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The effect of methyl testosterone on secondary sex characters and reproductive behaviour of gonadectomized… Wai, Evelyn 1962

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THE EFFECT OF METHYL TESTOSTERONE ON SECONDARY SEX CHARACTERS AND REPRODUCTIVE BEHAVIOUR OF GONADECTOMIZED STICKLEBACKS (GASTEROSTEUS ACULEATUS L.) by EVELYN WAI B. Sc., U n i v e r s i t y of B r i t i s h Columbia, i 9 6 0 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1°62 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Z^zr^-gg. ft The University of B r i t i s h Columbia,, Vancouver 8, Canada. Date t1 ^ Q v t ^ c I i i -ABSTRACT Treatment of gonadectomized adult male and female sticklebacks as well as normal juvenile, with methyl testos-terone, either by immersion i n a hormone solution or by implanting hormone p e l l e t s , induces the kidney c e l l s to develop into granular and mucous c e l l s , accompanied by an increase i n c e l l height. Increase i n c e l l height, up to a limit, i s a function of the length of hormone treatment. Prespawning aggressiveness and t e r r i t o r i a l i t y i s induced i n gonadectomized f i s h of both sexes by maintaining them under long photoperiod for four to f i v e weeks. Short photoperiod has no e f f e c t . Administration of methyl testosterone to the already aggressive f i s h showed no d e f i n i t e e f f e c t on t h i s behaviour. The combined ef f e c t of long photoperiod and methyl testosterone treatment induces the nest-building behaviour i n gonadectomized males and females with a much higher percentage i n the former than i n the l a t t e r . The component elements of the nest-building movement i n the treated gonadectomized male i s s i m i l a r q u a l i t a t i v e l y and quant i t a t i v e l y to that of the normal reproducing male. Treated gonadectomized females show deviations from the normal male i n t h e i r nest-building movements. Sexual behaviour i s the same i n castrates treated with methyl testosterone as i n the normal reproducing male, but completely absent i n the s i m i l a r l y treated gonadectomized females. ACKNOWLEDGEMENT The author would l i k e to express her gratitude to Professor W. S. Hoar, E. R. S. C , fo r . h i s guidance, c r i t i c i s m and encouragement throughout the project. Thanks are also due to Dr. P. Eord and Dr.. M. D. E. Udvardy for t h e i r advice on the general problem and to Dr. C. C. Lindsey for his sugges-tions i n the preparation of the manuscript. .The author also wishes to thank Mr. A. J". Wiggs, Mr. J". E. Mclnerney and Mr. J . G. Eales for t h e i r help i n the c o l l e c t i o n of the f i s h . The research was made possible through the f i n a n c i a l support of the National Research Council Studentship. - i i i -TABLE OF CONTENTS PAGE I. INTRODUCTION 1 I I . MATERIALS AND METHODS 4 A. COLLECTION AND GENERAL CARE OF FISH . . 4 B. GONADECTOMY OF ADULT FISH . . . . 4 C. PHOTOPERIOD AND TEMPERATURE CONTROL . . 6 D. ANDROGEN TREATMENT . . . . . . 6 1. Exogenous Method 6 2. Implantation of Hormone P e l l e t s . . 6 3. Injection of Aqueous Suspension . 7 E. OBSERVATION OF BEHAVIOUR 7 1. Pre-spawning Aggressiveness . . . 7 2. Nest-building Behaviour . . . . Q 3. Sexual Behaviour . . . . . 10 F. HISTOLOGICAL STUDIES . . . . . . 10 I I I . RESULTS . . • 12 A. HISTOLOGY OF THE KIDNEY . . . . . 12 1. Normal Adult Male and Female . . 12 2. Androgen Treated F i s h . . . . 12 a. Gonadectomized Adult Male and Female 12 b. Unoperated Juvenile Male and . Female . . . . . . . . 14 B. PRE-SPAWNING AGGRESSIVE BEHAVIOUR. . . 15 1. Photoperiod E f f e c t . . . . . . 16 - i v -T A B L E OF CONTENTS ( C o n t i n u e d ) P A G E 2 . S e x . 1 ° 3 . A n d r o g e n T r e a t m e n t . . . 20 C . N E S T - B U I L D I N G B E H A V I O U R 21 1. P h o t o p e r i o d a n d U n o p e r a t e d A d u l t M a l e s 23 2 . P h o t o p e r i o d a n d A n d r o g e n T r e a t m e n t o n G o n a d e c t o m i z e d M a l e s a n d F e m a l e s . . 23 a . E f f e c t o f P h o t o p e r i o d o n t h e I n t e n s i t y a n d T i m e t o R e s p o n s e o f t h e Two S e x e s t o t h e A n d r o g e n T r e a t m e n t 24 b . Q u a l i t a t i v e a n d Q u a n t i t a t i v e C o m p a r i s o n o f t h e N e s t - b u i l d i n g B e h a v i o u r i n N o r m a l M a l e s , T r e a t e d C a s t r a t e d M a l e s a n d G o n a d e c t o m i z e d a n d T r e a t e d F e m a l e s . . . . 24 D . S E X U A L B E H A V I O U R 30 I V . D I S C U S S I O N . . 33 A . GONADAL S T E R O I D S AND K I D N E Y D E V E L O P M E N T . 33 B . P R E - S P A W N I N G A G G R E S S I V E N E S S AND I T S E N D O C R I N E CONTROL . . . . . . 38 C . GONADAL S T E R O I D S AND THE N E S T - B U I L D I N G AND S E X U A L B E H A V I O U R . . . . . . 43 V . SUMMARY AND C O N C L U S I O N S 50 V T . B I B L I O G R A P H Y 52 LIST OF FIGURES FIGURE FOLLOWING PAGE 1. Height of e p i t h e l i a l c e l l s of the kidney tubules of castrated adult male and female immersed i n solution of methyl testosterone . 14 2. Height of e p i t h e l i a l c e l l s of the kidney tubules of unoperated juvenile male and female immersed i n solut i o n of methyl testosterone 14 J>. Mean number of attacks (with confidence l i m i t at 95% level) of gonadectomized males and females a f t e r having been maintained under long (16 hr) and short ( 8 h r ) photoperiod for 2 , 5 and 6 weeks . . . . . . 17 4. Mean number of attacks (with confidence l i m i t at 95% level) of gonadectomized males and females s i x weeks a f t e r gonadectomy. . . 19 - v i -LIST OF TABLES TABLE PAGE I. V a r i a t i o n i n mean height (- one standard de-v i a t i o n of mean) fo r the granular c e l l s i n kidneys of sticklebacks treated for di f f e r e n t periods with methyl testosterone. Each mean based on two f i s h with 25 measurements of kidney tubules from each f i s h ( t o t a l of 50 values). . 13 I I . Number and percent of tanks of f i s h maintained under long and short photoperiod, with and without t e r r i t o r y s i x weeks a f t e r gonadectomy . 18 I I I . Number and percent of tanks of gonadectomized f i s h with or without t e r r i t o r y s i x weeks af t e r gonadectomy . 18 IV. The effect of photoperiod and methyl testo-sterone treatment i n inducing nest-building behaviour (sexual maturation!, i n castrated males and females . . . . . . . . . . 22 V. Time of appearance of maximum a c t i v i t y f or di f f e r e n t behaviour patterns together with percent time so occupied by males maturing under natural conditions 25 VI. Time of appearance of maximum a c t i v i t y f or di f f e r e n t behaviour patterns together with percent time so occupied by androgen treated castrated males . . . . . . . 27 VII. Record of the percent time which four nest-building female castrates spent on each elements of the nest-building behaviour pattern on the di f f e r e n t days afte r nest-building was f i r s t started 29 I. INTRODUCTION The reproductive behaviour i n fishes shows much v a r i a t i o n , from the simplest patterns i n which eggs and sperms are broad-cast i n the water, as i n the cods, to the most complex behaviour, i n which nest-building, courtship and other pre- and post-spawn-ing a c t i v i t i e s are involved, as i n the sticklebacks, the c i c h l i d s and the anabantids. Whatever the reproductive behaviour, however, the patterns are stereotyped and innate, f i x e d by the genetic constitution, probably unaffected by experience, and released by the appropriate combination of i n t e r n a l and external conditions (Hoar, 1 9 5 5 ) . The reproductive behaviour of the stickleback, Gasterosteus aculeatus. which i s studied here, has been analyzed by Tinbergen (1953) as a complicated series of stereotyped res-ponse patterns, arranged i n a d e f i n i t e sequence and triggered only by the s p e c i f i c i n t e r n a l motivation and external s t i m u l i . Changes i n the hormonal l e v e l of the animal has been sug-gested to be one of the i n t e r n a l factors associated with the release of a p a r t i c u l a r behaviour pattern, e s p e c i a l l y reproductive behaviour. The exact mechanism through which t h i s endocrine sys-tem influences the behaviour elements i s s t i l l subject to further i n v e s t i g a t i o n . I t may modify the behaviour by a f f e c t i n g the metabolism of the animal (Hoar,et a£, 19-55; Fontaine & Xeloup, 1959) or i t may act d i r e c t l y on the central nervous system which releases the p a r t i c u l a r movement (Pickford, 1952? Harris.et a l . 1 9 5 8 ) . A t h i r d p o s s i b i l i t y i s that the hormone may modify the responsiveness of the animal to external s t i m u l i by increasing - 2 -the s e n s i t i v i t y of the receptor or effe c t o r organ (Beach .et a l . 