0 0 0 10 1 0 4 11 0  mean= 39.6  (4/11)  20 12 6 57 4 60 6 20 10 29 30 5 8 59 26 4 5 30 3 40 22 9 mean = 8.4 mean= 33.8  10 37 57 20 20 7 56 46 31 8 15 (11/11)  Ovulated Eggs and Ovipore Plug  % Restored  0 0 0 0 0 18 2 0 10 27 0  83 65 95 100 69 23 94 177 103 20 167  * = No. spawning acts i n themost active 20 minute period of spawning i n the test hour. No difference between groups i n pretreatment spawning behaviour (Mann-Whitney U t e s t ) .  28 ovulated donor females consistently induced normal spawning behaviour both i n females which had ovulated as much as a month or more before being tested and i n f i s h which had f a i l e d to ovulate after transfer to warm water. These results both support Yamazaki's' hypothesis concerning the s t i mulatory effect of ovulated eggs on spawning behaviour and demonstrate that the normally sequential processes of ovulation and oviposition can be e a s i l y dissociated.  The simple technique of egg i n j e c t i o n thus allows  the endocrine regulation of sexual behaviour to be examined independently of the control of ovulation.  This i s c r i t i c a l when i t i s realized that  the t r a d i t i o n a l approach of ovariectomy and replacement therapy i s rather inappropriate i n this case due to the dependence of the female's spawning behaviour on i n t e r n a l cues provided by eggs i n the ovarian lumen.  29 B.  Experiment 2.  Induction of Spawning Behaviour by Injection of Egg Substitutes  1.  Introduction Although  the technique of egg i n j e c t i o n i s a simple and r e l i a b l e  method for inducing spawning behaviour, i t i s associated with three obvious problems: (i) the necessity, and occasional d i f f i c u l t y , of inducing ovulation i n egg-donor females, ( i i ) the p o s s i b i l i t y donor females,  of differences i n stimulus quality of eggs from and  ( i i i ) the tendency for eggs to become adhesive  (bound) on contact  with water and thus to lose the a b i l i t y -.to stimulate spawning. Egg binding was  a common problem and reduced  experiments requiring egg i n j e c t i o n .  f i n a l sample sizes i n a l l  Binding s u f f i c i e n t to i n h i b i t  spawn-  ing usually occurs as hardened eggs attached to the withdrawan ovipore plug or as a small cluster of eggs which may be expressed with some d i f f i c u l t y from the ovipore by pressure on the b e l l y .  In some cases, binding may i n -  volve the hardening and adhesion of the entire mass of injected eggs.  In  cases where f i s h with bound eggs were not i n h i b i t e d from spawning, i t was assumed the binding had occurred after the onset of spawning behaviour. Data for a l l non-responding f i s h for which there was  any suggestion of egg  binding were discarded. Finding an injectable egg substitute f o r the induction of spawning would eliminate the d i f f i c u l t i e s of using eggs and greatly expedite experiments involving the a r t i f i c i a l induction of spawning behaviour ing without ovulation).  ( i . e . , spawn-  To this end, three p o t e n t i a l egg substitutes  30 have been tested; petroleum j e l l y , g e l a t i n , and Dow Corning 200 Silicone Fluid. 2.  Methods, Results, and Discussion The discouraging results of the preliminary tests did not j u s t i f y a  f u l l - s c a l e experiment to test egg substitutes.  Test conditions were not  standardized and varied i n such aspects as duration of the test period, the time elapsed since the previous ovulation of the test female, and whether or not an ovipore plug was used (Table I I ) . Petroleum j e l l y and Dow Corning 200 Silicone F l u i d were simply drawn up i n an egg i n j e c t i o n syringe and injected.  Gelatin was dissolved i n warm  dechlorinated tapwater at two concentrations, 0.3 g/ml and 0.6 g/ml, and stored overnight at 4 C.  Injection was as f o r petroleum j e l l y and the  Silicone F l u i d . As seen i n Table I I , both g e l a t i n and petroleum j e l l y induced spawning behaviour i n some f i s h , although the response to these substances was much less than that obtained with egg i n j e c t i o n . These limited results demonstrate that the a b i l i t y to induce spawning behaviour i s not a property unique to ovulated eggs and that i n some f i s h physical cues alone may be s u f f i c i e n t to induce the response.  The f a i l u r e  of the injected materials to duplicate the response to ovulated eggs i s believed to result from their i n a b i l i t y to duplicate the physical stimulus provided by eggs.  The i n a b i l i t y of p h y s i c a l l y altered (adhesive and hardened)  bound eggs to induce spawning supports this contention. An alternative explanation for the low stimulus q u a l i t y of the tested egg substitutes i s that they lack some chemical stimulant normally associated with ovulated eggs or f l u i d .  The fact that binding of only a few eggs  31  TABLE I I  EFFECT OF 'EGG SUBSTITUTES' (PETROLEUM JELLY, GELATIN, AND SILICONE FLUID) ON SPAWNING BEHAVIOUR OF RECEPTIVE FEMALE GOLDFISH  32  Treatment  Ovipore Plug Used  Condition of Fish  1 week POV  Ovulated Eggs  +  Petroleum Jelly  + + + + +  II  —  II  -  II II  G e l a t i n (low concentration)  G e l a t i n (high concentration)  Silicone Fluid  * Tested  -  *  II  it II II II  it II  —  M  -  II II  -  Duration of Observation Period (hours)  —  preovulatory  _  1 week POV  -  II  1 week a f t e r o v u l a t i o n  No. o f Spawning Acts  3 1.5  260 65  2 2 3 3 3 3 3  0 0 0 0 3 22 108  2 2 2  0 0 0  2 2 2 2  35 0 0 0  1.5 1.5  0 0  33  near the ovipore w i l l often completely i n h i b i t spawning even though the remainder of the i n j e c t e d eggs appears normal, would seem to argue against this alternative.  C.  Summary of Chapter I I I 1.  The duration of spawning behaviour of ovulated f i s h i s extended  by plugging the ovipore to prevent egg release. 2.  In ovulated f i s h , spawning behaviour i s terminated by removing  ovulated eggs.  Spawning behaviour i s restored by i n j e c t i n g ovulated eggs  through the ovipore and i n t o the ovarian lumen. 3.  I n j e c t i o n of ovulated eggs i n t o preovulatory and postovulatory  f i s h induces spawning behaviour w i t h i n several hours. 4.  On contact w i t h water, ovulated eggs become hard and adhesive and  lose the a b i l i t y to induce spawning behaviour. 5.  Several s u b s t i t u t e s f o r ovulated eggs ( g e l a t i n , petroleum j e l l y )  induce spawning behaviour when i n j e c t e d i n t o the ovarian lumen; these substances are l e s s e f f e c t i v e than ovulated eggs.  CHAPTER IV THE EFFECT OF STEROIDS ON THE SPAWNING BEHAVIOUR OF FEMALE GOLDFISH  36 A.  Experiment 3,  Steroid-Induced Spawning Behaviour i n Intact, Regressed Female Goldfish  1.  Introduction Normally, gonadal maturation and ovulation i n goldfish are accelera-  ted by r a i s i n g the water temperature to 20 C.  However, the ovaries of  spent or mature f i s h kept at 20 C for an extended period (2 to 3 months i s usually s u f f i c i e n t ) become regressed (Khoo, 1974); v i r t u a l l y a l l oocytes are i n the p r e v i t e l l o g e n i c stage although some may contain small yolk vesircles (Yamazaki, 1961, 1965).  Since v i t e l l o g e n e s i s resumes i f regressed f e -  males are returned to cold water (12 C) soon after spawning, temperature regression appears not to r e s u l t from an incapacity of the post-spawning reproductive system, but rather from an i n h i b i t i o n of the system by prolonged high temperature.  The mechanism by which high temperature prevents  ovarian recrudescence i s not known; however, there i s some evidence (Scruggs, 1951; Nagahama, 1973) that gonadotropin production i s low i n the post-spawning period.  Examination of a similar phenomenon i n another teleost,  G i l l i c h t h y s m i r a b i l i s , suggests the mechanism may operate by a decrease both i n gonadotropin secretion and i n the response of the gonad to gonadotropin (De Vlaming, 1972).  Presumably,  the functional significance of this  temperature-induced regression i s that the stimulatory effect of elevated temperature on gonadal maturation i s confined to the early part of the warm season when rearing conditions f o r the f r y are optimal. Injection of ovulated eggs into temperature-regressed f i s h invariably f a i l e d to induce spawning behaviour.  Khoo (personal communication) found  that the a c t i v i t y of steroid dehydrogenases  i s very low i n i n t a c t , regressed  female goldfish and L i l e y (1972) has shown that e s t r a d i o l i s e f f e c t i v e i n  37  restoring the sexual r e c e p t i v i t y of both hypophysectomized tomized female guppies.  and ovariec-  On the basis of these findings, i t seemed a  reasonable hypothesis that the lack of a spawning response to egg i n j e c t i o n was due to reduced s t e r o i d , i n p a r t i c u l a r to reduced e s t r a d i o l , production. To test this hypothesis, an experiment was carried out which showed that i n j e c t i o n of e s t r a d i o l restored the spawning of temperature-regressed f i s h i n response to egg i n j e c t i o n .  As there was no reason to believe that  e s t r a d i o l i s the only steroid capable of inducing spawning behaviour, the effects of other steroids were also examined; these additional  experiments  revealed an unexpected lack of s p e c i f i c i t y i n the behavioural response to steroid treatment. Ten steroids known to be synthesized by teleost ovaries (Eckstein, 1970; Eckstein and Katz, 1971; Lambert et_ al_. , 1971; Colombo and Belvedere, 1976; OZon, 1972) were tested; cholesterol, pregnenolone, 17'* -OHr-pregneno1  lone, progesterone, 17? -OH-progesterone,  dehydroepiandrosterone, androstene-  dione, testosterone, 11-ketotestosterone, and 17,6 - e s t r a d i o l .  The effects  of estrone, e s t r i o l , 5? -dihydrotestosterone, and androsterone were also examined.  Dehydroepiandrosterone, androstenedione, testosterone, e s t r i o l ,  estrone, and e s t r a d i o l were chosen as they induce sexual behaviour i n f e male mammals (Beyer et_ al., 1970, 1971) and because the l a t t e r three also stimulate receptive behaviour i n female guppies ( L i l e y , 1972). pregnenolone, 17°^ -OH-pregnenolone, and 17°?. -OH-progesterone  Cholesterol,  do not stimu-  late sexual behaviour i n female mammals and were not expected to induce spawning behaviour.  S i m i l a r l y , i t was thought that treatment with proges-  terone would not stimulate spawning behaviour.  F a c i l i t a t i o n by progesterone  of sexual behaviour i n female rodents requires estrogen pretreatment (je.g.,  38 Joslyn e_t a l . , 1971) and i n ovariectomized female guppies progesterone f a i l s to restore r e c e p t i v i t y ( L i l e y , 1972).  The 5  a  -reduced  androgens,  androsterone and dihydrotestosterone, were tested to examine the p o s s i b i l i t y that androgens stimulate spawning behaviour following aromatization to estrogens; there i s evidence that i n mammals 5^ -reduced steroids are not aromatized (Thompson et aT., 1971). B.  Materials and Methods Although this study i s presented as a single experiment, i t i s i n  fact a series of small experiments carried out over a period of a year and a half.  Tank f a c i l i t i e s were such that i t was not possible to obtain r e -  gressed f i s h and preovulatory egg-donor females i n s u f f i c i e n t numbers to perform the entire experiment at one time.  Each small experiment  the e f f e c t s of several steroids and a saline control i n j e c t i o n .  tested Test f i s h  had been kept at 20 C f o r 3 to 9 months and were assumed to be regressed. Assignment  to test groups was not random, but was arranged so that the 4-6  f i s h i n each 40 1 aquarium could be i d e n t i f i e d i n d i v i d u a l l y by morphological characters or color patterns.  Only one treatment group was kept i n each  tank. Steroids (Sigma) were ground to a fine powder i n a Misco homogenizer, suspended at a concentration of 2.5y g/pl i n 0.6% NaCl containing 4 drops Tween 80/100 ml and injected at a dosage of 20 Ug/g. Suspensions were made up 10 ml at a time and kept i n sealed centrifuge tubes at 4 C.  They  were discarded when depleted to 4-5 ml as a precaution against degradation and changes i n concentration. Fish were l i g h t l y anaesthetized i n MS^-222 p r i o r to. i n j e c t i o n and l a i d on.a wet paper towel.  The steroid suspension was agitated immediately p r i o r  39 to i n j e c t i o n , which was  always intraperitoneal through the right body  w a l l , s l i g h t l y above and behind the base of the p e l v i c fins... (25-30 gauge) was inserted at l e a s t 1/2" the e f f l u x of injected v e h i c l e .  The  needle  through the body w a l l to minimize  Following i n j e c t i o n , f i s h were immediate-  l y returned to the holding tank to recover. tions on alternate days over a 9 day period.  Each f i s h received 5 i n j e c On the morning of the day  following the f i n a l injectionX/ occasionally the following day i f ovulated eggs were not available) f i s h were anesthetized i n MS-222, injected with ovulated eggs (approximately  0.025 ml/g), f i t t e d with an ovipore plug, and  l e f t to recover i n 2 1 beakers for 30 to 60 minutes.  Fish were then  placed i n 60 1 observation aquaria containing a c t i v e l y courting males and spawning behaviour was  recorded for 3 hours.  In some of the l a t e r tests  i n which more females than males were placed i n each observation tank and the chance of injected eggs being expressed during chasing was  low, ovipore  plugs were not used. Following the 3 hour test period, females were removed from the observation tanks and anaesthetized and the ovipore plugs were c a r e f u l l y removed.  The f i s h were then checked for binding of the injected eggs by  gently squeezing the b e l l y near the ovipore with a moistened finger, and egg-bound, non-responding f i s h discarded from the sample.  A l l f i s h were  s a c r i f i c e d and the ovaries fixed and prepared for h i s t o l o g i c a l  C.  examination.  Results Of 191  'regressed' f i s h which received steroid or saline i n j e c t i o n s  i n this experiment, only 41 individuals achieved the requirements  of  successful egg i n j e c t i o n without binding, and complete absence of yolky  40 oocytes i n the ovaries.  The spawning data for these f i s h are presented  i n columns I and II of Table I I I .  Spawning scores i n columns I I I , IV,  and V are from incompletely regressed f i s h with ovaries i n the..early stages of yolk v e s i c l e formation.  Ovaries of f i s h i n column I I I contained some  yolk v e s i c l e stage oocytes; as they also contained early stages of degenerating yolk v e s i c l e oocytes, i t appeared that temperature regression had not yet been completed.  In contrast, as the ovaries of the few f i s h i n  column IV contained some oocytes i n the yolk v e s i c l e stage, but had only advanced a t r e t i c yolk v e s i c l e oocytes, i t appeared either that temperature regression had been arrested p r i o r to completion, or that v i t e l l o g e n e s i s had recommenced.  The f i s h i n column V showed no signs of a t r e s i a and were  i n the early stages of yolk v e s i c l e formation.  The h i s t o l o g i c a l differences  between v i t e l l o g e n i c (stages I I I , IV, and V) and nonvitellogenic ovaries (stages I and II) were much more obvious than were the differences between stages I and II or among stages I I I , IV, and V.  In v i t e l l o g e n i c ovaries,  oocytes containing yolk vesicles were usually quite numerous.  On the other  hand, the incidence of degenerating oocytes was nearly always low and i t i s quite l i k e l y that i f larger portions of the ovary had been examined, some stage IV and V ovaries would have been r e c l a s s i f i e d as stage I I I .  Data  from egg-bound f i s h and from f i s h with advanced yolk formation are not i n cluded i n Table I I I . None of the lOgregressed control females injected with saline (columns I and II) performed any spawning acts during the 3 hour test period.  Of the  5 saline-treated f i s h (column III) which had some early yolk v e s i c l e stage oocytes plus signs of ongoing regression, only one i n d i v i d u a l performed one spawning act.  In contrast, 4 of the 5 females i n columns IV and V performed  high levels of spawning during the test period.  TABLE I I I EFFECT OF STEROID TREATMENTS ON SPAWNING BEHAVIOUR OF FEMALE GOLDFISH WITH TEMPERATURE-REGRESSED OVARIES  42  Treatment  No. of spawning acts of f i s h with ovaries i n stage I  0 0 0 0 0  Saline  II III  0 .0 0 0 0  Cholesterol  1 0 0 0 0 0 0 0 46 29  IV  Pregnen ^ olone 1  17<*-0HPregnenolone Dehydroepiandrosterone  0 0 0 0 • 17 10  No. of spawning acts of f i s h with ovaries i n stage  V  I  19 16 0 32 95  Androstene- - 70 dione 77  0 0  0 0 0 0  Progesterone 17oc._oHProgesterone  Treatment  0  Testosterone Estradiol  II  109  36 7 4 100 119  0 Estriol  56 101  12 33  58  19  83  1 1 209 23  0  0  Estrone  23 32  Dihydrotestosterone 11-Ketotestosterone Androsterone  III  IV  V  77 44  0  80 176 314 33 0 3 131 14 56 38 0 6 14 0 33 32 8  54  86 1 s  77  32 106 7 52 59 18 55  21 17 28 83  14 38  86 40 43 6  24 16 40 36 48  45 82 0  Stage I (no yolk v e s i c l e s ; degenerating primary oocytes) and Stage II (no yolk v e s i c l e s ; no degenerating primary oocytes) ovaries are completely regressed. Stage I I I '(yolk v e s i c l e s ; degenerating secondary oocytes), Stage IV (yolk v e s i c l e s ; advanced degenerating secondary oocytes), and Stage V (yolk v e s i c l e s ; no degenerating oocytes) ovaries are not completely regressed. Spawning scores are t o t a l number of spawning acts per 3 hour test period. Data from f i s h with advanced stages of yolk formation not included i n table.  On the basis pf the above h i s t o l o g i c a l interpretation, and of the r e l a t i o n between spawning behaviour and ovarian histology i n s a l i n e treated f i s h , i t i s suggested that only data from f i s h with n o n v i t e l l o genic ovaries (stages I and II) be accepted as evidence for the action of exogenous steroids on spawning behaviour.  