1 9 5 0 , 1 9 5 2 ; Lehrman, 1 9 5 5 ) . Gonadal hormones and hormones from the p i t u i t a r y have been demonstrated to have marked effects i n e l i c i t i n g the reproductive behaviour i n higher vertebrates as well as i n f i s h e s . Tavolga (1955) showed that hypophysectomy r e s u l t s i n the cessation of a l l courtship behaviour i n the male Bathygobius while castration had no e f f e c t . Wilhelmi et a l (1955) were able to induce the spawn-ing r e f l e x i n the hypophysectomized or gonadectomized ffundulus by i n j e c t i o n of oxytocin or vasopressin. The d i r e c t e f f e c t of gonadal steroids on the reproductive behaviour has been demonstrated either by treatment with andro-genic or estrogenic hormones, or by gonadectomy. However, generalization concerning the hormone-behaviour r e l a t i o n i s impossible because of the varied and sometimes contradictory r e s u l t s obtained i n di f f e r e n t groups of animals. Several examples i l l u s t r a t e t h i s point. Boch (1928) and Ikeda (1933) showed that castrating male sticklebacks eliminated the nest-building behaviour. Hoar (1961) indicated the importance of the time of castration i n influencing these behaviour patterns. Noble et a l ( 1 9 3 6 ) , however, castrated male jewel f i s h and observed courtship, f e r t i l i z a t i o n and brooding behaviour i n the gonadectomized animals. Tavolga (1955) modified the discrimina-tory power of the male Bathygobius soporator by castration, but the courtship behaviour was hot affected. Male sexual behaviour was stimulated i n immature male and female black-crowned night herons by treatment with testosterone propionate (Noble & Wurm, 1 9 4 0 ) . Hiss et a l (1955) suggested that testosterone propionate - 3 -acted as an activator rather than a direct organizer for the sexual behaviour of the male guinea pig. Injection of testos-terone propionate to castrated adult male salmon, on the other hand, did not have any effect on the spawning behaviour (Jones & King, 1952). Hoar et al (1952) did not observe any new behaviour in chum salmon fry and coho fry treated with methyl testosterone. These examples, drawn from a rather vast litera-ture, show how variable the findings for different species have been. The present research is an attempt to identify the specific hormones concerned with the development of male secondary sex characteristics and reproductive behaviour of the stickleback. By using both gonadectomized males and females, an attempt was made to separate characteristics which were only dependent on hormones from those which were associated with other genetically controlled sexual characters. The stickleback was chosen be-cause of the many elements associated with its reproductive behaviour and also because this behaviour has been completely documented. Moreover, i t is possible to regulate pituitary activity (gonadotropic activity) by controlling the photoperiods and preliminary experiments have shown that these animals respond readily to androgens. - 4 -I I . MATERIALS AND METHODS A. COLLECTION AND GENERAL CARE OF FISH Fresh-water s t i c k l e b a c k s , type (Heuts, 1947), were c o l -l e c t e d d u r i n g May and June from backwaters of the s m a l l streams i n the Musquem I n d i a n R e s e r v a t i o n i n Vancouver; and from s i d e -streams of the Salmon R i v e r from September t o December. Both environments were s i m i l a r . A h i g h percentage of the f i s h c o l -l e c t e d i n May and June were mature or maturing w h i l e those c o l -l e c t e d i n the f a l l were mainly j u v e n i l e f i s h or those which had a l r e a d y spawned. The f i s h were t r a n s f e r r e d t o d e c h l o r i n a t e d water i n a l a r g e wooden tank, kept i n the l a b o r a t o r y under n a t u r a l p h o t o p e r i o d at 20 - 2 5 ° C, and f e d d a i l y w i t h f r o z e n b r i n e shrimp. They were u s u a l l y c a s t r a t e d f i v e t o s i x days a f t e r c o l l e c t i o n . P a r a s i t e s , p a r t i c u l a r l y I c h t h y o p h t h i r i u s , o f t e n developed, spread, and k i l l e d a h i g h percentage of the f i s h i f the l a t t e r were crowded i n the storage tank f o r two weeks or more. I f not too h e a v i l y i n f e c t e d , immersion i n sea water f o r f i f t e e n t o t h i r t y minutes cured the d i s e a s e . The m a j o r i t y of the f i s h c o l l e c t e d f o r experiment I had broken t a i l s and f i n s due t o the i n f e c t i o n of the f l u k e , G y r o d a c t v l u s on the epidermis. They were cured by immersing them i n g l a c i a l a c e t i c a c i d bath (l:j?00) f o r one minute. B. G0NADECT0MY OF ADULT FISH A d u l t s t i c k l e b a c k s (body l e n g t h > 4 cm), which were not i n b r e e d i n g c o n d i t i o n , were a n a e s t h e t i z e d l i g h t l y i n O.Olf. T r i c a i n e methane sulphonate (Sandoz MS 222) f o r approximately t h r e e - 5 -minutes u n t i l quiet even when taken out of the water. The anaesthetized f i s h was placed i n a suitable depression cut i n a wet p l a s t i c sponge, and a stream of water run through the mouth, over the g i l l s , and out through the operculum. Incisions, 1 -2 mm, were made f i r s t on one side and then on the other of the f i s h anterior to the anus, so that the gonads, which were along the dorsal l a t e r a l body wall, could be taken out with f i n e forceps. A binocular microscope (10 X 1.3) was used f o r the operation. Males and females were distinguished and separated by the d i f f e r e n t appearance of the t e s t i s and the ovary. The former i s white, with heavy black pigmented spots, and i s d i s t i n c t l y d i f -ferent i n appearance from the vas deferens which i s fi n e and thread-like. The ovary has a transparent, colourless appearance with l i t t l e or no pigment. Moreover, i t merges into the oviduct without a d i s t i n c t l i n e of demarkation. Generally, the ovaries were more d i f f i c u l t to remove com-p l e t e l y than the testes. The former extended quite f a r p o s t e r i -o r l y and necessitated a longer i n c i s i o n . Examination at the end of each experiment showed that about 20% of the castrated females s t i l l had a small part of the oviduct remaining. However, only 2 - 8% of these castrated females had regenerated a complete ovary. The t e s t i s was e a s i l y and completely removed with no regeneration. Gonadectomized f i s h were kept i n isotonic saline for approxi-mately twelve hours before they were transferred i n groups of four (Experiment V i n groups of two) to glass aquaria ( l 6 , f X 10" X 8") f o r observation and study. Weeds, rocks and sand were present i n a l l the tanks. - 6 -0 . PHOTOPERIOD AND TEMPERATURE CONTROL A l l the experimental f i s h were maintained under controlled photoperiod and temperature* They were transferred to the con-stant environment room a f t e r the ca s t r a t i o n and exposed to either eight hours of i l l u m i n a t i o n ( 5 0 - 5 5 ft.^-c.) alternating with sixteen hours of darkness;- or to sixteen hours of i l l u m i -nation alternating with eight hours of darkness throughout the experiment. The eight hours i l l u m i n a t i o n was used only i n Experiment V. The temperature was kept constant at 18+2° C. D. ANDROGEN TREATMENT Three methods of administration of the androgen, methyl te s t o s t e r o n e , ( B r i t i s h Drug House) were used. 1. Exogenous Method This method involved maintaining the experimental f i s h i n water containing the androgen throughout the experiment. A known quantity of methyl testosterone was suspended i n approxi-mately 500 ml of water i n a beaker, and then introduced into the d i f f e r e n t tanks every week. Thus the concentrations used were gradually increased so that the t o t a l amount of androgen present i n each tank, assuming no deterioration, would be: Total concentration i n 1st week 1:4 , 0 0 0 , 0 0 0 " " 11 2nd " 1 : 3 , 0 0 0 , 0 0 0 " " " 3 r d " 1:2 , 0 0 0 , 0 0 0 " " " 4th " 1:1 , 5 0 0 , 0 0 0 . 2. Implantation of Hormone P e l l e t s An i n c i s i o n 1 - 2 mm was made on one side of the abdomen of the f i s h a f t e r i t had been anaesthetized l i g h t l y i n ,-MS 222. A hormone p e l l e t , weighing approximately 2 mg, was introduced into the abdominal cavity, which was then closed - 7 -with, a s t i t c h to keep the p e l l e t i nside. F i s h were implanted only once at the beginning of the androgen treatment. 3. I n j e c t i o n of Aqueous Suspension 0.05 ml of an aqueous suspension of methyl testosterone, containing 0.5 mg/ml, was injected into the abdominal cavity of the experimental f i s h with a 0.25-ml syringe and 26-gauge needle. Each f i s h was injected once weekly on alternate side. A l l three methods were used i n the experiment f o r quanti-f y i n g the response of the kidney of the gonadectomized adults to the androgen treatment i n r e l a t i o n to the length of treatment. Only the exogenous method by the hormone bath was used i n the treatment of juvenile f i s h and i n the experiments where the reproductive behaviour was measured. E. OBSERVATION OF BEHAVIOUR 1. Pre-spawning Aggressiveness ' Observations were made beginning the second week a f t e r v gonadectomy, one week af t e r they had been maintained under the controlled photoperiod and temperature. Each tank was observed for f i v e minutes and the number of attacks recorded. This included changing, chasing and the actual nipping of another f i s h . Three observations were made weekly; and the mean number of attacks i n the three observations represented the a c t i v i t y f o r the whole week. Each tank was also assigned a l e v e l of aggressiveness f o r that p a r t i c u l a r observation based on the following c r i t e r i a : Level jfO - No attack during the observed period and no f i s h hides from any other L e v e l §• I - As above, but one or two a t t a c k s per minute between f i s h # I I - Three a t t a c k s per minute or more but no domi-nant or subordinate r e l a t i o n and no f i s h h i d e s from any other §• I I I - T e r r i t o r i a l r e l a t i o n e v ident i n which t e r r i - , t o r i a l f i s h remains w i t h i n a p r e f e r r e d area and any i n t r u d i n g f i s h i s a t t a c k e d . Outside the p r e f e r r e d t e r r i t o r y , however, a l l f i s h b i t e a t one another § IV - C l e a r l y marked dominant and subordinate r e l a -t i o n i n which one or two dominant f i s h a t t a c k s a l l o ther f i s h v i g o r o u s l y w i t h i n i t s own t e r r i t o r y . Subordinate f i s h h i d e i n c o r n e r s and behind r o c k s and weeds. The a c t u a l number of a t t a c k s i n t h i s l e v e l dropped markedly because of h i d i n g . Subordinate f i s h may be k i l l e d . I I n the l a s t experiment (Exp. V ) , an attempt was made t o s t a n d a r d i z e the a g g r e s s i v e behaviour more p r e c i s e l y as measured by r e c o r d i n g the number of a t t a c k s i n the f i v e minutes o b s e r v a t i o n . The number of a t t a c k s i n each tank observed de-pended, on the a g g r e s s i v e n e s s of a l l f o u r f i s h , so t h a t when one f i s h i n the tank became dominant i t would b i t e at a l l the f i s h which would h i d e among the rocks and weeds. The number of a t t a c k s r e c o r d e d when these f i s h were at l e v e l #IV, thus, dropped c o n s i d e r a b l y from t h a t recorded when the f i s h were i n l e v e l s #11 - I I I where th e r e was no dominance. I n Experiment V, a " s t a n d a r d " f i s h , an immature male or a female w i t h a s i l v e r y body., - 9 -which would a c t i v e l y swim up and down within the glass tube, was placed inside a glass tube four centimeters i n radius. The tube was introduced into the tank, approximately 10 cm from the nest of the dominant f i s h , or just within the t e r r i t o r y of a non-nesting but t e r r i t o r i a l f i s h . The number of attacks on th i s "standard" f i s h i n the f i v e minutes observation was recorded. 