Many f i s h with ovaries i n stages  III, IV, and V probably were receptive p r i o r to receiving steroid t r e a t ments . In contrast to the lack of response of the control group, regressed i n d i v i d u a l s i n groups treated with ly^-OH-pregnenolone,  androstenedione,  testosterone, e s t r a d i o l , dihydrotestosterone, 11-ketotestosterone, and androsterone performed high l e v e l s of spawning following egg i n j e c t i o n . regressed f i s h treated with pregnenolone responded  The  to egg i n j e c t i o n , sug-  gesting that this steroid also affects spawning behaviour.  Estriol  and  estrone appear to be i n e f f e c t i v e , although the sample sizes are small. IT*-OH-progesterone does not restore r e c e p t i v i t y i n regressed f i s h ; none of 5 individuals tested showed any spawning response.  There i s no i n -  formation as to the effectiveness of progesterone i n restoring r e c e p t i v i t y as none of the group receiving progesterone were completely regressed.  How-  ever, i t i s of interest that none of these four marginally v i t e l l o g e n i c f i s h displayed any spawning behaviour following egg i n j e c t i o n . may  simply indicate a period of poor egg i n j e c t i o n technique.  This result Alternatively,  i t suggests an i n h i b i t o r y effect of progesterone on responsiveness to egg i n j e c t i o n i n f i s h commencing ovarian maturation.  Two other progesterone-  treated f i s h with much more advanced yolk formation (not shown i n Table III) spawned normally following egg i n j e c t i o n . No f i s h i n the groups injected with cholesterol or dehydroepiandrosterone had completely regressed ovaries.  44 D.  Discussion The results of this experiment demonstrate that a variety of steroids  restores the responsiveness of ovulated eggs. not known.  of temperature-regressed  f i s h to i n j e c t i o n  The mechanism of action of the e f f e c t i v e steroids i s  They may  ual behaviour, or may  stimulate central nervous structures regulating sexhave some peripheral e f f e c t i n s e n s i t i z i n g the f i s h  to the stimulus of ovulated eggs i n the oviduct.  The only conclusion which  can be drawn regarding the d i v e r s i t y of e f f e c t i v e steroids i s that under the conditions of this experiment the mechanism c o n t r o l l i n g sexual behaviour does not appear to be highly steroid s p e c i f i c .  There are several obvious  explanations which could account for t h i s . One explanation may  simply be that the dosage of steroid used was  pharmacologically high and thus s p e c i f i c i t y was masked.  Certainly the  dosage used i n this study i s much higher than those found e f f e c t i v e i n i n ducing r e c e p t i v i t y i n female mammals.  However, i n a study ( L i l e y ,  1972)  which employed e s s e n t i a l l y the same dosage as i n the present work, e s t r a d i o l restored sexual behaviour of ovariectomized female guppies, and testosterone was  ineffective.  Another plausible explanation i s that the ovary of the temperatureregressed goldfish possesses an active steroidogenic pathway capable of converting exogenous steroids to one or more behaviourally active forms. Such an interpretation i s not at variance with the reported steroidogenic potential of the p r e v i t e l l o g e n i c ovary of the e e l , A n g u l l l a (Colombo and Belvedere, 1976). The results of this experiment also emphasize that f i s h with ovaries i n advanced stages of regression are responsive to the i n j e c t i o n of eggs,  45  provided that the ovaries contain at l e a s t some yolk v e s i c l e deposition. Thus i t i s evident that the mechanism r e g u l a t i n g spawning behaviour i s f u n c t i o n a l under endocrine conditions which l i k e l y are very d i f f e r e n t from those present at the time of o v u l a t i o n . The present experiment provides no evidence as to whether the exogenous steroids act d i r e c t l y to influence behaviour, or exert some a c t i o n on or with the p i t u i t a r y .  The f o l l o w i n g experiments, using hypophysectomized  f i s h , attempt to answer t h i s  question.  46 B.  Experiment 4.  Ineffectiveness of steroids on spawning behaviour of hypophysectomized female goldfish  1.  Introduction In Experiment 3 i t was shown that a number of steroids restore spawn-  ing i n response to i n j e c t i o n of ovulated eggs i n i n t a c t , nonreceptive, temperature-regressed female goldfish.  L i l e y (1972) has shown that p i t u i t a r y  hormones are not necessary for the expression of sexual behaviour i n the female guppy; e s t r a d i o l injections alone restore the r e c e p t i v i t y of hypophysectomized f i s h .  To determine whether the p i t u i t a r y i s involved i n the  action of steroids on female goldfish spawning behaviour, an experiment s i m i l a r to Experiment 3 was performed with hypophysectomized f i s h . steroids were tested.  E s t r a d i o l and testosterone were used as they were  known to be e f f e c t i v e i n intact f i s h . are  Seven  Although some evidence suggests they  i n e f f e c t i v e i n intact female goldfish, estrone and e s t r i o l were used as  they are e f f e c t i v e i n the female guppy (Liley, 1972).  As deoxycorticoster-  one has been shown to induce ovulation and oviposition i n hypophysectomized female Heteropneustes (Sundararaj and Goswami, 1966), t h i s steroid was also tested.  2.  Progesterone was also tested and cholesterol was used as a control.  Materials and Methods Females which had been kept at 20 C for 4 to 6 months and therefore  were assumed to have regressed ovaries, were hypophysectomized and kept i n 25% seawater for 4 to 9 weeks before beginning steroid treatment.  Prepara-  t i o n , dosage, and frequency of i n j e c t i o n of steroids was as described i n Experiment 3.  However, due to d i f f i c u l t i e s i n inducing ovulation i n donor  females, the duration of treatment varied from 7 days (4 injections) to 19 days (10 i n j e c t i o n s ) .  C o l l e c t i o n and i n j e c t i o n of ovulated eggs were as  47 described i n Experiment 1 and testing procedures were as described i n Experiment 3. 3.  Results As this experiment was  carried out before discovery of the treatment  for 'red disease', m o r t a l i t i e s were high and the sample sizes are small. Of 24 f i s h surviving the treatment, the data from s i x were discarded as the f i s h had bound eggs.  None of the remaining 18 ( e s t r a d i o l - 7;  e s t r i o l - 3; estrone - 2; testosterone - 1; progesterone - 3; cholesterol 2) displayed any spawning a c t i v i t y during the three hour test period. f i s h treated with deoxycorticosterone  4.  No  survived to be tested.  Discussion It appears that hypophysectomy interferes i n some way with the a b i l i t y  of exogenous steroids to potentiate the spawning response to. injected eggs. This may  indicate e i t h e r that the e f f e c t of steroids on spawning behaviour  of i n t a c t f i s h i s mediated by the p i t u i t a r y , or that a p i t u i t a r y factor i s required i n addition to the s t e r o i d .  I t i s also possible that the mechanism  regulating spawning behaviour requires stimulation by a steroid or steroids other than those used i n this experiment. In the female r a t , there i s evidence that the lordosis response to exogenous steroids i s decreased i f the hormonal treatment i s preceded by a prolonged period of hormonal deprivation (Damassa and Davidson, 1973; and Orndoff, 1974) . may  Beach  Thus the ineffectiyeness of steroids i n this experiment  have been due to a decline i n s e n s i t i v i t y to steroids i n the 4 to 9  week i n t e r v a l between hypophysectomy and the i n i t i a t i o n of treatment period of hormone deprivation may  have been longer as the f i s h most l i k e l y  were regressed p r i o r to hypophysectomy). this latter p o s s i b i l i t y .  (the  The following experiment  explores  48 C.  Experiment 5.  Ineffectiveness of Long-Term Steroid Treatment on Spawning Behaviour of Hypophysectomized  Female Gold-  fish 1.  Introduction In Experiment 4 i t was suggested that the ineffectiveness of steroids  i n inducing sexual behaviour of hypophysectomized  female goldfish might be  due to a decrease i n responsiveness to steroids following hypophysectomy. A similar phenomenon i s reported to occur i n female rats following ovariectomy (Beach and Orndoff, 1974; Damassa and Davidson, 1973). In goldfish, where ovarian development  i s normally very prolonged,  hormonal priming of sexual behaviour i s l i k e l y to occur over an extended period.  In addition, i f the hypothesis that unreceptive, temperature-  regressed f i s h are capable of i n vivo steroid conversion i s correct, i t i s l i k e l y that the hypophyseal-gonadal axis i s active, although operating at a l e v e l i n s u f f i c i e n t to induce.oocyte growth, vitellogeriesis, and sexual r e ceptivity.  Thus, the degree of hormonal deprivation and the time required  to rest-ore r e c e p t i v i t y by steroid treatment may be greater i n hypophysectomized than i n temperature-regressed f i s h .  To examine this p o s s i b i l i t y , I  carried out an experiment similar to Experiment 4, except that the i n t e r v a l between hypophysectomy and i n i t i a t i o n of steroid treatment was shorter and the duration of s t e r o i d treatment was longer.  Two steroids (estradiol and  dihydrotestosterone) were tested, each known to be e f f e c t i v e i n restoring r e c e p t i v i t y i n i n t a c t , temperature-regressed f i s h (Experiment 3). 2.  Materials and Methods Female goldfish which had been warmed to 20 C several weeks e a r l i e r  were hypophysectomized  and kept i n 25% seawater for a further 3 to 4 weeks  49 before being assigned to the steroid or saline treatment groups.  Prepara-  t i o n and i n j e c t i o n of steroids and procedures for handling and testing f i s h were as described i n Experiment 4,  However, the period over which  f i s h received i n j e c t i o n s was increased to 40 days; injections were given every fourth day for the f i r s t 20 days and on alternate days for the next 20 days. 3.  Results and Discussion Of 21 f i s h which survived the i n j e c t i o n schedule, one was too sick to  test and 5 had bound eggs.  None of the remaining 15 f i s h (saline - 5; es-  t r a d i o l - 5; dihydrotestosterone - 5) showed any spawning a c t i v i t y during the three hour observation period. It has been postulated (Experiment 4) that the prolonged steroid deprivation following hypophysectomy decreased the s e n s i t i v i t y of goldfish target tissues to exogenous steroids; a decrease i n the concentration of steroid receptor proteins may account for this proposed i n s e n s i t i v i t y .  There i s  evidence that the concentration of estrogen receptor i n the rodent uterus i s influenced by estrogen (see review by Milgrom et a l . , 1973).  However,  i t i s doubtful whether the lack of e f f e c t of the 40-day treatment i n the present experiment i s the result of i n s u f f i c i e n t steroid priming, as the binding capacity of mammalian tissue increases within hours of exposure to estrogen (Milgrom e t al_. * 1973) .  It i s emphasized, however, that i n the  present experiment treatment was not begun u n t i l as much as a month a f t e r hypophysectomy; the p o s s i b i l i t y that responsiveness to egg i n j e c t i o n might be maintained or restored by e a r l i e r treatment has not been examined. The f a i l u r e of exogenous steroids to restore sexual behaviour of hypophysectomized female goldfish d i f f e r s from the effects of similar treatments  50 i n female rats (Pfaff, 1970) and guppies ( L i l e y , 1972).  As steroids i n -  duce r e c e p t i v i t y i n intact but not i n hypophysectomized g o l d f i s h , i t i s suggested that some p i t u i t a r y factor(s) may be involved i n the regulation of spawning behaviour.  The experiments described i n Chapter V were car-  r i e d out to explore t h i s p o s s i b i l i t y .  D.  Summary of Chapter IV 1.  In female goldfish with 'regressed' (nonvitellogenic) ovaries,  i n j e c t i o n of ovulated eggs does not induce spawning behaviour. 2.  A number of steroids restore responsiveness to egg i n j e c t i o n i n  regressed f i s h . 3.  Hypophysectomy abolishes the spawning response to i n j e c t i o n of  ovulated eggs. 4.  Steroid treatments do not restore responsiveness to egg i n j e c t i o n  i n hypophysectomized  fish.  CHAPTER, V  ROLE OF THE PITUITARY IN THE SPAWNING BEHAVIOUR OF FEMALE GOLDFISH  A.  Experiment 6.  E f f e c t of P i t u i t a r y Replacement on the Spawning Behaviour of Hypophysectomized Female Goldfish  1.  Introduction In previous experiments, steroid injections restored the sexual be-  haviour of i n t a c t , temperature-regressed f i s h whereas similar treatment of hypophysectomized f i s h was without e f f e c t .  These results suggested the  involvement of the p i t u i t a r y i n the control of responsiveness to egg i n j e c tion.  To examine this p o s s i b i l i t y , hypophysectomized f i s h were injected  either with e s t r a d i o l or e s t r a d i o l plus homogenized  goldfish p i t u i t a r i e s ,  and tested f o r the a b i l i t y to spawn following the i n j e c t i o n of ovulated eggs.  2.  Materials and Methods Female goldfish were kept at 20 C for 1 to 2 weeks, hypophysectomized  (or sham operated), kept a further 2 weeks i n 25% seawater, and assigned to one of three treatment groups: (1) p i t u i t a r y replacement:  Females i n this group were injected i . p .  with fresh macerated goldfish p i t u i t a r i e s (2/day) on alternate days over a 20 day period, during the l a s t 8 of which they also received e s t r a d i o l (20 ug/g i . p . on alternate days).  Preparation  of e s t r a d i o l was as i n previous experiments. Injected p i t u i t a r i e s were dissected from freshly k i l l e d donors (males and females; 1015 g), ground i n a small mortar and pestle, and mixed with a small volume of cold 0.6% NaCl to give an i n j e c t i o n volume of 0:2-0.3 ml. As Experiments 4 and 5 showed that e s t r a d i o l alone does not restore responsiveness to egg i n j e c t i o n i n hypophysectomized f i s h , a l l f i s h were primed with e s t r a d i o l to eliminate the p o s s i b i l i t y that lack  of responsiveness could be due to i n s u f f i c i e n t e s t r a d i o l , rather than to the absence of some p i t u i t a r y factor. (2)  saline control:  Treatment was as f o r group (1) except that  roughly equivalent volumes of 0.6% NaCl were injected i n place of macerated p i t u i t a r i e s . (3)  sham control:  Females i n this group were sham hypophysectomized  and treated as i n group (2). On the morning of the day following the l a s t i n j e c t i o n , each f i s h was anaesthetized i n MS-222, injected with ovulated eggs (0.025 ml/g), f i t t e d with an ovipore plug, and l e f t i n a 2 1 beaker of 25% seawater f o r 40 to 80 minutes before being placed with one or two active males i n a 60 1 observation tank.  A f t e r a three hour observation period the f i s h were anaesthe-  t i z e d , checked for bound eggs, and checked by dissection for p i t u i t a r y remnants.  The ovaries were fixed i n Bouin's F l u i d and prepared for h i s t o l o g i c a l  examination. 3.  Results Sham hypophysectomy  behaviour.  had no obvious effect on female goldfish spawning  Five of seven sham f i s h spawned at a frequency of normal intact  f i s h (Table IV).  SH-3, which spawned only 3 times during the test period,  had s l i g h t l y bound injected eggs and regressed ovaries with very l i t t l e yolk v e s i c l e formation. Even though e s t r a d i o l was injected, hypophysectomy lished the response to injected eggs.  e s s e n t i a l l y abo-  Only 2 of the 8 females tested per-  formed any spawning a c t i v i t y and i n both f i s h the response was minimal. Injection of a homogenate of goldfish p i t u i t a r y restored the spawning behaviour of most of the test females.  Furthermore, t h i s treatment induced  54  TABLE TV EFFECT OF INJECTION OF GOLDFISH PITUITARY HOMOGENATE AND ESTRADIOL ON THE SPAWNING RESPONSE TO INJECTION OF OVULATED EGGS IN HYPOPHYSECTOMIZED  FEMALE GOLDFISH  55  II  Group  Treatment  No. Spawning Acts/3 hour Test Period  Hypophysectomy 20 day p i t u i t a r y treatment 8 day e s t r a d i o l treatment 56 2 72 57 19 0 12 2 11 111 123 (mean = 42.3)  Hypophysectomy 20 day saline treatment 8 day e s t r a d i o l treatment 2 0 0 0 0 0 1 0  III  Sham hypophysectomy 20 day saline treatment 8 day e s t r a d i o l treatment 25 155 3 67 25 0 137  (mean = 58.5)  No s i g n i f i c a n t difference between spawning behaviour of p i t u i t a r y injected (Group I) and sham hypophysectomized (Group III) f i s h (Mann-Whitney U-test).  56 yolk deposition ranging from early yolk v e s i c l e s to early yolk granules. 4.  Discussion The results of this experiment demonstrate a role for the p i t u i t a r y  i n the induction of spawning i n response to injected eggs, and support Yamazaki s T  (1965) finding concerning both the time course of second growth  stage a t r e s i a following hypophysectomy and the induction of yolk formation following p i t u i t a r y replacement. In the present experiment, 5 of the 8 hypophysectomized controls contained no yolk i n the ovaries. the  The s l i g h t yolk v e s i c l e deposition seen i n  other three f i s h (one of which spawned twice during the test period) i s  similar to that of some of Yamazaki's  9-week post-operative f i s h .  As i n  my experiment, Yamazaki apparently did not carry out h i s t o l o g i c a l checks for completeness of hypophysectomy and thus there i s a p o s s i b i l i t y that hypophysectomized f i s h with such minimal yolk deposition may have retained p i t u i tary fragments.  However, considering the high incidence of a t r e s i a of  second growth phase oocytes i n these ovaries and the  post-hvpophysectomv  persistence of yolk vesicles seen i n other experiments, i t seems more l i k e l y that post-hypophysectomy  regression i n these f i s h was incomplete.  57 B.  Experiment  7.  E f f e c t of Salmon Gonadotropin and Aminoglutethimlde  on  Spawning Behaviour of Hypophysectomized Female Goldfish 1.  Introduction In the preceding experiment, i n j e c t i o n of homogenized goldfish p i t u i -  tary material into hypophysectomized  f i s h demonstrated  a role for the  p i t u i t a r y i n spawning behaviour, but provided no information as to the nature or the mode of action of the p i t u i t a r y factor(s) involved, In the present experiment  an attempt was made to test the hypothesis that i t i s  gonadotropin which i s involved i n the regulation of spawning behaviour by treating hypophysectomized  females with a p a r t i a l l y p u r i f i e d spring salmon  (Oncorhynchus tshawytscha)  gonadotropin preparationSG-G100 ( g i f t of Dr.  E.M.  Donaldson).  To examine the p o s s i b i l i t y that gonadotropin might exert  i t s effect through stimulation of steroidogenesis, some gonadotropin-treated f i s h also were injected with the steroid enzyme i n h i b i t o r , aminoglutethimide (AG, Ciba).  As AG i n h i b i t s side-chain cleavage of cholesterol  (Gaunt  et a l . , 1968; Gower, 1974), i n j e c t i o n of this chemical should r e s t r i c t or prevent entry of precursors into the ovarian steroidogenic pathway. If gonadotropin induces spawning behaviour simply by stimulating o v a r i an steroid production, then concomitant treatment with AG should block or at least reduce the response; but i f gonadotropin influences behaviour by some mechanism not requiring steroids, then AG should have no i n h i b i t o r y effect.  However, i t i s possible that the mechanism regulating spawning be-  haviour requires both gonadotropin and s t e r o i d . with AG would also block spawning.  In this case, treatment  To control for the p o s s i b i l i t y that  spawning behaviour i s induced by a combination of gonadotropin and estrogen,  58 a l l f i s h receiving gonadotropin and AG also were injected with e s t r a d i o l , which previously had been found not to induce r e c e p t i v i t y i n hypophysectomized f i s h (Experiments 4, 5, and 6). The major f a u l t i n the design of this experiment i s that i f only gonadotropin and e s t r a d i o l are required to restore responsiveness to egg i n j e c tion, treatment with AG would appear to have noe e f f e c t .  If however, a  steroid other than e s t r a d i o l i s required, treatment with AG should block the e f f e c t of gonadotropin even though e s t r a d i o l i s present.  2.  Materials and Methods' Females were transferred from 12 C to 20 C for several days p r i o r to  hypophysectomy, a f t e r which they were kept i n 25% seawater for 23 to 27 days before beginning treatment. (i)  Six treatment groups were used (see Table V ) :  high dose gonadotropin (SG-G100; 15 yg/g) plus e s t r a d i o l ( E ; 20 yg/g) 2  (ii)  low dose SG-G100 (3 yg/g) plus E  (iii)  high dose SG-G100 plus AG (100 yg/g) plus E  (iv)  low dose SG-G100 plus AG plus E  (v)  control saline (0.6% NaCl) plus E  (vi)  control E  2  2  2  2  2  A l l groups received injections (either SG, AG, saline, or E ) on a l 2  ternate days over a 20 day period. E  2  Tn addition, groups 1 to v were given  on alternate days for the l a s t 9 days of treatment. SG-G100 was administered i n two dosages to establish a dose-response  relationship for groups i and i i ;  i t was thought that this dose effect might  also be evidence i n groups i i i and i v .  Group v served as control for the  E„ injections given to groups i to i v and group v i provided an additional  TABLE V  VEHICLE VOLUMES (PER INJECTION DAY AND TOTAL) INJECTED IN EXPERIMENT 7  Volumes of vehicles  (ml) injected per 20 g f i s h  on each of f i r s t 5 i n j e c t i o n davs Treatment Group  SG  i, i i  .10  i i i , iv  .10  AG  SAL  E  2  .06  vi  Total  SG  .10  .10  .16  .10  .10  .10  V  second 5 i n j e c t i o n days  .16  .16  AG  SAL  .06 .10  E  2  Total  Over 10 injection days  .08  .18  1.40  .08  .24  2.00  .08  .18  1.40  .16  .16  1.60  SG = salmon gonadotropin (SG-G100) AG = aminoglutethimide Sal = saline E„ = estradiol  o  61 control for the extended E^ exposure assumed to result from SG-G100 stimul a t i o n of steroidogenesis i n groups i and i i . Preparation and i n j e c t i o n of  was as described previously (Experi-  ment 3). SG-G100 was prepared immediately  before i n j e c t i o n by dissolving  the l y o p h i l i z e d hormone i n cold 0.6% NaCl to a concentration of 0.6 ug/ul (low dose) or 3.0 yg/ul (high dose).  AG was ground to a f i n e powder, sus-  pended at a concentration 30 ug/ul i n 0.6% NaCl containing 4 drops Tween 80/100 ml and injected i . p . at a dosage of 100 yg/g. To reduce handling to a minimum, no attempt was made to give equal numbers of injections to a l l groups.  However, concentrations of E^ suspen-  sions were adjusted so that groups receiving three chemicals at a time (groups i i i and iv) received only 50% more vehicle than group v i which was given only single i n j e c t i o n s .  As indicated i n Table V, the differences i n  t o t a l vehicle volumes injected over the duration of the experiment were even smaller. On day 20, each f i s h was anaesthetized i n MS-222, injected with ovulated eggs (0.02 ml/g), f i t t e d with an ovipore plug, and l e f t i n a 2 1 beaker of 25% seawater for 60-75 minutes before being placed with one or two active males (one female per 60 1 observation tank).  The f i s h were observed  continuously for three hours,, anaesthetized, checked f o r bound eggs, and sacrificed.  The heads were checked by dissection f o r completeness of-hypo^-  physectomy, and ovaries were fixed and prepared f o r h i s t o l o g i c a l examination.  3.  Results  As a r e s u l t of mortality and binding of injected eggs, the f i n a l  62 sample sizes i n this experiment were small.  The data are presented i n  Table VI. As i n previous experiments, e s t r a d i o l f a i l e d to restore r e c e p t i v i t y i n hypophysectomized of response.  f i s h (Group v ) ; one of four f i s h showed a low l e v e l  It i s not clear whether the response of this f i s h was due to  residual r e c e p t i v i t y or to the estrogen treatment.  No completely hypophy-  sectomized group v i (estradiol-treated) f i s h survived to be tested. At both the high and low dosages (Group 1 and i i ) , was  salmon gonadotropin  e f f e c t i v e i n restoring the response to injected eggs,  sponding i n the behavioural tests.  a l l 7 fish re-  In addition, the gonadotropin had a  marked effect on v i t e l l o g e n e s i s , ranging from the induction of the early yolk v e s i c l e stage i n most f i s h , to the early yolk granule stage i n one female.  These effects on oocyte development are consistent with those re-  ported by Yamazaki and Donaldson (1968) ; the greater degree of ovarian r e sponse i n t h e i r study l i k e l y was due to higher gonadotropin dosage and healthier f i s h .  Except for one degenerating oocyte i n one f i s h ,  gonadotro-  pin treatment also prevented the degeneration of f i r s t and second growth stage oocytes.  This i s i n contrast to the results of e s t r a d i o l treatment  (groups v and v i ) where 8 of 10 f i s h had d e s p e r a t i n g oocytes. Administration of aminoglutethimide was highly e f f e c t i v e i n i n h i b i t i n g spawning i n response to egg i n j e c t i o n i n hypophysectomized,  gonadotropin'  treated f i s h (Group i i i and i v ) ; only one of s i x f i s h responded i n the behavioural tests.  However, the ovarian histology of f i s h treated with gona-  dotropin and aminoglutethimide was similar to that of the f i s h receiving gonadotropin alone; yolk v e s i c l e s were present i n a l l ovaries and a t r e s i a of oocytes was i n h i b i t e d completely.  63  TABLE VI EFFECT OF SALMON GONADOTROPIN (SG-G100) AND AMINOGLUTETH1MIDE ON THE SPAWNING RESPONSE TO EGG INJECTION IN HYPOPHYSECTOMIZED FEMALE GOLDFISH  Group Treatment F i r s t 10 days Second 10 days  i  i i  Mi  iv  v  vi  SG - 15 yg/g  SG - 3 yg/g  SG - 15 yg/g AG - 100 yg/g  SG - 3 yg/g AG - 100 yg/g  saline  SG - 15 yg/g E - 20 yg/g  SG - 3 yg/g E - 20 yg/g  SG - 15 yg/g AG - 100 yg/g E - 20 yg/g  SG - 3 yg/g AG - 100 yg/g E - 20 yg/g  saline E E - 20yg/g  2  E  2  - 20 yg/g - 20 yg/g  Z  2  116  72  0  44  0  no  acts/3 hour  61  36  0  0  0  fish  test period  6  18  0  0  5  tested  No• spawning  38 SG = salmon gonadotrpin (SG-G100)  AG = aminoglutethimide  E  2  = estradiol  -  vn  4>  -I  LEAF 65 OMITTED IN PAGE NUMBERING.  66 4.  Discussion The r e s u l t s of this experiment demonstrate that i n hypophysectomized  female goldfish, treatment with a p a r t i a l l y p u r i f i e d salmon gonadotropin preparation (SG-G100) combined with e s t r a d i o l (I^) i s e f f e c t i v e i n restoring spawning i n response to the i n j e c t i o n of ovulated eggs. experiments, treatment with  alone was i n e f f e c t i v e .  As i n previous  These findings sug-  gest that gonadotropin i s the p i t u i t a r y factor responsible for restoring r e c e p t i v i t y i n the hypophysectomized homogenate i n Experiment 6.  f i s h injected with goldfish p i t u i t a r y  However, as the gonadotropin preparation used  i n this experiment l i k e l y contained some thyrotropic a c t i v i t y  (Donaldson  and McBride, 1974), i t i s possible that i t was thyrotropin rather than, or i n addition to, gonadotropin which restored r e c e p t i v i t y . When hypophysectomized  f i s h receiving SG-G100 and E^ also were injected  with the steroid enzyme i n h i b i t o r , aminoglutethimide (AG), the response to injections of eggs was strongly i n h i b i t e d .  This i n h i b i t i o n of spawning be-  haviour by AG may simply have been pharmacological and independent of any effects on steroidogenesis; however, neither the non-spawning behaviours nor the general condition of AG-treated f i s h were obviously d i f f e r e n t from those of the other groups.  I f AG affected spawning by i n h i b i t i n g steroidogenesis,  then i t appears the effect was due to i n h i b i t i o n of the production of an essential steroid (or steroids) other than e s t r a d i o l .  Whether or not AG  was administered, SG-G100 stimulated yolk production.  This i s to be expected,  as gonadotropin and e s t r a d i o l were present i n a l l cases.  C.  Summary of Chapter V 1.  Injection of homogenized goldfish p i t u i t a r i e s or p a r t i a l l y p u r i -  f i e d salmon gonadotropin into hypophysectomized  female goldfish restores  responsiveness to i n j e c t i o n of ovulated eggs. 2.  In hypophysectomized  f i s h treated with salmon gonadotropin and  e s t r a d i o l , i n j e c t i o n of the steroid enzyme i n h i b i t o r , aminoglutethimide, i n h i b i t s responsiveness to i n j e c t i o n of ovulated eggs, suggesting a steroid other than e s t r a d i o l may play a role i n spawning behaviour.  CHAPTER VI  THE ROLE OF PROSTAGLANDINS IN THE SPAWNING BEHAVIOUR OF FEMALE GOLDFISH  69 A.  Introduction The majority of the preceding experiments were designed to examine  the effects of various hormones on the spawning responses of female goldf i s h to the i n j e c t i o n of ovulated eggs.  These studies have been concerned  with r e l a t i v e l y long-term e f f e c t s of hormonal deprivation and replacement over periods of days or weeks.  In this section, the short-term regulation  of sexual behaviour i s examined i n experiments involving the mechanism by which egg i n j e c t i o n triggers spawning. In h i s study of the spawning behaviour of goldfish, Yamazaki (1965) noted that even though a female had ovulated, spawning would not occur  un-  less both aquatic vegetation and a sexually active male were present.  Re-  moval of the vegetation i n h i b i t e d spawning immediately, restored i t within minutes.  while replacement  My observations also show that i f an ovulated  female, isolated from other f i s h for days, i s placed with a sexually active male, spawning can occur within a minute.  However, a consistent feature  of spawning induced by egg i n j e c t i o n i s that there i s a variable latent period (as much as several hours) between the time eggs are injected and the time spawning begins.  Although this latent period may be nothing more  than an i n h i b i t i o n induced by anaesthetization and handling during egg i n j e c t i o n , some preliminary data suggest this i s not so. Seven highly receptive f i s h with second growth phase oocytes were anaesthetized and injected with ovulated eggs: two females (#1,2) were placed with active males immediately,  four (#3-6) were placed with the  males after 200 minutes 'incubation' i n i s o l a t i o n from other f i s h , and  one  (#7) was placed with males following a second anaesithetization at the end of the 200 minute incubation (Table VII).  In the f i s h placed with males  70  TABLE VII EFFECT OF INCUBATION TIME (DELAY BETWEEN EGG INJECTION AND PLACEMENT OF FEMALE WITH MALE) ON LATENCY TO FIRST SPAWNING ACT IN RECENTLY OVULATED (3-to 7-DAY BOSTOVULATORY) FEMALES.  71  Fish  Incubation (min)  Latency to first spawning act  No. spawning acts i n f i r s t 40 min of spawning  1 - POV-7  10  37  52  2 - P0V-4  4  55  25  3 - POV-7  200  3  74  4 - POV-7  200  9  19  5 - P0V-3  200  2  118  6 - POV-4  200  9  52  7 - P0V-3  200*  12  66  *Fish #7 received a second anaesthetization of 200 minutes.  72 immediately after recovery from anaesthetic, the latency to the f i r s t spawning act was 200 minutes.  considerably greater than that i n f i s h incubated for  This was  true even when an incubated f i s h was  anaesthetized  a second time. These limited results suggest that eggs i n the ovarian lumen stimulate spawning by a d i f f e r e n t mechanism than do the presence of male goldfish or aquatic vegetation.  Whereas presentation of the l a t t e r stimuli e l i c i t s  spawning within a few minutes i n f i s h which nay'e had ovulated eggs i n the ovarian lumen for some time, i n j e c t i o n of ovulated eggs i s followed by a latent period which apparently i s not due simply to anaesthetization.  Of  course, the second anaesthetization at 200 minutes i s a poor control as i t indicates only that MS-222 has l i t t l e e f f e c t on the behaviour of a f i s h which i s already receptive. It was  thought that the latent period might represent the time r e -  quired for oviduct stimulation to effect changes i n c e n t r a l neural structures c o n t r o l l i n g sexual behaviour.  As a number of workers have reported  that i n j e c t i o n of neurohypophyseal hormones induces a 'spawning r e f l e x ' , oviposition, or p a r t u r i t i o n i n a v a r i e t y of teleost species ( L i l e y ,  1969;  Macey et a l , , 1974), i t seemed possible that these hormones might be i n volved i n mediating of female goldfish.  the effects of egg i n j e c t i o n on the spawning behaviour No formal experiments were carried out to test this  hypothesis as i n a l l preliminary tests oxytocin was  completely  ineffective  i n inducing spawning behaviour i n female goldfish with v i t e l l o g e n i c or i n increasing the rate of spawning i n ovulated f i s h .  oocytes  Similar results  have apparently been obtained by Ptckford (unpublished results cited i n Macey e_t a l . , 1974) .  73 In female mammals, physical stimulation (stretch) of the reproductive tract and other smooth muscles (situations roughly comparable to ovulation o,r egg i n j e c t i o n i n goldfish) i s associated with the release of prostaglandins  (Poyser et a l . , 1971;  Piper and Vane, 1971;  Prostaglandins  (PG), the ' i n t r i n s i c uterine stimulant'  Csapo, 1973).  (Csapo, 1973), have  been implicated i n the oviposition of birds (Hertelendy, 1972, Heftelendy et a l . , 1974,  1975;  Wechsung and Houvenaghel, 1976), i n the  p a r t u r i t i o n of mammals (Liggins et a l . , 1973; 1974;  F l i n t et a l . , 1974;  Currie, 1975;  Umo  La'bhsetwar, 1974;  Aiken,  et a l . , 1976), and i n the  mechani sm of action of some IUDs (Spilman and Duby, 1972; The findings that steroids may  1973;  Chaudhuri, 1975).  a l t e r the release of PG from the genital  tract (Roberts et al_., 1975) , that the response o.f. the g e n i t a l tract to PG may may  be modified by steroids (Csapo, 1973;  Spilman, 1974), and that PG  play a role i n the action of LH on ovarian tissue (Kuehl et a l . , 1970;  Marsh et a l . , 1974)  a l l suggested that investigation of PG function i n the  female goldfish might shed l i g h t on the problem of the induction of spawning behaviour by egg i n j e c t i o n . sults of these investigations.  The following experiments present the re-  74  B.  Experiment 8.  I n h i b i t i o n by Indomethacin of Spawning Behaviour Induced by Injection of Ovulated Eggs  1.  Int ro due t i o n It was postulated that i f PGs were involved i n goldfish spawning  behaviour, their synthesis or release may be increased by the stimulus provided by egg i n j e c t i o n .  In female mammals, endogenous PG production  can be i n h i b i t e d by i n j e c t i o n of indomethacin (IM), a potent i n h i b i t o r of PG synthesis which apparently affects a complex of enzymes referred to as prostaglandin synthetase  (Vane, 1971).  As a f i r s t step i n demon-  s t r a t i n g a role for PG i n goldfish spawning behaviour, used to i n h i b i t endogenous PG production.  IM i n j e c t i o n  To examine the speed and  was dura-  tion of action of IM, injections were given at various'times i n r e l a t i o n to the i n j e c t i o n of eggs and the onset of spawning behaviour.  2.  Materials and Methods A l l female goldfish used i n this experiment had ovaries i n various  stages of v i t e l l o g e n e s i s and therefore could have been expected when injected with ovulated eggs. Table V I I I ) . pension  Four treatment  Indomethacin (IM, Sigma) was  to spawn  groups were used (see  injected i . p . as a saline sus-  (0.6% NaCl; 4 drops Tween 80/100 ml) at a dosage of 10 ug/g  (5yl/g).  This dosage had been e f f e c t i v e i n a study of goldfish ovulation (Stacey and Pandey, 1975). At 1000 h on the test day, f i s h were anaesthetized i n MS-222, weighed and injected with ovulated eggs (0.02 ml/g).  They were then l e f t for 1  hour, placed i n 60 1 observation tanks with a c t i v e l y courting males, and observed  continuously for three hours.  Females i n Group i had received  TABLE VIII EFFECT OF INDOMETHACIN ON SPAWNING IN RESPONSE TO INJECTION OF OVULATED EGGS  76  Group  i ii iii  Treatment  N  No. Responding  IM 10 h before eggs  7  0  0+  IM with eggs  5  0  0+  saline with eggs  Q  9  6-H-  IM after 20 minutes spawning iv  saline with eggs saline after 20 minutes spawning  * Based on responding i n d i v i d u a l s . + Mean no. per 3 h test period ++ Mean no. per 20 min +++ Mean no. after 2nd i n j e c t i o n IM = indomethacin  Mean • No. Spawning Acts* (range)  (2-11)  9 1 8  (!)