2 . Nest-building Behaviour Approximately two weeks a f t e r the f i r s t addition, of methyl testosterone, some of the f i s h began to c o l l e c t algae and b u i l d nest. During the f i v e minutes observation for the aggressiveness, the presence or absence of t h i s behaviour was noted. Detailed records of a 30 minute observation was then made of the nest-building i f present. The nest-building behaviour was separated into the following elements: c o l l e c t i n g material including the te s t i n g of the material, gluing, pushing and fanning (parental and displacement). A metronome was used while recording these elements. I t was set at a speed of 60 per minute. Each time the metronome ticked, the p a r t i c u l a r movement which the f i s h was performing at that second was recorded by a symbol, such as 'M' f o r c o l l e c t i n g material, "G» for gluing and fF' f o r fanning. Since the f i s h normally perform a p a r t i c u l a r movement f o r longer than a second, the appearance of a l l the elements would be recorded. For instance, a series of recordings such as, MMPGGF, would indicate that the f i s h c o l l e c t e d material f o r two successive seconds and then pushed the nest material f o r one second, followed by two seconds of gluing and then one second of fanning. This kind of record showed the amount of - 10 -time the f i s h spent on each movement as well as the sequence of the d i f f e r e n t movements. T h i r t y minutes observation was made on the f i r s t and second day when nest-building f i r s t appeared. F i f t e e n minute observations were then made on alternate days subsequently f o r a maximum of twenty days. 3. Sexual Behaviour A normal mature male or castrated but treated male usually f i n i s h e d i t s nest the f i r s t or second day a f t e r i t started the nest-building. The. end of the nest-building i s marked by the f i s h boring through the nest. To test the sexual motivation of the f i s h at the end of t h i s nest-building phase, a mature or p a r t l y mature female with enlarged abdomen was introduced into the tank on the second day of nest-building and on alternate days subsequently. About an hour a f t e r the i n t r o -duction, observation of the response of the nest-building f i s h to the gravid female was made. The absence or presence of courtship behaviour, including the zigzag dance, leading back to the nest, inducing the female to lay eggs, and f e r t i l i z a t i o n was noted. The female was then taken out. F. HISTOLOGICAL STUDIES The f i s h were k i l l e d i n MS 222 and fixed i n Bouin's a f t e r d i f f e r e n t i n t e r v a l s of androgen treatment. The kidneys were removed, dehydrated, and embedded i n p a r a f f i n following routine procedures. Sections of lOu were cut, mounted and stained i n E h r l i c h ' s haematoxylin and 2% aqueous eosin solution.. The height of the granular e p i t h e l i a l c e l l s of the convoluted tubules, which could be distinguished by t h e i r granular appearance as - 11 -well as the c i r c u l a r cross section of the tubule, was measured with the micrometer under high power magnification (40 X 10). The mean minimum height of the e p i t h e l i a l c e l l s i n 25 kidney-tubules was taken to represent the c e l l height of that p a r t i -cular f i s h . The average value f o r two f i s h was used to repre-sent the c e l l height f o r that number of days of treatment. The presence or absence of clear mucous c e l l s i n the c o l l e c t i n g tubules were also recorded. - 12 -I I I . RESULTS A. HISTOLOGY OF THE KIDNEY The kidney tubules of normal and gonadectomized males and females were studied. Table I summarized the data from the h i s t o l o g i c a l studies of the kidney tubules of androgen treated f i s h , k i l l e d and f i x e d at 0, 3, 6, 9, 14, 21, and 30 days a f t e r the commencement of the treatment. 1. Normal Adult Males and Females The height of the c e l l s of the convoluted tubules of sexually matured and non-mature females were about the same (918*1.2 u and 10.2±1.6 u r e s p e c t i v e l y ) . Mature male with n u p t i a l colour and showing the reproductive behaviour had these c e l l s stimulated to 31.0+2.9 u as compared to 11.9+2.4 u i n the adult but not sexually matured male (Table I ) . 2. Androgen Treated F i s h a. Gonadectomized Adult Male and Female ( l e n g t h s 4 cm) Three weeks a f t e r gonadectomy, p r i o r to the addi-t i o n of the androgen, the c e l l height i n the males and females i s at a minimum of 8.6±1.5 ji and 8 . i l l . 4 jx respectively - a s l i g h t decrease from the normal non-reproducing adult males (Table I ) . Mucous c e l l s were also absent. Three days of treatment by the addition of androgen to the water or by implants stimulated the c e l l height of both males and females to 14.9±1.9 P- and 12.5+1.6 p., an increase by approximately 50%. Mucous c e l l s were not present i n the convoluted tubules of the females, although some were evident i n those of the males. These mucous c e l l s appeared i n the male and female only, when TABLE I V a r i a t i o n i n mean h e i g h t {- one standard d e v i a t i o n of mean) f o r the g r a n u l a r c e l l s i n kidneys of s t i c k l e b a c k s t r e a t e d f o r d i f f e r e n t p e r i o d s with methyl t e s t o s t e r o n e . Each mean based on two f i s h w i t h 2 5 measurements o f kidney tubules from each f i s h ( t o t a l of 50 v a l u e s ) GONADECTOMIZED FISH JUVENILE UNOPERATED FISH NORMAL ADULTS Type No. 9<j. $g O Q of days C e l l Mu- C e l l Mu- C e l l Mu- C e l l Mu- C e l l Mu- C e l l Mu-t r e a t - t r e a t - h e i g h t cous h e i g h t cous height cous h e i g h t cous h e i g h t cous h e i g h t cous ment ment ji-S.D. c e l l u*S.D. c e l l u*S.D. c e l l u-S.D. c e l l u-S.D. c e l l p±S.D. c e l l 0 . 8 . 6 i .1 .49 - . 8 . 1 i 1 .43 - 4. 8 ' 6 n ± 2 . 0 8 - i 1.69 -3 . 1 4 . 9 ± 1.9 + mi - - . 1 2 . 6 - 1 . 1 — b x 1 6 . 9 ± 2 . 0 2 + + + 16.6 ± 2.62 + O . U •H CD 9 . 2 1 . 8 i 2 . 5 5 + + + ± 2 .85 + +3 O +3 •H -P CO <! 12 . 2 3 . 0 . i 2 . 2 6 + + 2 3 . 4 i 2 .93 + 14 . 2 2 . 9 ± 2.62 + 4 . 2 3 . 0 ± 2.71 + 21 + + . 2 4 . 1 ± 2.30 + ^24.6 ± 2 .70 + 3 0 + ± 2 ? : o 4 + ± 5 2!§8** + i ? :§** " 3 14.8 ± 2 .2 + 10.9 ± 1.33 --P CO .1772 ± 2.77 + + in -P CO <rH CD H O H 9 . 2 0 . 4 ± 2 . 1 1 + + Pi rH S <D : H Pi 14 ±ZH + . 2 2 . 1 . ± 3 . 0 6 24 + ± 3 . 1 5 + £n CO d o d o 3 ± 2.89 + . 1 1 . 4 ± 1.74 -•H O -H -P CD CO ^ i . - i i — • ± 1.43 -CD O O1 <D •r-3 CO Pi Cj CO 14 — ± 1 . 8 5 — * immature f i s h ** s e x u a l l y mature f i s h H d CO 21 ±4:14 — , .21.6 ± 2 . 5 5 + - 14 -the c e l l s of the convoluted tubules were stimulated to 14 u and above. Increasing the period of androgen treatment i n -creased the c e l l height correspondingly u n t i l the l a t t e r reached about 30 u a f t e r 30 days (Table I ) . The increase became more and more gradual a f t e r nine days of treatment and leveled o f f from about the 18 days onward suggesting that there would be a maximum height f o r these e p i t h e l i a l c e l l s around 30 u as indicated i n F i g . 1. The c e l l height of the treated male was consistently s l i g h t l y higher than that of the female as i n d i -cated. The c e l l s of male, castrates stimulated to nest-building a f t e r 14 days and 19 days of androgen treatment were about 24 u and 28 u which correspond to the other castrated male treated f o r the same number of days but which did not show the repro-ductive behaviour. As measured by the response of these e p i t h e l i a l c e l l s (Table l ) , treatment by androgen immersion was as e f f e c t i v e as implantation. Injection of aqueous solution, however, was not r e l i a b l e , as shown by the i r r e g u l a r i t y i n the response. This was probably because the suspension was not uniform and the p a r t i c l e s also tend to block the needle during the i n j e c t i o n so that one f i s h would receive a larger dose than the other. b. Unoperated Juvenile Male and Female (length< 3 . 