+++ 5-H-  19+4-  (1-9)  (5-37)  77 IM at 2400 h the previous evening, 10 hours before egg i n j e c t i o n , while those i n Group i i had received IM while anaesthetized f o r egg i n j e c t i o n . Groups i i i and i v received saline injections coincident with egg i n j e c tions as a control for the IM i n j e c t i o n given to Group i i .  In addition,  Group i i i received an IM i n j e c t i o n and Group i v a second saline i n j e c t i o n 20 minutes a f t e r spawning had commenced.  For this second i n j e c t i o n f i s h  were not anaesthetized, but simply netted from the observation tank, i n jected while hand held, and replaced immediately.  3.  Results and Discussion As shown i n Table VIIT, none of the females injected with IM 10 hours  priord to, or coincident with, i n j e c t i o n of ovulated eggs spawned during the 3 hour test period; i n contrast, a l l females injected with saline at the time of egg i n j e c t i o n (Groups i i i and iv) spawned i n the test period. Following IM i n j e c t i o n , one Group i i i  female performed one spawning act  (5 minutes p o s t - i n j e c t i o n ) , whereas a l l group i v females continued to spawn following the second control saline i n j e c t i o n . Injection of an IM suspension i s thus seen to have a rapid and r e l a t i v e l y long-lasting i n h i b i t o r y e f f e c t on the spawning response to injected eggs.  (In contrast, courting males, which were observed for up to three  hours p o s t - i n j e c t i o n , showed no obvious behavioural response to the i n j e c tion of IM; however, no quantitative measures were taken. whether treatment with IM i n h i b i t s the release of sperm).  I t i s not known  78 C.  Experiment 9.  E f f e c t of Prostaglandins on the Spawning Behaviour of Indomethacin-Treated Female Goldfish  1.  Introduction This experiment tested the a b i l i t i e s of injected prostaglandins  (PGE^, P^ 2' E  ^^2^^  t 0  o  v  e  r  c  o  m  e  t  ^  ie  spawning following egg i n j e c t i o n .  indomethacin-induced  i n h i b i t i o n of  These three PGs were chosen as they  were found to be e f f e c t i v e i n overcoming the indomethacin blockade of ovul a t i o n i n female goldfish (Stacey and Pandey, 1975).  2.  Materials and Methods Five treatment  was  groups were used and the i n i t i a l treatment  as for Group i i i n the preceding experiment.  Fish were anaesthetized,  weighed, and injected with ovulated eggs (0.02 ml/g) tween 900 and 1000 h on the test day.  for a l l  and IM (10 yg/g)  be-  A l l f i s h were then l e f t for one  hour to recover, netted, injected i . p . with the saline vehicle (Group i ) or one of the PGs  (Groups i i - v ) , placed i n the observation tanks with ac-  t i v e males, and observed PGE2  continuously f o r 2 hours.  PGE^  (Group i i ) and  (Group i i i ) were prepared by dissolving i n 95% ethanol (1 mg/0.1 ml)  and d i l u t i n g 9X with buffered saline (20 mg Na C0 /100 ml 0.6%  NaCl).  (Group iv) was  simply dissolved i n buffered saline (1 mg/ml).  Group v  received PGF,^  prepared i n the same manner as PGE^  2  3  and PGE  for possible behavioural effects of the ethanol solvent. were injected i . p . at a dose of 5 yg/g 3.  2  as a control  A l l PGs  (Upjohn)  (5 y l / g ) .  Results and Discussion None of the Group i f i s h treated with the saline vehicle spawned  during the 2 hour test period (Table IX).  PGF^  Though the sample size  was  79  TABLE IX EFFECT OF PROSTAGLANDINS ON THE SPAWNING RESPONSE TO INJECTION OF OVULATED EGGS.IN FEMALE GOLDFISH TREATED WITH INDOMETHACIN  80  Group  Treatment  N  IM, eggs and s a l i n e  6  0  0  ii  IM, eggs and P G E  1  5  0  0  iii  IM, eggs and P G E  2  7  2  5  IM, eggs and P G F ^  7  5  27  (7-62)  IM, eggs and PGF(alcohol vehicle;  4  3  52  (5-115)  i  iv V  No. r e sponding  * Based on i n d i v i d u a l s r e s p o n d i n g i n 2 h t e s t p e r i o d . Indpmetnaeini;,(IMi)i and eggs i n j e c t e d 1 h PGs i n j e c t e d  at s t a r t of t e s t  before t e s t ,  Mean No. Spawning Acts* (range)  (4,6)  81 small, PGE^ ade,  (5 f i s h ) had no e f f e c t i n overcoming the indomethacin block-  and PGE was 2  only s l i g h t l y active, inducing low l e v e l s of spawning  i n 2 of the 7 f i s h tested.  In contrast, PGF^x was  quite e f f e c t i v e i n  inducing spawning behaviour i n IM-treated f i s h ; presence of ethanol i n the vehicle does not seem to account for the ineffectiveness of PGE^ PGE^,  and  as 3 of the 4 Group V f i s h spawned normally. The latencies from the i n j e c t i o n of PG to the onset of spawning were  quite variable (5-100 minutes; mean 40 minutes).  There were no s i g n i f i -  cant differences among the latencies of the responding f i s h i n groups i i i , i v , and v.  Of the 8 f i s h which spawned following PGF2  0Q  i n j e c t i o n , a l l but  one continued to spawn for the remainder of the observation  period.  82 D.  Experiment 10.  Effect of Prostaglandins Alone (Without Egg Injection) on the Spawning Behaviour of Female Goldfish  1. Introduction Based on the findings that indomethacin (IM) blocked the spawning response to injected eggs, and that PGs  (especially PGF ) were e f f e c t i v e 2a:  i n overcoming this blockade, this experiment was conducted to determine whether PG i n j e c t i o n alone ( i . e . , without p r i o r treatment with ovulated eggs) was s u f f i c i e n t to induce spawning behaviour, and whether the order of PG potenciess was as found i n Experiment 9 (PGF^  2.  > PGE  2  > PGE^ .  Methods, Results, and Discussion Female goldfish were injected i . p . (no anaesthetization) with either  PGF„ , PGE.. , or PGE_ 1  at 5 yg/g, placed immediately with active males, and  2  observed continuously.for 3 hours. As shown i n Table X, PGF  2co  No control group was  used.  was highly e f f e c t i v e i n inducing spawning  behaviour even though ovulated eggs were not present i n the ovarian lumen. Several of the f i s h i n this group were injected with IM 20 minutes after spawning had commenced; there was no apparent effect on spawning behaviour, PGE was marginally e f f e c t i v e i n inducing spawning behaviour (2 o.f 10 f i s h 2  responding) and PGE^ was without e f f e c t . As i n Experiment 9, the latencies to the onset of spawning were quite variable (5-135 minutes; mean 45 minutes). i n the f i s h responding to PGF.  Also, the duration of spawning  was considerable, several individuals r e -  sponding regularly f o r longer than 2 hours. These results suggest that the effect which ovulated eggs i n the o v a r i an lumen have "an spawning behaviour i s mediated through the release of  TABLE X  EFFECT OF PROSTAGLANDINS ON THE SPAWNING BEHAVIOUR OF FEMALE GOLDFISH WITHOUT OVULATED EGGS TN THE OVARIAN LUMEN  84  Group  Treatment  N  No. Responding  Mean TCHQ. Acts*  Spawning (Range)  i  PGE^  10  0  0  ii  PGE  10  2  11  (7,15)  13  13  59  (11-208)  iii  2  PGP.  Based on individuals responding i n 3 h test period. PGs injected at start of test.  85  p r o s t a g l a n d i n s , and t h a t i t i s some a c t i o n o f p r o s t a g l a n d i n s , o f the presence of eggs, which induces spawning courtship.  independent  i n response to male  86 E.  Experiment 11.  E f f e c t of Hypophysectomy and Gonadotropin Replacement on Prostaglandin-lnduced Spawning Behaviour i n Female Goldfish  1. Introduction In experiments presented i n Chapters IV and V, treatments with p i t u i t a r y and ovarian hormones were found to a f f e c t the responsiveness of female goldfish to egg i n j e c t i o n .  A variety of steroid hormones were  e f f e c t i v e i n inducing r e c e p t i v i t y i n i n t a c t , temperature-regressed f i s h , whereas i n hypophysectomized totally ineffective.  f i s h , a l l steroids tested were found to be  Treatment with g o l d f i s h p i t u i t a r y homogenate or a  p a r t i a l l y p u r i f i e d salmon gonadotropin preparation (SG-G100) was e f f e c t i v e i n restoring spawning behaviour i n hypophysectomized  highly  fish.  As  the i n j e c t i o n of the steroid enzyme i n h i b i t o r , aminoglutethimide, inhibited the response to SG-G100, i t was suggested that the mode of action of gonadotropin on spawning behaviour may be through the stimulation of ovarian steroidogenesis.  A number of interpretations of these results i s possible.  In many mammalian studies, prostaglandin release has been shown to be correlated with reproductive cycles and to be influenced by gonadotropin and steroids (see references i n section A, this chapter).  As PG i s ap-  parently essential for spawning behaviour to occur, i t seemed a plausible explanation that the i n a b i l i t y of steroids to restore r e c e p t i v i t y i n hypophysectomized f i s h i s due to the absence of PG synthesis following hypophysectomy.  The following experiment examined this hypothesis by  the response to PGF^  i n j e c t i o n i n hypophysectomized  comparing  f i s h treated either  with steroids, salmon gonadotropin, or a saline v e h i c l e .  87  2.  Materials and Methods Females which had been hypophysectomized 3 to 4 months e a r l i e r were  injected i . p . (no anaesthetization)  as follows:  Group i  - saline vehicle (0.6% NaCl; 4 drops Tween 80/100 ml),  Group i i  - 10 yg/g  spring salmon (Oncorhynchus tshawytscha)  gonadotropin (SG-G100, l o t #BCR-3) dissolved i n saline, Group i i i  ^ 20 yg/g  17$-estradiol suspended i n s a l i n e .  A l l treatment groups received i n j e c t i o n s at 5 y l / g on alternate days for a 15 day period.  On the morning of the day following the l a s t i n j e c t i o n ,  f i s h were injected i . p . with 5 yg/g PGF^, courting males, and observed continuously  placed immediately with a c t i v e l y f o r 3 hours.  F i s h i n groups i  and i i were then s a c r i f i c e d and the heads examined by dissection for p i t u i tary remnants.  Ovaries were fixed and prepared for h i s t o l o g i c a l examina-  tion . To examine the p o s s i b i l i t y that gonadotropin-induced r e c e p t i v i t y was due to steroids other than e s t r a d i o l , Group i i i was  divided into two groups;  Group i i i A received e s t r a d i o l injections on alternate days for a further 15 day period, while Group i i i B received injections of a mixture of s t e r oids ( e s t r a d i o l , 20 yg/g;  testosterone, S^dihydrotestosterone, 11-keto-  testosterone, progesterone, and deoxycorticosterone same period of time.  a l l at 5 yg/g)  Testing and related procedures following this second  i n j e c t i o n period were the same as those following the 3.  for the  first.  Results Injection of PGF^  f a i l e d to induce spawning behaviour i n female  goldfish which had been hypophysectomized for 3 to 4 months; a 2 week  88 pretreatment  with salmon gonadotropin  restored PG-induced spawning be-  haviour i n 8 of 13 hypophysectomized f i s h tested (Table XI). .with e s t r a d i o l or a combination of steroids was without ing the responsiveness  Treatment  effect i n r e s t o r -  to PG, only 1 of 12 f i s h showing a low l e v e l of  spawning i n each testing session. As i n the two preceding experiments, the latency to spawning was variable (20-160 minutes; mean 60 minutes) and the duration of spawning was  often considerable, 4 of the 8 responding  f i s h i n Group i i spawning  for longer than 2 hours. H i s t o l o g i c a l examination of the ovaries revealed no yolk deposition i n any Group i or Group i i i f i s h .  The ovaries of the 6 most active spawn-  ers i n Group i i contained small numbers of oocytes i n the early yolk v e s i cle stage.  Of the 5 non-responding Group i i f i s h , one had ovaries with  early yolk v e s i c l e s , three had no yolk v e s i c l e stage oocytes, and  the  ovaries of one contained only a^-stage corpora lutea (Khoo, 1975).  No  oocytes containing yolk granules were observed.  The greater ovarian r e -  sponse to SG-G100 reported for hypophysectomized g o l d f i s h by Yamazaki and Donaldson (1968) may  have been due e i t h e r to a longer i n j e c t i o n  schedule  (3 weeks rather than 2) or a shorter period following hypophysectomy (2 months rather than 3 or 4). SG-G100 was  also e f f e c t i v e i n preventing oocyte degeneration:  where-  as 13 of 16 Group I and 8 of 12 Group i i i f i s h had ovaries containing degenerating p r e v i t e l l o g e n i c oocytes, these were found i n only 3 of  13  Group i i f i s h . 4.  Discussion In hypophysectomized f i s h , salmon gonadotropin  restores the spawning  TABLE X I  EFFECT OF SALMON GONADOTROPIN (SG-G100) AND STEROIDS ON SPAWNING BEHAVIOUR IN RESPONSE TO INJECTION OF PROSTAGLANDIN F ^  IN HYPOPHYSECTOMIZED  FEMALE GOLDFISH  90  Group  N  No. r e sponding  saline  16  9 ,  salmon gonadotropin  13  8  26  i n  estradiol  12  1  (3)  iiiA  estradiol  6  1  (5)  iiiB  steroid mixture  6  0  0  i ii  Treatment  Mean i.oNo. Spawning Acts* (range)  Based on individuals responding i n 3 h test period PGs injected at start of test  0 (5-53)  91 response  to p r o s t a g l a n d i n ( P G F ^ ) i n j e c t i o n , w h i l e treatment  estradiol of  or a mixture o f s t e r o i d s has no e f f e c t .  estradiol  steroid  i s not l i k e l y  to be due  The  to an i n h i b i t o r y  lack of  dosage, as the same dosage g i v e n to i n t a c t r e g r e s s e d f i s h 3)  and w i t h salmon gonadotropin to hypophysectomized  periment  7)  d i d not i n h i b i t  PG sis  effect  e f f e c t of high  periment  The  with  results  fish  (Ex(Ex-  spawning.  o f t h i s experiment  demonstrate  that r e s p o n s i v e n e s s to  i s modulated by g o n a d o t r o p i n ; however, they do not support the  hypothe-  t h a t the i n e f f e c t i v e n e s s of s t e r o i d s on the r e s p o n s i v e n e s s of hypo-  physectomized  f i s h to egg i n j e c t i o n i s due  hypophysectomized effects jection, respond  to the absence of PG.  In  f i s h t r e a t e d w i t h salmon g o n a d o t r o p i n o r s t e r o i d s ,  o f PG on spawning b e h a v i o u r are s i m i l a r s u g g e s t i n g that f i s h which f a i l to endogenous PG,  to the e f f e c t s  the  of egg i n -  to respond, to eggs simply may  not  92  F.  Summary of Chapter VI 1.  Injection of indomethacin, an i n h i b i t o r of prostaglandin synthe-  s i s , blocks the effect of ovulated eggs on spawning behaviour. 2.  Injection of prostaglandins restores spawning behaviour i n egg-  injected f i s h treated with indomethacin and induces spawning i n f i s h which have not been injected with ovulated eggs. 3.  The spawning response to prostaglandin i n j e c t i o n i s abolished by  hypophysectomy and restored i n hypophysectomized salmon gonadotropin.  f i s h by treatment with  Steroid treatment i s i n e f f e c t i v e i n restoring the  response to prostaglandin i n j e c t i o n i n hypophysectomized  fish.  CHAPTER VII  GENERAL DISCUSSION  94  A. Introduction This study not only raises a number of questions as to the nature of the roles of the hormones examined; concerning  i t also suggests fascinating problems  the evolution of the regulation of sexual behaviour i n female  vertebrates i n general. There i s much scope for speculation on the mechanism which regulates spawning behaviour i n female goldfish.  However, i t must be acknowledged  that a l l conclusions derived from this study are biased to the extent that they are based on results obtained i n an experimental signed to measure only oviposition behaviour. hormones may  s i t u a t i o n de-  As detailed by Beach (1976),  influence three basic aspects of female reproductive be-  haviour; ( i ) a t t r a c t i v i t y - measured i n terms of the appetitive sexual responses evoked i n conspecific males, ( i i ) p r o c e p t i v i t y - appetitive sexual behaviours  evoked i n females by males, and  the consummatory phase of the mating sequence. (page 16  ( i i i ) receptivity -  As mentioned above  ), i n situations where the male goldfish i s r e l a t i v e l y sexually  i n a c t i v e , females may  exhibit proceptive behaviour including components  which would f a l l into the categories Beach (1976) refers to as a f f i l i a t i v e , s o l i c i t a t i o n a l , approach-withdrawal, and contact responses. non-behavioural  One aspect of  stimuli contributing to the a t t r a c t i v i t y of female gold-  f i s h consists of unidentified substances which apparently are released by the ovaries of preovulatory and ovulated f i s h and function as o l f a c t o r y stimulants of appetitive sexual responses i n males (Partridge et al.,1976). The present study has dealt solely with the endocrine control of the spawning act, the consummatory phase of female goldfish sexual  behaviour;  95  i t i s possible that had data been gathered on the e f f e c t of hormones on the stimulus quality of females or on the tendency of females to approach males or engage i n other types of proceptive behaviour, the results and conclusions might have been d i f f e r e n t . The results of experiments presented i n this thesis i d e n t i f y four endogenous factors believed to play major roles i n spawning behaviour; (i) p i t u i t a r y hormones (apparently gonadotropin), ( i i ) ovarian steroids, ( i i i ) stimuli from ovulated eggs i n the oviduct, and (iv) prostaglandins. As so l i t t l e i s known of the endocrine regulation of female reproductive behaviour i n other teleostean species, speculation concerning the function of these four factors i n the control of spawning behaviour i n goldfish i s based largely on information from studies of reproduction and sexual behaviour i n higher vertebrates, p a r t i c u l a r l y mammals. B.  The Role of the P i t u i t a r y The results of hypophysectomy and p i t u i t a r y replacement therapy demon-  strate that the p i t u i t a r y plays at least an i n d i r e c t role i n regulating spawning behaviour i n female goldfish.  That p i t u i t a r y gonadotropin i s  involved i n this regulation i s suggested by the effectiveness of salmon gonadotropin i n restoring r e c e p t i v i t y i n hypophysectomized  fish.  It i s possible that the effect of salmon gonadotropin on spawning behaviour was due to, or enhanced by, contamination of the gonadotropin preparation with thyrotropin.  Donaldson and McBride  (1974) found evidence  that salmon gonadotropin i n j e c t i o n i n adult salmon led to increased thyroid a c t i v i t y , an effect which they attributed to thyrotropic a c t i v i t y i n the gonadotropin preparation. I t i s not clear how thyrotropin or thyroxin might be involved i n stimulating r e c e p t i v i t y .  I f these hormones were  96  r e q u i r e d to mediate  the e f f e c t o f s t e r o i d s on b e h a v i o u r , then the l a c k o f  e f f e c t o f s t e r o i d s i n hypophysectomized  f i s h would be  understandable.  However, t h e r e a p p a r e n t l y i s no evidence t h a t t h y r o t r o p i n or t h y r o x i n hances the a c t i o n o f s t e r o i d s on t a r g e t I t i s not known how  issues.  gonadotropin r e s t o r e s r e s p o n s i v e n e s s to egg  i n j e c t i o n i n hypophysectomized  fish.  o r both o f two b a s i c mechanisms may 1. gonadotropin may  en-  However, i t i s suggested t h a t be  either  involved:  directly affect  central neural structures  c o n t r o l l i n g sexual behaviour, or 2. gonadotropin may  e x e r t an i n d i r e c t  c o n t r o l over b e h a v i o u r by  s t i m u l a t i n g the f o r m a t i o n o f s t e r o i d hormones which i n t u r n a c t d i r e c t l y on the mechanism r e g u l a t i n g s e x u a l r e s p o n s i v e n e s s . There i s no d i r e c t evidence from t h i s o r o t h e r s t u d i e s of t e l e o s t s t h a t gonadotropin e x e r t s a d i r e c t c o n t r o l over female s e x u a l b e h a v i o u r . However, i t has been suggested (Hoar, 1962;  Baggerman, 1966)  g o n a d o t r o p i n may  that i n male G a s t e r o s t e u s a c u l e a t u s and Cymatogaster  aggregata  (Wiebe,  1967)  a f f e c t b e h a v i o u r a s s o c i a t e d w i t h r e p r o d u c t i o n through a  mechanism independent  of the gonads.  Gonadotropin has been suggested to  e x e r t a d i r e c t c o n t r o l over s e x u a l b e h a v i o u r i n the female guppy ( L i l e y , 1968); more r e c e n t evidence L i l e y , 1972)  fails  There appears  ( L i l e y and Donaldson,  1969;  to p r o v i d e support f o r t h i s h y p o t h e s i s . to be no evidence t h a t gonadotropins enhance s e x u a l  b e h a v i o u r i n female mammals except through s t i m u l a t i o n o f s t e r o i d o g e n e s i s . In o v a r i e c t o m i z e d r a t s , hypophysectomy does n o t a f f e c t l o r d o s i s induced by h i g h e s t r o g e n dosage ( P f a f f , 1970)  and f a c i l i t a t e s  female s e x u a l  b e h a v i o u r when t h r e s h o l d dosages o f e s t r o g e n are employed  (Crowley e t a l . , 1 9 7 6 ) .  97  As i n j e c t i o n of l u t e i n i z i n g hormone supresses lordosis responses i n hypophysectomized-ovariectomized  individuals, Crowley e_t al.(1976)  suggest •'that this gonadotropin may affect sexual behaviour by i n h i b i t i n g the release of l u t e i n i z i n g hormone-releasing hormone (LH-RH).  LH-RH  stimulates lordosis i n the female rat (Pfaff, 1973; Moss and Foreman,1976). There i s some evidence that gonadotropin induces r e c e p t i v i t y i n female goldfish by stimulating steroidogenesis.  As estrogen enhances yolk  formation i n other teleosts (Campbell and I d l e r , 1976; Emmersen and Petersen, 1976), i t i s l i k e l y that salmon gonadotropin, which induces yolk formation i n hypophysectomized  goldfish (Yamazaki and Donaldson, 1968) ,  also stimulates the production of estrogen and other steroids.  Thus, the  a b i l i t y of aminoglutethimide to i n h i b i t spawning behaviour of salmon gonadotropin-treated goldfish suggests that gonadotropin influences recepti v i t y by stimulating steroidogenesis.  A similar mechanism has been proposed  to explain the e f f e c t of salmon gonadotropin on sexual behaviour i n the female guppy (Liley and Donaldson,  1969).  In addition to i n h i b i t i n g side-chain cleavage of cholesterol (Gaunt et a l . , 1968; Gower, 1974), aminoglutethimide has been shown to i n h i b i t aromatization (Thompson and S i i t e r i , 1973) and to block the stimulatory e f f e c t of testosterone on sexual behaviour i n the male rat (Beyer e_t a l . , 1976).  It i s not l i k e l y that aminoglutethimide suppressed  spawning behaviour by i n h i b i t i n g aromatization, as a l l f i s h receiving gonadotropin and aminoglutethimide were also injected with e s t r a d i o l .  The  fact that e s t r a d i o l did not overcome the i n h i b i t o r y e f f e c t of aminoglutethimide suggests that spawning behaviour may be regulated by steroids other than (or i n addition to) e s t r a d i o l .  98  The p o s s i b i l i t y that the effect of aminoglutethimide on spawning behaviour i s pharmacological, and not related to an e f f e c t on steroidogenesis, cannot be excluded.  In mammals, aminoglutethimide affects the  synthesis of thyroid (Rallison e_t a l . , 1967) and adrenocortical hormones (Philbert et a l . , 1968) and depresses brain a c t i v i t y (Elazar and Blum, 1971).  However, neither i n the present study nor i n that of Beyer et a l .  (1976) did aminoglutethimide treatment produce any obvious changes i n nonreproductive behaviour. Salmon gonadotropin restores r e c e p t i v i t y i n hypophysectomized  female  guppies but not i n f i s h which have also been ovariectomized ( L i l e y and Donaldson, 1969) , suggesting that the hormone exerts i t s effect on behaviour by stimulating ovarian steroidogenesis.  The subsequent  that estrogen treatment induces r e c e p t i v i t y i n hypophysectomized guppies ( L i l e y , 1972) demonstrates  finding female  that i n this teleost gonadotropin i s not  essential for the expression of sexual behaviour.  As i n the guppy, steroid  hormone restores r e c e p t i v i t y i n the hypophysectomized  female rat (Pfaff,  1970) ; i n fact, hypophysectomy has been found to enhance behavioural responsiveness to estrogen treatment i n the rat (Crowley et a l . ,  1976),  possibly by removing short-loop feedback i n h i b i t i o n of LH-RH release (see Kuhl and Taubert, 1975). In female g o l d f i s h , the results of steroid treatment of i n t a c t , r e gressed f i s h and of aminoglutethimide injections i n hypophysectomized, gonadotropin treated individuals indicate that steroids may be involved i n regulating spawning behaviour.  However, the f a i l u r e of steroid treatments  alone to restore r e c e p t i v i t y of hypophysectomized  f i s h suggests that, i n  contrast to the s i t u a t i o n i n rats and guppies, gonadotropin may play an  99  indispensable role i n the sexual behaviour of female goldfish.  Whether  this apparent requirement f o r gonadotropin i s of physiological s i g n i f i cance, or simply results from inappropriate steroid therapy applied to hypophysectomized C. 1.  f i s h , cannot be determined without further study.  The Role of Steroids Introduction This study provides the f i r s t demonstration that steroids stimulate  spawning behaviour i n an oviparous female f i s h *  In i n t a c t g o l d f i s h with  regressed, nonvitellogenic ovaries, a variety of steroids restores spawning behaviour i n response to i n j e c t i o n of ovulated eggs; hypophysectomy abolishes this effect of steroids on behaviour.  These findings  suggest that the p i t u i t a r y i s involved i n the effects of steroids i n intact f i s h , but do not c l a r i f y the r e l a t i v e contribution of steroid and p i t u i t a r y hormones to the regulation of spawning behaviour.  On the basis  of the results presented i n this study, i t i s not possible to determine whether the induction of spawning behaviour results from the combined action of p i t u i t a r y and ovarian hormones, or requires endocrine input from only one of these organs. In the following discussion, speculations concerning the r o l e of steroids i n goldfish spawning behaviour are based on the effects of steroids on reproductive physiology and behaviour of other female vertebrates.  Therefore, i t i s emphasized  at the outset that the relationship  between ovarian development and the onset of sexual r e c e p t i v i t y i n goldfish appears to be unique. The onset of estrous behaviour i n mammals has been attributed to  100  periovulatory fluctuations i n steroid hormones (Davidson and Levine, 1972); also i n the guppy, female sexual behaviour normally occurs when the ovary i s i n an advanced stage of development ( L i l e y , 1968).  In female g o l d f i s h ,  however, spawning behaviour may be induced i n the absence of endocrine stimuli associated with the l a t e r stages of ovarian development; f i s h with p a r t i a l l y regressed ovaries (containing minimal yolk v e s i c l e formation) perform normal spawning behaviour when injected with ovulated eggs.  It  could be argued that this finding i n g o l d f i s h i s analogous to the results of experiments i n female rats i n which high l e v e l s of sensory stimulation (manual palpation of flanks and perineum) induce lordosis responses i n the absence of or with low doses of estrogen (see discussion by Crowley et al_. , 1976).  However, the fact that normal sensory stimuli (presence of  ovulated eggs i n the genital t r a c t , courtship of male goldfish) induce spawning behaviour at any time during an extended period of r e c e p t i v i t y (when yolk v e s i c l e s or granules are present i n the oocytes) raises the p o s s i b i l i t y that, i f steroids are involved i n regulating spawning behaviour, the role of these hormones may be fundamentally d i f f e r e n t from the role they play i n reproductive behaviour of other female vertebrates. 2.  Effects of Steroids i n Intact Female Goldfish Two obvious explanations could account for the d i v e r s i t y of steroids  which induce spawning behaviour i n i n t a c t , regressed g o l d f i s h .  The mechanism  c o n t r o l l i n g ;receptivity may  Alternatively,  respond to a variety of steroids.  steroid conversion may occur i n regressed f i s h ;  some of the steroids shown  to restore responsiveness to egg i n j e c t i o n may be metabolized to one or more active forms which influence behaviour.  101  .There i s no published information to indicate whether mechanisms cont r o l l i n g female sexual behaviour i n other species of oviparous f i s h are sensitive only to certain steroids.  However, i n the viviparous female  guppy, only estrogens have been shown to induce r e c e p t i v i t y ( L i l e y , 1972). In this species, treatment with e s t r a d i o l , estrone, e s t r i o l , or d i e t h y l s t i l b o e s t r o l restores sexual behaviour while C o r t i s o l , corticosterone and progesterone are without e f f e c t ;  females treated with testosterone  exhibit male sexual behaviour. The results of mammalian studies indicate that, where conversion of androgens to estrogens can be ruled out, sexual behaviour i n females i s induced only by estrogens (progesterone i s also required i n some species to f a c i l i t a t e the priming effects of estrogen:,- Ciaccio and L i s k , 1967; Joslyn et a l . , 1971  ).  Dihydrotestosterone, which cannol be aromatized, f a i l s  to induce estrous behaviour i n female rabbits (Beyer e_t al_. , 1970) or to increase sexual motivation i n female rats (McDonald and Meyerson, 1973). On the other hand, a number of estrogens ( e s t r a d i o l , estrone, e s t r i o l ) are capable o-f'inducing r e c e p t i v i t y i n rats (Beyer et a l . , 1971) and guinea pigs (Feder and S i l v e r , 1974).  As e s t r i o l apparently i s not converted to  e s t r a d i o l (Ruh et a l . , 1973), i t appears that more than one naturally occurring estrogen may  d i r e c t l y influence sexual behaviour i n female mammals.  Sexual behaviour i n female mammals and i n the female guppy appear to be induced s p e c i f i c a l l y by estrogens.  Therefore i t i s u n l i k e l y that the  effectiveness of the wide variety of steroids ( e s t r a d i o l , androgens, 17*?; -OHr-pregnenolone,  and possibly pregnenolone) which restores responsive-  ness to egg i n j e c t i o n i n female goldfish i s due to a lack of s p e c i f i c i t y i n the response'to steroids. Rather, the effects of steroids i n goldfish are  102  more consistent with the concept that many of the exogenous steroids are converted i n vivo p r i o r to affecting In some mammalian studies (e.g.  behaviour. Beyer e_t al_. , 1970; Whalen et a l . ,  1972), the e f f e c t s of non-estrogen steroids on female sexual behaviour have been attributed to i n vivo conversion of the exogenous steroids to estrogens.  The capacity for extragonadal conversion of behaviourally  active exogenous steroids has been demonstrated i n several species (review by Ryan et a l . , 1972)  and much recent work has attempted to  correlate the metabolism of androgens i n central neural tissue with the effects of androgens and estrogens on sexual behaviour et^ al_. , 1975; N a f t o l i n and Ryan, 1975). and Clemens, 1976; Beyer et a l . , 1976)  (Perez-Palacios  Recent demonstrations (Christensen that i n h i b i t i o n of aromatizing  enzymes blocks the stimulatory effect of testosterone on sexual behaviour of male rats further support the concept that the e f f e c t s of some steroid treatments  on behaviour may be mediated by i n vivo steroid conversion.  Although  there i s no evidence that i n vivo steroid conversion occurred  under the conditions of my experiments, there i s evidence that the capacity for conversion i s present i n goldfish (Khoo, 1974, (Ozon, 1972).  1975)  and other teleosts  Of relevance i s a study by Colombo and Belvedere  (1976) who  examined ovarian steroid synthesis i n sexually immature Anguilla i n which the oocytes were about to commence v i t e l l o g e n e s i s .  In v i t r o incubation of  the p r e v i t e l l o g e n i c ovarian tissue with pregnenolone or  progesterone  yielded testosterone plus several intermediate metabolites.  If the  steroidogenic properties of the immature Anguilla ovary are similar to those of the temperature-regressed  g o l d f i s h , then the effects of pregnenolone and  103  17<x -OH-pregnenolone on spawning b e h a v i o u r c o u l d be e x p l a i n e d as the r e s u l t of c o n v e r s i o n o f these s t e r o i d s to androgens o r e s t r o g e n s . D i f f i c u l t i e s a r i s e when the concept o f i n v i v o c o n v e r s i o n i s used to i n t e r p r e t  the b e h a v i o u r a l e f f e c t s of androgens and e s t r a d i o l .  For  example, i f i t i s assumed t h a t e s t r a d i o l s t i m u l a t e s the mechanism cont r o l l i n g spawning b e h a v i o u r , then the e f f e c t s o f androstenedione  and  t e s t o s t e r o n e c o u l d be e x p l a i n e d as the r e s u l t o f a r o m a t i z a t i o n .  However,  if  the g o l d f i s h i s s i m i l a r to mammals i n b e i n g unable to aromatize 5  duced  steroids  (Thompson et^ al_. , 1971),  the e f f e c t s of androsterone  a  -re-  and  d i h y d r o t e s t o s t e r o n e c o u l d n o t be e x p l a i n e d as the r e s u l t o f c o n v e r s i o n o f t h e s e s t e r o i d s t o e s t r o g e n ( i t i s not known whether 1 1 - k e t o t e s t o s t e r o n e can be c o n v e r t e d t o e s t r o g e n ) .  On the o t h e r hand, i f i t i s assumed t h a t  androgens r e g u l a t e r e c e p t i v i t y i n the female g o l d f i s h , then the r e s u l t s of aridrogenstreatments c o u l d be i n t e r p r e t e d as evidence t h a t the mechanism c o n t r o l l i n g spawning b e h a v i o u r responds  to a v a r i e t y o f androgens;  and mammals (Beyer e t a l . ,  female guppies  ( L i l e y , 1972)  S i l v e r , 1974),  s e x u a l r e c e i p t i v i t y i s induced by more than one e s t r o g e n .  This l a t t e r i n t e r p r e t a t i o n f a i l s  to account  1971;  Feder  in and  f o r the b e h a v i o u r a l a c t i v i t y o f  estradiol. That e s t r o g e n i s l i k e l y  t o be d i r e c t l y i n v o l v e d i n s t i m u l a t i n g spawn-  i n g b e h a v i o u r i s suggested by the e f f e c t s o f e s t r o g e n on s e x u a l b e h a v i o u r of female guppies  ( L i l e y , 1972), Mzardsm,(.Crews, 1975), b i r d s  and many mammals (Young, 1961; Beach, 1964; However, androgen appears  (Noble,  Davidson and L e v i n e ,  to c o n t r o l r e c e p t i v i t y i n the female  monkey ( E v e r i t t and H e r b e r t , 1975),  1972),  1972).  rhesus  T h e r e f o r e , c o n s i d e r i n g b o t h the  104  behavioural effects of the androgens used i n this study and the fact that plasma androgens increase p r i o r to spawning i n female goldfish (Schreck and Hopwood, 1974) and i n other female teleosts (Schmidt and I d l e r ,  1962;  Schreck et a l . , 1972; Katz and Eckstein, 1974; Campbell et a l . , 1976), the p o s s i b i l i t y that androgens may  regulate spawning behaviour should not be  ignored. As female sexual behaviour i n the guppy and i n many mammals'is i n duced only by estrogens (and androgens which can be metabolized to estrogens) i t i s unlikely that both estrogens and non-aromatizable androgens act d i r e c t l y to stimulate r e c e p t i v i t y i n female g o l d f i s h .  A more reason-  able explanation of the effects of estrogens and androgens on spawning behaviour of i n t a c t , regressed goldfish i s that either estrogen or androgen (perhaps both) acts i n d i r e c t l y by stimulating gonadotropin release.  This  p o s s i b i l i t y i s considered i n the following section of the discussion. In summary, no single explanation i s l i k e l y to account for the diversi t y of steroids which induce responsiveness to egg i n j e c t i o n i n i n t a c t , regressed goldfish. converted i n vivo  Pregnenolone  and 17* -OH-pregnenolone probably are  to behaviourally active metabolites.  S i m i l a r l y , andros-  tenedione and testosterone may be metabolized p r i o r to a f f e c t i n g behaviour. However, the effectiveness of e s t r a d i o l , and of androsterone and dihydrotestosterone (which l i k e l y cannot be aromatized), indicates that both androgen and estrogen restore r e c e p t i v i t y i n intact f i s h . may  These steroids  act d i r e c t l y on the mechanism c o n t r o l l i n g spawning behaviour.  n a t i v e l y , androgen and / or estrogen may stimulating gonadotropin release.  Alter-  induce r e c e p t i v i t y i n d i r e c t l y by  105  3.  