5 cm) Juvenile sticklebacks, c o l l e c t e d in.July, immersed i n the methyl testosterone bath, showed a s i m i l a r response. C e l l s of the convoluted tubules were stimulated to form granular c e l l s with an increase i n the c e l l height while that of the c o l l e c t i n g tubules developed into clear mucous c e l l s . The T o f o l l o w p a g e 14 F i g u r e 1. H e i g h t o f e p i t h e l i a l c e l l s o f t h e k i d n e y t u b u l e s o f c a s t r a t e d a d u l t m a l e a n d f e m a l e i m m e r s e d i n s o l u t i o n o f m e t h y l t e s t o s t e r o n e To follow page 14 Figure 2. Height of e p i t h e l i a l c e l l s of the kidney tubules of unoperated male and female immersed i n solution of methyl testosterone. S o l i d c i r c l e s , juvenile male; open c i r c l e s , juvenile female - 15 -the response was s i m i l a r i n these unoperated males and females as summarized i n Table I. Like the gonadectomized adults the response was much more rapid at the beginning of the treatment, with an increase by about 40% a f t e r the f i r s t three days, declining a f t e r they had been maintained i n the hormone bath for 12 days (Fig. 2 ) . In both groups, mucous c e l l s were absent when the granular c e l l s were 12 - 13 u, although they were present when the l a t t e r were 16 - 18 p.. They probably appear when c e l l s were around 14 p. as i n gonadectomized adults although no f i s h with c e l l s of that height was available. None of the treated juvenile showed the nest-building behaviour during the 21 days even though maintained under the 16 hours photoperiod. The above studies indicated that methyl testosterone treatment induces the development of the kidney tubules, a male secondary sexual c h a r a c t e r i s t i c , i n gonadectomized adult male and female as well as i n non-operated juveniles. The response was s i m i l a r i n both sexes i n the two groups, and when maximally stimulated i t was comparable to the mature male i n nature with the development of the granular and mucous c e l l s . The increase i n c e l l height was a function of the period of treatment reaching a possible maximum height similar to the mature male a f t e r 30 days. B. FRE-SPAWNING AGGRESSIVE BEHAVIOUR Gonadectomized f i s h , transferred to the constant environ-ment room the f i r s t week af t e r gonadectomy, showed varied l e v e l s of aggressiveness. No f i s h , i n i t i a l l y i n l e v e l $IV of - 16 -aggressiveness was used i n the experiment. Those used varied from #0 -#III. Moreover, they were introduced into a com-p l e t e l y new environment and were also s t i l l recovering from the operation. Thus, regular observations were not made u n t i l the second week af t e r the gonadectomy when most of them were at a low l e v e l of aggressive'ness of #0 -#II with 10 - 13 attacks per f i v e minutes observation, whether under 8 hours or lb hours il l u m i n a t i o n . The pre-spawning aggressive behaviour was studied i n r e l a t i o n to photoperiod, androgen treatment and genetic, sex. !• Photoperiod E f f e c t In one experiment (Experiment V), gonadectomized f i s h maintained under long and short photoperiod were compared. Eight tanks of males and eight of females were kept under each i l l u m i n a t i o n . A gradual increase i n the number of attacks from 2 - 12 per f i v e minutes at the second week to 15 - 33 per f i v e minutes observation on the f i f t h week occurred i n f i s h main-tained under long photoperiod. The l e v e l of aggressiveness also changes from #0 - #1 to #111 - #IV on the f i f t h week. However, when 80 - 90% of the f i s h were at l e v e l #IV on the s i x t h week, the number of attacks observed dropped markedly to 2 - 1 2 because the subordinate f i s h were a l l hiding among the weeds and rocks and the dominant f i s h no longer found any "cause" f o r attack. When the "standard f i s h " was introduced i n Experiment V i n four of the tanks, the number of attacks remained at the high l e v e l , above 30 attacks i n f i v e minutes, since the "standard f i s h " could not hide from the dominant f i s h . Figure 3 i l l u s t r a t e d the increase followed by the sudden - 17 -drop i n t h e mean number of a t t a c k s i n the. males and f e m a l e s two, f i v e and s i x weeks a f t e r gonadectomy when m a i n t a i n e d under l o n g p h o t o p e r i o d . I n b o t h males and f e m a l e s , t h e l o n g p h o t o p e r i o d produced an e v i d e n t i n c r e a s e i n a g g r e s s i v e n e s s w i t h i n t h e f i r s t f i v e weeks. A comparison o f F i g . 3A and B showed t h a t the a g g r e s s i v e b e h a v i o u r e l i c i t e d by the. l o n g p h o t o p e r i o d was s i m i l a r i n b o t h s e x e s . There was no p a r a l l e l i n c r e a s e i n f i s h m a i n t a i n e d under s h o r t p h o t o p e r i o d ( F i g . J ) . There was o n l y a s l i g h t i n c r e a s e i n f i s h o f b o t h ' s e x e s on t h e 5 t h week under t h i s s h o r t p h o t o p e r i o d w i t h a d e c r e a s e on t h e s i x t h week; but t h e d i f f e r e n c e was not s i g n i f i c a n t . There was much o v e r l a p p i n g i n t h e number of a t t a c k s , e s p e c i a l l y i n t h e male c a s t r a t e s . Thus, th e s l i g h t d i f f e r e n c e may j u s t be due t o some random v a r i a t i o n o r i n c r e a s e d f a m i l i a r i t y w i t h t h e s u r r o u n d i n g s r a t h e r t h a n a fundamental change i n b e h a v i o u r . By t h e 6 t h week, 80 - 90% o f t h e f i s h under t h e l o n g p h o t o p e r i o d showed d e f i n i t e d o m i n a n t - s u b o r d i n a t e r e l a t i o n among t h e f i s h i n each t a n k ; w h i l e o n l y 10 - 25% o f t h o s e under t h e s h o r t p h o t o p e r i o d had r e a c h e d t h e same l e v e l o f a g g r e s s i v e n e s s . T a b l e I I showed a comparison between t h e number of t a n k s of f i s h under 8 hours w h i c h r e a c h e d l e v e l #Iv" and t h e c o r r e s p o n d i n g number under 16 hours i l l u m i n a t i o n , A C h i Square t e s t ( i n c l u d i n g Y a t e s c o r r e c t i o n ) o f t h e number o f t a n k s showing and not showing t h e t e r r i t o r i a l b e h a v i o u r between t h e s h o r t and l o n g h o u r s f i s h , f i r s t i n t h e male and t h e n i n t h e f e m a l e c a s t r a t e , showed t h e f i s h under s h o r t p h o t o -p e r i o d was s i g n i f i c a n t l y d i f f e r e n t f r om t h a t under l o n g To follow page 17 Figure 3. Mean number of attacks (with confidence limit at 95% level) of gonadectomized males and females after having been maintained under long (16 hr) and short (8hr) photoperiod for 2, 5 and 6 weeks. Values are average levels of activity (week indicated) of the 8 tanks of each sex. Level of activity determined from three ^-^iB-dte observations of each tank. A = males B - females A . 3 0 + 20 + (J < O I B. 3 0 4 204 IO + I 16 H O U R S I H 1-I -I 1-I 8 H O U R S i 1 1 •4 r W E E K S TABLE I I Number and percent of tanks of f i s h maintained under long and short photoperiod, with and without t e r r i t o r y s i x weeks a f t e r gonadectomy GONADECTOMIZED MALE GONADECTOMIZED FEMALE Photo-period No. tanks with t e r r i t o r y No. tanks % tanks without with t e r r i t o r y t e r r i t o r y No. tanks with t e r r i t o r y No, tanks without t e r r i t o r y % tanks wi th t e r r i t o r y 8 hr. 2 6 X" 4 . 0 6 25 % 1 7 ^ - . 381 12% 16 hr. 7 1 p < . 0 5 88 % 7 3 p ™ . 0 5 70% Number and TABLE I I I . ..... percent of tanks of gohadect6mi-zed Afish with or without s i x weeks a f t e r gonadectomy t e r r i t o r y UNDER 16 HOUR ILLUMINATION UNDER 8 HOUR ILLUMINATION SEX No. tanks with t e r r i t o r y No. tanks % tanks without with t e r r i t o r y t e r r i t o r y No. tanks with t e r r i t o r y No. tanks without t e r r i t o r y % tanks with t e r r i t o r y 20 6 * a 0 . 0 5 77 % 2 6 ^ = 0 25% 27 11 p > 0.9 71 % 1 7 P ^  0 . 9 14% - 19 -photoperiod which i n d i c a t e d t h a t the l o n g p h o t o p e r i o d e f f e c t was d i f f e r e n t from t h a t of the s h o r t i l l u m i n a t i o n . 2. Sex There was no a p p r e c i a b l e d i f f e r e n c e between the changes i n a g g r e s s i v e n e s s i n the male and female c a s t r a t e s . A t o t a l of 25 tanks of male and 35 tanks of female c a s t r a t e s were exposed t o 16 hours i l l u m i n a t i o n i n the f o u r d i f f e r e n t experiments whose changes i n a g g r e s s i v e behaviour c o u l d be : compared. Both sexes were at a low l e v e l i n i t i a l l y on the 2nd week, i n c r e a s i n g g r a d u a l l y t o a maximum on the 5 t h week and th e n dropped suddenly on the 6 t h week when they were kept under the longer.hours of I l l u m i n a t i o n . N e i t h e r the 8 tanks of male nor the s i m i l a r number of female c a s t r a t e s showed such a marked i n c r e a s e when maintained under the sho r t p h o t o p e r i o d , although t h e r e was some i n c r e a s e among the females. F i g u r e 4 showed the number of a t t a c k s among the c a s t r a t e d males and females on the 5 t h week when the a g g r e s s i v e behaviour was at a maximum. I t i s apparent t h a t t h e r e i s no s i g n i f i c a n t d i f f e r e n c e between the two l e v e l s under the long or the sho r t p h o t o p e r i o d . Seventy per cent of the gonadectomized females and 75% of the males showed the dominant-subordinate r e l a t i o n ( L e v e l § IT) s i x weeks a f t e r gonadectomy under long photo-p e r i o d w h ile 13% females and 25% males i n the group under sho r t p h o t o p e r i o d showed t h i s c o n d i t i o n as recorded i n T a b l e I I I . The Ghi Square a n a l y s i s showed t h a t the males and females r e s -pond s i m i l a r l y i n i t s development of t e r r i t o r i a l behaviour no matter whether under long or s h o r t hours of i l l u m i n a t i o n and the d i f f e r e n c e s are probably due t o chance v a r i a t i o n . To follow page 19 Figure 4 . Mean number of attacks (with confidence l i m i t at 95% level) of gonadectomized males and females si x weeks afte r gonadectomy. Values are average l e v e l of a c t i v i t y on the 6 t h week of 8 tanks of both sexes under 8 hours photoperiod and 26 tanks of males and 38 tanks of females under 16 hours photoperiod ATTACKS 2 > m co T l m > r" rn co m co m > i — m co - 20 -3. Androgen Treatment Whether the a d m i n i s t r a t i o n of androgen had any e f f e c t on the a g g r e s s i v e behaviour of the c a s t r a t e s was not c o n c l u -s i v e l y shown. I t was e v i d e n t , however, t h a t under the l o n g photoperiod, androgen treatment was not e s s e n t i a l f o r stimu-l a t i n g t he c a s t r a t e s t o h i g h l e v e l of a g g r e s s i v e behaviour -as i n d i c a t e d by the i n c r e a s e number of a t t a c k s or by the d e v e l -opment of t e r r i t o r y . I n a l l the experiments under l o n g photo-p e r i o d , androgen was i n t r o d u c e d i n t o the tank a f t e r .80% of them co n t a i n e d t e r r i t o r i a l f i s h , u s u a l l y about s i x weeks a f t e r gonadectomy. A f t e r the a d d i t i o n of methyl t e s t o s t e r o n e , the dominant f i s h remain dominant w h i l e the r e s t of the f i s h remain h i d i n g so