Possible Mechanisms of Steroid Action Exogenous steroids may  restore responsiveness to egg i n j e c t i o n i n -  d i r e c t l y , by stimulating gonadotropin secretion, or d i r e c t l y , by acting on central neural or peripheral structures c o n t r o l l i n g spawning behaviour. Implicit i n the hypothesis that steroids induce r e c e p t i v i t y by stimulating gonadotropin release i s the assumption  that i n gegressed, un-  receptive f i s h plasma gonadotropin i s absent, or present i n low trations.  concen-  Plasma gonadotropin has been measured i n preovulatory (Breton  et a l , , 1972) but not i n regressed g o l d f i s h ; however, Nagahama (1973) described signs of nuclear and cytoplasmic degeneration i n p i t u i t a r y gonadotrophs from female goldfish with regressed ovaries. m i r a b i l i s , which undergoes a temperature-induced  Also i n G i l l i c h t h y s  gonadal regression simi-  l a r to that of goldfish (De Vlaming, 1972), u l t r a s t r u c t u r a l examination of gonadotrophs indicates that secretory a c t i v i t y of these c e l l s i s depressed i n regressed f i s h (Zambrano, 1972).  In female brook trout and  sockeye  salmon with ovaries i n early stages of development, plasma gonadotropin levels are low or undetectable (Grim e_t al_. , 1975) . As p a r t i a l l y regressed goldfish with minimal yolk v e s i c l e deposition perform spawning behaviour when injected with ovulated eggs, low plasma gonadotropin t i t r e s may s u f f i c i e n t to induce sexual r e c e p t i v i t y .  be  Thus, i n j e c t i o n of steroids may  restore responsiveness i n regressed f i s h by stimulating only small increases i n gonadotropin release. In female rats, steroids have been shown both to i n h i b i t and to f a c i l i tate release of gonadotropin (Davidson; 1969; Everett, 1969).  Although  some of these effects are exerted at the l e v e l of the hypothalamus, estrogen (Cooper and McCann, 1975) and testosterone (Perez-Palacios ejt a l . , 1976)  106  also increase the response of the p i t u i t a r y to l u t e i n i z i n g hormone-releasing hormone.  Steroids may  act on the teleost hypothalamus and p i t u i t a r y  to regulate gonadotropin secretion; i n the male sunfish (Lepomis cyanellus), P f a f f e_t al_. , (unpublished r e s u l t s c i t e d i n Morrell et. a l . , 1975) strated retention of l a b e l l e d testosterone  demon-  i n the anterior p i t u i t a r y and  nucleus l a t e r a l i s tuberis (an infundibular region suggested to regulate gonadotropin secretion i n goldfish [Peter, 1970]  ).  Generally, exogenous steroids exert i n h i b i t o r y e f f e c t s on the ovaries of i n t a c t f i s h (see review by Pickford and Atz, 1957). Heteropneustes f o s s i l i s , e s t r a d i o l or testosterone  In the c a t f i s h ,  treatments reduce both  the s i z e and number of p i t u i t a r y basophils, suggesting that ovarian a t r e s i a following administration of these steroids results from i n h i b i t i o n of gonadotropin secretion (Sundararaj and Goswami, 1968).  Similar i n d i r e c t e v i -  dence indicates that exogenous steroids i n h i b i t gonadotropin secretion i n female g o l d f i s h ; i n j e c t i o n of estrogens or testosterone v i t e l l o g e n i c oocytes (Khoo, 1974)  induces a t r e s i a of  and estrogen treatment produces degener-  ative changes i n gonadotrophs (Nagahama, 1973).  The r e s u l t s of these  studies suggest that, i n f i s h with maturing ovaries, estrogen and  testos-  terone lower gonadotropin output to l e v e l s i n s u f f i c i e n t f o r maintenance of yolky oocytes; however, these findings do not eliminate t h e t p o s s i b i l i t y that i n regressed tropin release.  f i s h these steroids may  stimulate a low rate of gonado-  Thus whether such a mechanism can account for the e f f e c t s  of steroids on spawning behaviour of regressed  f i s h remains an open question.  As noted above, i f steroids are d i r e c t l y involved i n the regulation of spawning behaviour, they may  act on central neural structures or may  affect  the responsiveness of the genital tract to the presence of ovulated eggs.  107  Estrogen stimulates sexual behaviour i n female mammals by actions on s p e c i f i c areas of the brain, i n p a r t i c u l a r the region of the preoptic nucleus - anterior hypothalamus.  There i s evidence that the preoptic  area i s involved i n the control of reproductive behaviour i n t e l e o s t s . E l e c t r i c a l stimulation of the preoptic region evokes courtship behaviour i n male b l u e g i l l sunfish, Lepomis macrochirus (Demski and Knigge, 1971), r e lease of milt i n male sunfish and g o l d f i s h , and release of eggs i n ovulated female goldfish (Demski et a l . , 1975). Furthermore, e l e c t r o l y t i c lesions of the preoptic area i n Fundulus  abolish the spawning r e f l e x response to  i n j e c t i o n of neurohypophyseal hormones (Macey et a l . , 1974). There i s no evidence i n teleosts that steroids are involved i n the function of the preoptic area or of other brain areas which may Although P f a f f et a l . (unpublished  regulate reproductive behaviour.  results cited i n Morrell et al_. 1975)  demonstrated binding of testosterone i n the nucleus l a t e r a l i s tuberis of the male sunfish, Lepomis cyanellus, the functional s i g n i f i c a n c e of this finding i s not known. Steroids may tract.  influence spawning behaviour by an action on the g e n i t a l  In the female r a t , physical stimulation of the p e r i g e n i t a l area  plays a role i n sexual behaviour (Pfaff et a l . , 1973;  Kow  and estrogen increases the s e n s i t i v i t y of this region (Kow S i m i l a r l y , steroids may  1976)  and P f a f f , 1973-4).  a f f e c t spawning behaviour i n g o l d f i s h by s e n s i t i z -  ing the oviduct to the stimulus provided by ovulated eggs. follow  and P f a f f ,  In discussion to  i t i s suggested that ovulated oocytes are transported  to the  ovipore  by c i l i a r y action and that i t i s the portion of the oviduct near the  ovipore  which i s sensitive to ovulated eggs. c i l i a r y ova transport;  Steroids may  regulate this proposed  treatment of g o l d f i s h f r y with e t h i n y l e s t r a d i o l or  108  methyltestosterone induces hypertrophy and extensive c i l i a t i o n of o v i duct epithelium, processes which are normally associated with v i t e l l o g e n e s i s (Takahashi and Takano, 1971).  In female mammals, the effects of e s t r a d i o l  on p r o l i f e r a t i o n of c i l i a i n the genital tract are w e l l known (More and Masterton, 1976). In summary, evidence that exogenous steroids i n h i b i t gonadotropin secretion i n goldfish and other teleosts suggests that steroids stimulate spawning behaviour i n i n t a c t , regressed female goldfish by an action on the genital tract or on the central nervous system.  Steroids may influence  spawning behaviour solely through an action on the genital t r a c t , a c t i v a t ing a mechanism which detects ovulation and relays this information to the central nervous system.  I t i s suggested that i f steroids influence  spawning behaviour by an action on the central nervous system, the mechanism involved may be d i f f e r e n t from that i n female mammals.  For example, i n  female mammals, elevated plasma estrogen l e v e l s induce a b r i e f period of r e c e p t i v i t y near the time of ovulation; endocrine stimuli associated with ovulation are not necessary to induce r e c e p t i v i t y i n g o l d f i s h , as females are  responsive to i n j e c t i o n of ovulated eggs at any time during v i t e l l o -  genesis.  Thus, steroids i n mammals function as chemical messengers from  the ovaries to the brain, s i g n a l l i n g that the ovaries are prepared f o r ovulation.  In female g o l d f i s h , stimuli generated by intraovarian ovulated  eggs synchronize spawning behaviour with ovulation; steroids ( i f they are involved) appear only to prime the mechanism regulating r e c e p t i v i t y , and not to transmit s p e c i f i c information regarding the state of ovarian development. D.  The Role of Ovulated Eggs. Spawning behaviour of female goldfish i s temporally linked with ovulation  J  109  by the stimulus of ovulated eggs i n the genital t r a c t .  The a b i l i t y of  ovulated eggs to induce spawning behaviour i s not r e s t r i c t e d to the day of ovulation but i s seen i n a l l f i s h with ovaries i n any stage of v i t e l l o genesis.  As discussed i n d e t a i l above, both p i t u i t a r y and ovarian hormones  may a f f e c t responsiveness  to ovulated eggs.  The s i t e of action of ovulated eggs i n inducing spawning behaviour i s not known; however, several observations suggest that the terminal portion of the oviduct may be involved.  For example, whereas stripping of a l l  e a s i l y removed eggs (believed to be those i n the oviduct and posterior ovisac) usually terminates spawning f o r 10 to 15 minutes, removing only a portion of these eggs, and thus leaving additional eggs at the ovipore, has l i t t l e e f f e c t on spawning behaviour.  I t was also observed  that i n f e -  males from which a l l accessible eggs had been removed, resumption of spawning generally was r e s t r i c t e d to individuals which, on further s t r i p p i n g , were found to have eggs at the ovipore. spawning behaviour  Furthermore, i n some cases where  following egg i n j e c t i o n i s inhibited by egg binding  (hardening and adhesion of eggs i n contact with water),, only a small number of eggs at the ovipore are bound and the remainder of the injected eggs i n the oviduct and ovisacs are apparently normal. It i s not known how ovulated eggs are transported to the ovipore. Eggs may be moved by the a c t i v i t y of smooth muscles or, more l i k e l y , by coordinated c i l i a r y motion as has been suggested  to occur i n ovum transport i n  the rabbit oviduct (Halbert et a l . , 1976) and i n the frog coelom (Suvarnalatha and Sarkar, 1972).  In goldfish, both the ovarian lamellae  and the ovisacs possess conspicuously c i l i a t e d epithelium. The e f f e c t of ovulated eggs on behaviour  i s thought to be mediated at  110  least p a r t i a l l y by physical cues, as substitutes for eggs ( g e l a t i n , petroleum j e l l y ) a r e marginally e f f e c t i v e .  The decreased a b i l i t y of bound  eggs to induce spawning may be due either to inappropriate physical c h a r a c t e r i s t i c s or to l o c a l i z e d 16ss of, aachemisal - stimuMnt. There-is^evidence that stimulation of the genital tract influences female sexual behaviour not only i n teleosts but also i n other vertebrate classes.  The close temporal correlation between ovulation and spawning  ( L i l e y , 1969) suggests that a mechanism whereby ovulated eggs stimulates spawning behaviour may be widespread i n this group.  In previous studies  of oviparous f i s h (see review by L i l e y , 1969),failure to induce female sexual behaviour by steroid therapy may have been due to the absence of the requisite stimuli from i n t e r n a l sexual structures. ovulated eggs may Anura.  Stimuli from  function i n the reproductive behaviour of some female  Intraperitoneal saline injections (10-30 ml) induce o v i p o s i t i o n  behaviour i n preovulatory and recently spent Rana pipiens (Noble and Aronson, 1942) i n d i c a t i n g that abdominal distension may s i m i l a r to oviduct distension i n g o l d f i s h .  serve a function  Noble and Aronson (1942) suggest  that, as saline i n j e c t i o n f a i l s to induce oviposition behaviour i n females treated more than thirteen days after ovulation, the behavioural response to abdominal distension may be influenced by hormones.  Although physical s t i -  mulation of the vagina and cervix f a c i l i t a t e s lordosis responses i n the female rat (Rodriguez-Sierra e_t a l . , 1975), this phenomenon d i f f e r s i n several aspects from the effects of ovulated eggs on spawning behaviour i n goldfish.  For example, the f a c i l i t a t o r y effect of c e r v i c a l probing occurs  within a minute, p e r s i s t s for several hours after withdrawal of the stimul u s , and i s neither f a c i l i t a t e d by estrogen treatment nor reduced by hypophysectomy.  Ill  E.  The Role of Prostaglandins Prostaglandins  of female g o l d f i s h .  (PGs) appear to play a role i n the spawning behaviour The results of the present  study suggest that stimu-  l a t i o n of the oviduct following ovulation 'or i n j e c t i o n of ovulated eggs or egg substitutes causes the release of PGs (most l i k e l y PGF2 ••) which a  then act d i r e c t l y or i n d i r e c t l y to induce spawning behaviour. The  fact that indomethacin (an i n h i b i t o r of PG synthesis)  eliminates  a l l spawning within minutes suggests that endogenous PG i s u t i l i z e d rapidly and that spawning behaviour following ovulation o-t egg i n j e c t i o n results from PG released over extended periods.  The often considerable duration of  spawning following i . p . PG i n j e c t i o n indicates a slow uptake from the peritoneal cavity.  The v a r i a b i l i t y i n the latency to the onset of the  behavioural response probably r e f l e c t s the imprecision of this route of administration.  The fact that some f i s h begin to spawn within a few minutes  of receiving PG i n j e c t i o n suggests that the latent period  preceding  spawning behaviour induced by egg i n j e c t i o n may be the time required f o r the stimulus of eggs i n the oviduct to elevate PG to e f f e c t i v e l e v e l s . The capacity for PG biosynthesis has been demonstrated i n several tissues of the carp  (Christ and Van Dorp, 1972) and PGE and PGF have been  i s o l a t e d from the testes of several teleost species (Nomura et al_., 1973) . The source of endogenous PG which induces spawning behaviour i n goldfish i s not known.  PG may be released from the oviduct following physical stim-  ulation, as has been shown to occur i n mammalian u t e r i (Poyser et a l . , 1971), or i t may be released i n the brain i n response to afferent signals generated i n the oviduct. It i s also possible that PG i s released by ovarian macrophages i n  112  response to egg i n j e c t i o n .  Macrophages have been implicated i n the action  of IUDs (Myatt et a l . , 1975) 1975)  and i t has been suggested  (Higgs and Youlten,  that PG production by leukocytes might regulate the emigration of  leukocytes from blood vessels during acute inflammation. mechanisms were activated  If similar  egg i n j e c t i o n i n goldfish, the latency from  egg i n j e c t i o n to onset of spawning might represent the time required for s u f f i c i e n t numbers of c e l l s to aggregate at the injected eggs.  Macrophage  aggregations i n the v i c i n i t y of injected eggs are observed frequently i n h i s t o l o g i c a l preparations (Fig.29 i n the Appendix).  The fact that there ,!  i s much evidence that the number and phagocytic a c t i v i t i e s of macrophages are greatly increased by estrogen and fluctuate with the female reproductive cycle (Vernon-Roberts,  1969)  suggests that the p o s s i b i l i t y of macro-  phage involvement i n prostaglandin production and spawning behaviour merits investigation. Recent unpublisheddstudies of female spawning behaviour i n two other oviparous teleosts, (Jordanella floridae (Crawford, 1975) and Gasterosteus (Lam, Chan and Pandrey,1976) , support the present findings that PGs are i n volved i n oviposition behaviour.  Indomethacin i n j e c t i o n abolished the re-  sponses of ovulated female sticklebacks; following PGF^  i n j e c t i o n , these  responses were at least p a r t i a l l y restored and i n some cases oviposition occurred.  Though i n j e c t i o n of PGF„  into female Jordanella f a i l e d to i n -  duce oviposition or even behavioural coordination with courting males, spawning r e f l e x responses s i m i l a r to those seen i n the spawning behaviour of this and related cyprinodonts were usually e l i c i t e d . Both i n Fundulus (Pickford, 1952; Wilhelmi e_t a l . , 1955)and i n female Jordanella (Crawford, 1975), injections of neurohypophyseal  hormones induce  113  a spawning r e f l e x response s i m i l a r to that observed i n female Jordanella following administration of PGF„  .  In contrast, treatment with neuro-  hypophyseal hormones f a i l s to stimulate spawning behaviour i n goldfish (present study; Pickford, unpublished results cited i n Macey et a l . , 1974) and i n several other teleosts (see Macey e_t a l . , 1974). the e a r l i e r work on Fundulus (Pickford, 1952; which demonstrated that pharmacologically  On the basis of  Wilhelmi e_t a l . , 1955),  high doses of neurohypophyseal  hormones (injected intraperitoneally) are required to induce the spawning r e f l e x response and that these hormone treatments are equally e f f e c t i v e i n intact and gonadectomized f i s h , Macey et_ al_. (1974) proposed that neurohypophyseal hormones may  act on some brain centre to e l i c i t the  response; the finding (Macey et _al., 1974)  behavioural  that e l e c t r o l y t i c lesions of the  nucleus preopticus impaired or abolished the spawning r e f l e x response to hormone i n j e c t i o n provided  support for this hypothesis.  However, i t has  recently been shown (Peter and Knight, unpublished r e s u l t s cited i n Peter, 1976)  that, even when neurohypophyseal hormones are administered  by i n t r a -  v e n t r i c u l a r i n j e c t i o n , the dosage required to e l i c i t the spawning r e f l e x response i s s i m i l a r to that required i n t r a p e r i t o n e a l l y . Peter (1976) i n terprets these r e s u l t s as evidence of a peripheral rather than a central effect of these hormones and further suggests that a peripheral action of 'neurohypophyseal hormones i s probably not a part of the normal mechanism for triggering spawning behaviour i n t e l e o s t s ' . The response of Fundulus to neurohypophyseal hormones i s s i m i l a r to that of female Jordanella injected with either oxytocin of YGF^^ neither species do treated f i s h show behavioural opposite  sex.  ; in  coordination with the  Thus i t i s questionable whether the behavioural  e f f e c t of  114  PGF2  AC  i n female Jordanella i s p h y s i o l o g i c a l .  Furthermore, i t i s stressed  that the e f f e c t of neurohypophyseal hormones i n Eundulus, where both gonadectomized  (Wilhelmi e_t a l . , 1955) and hypophysectomized  fish  (Pickford, 1952) show a spawning response without coordination of the sexes, contrasts sharply with the a b i l i t y of "PGF^^  t  o  induce a l l aspects of  spawning behaviour i n female g o l d f i s h , but only i n the normal  spawning  environment ( i n the presence of males and aquatic vegetation) and under p i t u i t a r y stimulation. The mode of action of PG i n inducing spawning behaviour i n female goldfish i s not known. H a l l et a l . (1975)showed that i n j e c t i o n of  PGE2  into the t h i r d v e n t r i c l e of ovariectomized, estrogen-primed female rats induced r e c e p t i v i t y similar to that following treatment with e s t r a d i o l and progesterone.  As LH-RH, shown to induce or increase lordosis i n hypo-  physectomized, estradiol-primed female rats (Pfaff, 1973), may be released under the stimulation of PGE  2  (Eskay et a l . , 1975; Ojeda et a l . , 1975),  H a l l e_t al_. suggest that the mechanism by which  PGE2  increases sexual be-  haviour i n female rats may involve the release of LH-RH.  Hypothalamic  implants of LH-RH have been shown by Dyer and Dyball (1974) to affect e l e c t r i c a l a c t i v i t y of hypothalamic neurons, and these workers stress the p o s s i b i l i t y that i f this hypothalamic peptide functions both as a neurotransmitter and as a releasing factor, a single neuron or group of neurons could influence both behaviour and p i t u i t a r y function. that the goldfish hypothalamus  There i s evidence  contains the teleost equivalent to mammalian  LH-RH (Peter, 1973; Crim et a l . , 1976); release of this hypothalamic factor may be involved i n the action of PG on goldfish spawning behaviour. In the only report of an e f f e c t of PG on the sexual behaviour of male  115  mammals, twice-daily  injections of PGF^  la:  (PGE^ was without effect) i n -  duced at.70% increase i n mean number of ejaculations 9 days of treatment (Agmo, 1975).  of male rabbits  after  The long latency of t h i s response suggests  a less direct effect on behaviour than i s l i k e l y to occur i n the female goldfish. PG (and, i n d i r e c t l y , ovulation and egg injection) may exert some effect on ovarian or oviductal afferent stimulation.  smooth muscle which i s monitored centrally through Portions of the female goldfish reproductive tract  may be s i m i l a r to mammalian u t e r i which, as well as responding to PG (Aiken, 1974), w i l l also release PG when physically stimulated by stretch (Poyser et a l , , 1971) or by i n s e r t i o n of a foreign object (Spilman and Duby, 1972) . The finding that PGP 2 * * stimulating  s  c o n s  W e r a b l y more e f f e c t i v e than PGE^^.. \ • ±r  spawning behaviour may be due to d i f f e r e n t i a l a c t i v i t y at a  single s i t e of action.  A l t e r n a t i v e l y , VGF^  s i t e s , the lower potency of PGE  2  a n  ^ P 2cx y GE  ma  a  c  t  a t  different  r e s u l t i n g from i n s u f f i c i e n t concentration  of t h i s prostaglandin i n i t s target  tissue following  intraperitoneal i n -  jection. The effects of PG i n j e c t i o n on hypophysectomized  female goldfish which  have been treated with salmon gonadotropin or steroids p a r a l l e l the effect of egg i n j e c t i o n on f i s h receiving similar treatment.  Salmon gonadotropin  replacement therapy restores the spawning response to i n j e c t i o n of either eggs or PG, while steroid treatments are without e f f e c t . inducessspawning behaviour by stimulating  I f egg i n j e c t i o n  PG synthesis and release,  the f a i l u r e of egg i n j e c t i o n to evoke a response i n hypophysectomized may simply r e f l e c t t h e t i n a b i l i t y of hypophysectomized  then fish  f i s h to respond to PG.  116  A l t e r n a t i v e l y , p i t u i t a r y removal may  disrupt many steps i n a chain of  events leading from the distension of the oviduct, by ovulation or egg i n j e c t i o n , to the onset of spawning behaviour. F.  The Regulation of Spawning Behaviour In the foregoing discussion ,' more than one mechanism has been suggested 1  to explain the function of each of the endogenous components which i n f l u ence spawning behaviour.  In the present  section, the control of spawning  behaviour i s described by a model ( F i g . l ) based on what i s believed to be the most l i k e l y set of mechanisms involved. I m p l i c i t i n the construction of the proposed model i s the assumption that there are fundamental simil a r i t i e s i n the endocrine control of sexual behaviour i n female g o l d f i s h and mammals; without this constraint, the regulation of spawning behaviour could be described by other models consistent with the data presented i n this study. As indicated i n Figure 1, gonadotropin stimulates oocyte~development and synthesis of steroids which both maintain and s e n s i t i z e the oviduct and prime hypothalamic (and/or preoptic) areas c o n t r o l l i n g spawning behaviour. Following ovulation, signals generated by stimulation of the oviduct induce changes i n the steroid-primed  hypothalamus; this mechanism may  involve pro-  staglandin (PG) at the l e v e l of the oviduct and the hypothalamus.  Spawn-  ing occurs when s p e c i f i c external cues (sexually active male; substrate for egg deposition) are  present.  The model attributes the lack of effect of steroids i n hypophysectomized f i s h to the f a i l u r e of exogenous steroids to duplicate the action of endogenous s t e r o i d production and predicts that, i n the absence of gonadotropin,  117  Figure 1.  Model of the regulation of spawning behaviour i n female goldfish (a) gonadotropin stimulation of oocyte growth and steroidogenesis (b) steroid stimulation of ovisac and oviduct (c) steroid action on s e n s i t i v i t y of oviduct (d) steroid priming of spawning centre GRH -  gonadotropin releasing hormone, OE -  ovulated egg, P - p i t u i t a r y , PG - prostaglandin, SC - spawning centre  118  external stimuli  gonadotropi n  M O D E L OF THE REGULATION OF S P A W N I N G IN THE FEMALE G O L D F I S H  BEHAVIOUR  119  some exogenous steroid treatment of hypophysectomized  f i s h should be cap-  able of restoring responsiveness to Injection of eggs or PG.  This model  also attributes the behavioural action of gonadotropin s o l e l y to an e f f e c t on steroidogenesis.  I f i t could be shown that the a b i l i t y of PG to induce  spawning behaviour p e r s i s t s a f t e r ovariectomy, then PG treatment of gonadectomized f i s h could provide valuable information regarding the roles of gonadotropin and steroids on spawning behaviour. The model proposes that PG acts both on the oviduct and the hypothalamus  to stimulate spawning behaviour.  I n h i b i t i o n of PG^-induced spawning  ,by s u r g i c a l removal of the oviduct would provide evidence that PG acts at this peripheral s i t e .  Both induction of spawning by i n t r a v e n t r i c u l a r i n -  j e c t i o n of PG and i n h i b i t i o n of egg injections-induced spawning by i n t r a v e n t r i c u l a r i n j e c t i o n of indomethacin would provide support for a central action of PG on spawning behaviour. As indicated i n the proposed model, gonadotropin releasing hormone (GRH) i s suggested to play a role i n spawning behaviour; stimulation of the oviduct following ovulation generates afferent input to the hypothalamus,  inducing the release of GRH which then acts on the central neural  centre(s) c o n t r o l l i n g spawning behaviour.  The speculation that GRH i s i n -  volved i n spawning behaviour i s based mainly on the results of mammalian studies.  In the r a t , vaginal stimulation induces changes i n neuronal a c t i v -  i t y i n brain centres c o n t r o l l i n g sex behaviour and LH-RH release (Blake and Sawyer, 1972), changes i n hypothalamic LH-RH content (Takahashi et a l . , 1975), and persistent l o r d o s i s to manual stimulation (Rodriguez-Sierra et a l . , 1975); there i s much evidence that mechanisms by which genital s t i m u l i induce gonadotropin (and presumably LHr-RH) release are widespread  120  i n mammals (reviews by Jochle, 1973, 1975).  Furthermore, LH-RH has  been shown to induce lordosis i n female rats (Pfaff, 1973; Moss and Foreman, 1976) and the effect of  on lordosis has been  suggested  (Hall et a l . , 1975) to be due to release of LH-RH (see Eskay et a l . . 1975,  and Ojeda et a l . , 1975, for effects of PG on LH-RH release).  These findings, which indicate that genital stimuli may enhance sexual behaviour i n female mammals by releasing LH-RH, raise the p o s s i b i l i t y that ovulated eggs may induce spawning behaviour by a similar mechanism. There i s no published information on the effect of GRH on spawning behaviour of teleosts.  However, the findings that plasma  gonadotropin  t i t r e s are greatly elevated i n ovulated females of several salmonid species and i n ovulating and p a r t i a l l y spawned female sockeye salmon (Crim ejt al_., 1975) suggest not only that GRH release may increase during ovulation but also that high rates of release may p e r s i s t i n ovulated f i s h , perhaps due to the stimulus provided by ovulated eggs. of plasma gonadotropin  In goldfish, l e v e l s  are high on the day of ovulation and return to pre-  ovulatory values the following day (Breton et_ al_., 1972). ence of ovulated eggs stimulates GRH and gonadotropin  I f the pres-  release i n goldfish,  the r e l a t i v e l y transient increase i n plasma gonadotropin  observed by  Breton et a l . (1972) may have resulted from a decrease i n stimulus quality of the ovulated eggs (my observations show that i n most cases where ovulated females are not placed with males'until the day a f t e r ovulation, at least s l i g h t egg binding occurs and few or no spawning acts are performed). The hypothesis that GRH plays a role i n spawning behaviour tested i n d i r e c t l y by determining changes i n plasma gonadotropin following i n j e c t i o n of ovulated eggs.  could be levels  Behavioural observation following  121  i n t r a v e n t r i c u l a r i n j e c t i o n of mammalian LH-RH would provide a more d i r e c t approach; t h i s technique has been used i n examining the effects of neurohypophyseal hormones on the spawning r e f l e x response of Fundulus (Peter, 1976).  However, as there i s evidence that mammalian LH-RH and  teleost GRH may be d i f f e r e n t (Deery, 1974), f a i l u r e to induce spawning behaviour with mammalian LH-RH would not eliminate the p o s s i b i l i t y that endogenous GRH i s involved. G.  A Comparative Approach to the Study of Female Sexual Behaviour In the preceding discussion, which considered the regulation of  spawning behaviour i n terms of mechanisms proposed to regulate sexual behaviour i n female mammals, i t has been assumed that s i m i l a r i t i e s i n the endocrine control of reproduction i n mammals and teleosts may be paral l e l e d by s i m i l a r i t i e s i n the endocrine control of reproductive  behaviour.  Although this approach has been useful i n i n t e r p r e t i n g experimental r e s u l t s , i t affords only a limited view of the relationship between mechanisms c o n t r o l l i n g reproductive behaviour i n goldfish and mammals.  A more  comprehensive view of this r e l a t i o n s h i p may r e s u l t from considering these behaviours  i n an evolutionary context.  In the course of vertebrate evolution, reproductive strategies have changed greatly, the presumed ancestral mode of reproduction, external f e r t i l i z a t i o n , giving r i s e to i n t e r n a l f e r t i l i z a t i o n associated with o v i p a r i t y , v i v i p a r i t y , etc.  Regardless  of the strategy employed, however,  the formation and fate of the oocytes involves a s i m i l a r series of stages: pituitary-stimulated development, ovulation, passage of ova or embryos through the coelomic cavity (gymnovarian teleosts) or reproductive tract (cystovarian t e l e o s t s , mammals) where they may be held for varying periods,  122  expulsion of ova or foetuses.  What has changed d r a s t i c a l l y i s the stage  at which the male intervenes and sexual behaviour occurs.  For example, i n  female goldfish and frogs, where f e r t i l i z a t i o n i s external, sexual behaviour and the release of ova are synchronous.  In oviparous l i z a r d s  and b i r d s , and i n viviparous guppies and mammals, the advent of i n t e r n a l f e r t i l i z a t i o n has been accompanied by an advancement i n the timing of sexual behaviour i n r e l a t i o n to oviposition and p a r t u r i t i o n . Thus, as the role of the female i n reproductive behaviour changed from donator of ova  (external f e r t i l i z a t i o n ) to recipient of sperm ( i n t e r n a l f e r t i l i z -  ation) , the occurrence of sexual behaviour became temporally from the expulsion of sexual products;  dissociated  i n contrast, the persistent role  of the male as gamete donator necessitated a close temporal association between sexual behaviour and gamete release i n a l l male vertebrates. It i s proposed that i n the ancestral, externally f e r t i l i z i n g female vertebrate  ( i . e . goldfish) a single mechanism, primed by endocrine factors  associated with ovarian development and activated by physical stimulation of the genital tract following ovulation, performed the dual function of regulating sexual behaviour and releasing gametes.  As the timing of  sexual behaviour s h i f t e d with the evolution of i n t e r n a l f e r t i l i z a t i o n , the mechanism c o n t r o l l i n g female sexual behaviour came to be influenced by hormonal factors associated with e a r l i e r stages i n oocyte development. For example, s t i m u l i from ovulated eggs are required to induce spawning behaviour i n female g o l d f i s h , whereas estrogen alone i s s u f f i c i e n t to stimulate r e c e p t i v i t y i n female mammals i n which sexual behaviour precedes ovulation.  However, as stimulation of the genital tract affects sexual  behaviour i n female mammals, the a b i l i t y of female mammals to respond to  123  s t e r o i d s alone may be due to e l a b o r a t i o n of a b a s i c mechanism r e g u l a t i n g s e x u a l b e h a v i o u r i n a l l female  vertebrates.  These h i g h l y s p e c u l a t i v e p r o p o s a l s , plex processes,  which g r e a t l y s i m p l i f y com-  are o f f e r e d i n the hope of p r o v i d i n g a  conceptual  b a s i s f o r comparing the r e g u l a t i o n of sexual b e h a v i o u r and r e l a t e d phenomena i n female  vertebrates.  124  BIBLIOGRAPHY  125  Agmo, A. 1975. E f f e c t s of prostaglandin E^ and F on serum l u t e i n i z i n g hormone concentration and on some sexual functions i n male rabbits. Prostaglandins, 9_: 451-457. Aiken, J.W. 1974. Prostaglandins and prostaglandins synthetase i n h i b i tors: studies on uterine m o t i l i t y ^ function. 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The r e p r o d u c t i v e p h y s i o l o g y o f the v i v i p a r o u s Cymatogaister aggregata CGihbb'ns. Ph.D. T h e s i s , U.B.C.  seaperch,  136 Wilhelmi, A.E., G.E. Pickford, and W.H. Sawyer. 1955. I n i t i a t i o n of the spawning r e f l e x responses i n Fundulus by the administration off i s h and mammalian neurohypophyseal preparations and synthetic oxytocin. Endocrinology, 57: 243-252. Yamamoto, K., Y. Nagahama, and F. Yamazaki. 1966. A method to; induce a r t i f i c i a l spawning of goldfish a l l through the year. B u l l . Jap. Soc. S c i . F i s h e r i e s , 32: 977-983. Yamazaki, F. 1961. The e f f e c t s of hypophysectomy on the ovary of the g o l d f i s h , Carassius auratus, B u l l . Fac. F i s h . Hokkaido Univ., 12: 167-180. Yamazaki, F. 1965. Endocrinological studies on the reproduction of the female g o l d f i s h , Carassius auratus L., with s p e c i a l reference to the function of the p i t u i t a r y gland :. Mem. Faculty of F i s h e r i e s , Hokkaido University, 13: 1-64. r  Yamazaki, F., and E.M. Donaldson. 1968. The effects of p a r t i a l l y p u r i f i e d salmon p i t u i t a r y gonadotropin on spermatogenesis, v i t e l l o g e n e s i s , and ovulation i n hypophysectomized goldfish (Carassius auratus). General and Comparative Endocrinology, 11: 292-299. Young, W.C. 1961. The hormones and mating behaviour.ialn: Sex and Internal Secretions. W.C. Young (Ed.). 3rd E d i t i o n , Vol. 2, pp. 1173-1239. Williams and Wilkins, Baltimore. Zamb.rano;} j D. 1971. The nucleus latercalis tuberis system of the gofrlid f i s h G i l l i c h t h y s m i r a b i l i s - I I I . Functional modifications of the neurons and gonadotropic c e l l s . General and Comparative Endocrinology, 17_: 168-182.  137  APPENDIX  138  SOME ASPECTS OF THE  In t h i s study,  HISTOLOGY OF REGRESSED OVARIES  the o v a r i a n h i s t o l o g y of e x p e r i m e n t a l  f i s h has  been  used both i n a s s e s s i n g the e f f e c t s of v a r i o u s i n j e c t e d hormones and determining  the completeness of hypophysectomy and  The  o f s e l e c t i n g o v a r i a n c h a r a c t e r i s t i c s which c o u l d  process  l a t e d w i t h v a r i o u s endocrine  ovarian regression. be  s t a t e s r e v e a l e d s e v e r a l a s p e c t s of  h i s t o l o g y which a p p a r e n t l y have not been r e p o r t e d p r e v i o u s l y . t i o n of p r e v i t e l l o g e n i c ( f i r s t  growth phase) oocytes was  e f f e c t s of s t e r o i d s and e a r l y y o l k v e s i c l e stage As was  a l s o are  not d i s c o v e r e d u n t i l completion  ation.  concerning  the  discussed.  p r e v i t e l l o g e n i c oocytes  of a l l experiments, no  (DPVOs)  special  were employed i n p r e p a r i n g o v a r i e s f o r h i s t o l o g i c a l  examin-  A l l o v a r i e s w i t h DPVOs were r o u t i n e l y f i x e d i n Bouin's F l u i d  embedded ( P a r a p l a s t ) .  h a e m a t o x y l i n and 1962), or PAS As  of degener-  C y t o l o g i c a l f e a t u r e s of p r e a t r e t i c  the e x i s t e n c e of d e g e n e r a t i n g  techniques  wax  oocytes  ovarian  observed many  some i n f o r m a t i o n i s p r o v i d e d  gonadotropin.  corre-  Degenera-  times i n i n t a c t and hypophysectomized f i s h ; a s e r i e s of stages a t i o n i s d e s c r i b e d below and  in  and  B l o c k s were cut at 5 u and  e o s i n , M a l l o r y trichrome light  connective  o v a r i a n h i s t o l o g y was  not  (Gurr,  of the ovary and  as  the  undertaken.  