t h a t the number of a t t a c k s was low as b e f o r e . I n Experiment Y where the "standard f i s h " was i n t r o d u c e d to eval u a t e the number of a t t a c k s , v a r y i n g changes occu r r e d . Among the c a s t r a t e d males, t h e r e was a decrease i n the number of a t t a c k s i n f o u r tanks, no apparent change i n one, tank, and an i n c r e a s e i n one tank. Among the females, two tanks showed decrease w h i l e one tank remained unchanged. T h i s seem t o suggest a decrease i n the number of a t t a c k s on the "standard f i s h " w i t h the a d d i t i o n of methyl t e s t o s t e r o n e although the r e s u l t was not c o n c l u s i v e because of the few number of tanks of f i s h used. A p o s s i b l e reason f o r t h i s c o n s i d e r e d i n the d i s -c u s s i o n . The low l e v e l of aggressiveness i n s i x tanks of males and seven of females ( t o t a l of e i g h t tanks o f each) under the sho r t p h o t o p e r i o d was not a f f e c t e d by androgen a d m i n i s t r a t i o n . There was n e i t h e r any apparent i n c r e a s e i n the number of a t t a c k s - 21 -nor a development of t e r r i t o r y when methyl testosterone was introduced. However, i n two tanks of treated male castrates and one of female, where t e r r i t o r y was developed before the addition, there was a loss of the t e r r i t o r i a l i t y a week a f t e r the f i r s t addition. In summary, the pre-spawning aggressive behaviour of castrated male and female sticklebacks leading to the develop-ment of t e r r i t o r y was increased by being maintained under the long photoperiod but not by the short photoperiod. The response was sim i l a r i n both sexes. Methyl testosterone had no apparent e f f e c t on t h i s aggressiveness although there was s l i g h t i n d i c a -t i o n of suppressing e f f e c t . C. NEST-BUILDING BEHAVIOUR Var i a t i o n i n photoperiod i t s e l f had no effect i n inducing the nest-building behaviour i n gonadectomized adult sticklebacks. Gonadectomized f i s h never b u i l d nests unless stimulated by androgen. Methyl testosterone treatment was es s e n t i a l i n s t i -mulating the response i n both sexes. Table IV summarized the e f f e c t s of long or short photoperiod combinediwith androgen ^treatment i n e l i c i t i n g the nest-building behaviour. One of the f i r s t points of intere s t i s what appears to be a seasonal e f f e c t . I t was noted that i n Experiments I and IV, which were performed i n the summer, the time to respond was comparatively shorter than i n Experiments I I and V, performed i n the f a l l and winter. In most situations, the"aggressive f i s h showed nest-building, although t h i s was not always true. A subordinate f i s h might start to b u i l d a nest i n an obscured corner of the tank where TABLE IV The e f f e c t of photoperiod and methyl testosterone treatment i n inducing nest-building behaviour (sexual maturation), i n castrated males and females Exper--iment Date Under 16 hour illumination Castrated Male Castrated Female No. tanks No. f i s h fo f i s h with gonad regene-r a t i o n i f i s h mature Time range i n day No. tanks No. f i s h f0 f i s h with gonad regene-ration % f i s h mature Time range i n days I June-Aug. I960 6 12 7 58 13-23 6 12 7 8 20 IV May-J u l y 1961 7 28 0 57 9-27 12 48 2 6 2 5 - 3 0 II Sept-Nov. I 9 6 0 6 24 0 50 22-30 6 14 7 7 26 V Sent-7 14 7 64 19-36 8 12 8 0 Nov. * 1961 - 4 8 10 25 31-40 6 12 17 0 These f i s h under 8 hour illumination. - 23 -i t would be least noticed by the dominant f i s h . I t would i swim out occasionally to c o l l e c t nest material while the dominant f i s h was occupied elsewhere i n the tank. The pre-sence of other subordinate f i s h near i t s nest did not bother i t . In two of the experiments, however, three very aggressive f i s h which were building nests themselves did suppress a sub-ordinate f i s h i n the same tank from building a nest. The l a t t e r started to c o l l e c t material, but i t was attacked f i e r c e l y by the dominant f i s h , and a f t e r a few hours i t ceased building and hid i n the corner of the tank. 1. Photoperiod and Unoperated Adult Males Normal male adults ( > 4 cm) co l l e c t e d i n winter matured and showed nest-building when maintained under 16 hours i l l u m i n a t i o n . In an experiment i n January - March, with 8 tanks of f i s h (among which were 10 males), 80% of the males matured i n 16 - 49 days while they were kept under the long photoperiod. In the 8 tanks under short photoperiod (with t o t a l of 8 males), only one f i s h matured i n 47 days. This experiment can be compared only with Experiment V because only two f i s h were present i n each tank ( i n a a l l the other experi-ments there were 4 f i s h ) . Sixty-four percent of androgen treated male castrates matured i n Experiment V i n 19 - 36 days while 80% matured i n 16 - 49 days i n the normal f i s h . A s l i g h t difference i n the response i n the two groups i s evident. 2 . Photoperiod and Androgen Treatment on Gonadectomized Males and Females a. E f f e c t of Photoperiod on the Intensity and Time to Response of the Two Sexes to Androgen Treatment - 24 -The difference i n photoperiod modified the per-centage and the time to response of the f i s h treated with methyl testosterone. Only 25% of the male castrates ( 2 f i s h ) and no females were induced to b u i l d a nest within a period of 31 - 40 days a f t e r androgen treatment when under short photoperiod, whereas 64% (9 f i s h ) of the castrated male, but also no females, responded a f t e r 19 - 3 6 days of androgen treatment. In a l l the experiments under the long photo-period, a higher percentage of castrated treated males showed nest-building than s i m i l a r l y treated females. An average of 50 - 6 0 % of males responded a f t e r 9 - 3 6 days of androgen administration while only 5 - 10% of females showed the nest-b u i l d i n g a f t e r 20 - 30 days of treatment. b. Q u a l i t a t i v e and Quantitative Comparison of the  Nest-building Behaviour in Normal Males, Treated  Castrated Males and Gonadectomized and Treated  Females A comparison of the actual nest-building move-ments i n the n a t u r a l l y matured male and the treated castrated male was made. Table V was a record for 19 n a t u r a l l y matured nest-building males (NA ,to NT). The elements of nest-building behaviour were categorized as mentioned previously with sand-digging omitted because t h i s phase was very short and thus usually not noticed. The "percent time" recorded i n the table was the per cent of time during that p a r t i c u l a r observation i n which t h i s p a r t i c u l a r element was performed; and the "day" was the number of day from the f i r s t day of nest-building (except for fanning which i s number of days from spawning) - 25 -TABLE V Time of appearance of maximum a c t i v i t y f o r d i f f e r e n t behaviour patterns•together with percent time so occupied by males maturing under natural conditions. F i s h above double l i n e a c t u a l l y spawned. Further de s c r i p t i o n i n text Fanning Fanning N C o l l e c t i n g (after (no material Gluing Pushing spawning ;) spawning) i i 1 % % Day Time Day Time Day Time Day Time Day Time NA 1 23 1 4 4 19 4 55 NB 1 28 2 7 5 23 6 30 NC 1 16 2 5 1 16 4 44 ND 1 22 2 7 1 18 6 30 NE 1 20 2 6 2 14 6 41 NG 1 19 2 8 2 22 6 27 NL 1 23 2 4 2 13 6 32 NP 1 26 2 5 2 5 7 51 NKi 1 21 1 3 1 8 7 37 NKg 1 18 1 6 1 22 6 39 NF 2 28 2 4 3 14 2 m 2 39 2 3 1 11 8 NJ 1 20 1 3 3 12 6 NM 1 17 2 3 2 - 18 7 NN 2 21 2 4 1 6 4 NQ 1 25 1 6 1 17 7 m 1 28 3 • 5 1 11 6 NS 1 21 1 5 1 8 5 NT 1 27 1 3 2 9 4 Average 1-2 1-2 i 1-5 4-7 i n t e n s i t y days 24% days 5% days 15% days 39% 5% - 26 -dn which that element was performed f o r the maximum percentage of time i . e . performed at a maximum i n t e n s i t y . Thus, fo r f i s h NA, there was maximum of c o l l e c t i n g nest material (23% of the time) and gluing (4% of the time) on the f i r s t day of nest-b u i l d i n g . However, the f i s h spent maximum time on pushing nest material on the fourth day. Fanning reached a maximum on the fourth day af t e r spawning. Table vT was a si m i l a r record f o r 23 castrated androgen treated males (GA to GW). The average maximum i n -t e n s i t y of the d i f f e r e n t elements f o r the naturally matured males was consistently s l i g h t l y higher than that of the experi-mental males; although the difference was not great. The d i f -ferences i n the i n t e n s i t y of c o l l e c t i n g material and f o r parental fanning were most evident. The number of days a f t e r the f i r s t day of nest-building i n which the d i f f e r e n t movements reached a maximum was also s i m i l a r i n the normal and castrated male except f o r the parental fanning which reached a much higher i n t e n s i t y and also l a t e r i n the intact than i n the castrated male. The nest-building of the treated castrated male was s i m i l a r to that of the normal male i n that maximum nest-building occurred on the f i r s t or second day a f t e r which the f i s h reached the sexual phase and were ready to court any mature female introduced. I f no female was available, the sexually mature f i s h remained i n the nest maintaining stages showing occasional c o l l e c t i o n of material, displacement gluing or fanning, and pushing nest material. Thus, one could observe some elements associated with nest-building everyday, although - 27 -TABLE VI Time of appearance of maximum a c t i v i t y f o r d i f f e r e n t behaviour patterns together with percent time so occupied by androgen treated castrated males. F i s h above double l i n e a c t u a l l y spawned. Further description i n text. Fanning Fanning GM C o l l e c t i n g (a f t e r (no material Gluing Pushing spawning) spawning) 1 1 1 Day Time Day Time Day Time Day Time Time GA 2 18 3 3 3 5 2 16 GB 1 16 2 3 1 11 2 19 GD 1 15 3 8 5 26 2 18 GE 1 19 2 6 1 11 2 19 GI 2 23 2 2 3 7 3 15 GM 2 10 3 2 3 7 2 18 GP]. 1 18 2 5 2 10 3 18 GP? 1 15 1 3 3 11 3 20 GQ 1 14 . 1 2 1 7 5 20 GS 1 16 2 2 1 16 4 18 GC 1 17 4 3 1 29 9 GF 2 19 2 4 3 8 3 GH 1 14 2 2 1 11 4 GJ 1 16 2 1 2 8 5 GK 1 28 2 4 2 6 4 GL 2 13 1 2 2 7 7 GN 1 18 3 3 3 12 8 GP 1 18 3 4 3 10 1 G P z 1 17 1 2 1 9 2 GR 2 21 1 3 3 13 6 GT 1 22 1 2 1 7 4 GU 2 17 1 1 3 8 1 GV 1 20 1 2 1 9 3 GW 1 15 2 1 2 12 5 Average 1-2 1-3 1-3 i n t e n s i t y days 197° days 3% days 11% days 20% • 4% - 2 8 -at a r e l a t i v e l y low I n t e n s i t y . Q u a l i t a t i v e l y , the nest of castrated male was similar to that of the normal male, consist-ing of a neat p i l e of algae glued smoothly together. The sides were securely embedded i n the sand. The castrated male, l i k e the normal f i s h , bored through the nest to make i t a tunnel-l i k e structure when the nest was f i n i s h e d . Only 5 - 8 f . of females were stimulated to b u i l d nests by the methyl testosterone treatment under the long photo-period. Q u a l i t a t i v e l y , the nests of the females were d i f f e r e n t from those of castrated or normal males. The nests were merely loose masses of algae p i l e d together - neither pushed together t i g h t l y nor glued together smoothly. They dug a shallow p i t i n which the algae were co l l e c t e d and l a i d loosely together. No d e f i n i t e entrance exists i n these nests because none of the nest-building females bored through the mass of algae which was a f l a t mat i n the shallow depression and not a tunnel-shaped structure l i k e that of the normal male. Five treated castrated females had a nest i n t h e i r tanks although records of actual nest-building was ob-tained with only four of them. Table VII recorded the per cent of time, i n each observation, i n which the d i f f e r e n t elements belonging to the nest-building behaviour was performed. For f i s h GFA, nest-building movements were observed on the f i r s t , second, sixth, eigth and nineth day, counting from the f i r s t day of nest-building, but absent i n a l l the other days. For GFB, nest-building was seen only f o r three days, GFG, one day only and GFD, f o r three days. Thus i n a l l the f i s h , there were days i n which nest-building was completely absent, reappearing - 29 -T a b l e V I I Record of the percent time which f o u r n e s t - b u i l d i n g female c a s t r a t e s spent on each elements of the n e s t - b u i l d i n g behaviour p a t t e r n on the d i f f e r e n t days a f t e r n e s t - b u i l d i n g was f i r s t s t a r t e d No. day % time Code no. of n e s t - on sand C o l l e c t i n g f i s h b u i l d i n g d i g g i n g m a t e r i a l Cluing Pushing Fanning GFA 1st 1 26 1 13 2 2nd 0 8 4 . 4 1 3rd-5th no nest-• b u i l d i n g elements observed 6 t h 2 7 4 10 1 7th no nest-• b u i l d i n g 8 t h 1 1 2 2 3 9th 1 1 1 1 4 GFB 1st 10 24 0 7 0 2nd 1 2 2 1 1 3rd no nest-• b u i l d i n g 4th 2 13 0 1 0 no more n e s t - b u i l d i n g observed GFC 1st 4 4 2 . 8 0 no more n e s t - b u i l d i n g observed . GFD '. 1st 5 5 0 0 1 2nd-5th no nest-• b u i l d i n g observed 6th 3 3 0 1 0 7th-9th no nest-• b u i l d i n g observed < 10th 2 1 0 7 2 - 30 -suddenly a few days l a t e r . This seldom occur i n normal or androgen treated castrated males which show continuous a c t i v i t y f o r many days. From these records, however, i t i s evident that females can show a l l the elements associated with nest-building i n the normal or treated castrated males, except boring through the nest at the end; no gluing was observed i n one of the f i s h . The maximum i n t e n s i t y of the d i f f e r e n t movements i n the f i s h were also s i m i l a r to the normal male f i s h . The above studies showed that long photoperiod together with methyl testosterone treatment induced the nest-building behaviour i n castrated males and females, at a much higher percentage i n the former and a r e l a t i v e l y low percentage i n the l a t t e r . There was no apparent difference i n the nest-building movements i n the experimental males and normal males matured i n nature. Treated castrated females, however, seemed to be d i f f e r e n t as indicated by the q u a l i t y of the ' f i n i s h e d " nest as well as the absence of the f i n a l elements of nest-building, v i z . boring through the nest. D. SEXUAL BEHAVIOUR Mature male sticklebacks i n nature become highly motivated sexually as soon as the nest i s completed. The male w i l l then court any gravid female by the zigzag dance, lead the l a t t e r to i t s nest, induce her to lay the eggs which he f e r t i l i z e s , usually immediately. The male w i l l court and spawn with two or - 31 -three more females; and i n subsequent days w i l l fan the eggs . u n t i l the young hatched. I f no mature females are available, displacement a c t i v i t y such as fanning and gluing w i l l be ob-served. No quantitative comparison of the sexual behaviour between the normal and treated castrated males was made except for the displacement and parental fanning. Androgen treated castrated males courted gravid females with the zigzag dance, led the l a t t e r to the nest, and stimu-lated her to lay eggs just as did the normal males. Then, similar to the normal male, the castrate also showed movements of " f e r t i l i z i n g " the eggs by entering the nest a f t e r the female had l a i d the eggs and performed.the appropriate movements? although presumably no sperm would be emitted. Castrates repeat the same courtship with several gravid females available just as normal,males. On subsequent days, male castrates also show the parental fanning. However, as shown i n Table VI, the maxi-mum parental fanning performed by these castrates was only up to approximately 20% on the 2nd - 3rd day af t e r spawning had occurred. This was much lower than the maximum parental fan-ning performed by the normal male, an average of 40% on the 4th - 7th.day af t e r spawning. Displacement fanning and gluing were also shown by male castrates i f no gravid females were ava i l a b l e . Nest-building females never show any sexual behaviour. Sgme fanning was observed which was probably the low l e v e l of fanning associated with nest-building, also regularly observed i n male castrates and normal males. The nest-building female paid no attention to the gravid female introduced andv.would ignore the l a t t e r or attack her as i f attacking any; f i s h intruding into i t s t e r r i t o r y . No zigzag dance or leading or> any other elements of the courtship behaviour was ever observed with these androgen treated females. In summary, castrated males could be induced to perform a l l the elements associated with the sexual behaviour of normal male stickleback, although the castrated females seem to be g e n e t i c a l l y incapable of performing them. - 3 3 -IY. DISCUSSION A. GONADAL STEROIDS AND KIDNEY DEVELOPMENT The normal functions of the kidney of the vertebrates are excretion and maintenance of e l e c t r o l y t e balance. However, i n the male sticklebacks, as well as i n a number of other lower vertebrates such as l i z a r d s and snakes, the kidney assumes a t h i r d function i n the breeding season associated with reproduc-t i o n . Bishop ( 1 9 5 9 ) reported the presence of the "reproductive segment" i n the kidney tubule of the garter snake. The exact function of t h i s segment i s s t i l l unknown although i t has been demonstrated to be under the dir e c t control of androgens. The kidney tubule i n the sexually matured male stickleback i s responsible f o r secreting the mucous substance which i s used to glue the nest material together. ; Oourrier ( 1 9 3 2 ) i d e n t i f i e d f i v e d i f f e r e n t parts i n the kidney nephron of the stickleback and described changes i n some segment during reproduction! The glomerulus and the neck re-gion, he pointed oyt, were the same i n breedingand non-breeding males and females; while the d i s t a l and proximal convoluted tubules were stimulated to form granular c e l l s ; and f i n a l l y the c o l l e c t i n g tubules to form clear mucous c e l l s i n the spawning male during the breeding season. In the sexually matured male, the glomerulus and the neck region of the nephron probably perform the normal physiology of excretion and e l e c t r o l y t e balance while the tubules assume the secretory function f o r reproduction. Castration of spawning male f i s h resulted i n the atrophy - 34 -of these secretory c e l l s which gradually returned to the immature condition as i l l u s t r a t e d by Ikeda ( 1 9 3 3 ) . Ikeda recorded the height of the secreting c e l l s d a i l y a f t e r the castration for 50 days during which the c e l l s decreased i n height by 65%. This was comparable to the present data i n which the height ef the granular convoluted tubule c e l l s of the mature male was reduced by approximately 60% i n a shorter period - three weeks af t e r castration. Tubules of the gonad-ectomized females, however, remained the same as the gravid or non-gravid females - similar to those of the males three weeks a f t e r castration. This suggests that-estrogen from the normal female has l i t t l e or no effect on stimulating the kidney t i s s u e of the f i s h , whereas the androgen, secreted at a p a r t i -c u l a r l y high l e v e l i n the breeding season, i s responsible f o r the hypertrophy of the tubules. That the development of the kidney tubule c e l l s was under the d i r e c t control of the male gonadal stero i d was demonstrated' e a r l i e r by the administration of the hormone to castrated i n d i v i d u a l s . Oguro (1958) implanted methyl testosterone and estrogen into normal and castrated male and female sticklebacks and measured the height of the stimulated c e l l s ten days and 30 days a f t e r the implant. Estrogen had no e f f e c t ; but methyl testosterone stimulated the c e l l s of castrates of both sexes as well as i n normal females; although the height i n the l a t t e r was about 14% lower than that of the gonadectomized females. This seemed to indicate a possible suppressing effect of the estrogen, secreted by the normal female, counteracting that of the androgenic e f f e c t . The present data provide a more det a i l e d study of the effects of administration of methyl testosterone on the kidney tubules using castrated adult and normal juvenile f i s h . The Increase i n the c e l l height was measured at three days i n t e r v a l so that the response of the kidney could be quantified i n terms of increasing number of days of androgen treatment. As i n d i -cated i n F i g . 