i n hypophysectomized f i s h ,  h i g h e r i n the l a t t e r  tissue stain  s t a n d a r d i z e d , no q u a n t i t a t i v e assessment of  DPVOs were found i n the o v a r i e s o f i n t a c t , females and  stained with e i t h e r  green.  t i s s u e s were sampled from v a r i o u s areas  c u t t i n g o r i e n t a t i o n was  and  temperature-regressed  though the i n c i d e n c e was  much  group.  In the e a r l y stages  of a t r e s i a  ( F i g . 2 ) , DPVOs were v e r y  difficult  139 to distinguish from degenerating early yolk v e s i c l e stage oocytes (DEYVOs).  However, as a t r e s i a progresses, the f o l l i c l e s of the DEYVOs  develop into corpora a t r e t i c a , passing through stages  1  to { as described  by Khoo (1975) f o r more advanced oocytes: i . e . the f o l l i c u l a r layer hypertrophies, the oocyte contents are taken up by the granulosa and invading macrophages (Figs. 3 and 4), the hypertrophied f o l l i c l e f i r s t surrounds an empty cavity (Figs. 4 and 5), and f i n a l l y collapses into an i r r e g u l a r mass of c e l l s .  On the other hand, DPVOs do not form corpora  a t r e t i c a , there i s very l i t t l e or no f o l l i c u l a r hypertrophy, and although macrophages invade and remove the contents of the oocyte, there i s no collapse of the f o l l i c l e , which eventually surrounds a space approximately the size of the evacuated oocyte (Fig. 6). It i s d i f f i c u l t to be certain that a l l small degenerating f o l l i c l e s that form corpora a t r e t i c a also contain yolk v e s i c l e s , as the amount of yolk v e s i c l e material may be very small (one vesicle/5y section). ing with PAS and l i g h t  Stain-  green has proven e f f e c t i v e i n detecting small  amounts of yolk v e s i c l e s , but this method i s complicated by the fact that the yolk nucleus (an accumulation of mitochrondria) not only i s taken up by the hypertrophied f o l l i c l e c e l l s  (as are the yolk v e s i c l e s ) , but, as  with the yolk v e s i c l e s , i s strongly PAS-positive (Fig. 7). The f i r s t recognizable stage of a t r e s i a i n p r e v i t e l l o g e n i c oocytes involves migration of the nucleus toward the egg membrane, breakdown of the nucleus, disappearance of any difference i n staining properties of nucleoplasm and cytoplasm, and a general decrease In the basophilia of the two  (Fig. 8).  The early stages of the loss of staining may be  simply to the d i l u t i o n of the cytoplasm with the weakly staining  due  nucleoplasm;  140  i n l a t e r stages, the weaker staining i s l i k e l y due to changes i n cytoplasmic constituents as the cytoplasm takes on a more granular appearance and eventually i s composed of aggregated debris.  Whether other  f o l l i c u l a r events precede these early changes i s not known.  However, a  common finding i n ovaries that contain DEYVOs i s that the yolk v e s i c l e s i n the non-degenerating oocytes no longer maintain a normal cytoplasmic d i s t r i b u t i o n (Figs. 9 and 10), but are very close to, i f not touching, the oocyte membrane (Figs. 11 and 12). appear normal.  In a l l other aspects the oocytes  As the yolk v e s i c l e s i n oocytes which have j u s t become  a t r e t i c are not v i s i b l e i n the cytoplasm but can be found i n the hypertrophied f o l l i c u l a r layer (Fig,13), i t appears that yolk v e s i c l e s migrate to the extreme periphery of the cytoplasm p r i o r to the onset of a t r e s i a i n early yolk v e s i c l e stage oocytes.  No other cytoplasmic or nuclear  abnormalities are evident at this time. Migration of the nucleus toward the periphery i s seen commonly i n DPVOs and DEYVOs and has been reported by Yamazaki '(1961) and observed i n the present study to occur i n oocytes with more advanced yolk v e s i c l e formation (Fig.14).  In the early v i t e l l o g e n i c and p r e v i t e l l o g e n i c oocytes,  the nucleus moves as a unit toward the egg membrane, at which point some or a l l of the n u c l e o l i are released. may not (Fig.16) be hypertrophied.  The f o l l i c u l a r layer may  (Fig.15) or  In many i f not a l l instances, the  nuclear membrane, often retaining a large (10 - 12u) spherical structure with highly variable staining properties (Figs. 17 and 18), returns to the centre of the oocyte, where i t may p e r s i s t after f o l l i c u l a r trophy has begun.  hyper-  It i s believed that i t i s this stage which has been  interpreted by Khoo (1974) as evidence of pregnenolone-induced  yolk  141  granule formation. It i s not known whether nuclear migration occurs i n DPVOs which have no detectable f o l l i c u l a r development.  However, i n oocytes degener-  ating at this stage, the n u c l e o l i do not remain at the periphery of the c e l l but are found i n the general cytoplasm after nuclear breakdown.  In  oocytes which have become a t r e t i c at a s l i g h t l y more advanced stage i n which the f o l l i c l e i s p a r t i a l l y developed, small numbers of n u c l e o l i can be found within the scattered hypertrophied f o l l i c u l a r c e l l s , the rest remaining i n the cytoplasm (Fig.19). As degenerating oocytes are found i n which hypertrophy of f o l l i c u l a r c e l l s and uptake of n u c l e o l i occur and yet i n which no yolk v e s i c l e s can be detected, i t appears that f o l l i c u l a r envelopment of the oocyte i s completed p r i o r to yolk v e s i c l e formation.  It seems that f o l l i c u l a r hypertrophy i n the p a r t i a l l y envel-  oped p r e v i t e l l o g e n i c oocyte i s transient and that no corpus atreticum i s formed. In the early a t r e t i c stages of oocytes with developed  follicles,  n u c l e o l i recently deposited at the periphery are usually, clumped i n a small area;  the degenerating nucleus often i s seen close to these  nucleolar concentrations (Figs. 8 and 15).  Eventually the n u c l e o l i are  distributed f a i r l y evenly within the hypertrophied f o l l i c u l a r c e l l s (Fig.3); i t i s not known how  this i s accomplished.  Unlike the s i t u a t i o n  i n degenerating oocytes without developed f o l l i c l e s , n u c l e o l i are not found free i n the cytoplasm. In DPVOs and DEYVOs, the stage of nuclear migration i s also characterized by invasion of macrophages which i n i t i a l l y tend to accumul a t e i n and around the degenerating nucleus.  In some instances,  142  the n u c l e o l i at thisvstage are found i n clusters (Fig.20).  Two readily  distinguishable types of macrophage are found both i n DPVOs and i n DEYVOs. The most common, which w i l l be referred to as M-l type macrophages, are small, usually spherical but occasionally e l l i p s o i d a l c e l l s with clear cytoplasm and small central n u c l e i (Figs. 6, 20, and 21). The second type (M-2) i s a larger, roughly spherical c e l l with granular cytoplasm and a large, eccentric, bilobed nucleus (Fig,s.6 and 22). As c e l l s similar to both ,M-l and M-2 macrophages are usually very numerous i n the channels between, the ovarian lamellae and often are found adjacent degenerating oocytes (Figs. 23 and 24), i t appears that the free macrophages of DPVOs and DEYVOs are of e x t r a f o l l i c u l a r o r i g i n . Although this was not quantified, i t appeared that the less common M-2 macrophage occurred most frequently i n the less developed a t r e t i c oocytes. Macrophages are present i n DPVOs from the time of nuclear degeneration u n t i l a l l oocyte constituents have been removed.  When t h i s has  occurred, the non-hypertrophied f o l l i c u l a r envelope may remain i n t a c t around the cavity (Fig.25); however, many of these envelopes apparently disintegrate.  Thus, i h many long-term hypophysectomized  , and i n several  i n t a c t , regressed f i s h , i t appeared that extensive a t r e s i a of p r e v i t e l l ogenic oocytes had created large open spaces i n the ovary (Figs. 26 and 27).  This was not simply the result of a t r e s i a of large yolky oocytes  and shrinkage of the resulting corpora a t r e t i c a , as i n that process the entire ovary decreases i n volume while maintaining close association of normal and a t r e t i c oocytes (Fig.28).  In addition, corpora a t r e t i c a are  persistent structures which are evident for at least several months:,after t h e i r formation.  143  A f t e r cytoplasmic debris has been removed, DEYVOs resemble the Y - stage a t r e t i c f o l l i c l e s described by Khoo (1975), except that they are smaller (usually less than 50y i n cross section).  Eventually these  small structures collapse to form persistent 6- stage corpora a t r e t i c a . No experiments were conducted to investiage the endocrine basis of degeneration of early yolk v e s i c l e or p r e v i t e l l o g e n i c oocytes.  However,  some incidental observations are worth noting. In none of the experiments reported i n this study was there any i n d i c a t i o n that treatment with exogenous steroids induced the formation of yolk v e s i c l e s .  There, was, however, some i n d i c a t i o n that i n j e c t i o n  of e s t r a d i o l or 5°= - dihydrotestosterone delayed onset of a t r e s i a i n early yolk v e s i c l e oocytes following hypophysectomy (Experiment 5). For example, whereas 5 of 8 f i s h receiving e s t r a d i o l and 4 of 8 f i s h receiving 5  03  - dihydrotestosterone had small numbers of normal yolk  v e s i c l e s i n their oocytes, there were no normal yolk v e s i c l e s i n any of 6 saline-treated f i s h .  That this indicated i n h i b i t i o n of a t r e s i a rather  than induction of yolk v e s i c l e formation i s suggested by the fact that whereas a l l 6 s a l i n e - i n j e c t e d females had many DEYVOs, these were found i n low numbers i n only 4 of the e s t r a d i o l and 4 of the dihydrotestosterone groups.  In addition, yolk v e s i c l e s associated with the egg membrane  were abundant i n 6 of the f i s h receiving e s t r a d i o l and i n 5 of the f i s h receiving dihydrotestosterone, whereas these structures were seen i n only 3 of the f i s h receiving s a l i n e , and then only i n low numbers.  This i s  interpreted to mean that i n f i s h treated with saline, the proposed t r a n s i t i o n of normal early yolk v e s i c l e oocytes to oocytes with membrane vesicles and f i n a l l y to DEYVOs has proceeded more rapidly than i t  144  has i n f i s h receiving e s t r a d i o l or dihydrotestosterone. In a l l three experiments involving hypophysectomy and i n j e c t i o n of p i t u i t a r y material, there was good evidence that both homogenized goldfish p i t u i t a r i e s and p a r t i a l l y p u r i f i e d salmon gonadotropin (SG-G100) inhibited a t r e s i a following hypophysectomy.  In the two experiments i n which replace-  ment therapy began within one month of hypophysectomy, none of J.11 f i s h receiving homogenized p i t u i t a r i e s (Experiment 6) and only 1 of 10 f i s h receiving SG-G100 (Experiment 7) had DPVOs i n their ovaries.  In contrast,  following e s t r a d i o l treatment, 7 of 8 f i s h i n Experiment 6 and 8 of 10 f i s h i n Experiment 7 had DPVOs.  SG-G100 appeared to be less e f f e c t i v e i n  i n h i b i t i n g a t r e s i a i n long-term hypophysectomized  goldfish, as the ovaries  of 4 of 15 females treated with SG-G100 i n Experiment 11 contained DPVOs; however, this was s t i l l a lower incidence of a t r e s i a than that found i n f i s h treated with estrogen (8 of 12 fish) or saline (13 or 16 f i s h ) .  As  females receiving SG-G100 also had more yolk v e s i c l e s associated with the egg membrane, i t i s possible that some of the atresia involved oocytes i n which yolk vesicles were induced by the gonadotropin. Most studies of oocyte a t r e s i a i n teleost ovaries have dealt with the degeneration of second growth stage (vitellogenic) oocytes (references c i t e d by Khoo, 1975) which are generally accepted to be dependent on the p i t u i t a r y (review by Dodd, 1972).  P r e v i t e l l o g e n i c oocytes have been sug-  gested to be independent of p i t u i t a r y influence (Dodd, 1972) and a t r e s i a of these oocytes has received scant attention i n the l i t e r a t u r e .  Beach  (1959) described corpora a t r e t i c a i n goldfish which he believed were derived from p r e v i t e l l o g e n i c oocytes; however, as these structures had a hypertrophied granulosa layer, they may have developed from early yolk  145 v e s i c l e stage oocytes.  MacKay (1973) reported 'small corpora a t r e t i c a  apparently derived from degeneration of p r e v i t e l l o g e n i c oocytes' i n ovari e s of f i r e t a i l gudgeons (Hypseleotris g a l I i i ) r e c e i v i n g methallibure treatment.  As these s t r u c t u r e s , found only i n females given long-term  (2 month) methallibure treatment, appeared to have no granulosa l a y e r , i t i s l i k e l y they were DPVOs. The r e s u l t s of the present study i n d i c a t e that the C o r r e l a t i o n between the post-hypophysectomy degeneration of t e l e o s t oocytes and the presence of y o l k d e p o s i t i o n i s not as simple as has been suggested i n e a r l i e r work.  Although these f i n d i n g s are only suggestive, they point to  a greater p i t u i t a r y influence on p r e v i t e l l o g e n i c oocytes than has been recognized p r e v i o u s l y .  APPENDIX FIGURES  147  Figure 2:  Early stage of degenerating early yolk v e s i c l e (DEYVO) or previtellogenic (DPVO) oocyte.  A  few red n u c l e o l i (N) are seen at the periphery. Mallory  Figure 3:  trichrome.  F o l l i c u l a r hypertrophy more advanced than i n Figure 2.  Figure 4:  Mallory  trichrome.  Advanced DEYVO from intact regressed f i s h . trophied f o l l i c l e surrounds empty cavity.  Hyper^ NSte  that presumptive n u c l e o l i (N) are s t i l l numerous. Mallory  trichrome.  149  Figure 5:  Advanced DEYVO from female goldfish for 2 months.  hypophysectomized  PAS-positive yolk v e s i c l e s (V) and  green n u c l e o l i (N) are s t i l l obvious.  Figure 6:  PAS-light green.  Advanced DPVO from f i s h hypophysectomized  for 2 months.  Most oocyte contents have been removed and several M-l (1) and M-2 (2) macrophages remain. f o l l i c u l a r hypertrophy.  Figure 7:  Mallory trichrome.  Yolk nucleus i n oocyte of f i s h hypophysectomized for 4 months,  Figure 8:  Note absence of  PAS-light green.  Nuclear migration stage i n oocyte from f i s h tomized for 4 months.  hypophysec-  Note breakdown of nuclear structure  and loss of cytoplasmic basophilia.  Mallory trlchrome.  151  Figure 9;  Normal configuration of yolk vesicles (V). Note vesicles tend to form a ring and do not contact the oocyte membrane.  Mallory  trichrome.  Figure 10\  As i n Figure 9 but stained with PAS-light green.  Figure 11;  Yolk vesicles (V) associated with membrane of presumptive p r e - a t r e t i c oocyte.  From  f i s h hypophysectomized for 2 months. Mallory  Figure 12:  trichrome.  As i n Figure 11 but stained with PAS-light green.  153  Figure 13:  Yolk v e s i c l e s (V) and presumptive n u c l e o l i (N) i n hypertrophied  f o l l i c u l a r layer from  f i s h hypophysectomized for 2 months. PAS-light green.  Figure 14:  Presumptive pre-nuclear breakdown stage oocytes  (0) i n ovary of f i s h hypophysectom-  ized for 2 months.  Note abnormal yolk  v e s i c l e d i s t r i b u t i o n . Mallory  Figure 15:  trichrome.  Degenerating nucleus at early stage of a t r e s i a . Spherical body (S) appears outside nuclear membrane and i s very l i g h t l y stained. Presumptive n u c l e o l i (N) are seen both within the nuclear membrane and within the presumptive granulosa c e l l s  (G) i n the s l i g h t l y hyper-  trophied f o l l i c l e .  From intact regressed  female.  Figure 16:  Mallory  trichrome.  Peripheral nucleus with spherical body (S) and several n u c l e o l i (N). hypertrophied.  Mallory  F o l l i c l e i s not  trichrome.  Presumptive DEYVO from f i s h hypophysectomized for 2 months.  Nucleus i s unusual as i t stains  d i f f e r e n t l y than the cytoplasm and appears to r e t a i n some n u c l e o l i .  Mallory trichrome.  Advanced DEYVO from i n t a c t , regressed f i s h . Degenerated nucleus with spherical body (S) i s present.  Hypertrophied granulosa c e l l s (G)  and presumptive n u c l e o l i (N) can also be seen. Mallory trichrome.  Advanced DPVO from i n t a c t , regressed f i s h . Presumptive n u c l e o l i (N) are seen both i n the s l i g h t l y hypertrophied f o l l i c l e and i n the cytoplasm.  Spherical body (S) and M-l macro-  phages (1) also are present.  Mallory trichrome  Clumped n u c l e o l i (N) from degenerating nucleus a t r e t i c oocyte of f i s h hypophysectomized months-M-l macrophages (1) can be seen. Mallory trichrome.  for 4  157  Figure 21:  M-l macrophage (1) and presumptive nucleoli  (N) i n nuclear debris of oocyte  of f i s h hypophysectomized f o r 4 months. PAS-light green.  Figure 22:  M-2 macrophage (2) i n a t r e t i c oocyte of i n t a c t , regressed f i s h .  Mallory  tri-  chrome .  Figure 23:  Macrophage aggregation adjacent to early a t r e t i c oocyte.  Figure 24:  Mallory  trichrome.  Macrophages entering early a t r e t i c oocyte i n ovary of intact regressed Mallory  trichrome.  fish.  159  Figure 25:  Ovary of female goldfish hypophysectomized for 4 months.  Two nearly evacuated DPVOs  (E) can be seen.  Older presumptive DPVOs  (D) are apparently Mallory  Figure 26:  disintegrating.  trichrome.  Ovary of f i s h hypophysectomized for 4 months showing open spaces apparently caused by extensive a t r e s i a of previtellogenic oocytes.  Figure 27:  Mallory  trichrome.  As i n Figure 26 but at a lower magnification.  Mallory  trichrome.  161  F i g u r e 28:  Ovary of female  g o l d f i s h hypophysectomized  f o r 2 months showing c l o s e a s s o c i a t i o n of normal (N) and l a r g e oocytes another  (A) o o c y t e s .  The  (0) are o v u l a t e d oocytes  female) which were i n j e c t e d  the o v i p o r e .  F i g u r e 29:  atretic  two  (from  through  Mallory trichrome.  Macrophage a g g r e g a t i o n a d j a c e n t to i n j e c t e d ovulated oocyte. Mallory  D e t a i l from F i g u r e  trichrome.  28.  

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