1 , the i n i t i a l response of the kidney tissue was r e l a t i v e l y rapid during the f i r s t three days of treatment, becoming more and more gradual with increasing periods of treatment, eventually l e v e l i n g o f f , thus suggesting a maximum height, comparable to the normal mature male. Oguro ( 1 9 5 8 ) found no apparent difference i n the c e l l height of the gonadectomized males and females a f t e r 3 0 days of treatment. The present data showed no s i g n i f i c a n t d i f f e r -ence i n the c e l l height either at the beginning of the t r e a t -ment ( 3 days) or af t e r 3 0 days. This suggests that the female kidney i s as sensitive as the male and responds at the same rate as well as to the same degree with androgen treatment. Thus the p o t e n t i a l i t y of the kidney tissue to assume the se-cretory function i s present equally i n both sexes and not i n any way sex-linked. The gonadal steroids are apparently responsible f o r the development of the tissues, with the high l e v e l of androgen secreted i n the breeding season stimulating the c e l l s to secretory a c t i v i t y . The estrogen seems to have no d i r e c t action on the tubules so that, i n normal females,, without the influence of androgen, the kidney tubules remain i n their-quiescent state with the c e l l height si m i l a r i n gonadectomized or normal females. - 36 -The c e l l height of the treated male castrates was con-s i s t e n t l y s l i g h t l y higher than that of the gonadectomized female as F i g . 1 indicates. As mentioned previously, the difference was not s t a t i s t i c a l l y s i g n i f i c a n t , although i t was a very consistent phenomenon; and i t seemed to suggest the possible priming ef f e c t of the gonadal steroids on the kidney tissue. The androgen,secreted by the t e s t i s previously might have a s e n s i t i z i n g effect on the kidney while previous effect of the estrogen might have an opposing effect of suppressing the c e l l development, so that with the same dosage of methyl testosterone the height of the c e l l s i n the females i s consistently lower. On the other hand, the observed differences may be related to differences between the natural and synthetic hormone. The response of the kidney tissue of the juveniles was i d e n t i c a l f o r both sexes with the points coinciding as i n F i g . 2. It was also comparable to that of the gonadectomized adults. There were underyearlings which hatched i n the spring and were col l e c t e d i n early July. Their gonads, thus, had never reached sexual maturity and were l i k e l y to be i n the phase $0 of development with both the germ epithelium and the i n t e r s t i t i a l c e l l s not yet developed (Baggerman, 1957). Thus, the kidney tissue of these f i s h had probably never been affected by gonadal steroids u n t i l androgen was administered to them i n the experi-ment, i n contrast to the adults which had been under some influence of androgen or estrogen u n t i l the gonadectomy. Since the juveniles of both sexes responded i d e n t i c a l l y to the androgen treatment while there was a s l i g h t difference between adult males and females, i t seems to agree with the p o s s i b i l i t y of some - 37 -priming effect of previous influence of the gonadal steroids. The administration of the steroid by implanting the hormone pellets, or by simply adding i t to the water was equally e f f i -cient as measured by the degree of response in the kidney tissue. Each method, however, also had i t s limitations. Adding the known concentration of hormone to the water was simple and direct; but a l l that was known about the actual concentration in the water after the addition was that i t had a concentration which would induce a response in the kidney tissue and the reproductive behaviour of the f i s h . The solubility of methyl testosterone in water is very low and not yet known. It might be that only part of the added androgen went into solution in the tanks. Moreover, with the sand, weed, rocks and the f i l t e r -ing system used in the tanks, not to mention the presence of the f i s h themselves, the rate of deterioration of the hormone might be considerable. The significance of these factors, i s s t i l l unknown. Attempts were made to analyze the actual content in the water each week by paper chromatography, but no satisfactory results have yet been obtained. Implanting the androgen pellets was a more precise tech-nique. One can introduce a known weight of excess androgen into the abdominal cavity, weigh the pellet again at the end of the experiment and thus obtain an estimation of the quantity of the androgen used. However, this involved accurate weighing of the minute quantity that was l i k e l y to be absorbed, the drying of the pellet at the end of the experiment, and the additional surgery of the f i s h . In practice, the method of adding hormone to the water was used in preference because the implant - 38 -necessitate a second operation, following gonadectomy. This i n e v i t a b l y disturbed the behaviour which was under observation during the period when hormone treatment was started. Once the. association between the effect of androgen and the development of the kidney tissue has been accepted and.the quantitative r e l a t i o n between the dosage and and degree of response established, the appearance of t h i s secondary sexual c h a r a c t e r i s t i c can be used as an i n d i c a t i o n of the e f f i c i e n c y of u t i l i z a t i o n or absorption of the hormone by the f i s h . By observing whether there i s any changes induced on the kidney t i s s u e , one can determine whether the f i s h i s getting access to and absorbing the androgen or not. S i m i l a r l y i t might also be used f o r the bioassay of material suspected to have andro-genic a c t i v i t y by administering the l a t t e r to the f i s h and then measuring the effect that i t w i l l have on the kidney tissue. B. PRE-SPAVJNING AGGRESSIVENESS AND ITS .ENDOCRINE CONTROL Generally, the gonadal steroids are believed to have a d i r e c t e f f e c t on the reproductive behaviour of fishes'} while the p i t u i t a r y hormones mediate only through the stimulation of the gonads (Baggerman, 1 9 5 7 ) . In c e r t a i n species, t h i s has been demonstrated to be p a r t i c u l a r l y true of aggressiveness whether or not associated with reproduction. Low ranking in d i v i d u a l s i n flocks of hens rose i n t h e i r s o c i a l status a f t e r the i n j e c t i o n of the male steroids as i l l u s t r a t e d by A l l e e et a l ( 1 9 3 9 , 1 9 5 5 ) . This p o s i t i o n i n the s o c i a l order was mainly dependent on the r e l a t i v e aggressiveness of the i n d i v i -duals, so that the importance of the hormone administered i n augmenting the aggressiveness was evident. - 39 -In some mammals, females treated with androgen become more aggressive. B a l l (1940) found that rats injected with testosterone propionate f i g h t more frequently. This was con-firmed by Beach (1942) on castrated i n d i v i d u a l s . Ivans (1952) observed an increase i n aggressiveness and restlessness i n the juvenile box t u r t l e , Terrapeae Carolina, implanted with the male gonadal steroid. Numerous other references, p a r t i c u l a r l y works on the higher vertebrates t e s t i f y to the dir e c t effect of the gonadal steroids, especially androgens, i n increasing the aggressiveness of the individuals (Beach, 1948). Much less l i t e r a t u r e points to direct effects of p i t u i t a r y hormones on aggressiveness; although evidence for the dir e c t influence on other reproductive behaviour i s abundant, some of which might be mentioned. Noble and Kumpf (193b) induced the f i r s t stages of court-ship i n the spayed jewel f i s h by implanting frogs' p i t u i t a r y gland. Pickford (1952, 1954a) observed the spawning r e f l e x i n the hypophysectomized or castrated male Fundulus when she injected p u r i f i e d fractions of pollack p i t u i t a r y glands. This hormonal material was l a t e r demonstrated to be oxytocin and vasopressin (Wilkelmi et a l . 1955). Recent work on birds by Lehrman (1959) also suggested the possible direct effect of the gonadotrophin i n tr i g g e r i n g some elements of the repro-ductive behaviour such as courtship and nest-building. The direct effect of p i t u i t a r y hormone on the aggressive behaviour i n the male gobiid f i s h Bathygobius was demonstrated by Tavolga (1955). He separated the actual courtship and the combat response of the sexually matured male from i t s - 40 -discriminatory power f o r gravid females. The hypophysectomized male l o s t i t s courtship and combat responses while the castrated male retained i t , except that i t would court gravid and non-gravid female as well as other males. This indicated the d i r e c t e f f e c t of the gonadotrophins, or some other elements from the p i t u i t a r y , on e l i c i t i n g the combat and courtship response while the gonadal steroids was involved only i n the discriminatory power of the f i s h . Hoar ( l Q 6 l ) showed that male stickleback castrated during i t s pre-breeding phase retained i t s aggressive behaviour when maintained under long photoperiod, but l o s t i t when maintained under short photoperiod. This was interpreted as due to the high p i t u i t a r y a c t i v i t y i n the former group (stimulated by the long photoperiod) and the low p i t u i t a r y a c t i v i t y i n the l a t t e r group (suppressed by the short photoperiod). Thus, gonadotrophins was suggested to have a di r e c t effect on the pre-breeding aggressiveness of the male castrates, stimulating the male to elevated aggressiveness when present i n high con-centration, and maintaining the f i s h at the r e l a t i v e l y low l e v e l of aggressiveness when at a low hormonal l e v e l . The present data with gonadectomized male and female sticklebacks confirmed the above findings, with the gradual increase i n aggressiveness leading to the development of t e r r i t o r y under long photoperiod and a decrease or retaining the same l e v e l of aggressiveness under the short photoperiod. The castration eliminated the p o s s i b i l i t y of the eff e c t of gonadal s t e r o i d and favours the idea of d i r e c t control by the p i t u i t a r y . Moreover, the addition of androgen to these castrates - 41 s i x to seven weeks af t e r they had been maintained under the constant photoperiod did not greatly a f f e c t the l e v e l of aggressiveness. I t did not increase the aggressiveness of the already aggressive castrates, and even suppress i t i n some cases. Under the short photoperiod, i t did not a f f e c t most of the f i s h and a c t u a l l y eliminated the t e r r i t o r i a l behaviour i n two tanks of male castrates. Thus, the data suggested that the dosage of methyl testosterone administered i n the present experiment di d not a f f e c t the aggressiveness of the castrates either under r tor short photoperiod. I t might have a s l i g h t suppressing e f f e c t , possibly due to the suppression of p i t u i t a r y a c t i v i t y by the feed-back mechanism. Gonadectomized f i s h of both sexes responded i n the same way to the photoperiod i n t h e i r increase i n the number of attacks among themselves and i n the development of t e r r i t o r i a l be-haviour. This indicated that the aggressive behaviour, which was normally c h a r a c t e r i s t i c of the spawning male only, was g e n e t i c a l l y present i n both male and female, that i s , the p o t e n t i a l i t y to become aggressive and to develop a' t e r r i t o r y was not i n any way sex-linked. However, the p h y s i o l o g i c a l d dosage of the androgen i n the breeding male probably tr i g g e r s the aggressiveness or even enhance i t by acting through the p i t u i t a r y , while the estrogen l e v e l i n the gravid or non-gravid female suppresses i t . Hoar (>196l) separated the pre-spawning and spawning ag-gressiveness and suggested that they might be under d i f f e r e n t hormonal control. He showed that gonadeetomy during the pre-spawnin phase did not i n t e r f e r e with the aggressive behaviour of the - 42 -f i s h i f t h e l a t t e r was m a i n t a i n e d under l o n g p h o t o p e r i o d . Baggerman, however, t e s t e d t h e a g g r e s s i v e n e s s o f males c a s -t r a t e d i n t h e n e s t - b u i l d i n g o r s e x u a l phase and r e p o r t e d a marked d e c r e a s e i n t h e number o f a t t a c k s by 5-10 t i m e s f o r a p e r i o d o f a t l e a s t 10 weeks. From t h e s e two o b s e r v a t i o n s , i t was s u g g e s t e d t h a t t h e p i t u i t a r y hormones were r e s p o n s i b l e f o r t h e pre-spawning a g g r e s s i v e n e s s w h i l e t h e a n d r o g e n i c a c t i v i t y was s t i l l l ow i n t h e f i s h . However, as the male becomes s e x u a l l y mature, t h e androgen l e v e l r o s e w h i c h t h e n s u p p r e s s e d t h e p i t u i t a r y a c t i v i t y i n t h e f i s h by t h e feed-back mechanism. A t t h i s l a t e r s t a g e , i t i s i n d i c a t e d t h a t androgen t a k e s o v e r c o n t r o l o f t h e whole r e p r o d u c t i v e b e h a v i o u r i n c l u d i n g t h e a g g r e s s i v e n e s s . Thus, c a s t r a t i o n a t t h i s s t a g e e l i m i n a t e s t h e a g g r e s s i v e b e h a v i o u r . The a c t i v e p i t u i t a r y p r oduces two g o n a d o t r o p h i n s , ( t h e l u t e i n i z i n g hormone (L.H.) and t h e f o l l i c l e s t i m u l a t i n g : hormone (F.S.H.), as w e l l as t h e l a c t o g e n i c hormone w h i c h a r e i n v o l v e d i n r e p r o d u c t i o n . These f a c t o r s have been i d e n t i f i e d i n the f i s h ( P i c k f o r d & A t z , 1 Q 5 7 ) . On a p p r o a c h i n g t h e b r e e d i n g season i n t h e s p r i n g , t h e n a t u r a l p h o t o p e r i o d g r a d u a l l y i n c r e a s e s . T h i s p r o b a b l y s t i m u l a t e s t h e p i t u i t a r y t o produce t h e s e h o r -mones (perhaps a l s o o t h e r f a c t o r s ) w h i c h t h e n a c t on t h e gonads t o p r e p a r e them f o r r e p r o d u c t i o n . A t t h e same-time, t h e g o n a d a l s t e r o i d s may t h e n i n f l u e n c e t h e p i t u i t a r y by t h e f e e d -back mechanism. The e q u i l i b r i u m between t h e l e v e l o f t h e s e p i t u i t a r y hormones and t h e g o n a d a l s t e r o i d s f r o m the t e s t i s o r t h e o v a r y r e s u l t s i n i n c r e a s i n g o r d e c r e a s i n g t h e a g g r e s s i v e -n e s s of t h e i n d i v i d u a l , t h u s d e t e r m i n i n g t h e l e v e l o f i t s - 43 -pre-spawning aggressiveness. ...j The high l e v e l of pre-spawning aggressiveness and the development of t e r r i t o r y i s of great b i o l o g i c a l importance to the maturing male, even at t h i s stage when i t s gonad was not quite ready f o r the actual spawning act; Aronson (1957) sug-gested some adaptive advantages of t e r r i t o r i a l i t y such as providing food reserve for the parent and young, regulate population, expansion of the range of the species, ,and pro-viding an undisturbed and protected place f o r spawning. Carpenter (1958) also gave a detailed discussion of the bio--l o g i c a l s i gnificance of t e r r i t o r i a l i t y i n animal l i f e . C. GONADAL STEROID AND THE NESTrBUILDING AND SEXUAL BEHAVIOUR The present data on the nest-building and sexual behaviour of the stickleback are i n l i n e with the d i r e c t e f f e c t of the gonadal hormones on reproductive behaviour (Boch, 1948; Eversole, 1941; Cohen, 1946; Tavolga, 1940). Castrated male stickleback did not show any elements of the nest-building or sexual behaviour whether maintained under long or short photoperiod. Administration of methyl testos-terone, however, induced the nest-building behaviour i n both gonadectomized males and females although sexual behaviour was e l i c i t e d only i n the males. Long photoperiod, combined with androgen treatment increased the percentage of animal respond-ing as well as decreased the time of treatment required to i n -duce the response. Thus, although the p i t u i t a r y a c t i v i t y alone w i l l not e l i c i t nest-building and sexual behaviour, i t i s apparently involved i n the more complete expression of these - 44 -"behaviour patterns (Hoar, l Q 6 l ) . A lower percentage of castrated males were induced to per-form the nest-building and courtship behaviour by the combined treatment of long photoperiod and androgen administration than the percentage of normal adult males brought to sexual matura-t i o n by the a r t i f i c i a l l o n g photoperiod i n winter. This might be due to the difference between the dosage of methyl testos-terone administered and the ph y s i o l o g i c a l dosage i n the normal f i s h . A more del i c a t e balance between the p i t u i t a r y and the gonadal hormones might exist i n the normal f i s h which i s more e f f i c i e n t i n t r i g g e r i n g the behaviour patterns. I f the dosage of androgen administered i s too high, i t might suppress the p i t u i t a r y a c t i v i t y by the feed-back mechanism which would then re s u l t i n reducing the nest-building behaviour. Moreover, the methyl testosterone administered was a synthetic product which i s probably d i f f e r e n t from that present i n the stickleback i t -s e l f . Thus, although i t i s e f f e c t i v e , to a c e r t a i n extent, i n e l i c i t i n g the behaviour, i t might not be the substance f o r the complete control of the movements. The nest-building movements performed by the treated male castrates were si m i l a r to those of the normal mature male. The same elements - sand-digging, c o l l e c t i n g material, gluing and so on, were a l l present i n these castrates. The sequence of the d i f f e r e n t elements was also s i m i l a r , co-ordinated and directed i n such a way that a complete nest was b u i l t , i d e n t i c a l with that of a normal male. The i n t e n s i t y of the d i f f e r e n t movements, however, was s l i g h t l y lower than that of normal f i s h which again-might be due to the non-physiological dosage of - 45 -methyl t e s t o s t e r o n e a d m i n i s t e r e d . T h i s i n d i c a t e d t h a t these d i f f e r e n t s t e r e o t y p e d movements were g e n e t i c a l l y determined i n the male f i s h and the gonadal s t e r o i d a c t e d only i n e l i c i t -i n g i t and perhaps a l s o i n a d j u s t i n g the i n t e n s i t y w i t h which they were performed. Three d i f f e r e n t types of f a n n i n g movements were d i s t i n -guished by Van I e r s a l (1?53>). A low i n t e n s i t y f a n n i n g occurred near the end of the n e s t - b u i l d i n g phase which might be regarded as p a r t of the n e s t - b u i l d i n g behaviour. Then t h e r e was the displacement f a n n i n g which was performed when the male f i s h was h i g h l y motivated s e x u a l l y , but was p r o v i d e d w i t h no o u t l e t f o r the sexual behaviour. The p a r e n t a l f a n n i n g o c c u r r e d a f t e r the normal male had spawned and the f e r t i l i z e d eggs were d e v e l o p i n g . T h i s p a r e n t a l f a n n i n g was the most important type of f a n n i n g movement and the most i n t e n s e . Normal and c a s t r a t e d males which were not allowed t o spawn showed onl y the low l e v e l o f f a n n i n g , p r o b a b l y c o n s t i t u t e d mainly of n e s t - b u i l d i n g and displacement f a n n i n g , and t h e r e was no d i f f e r e n c e between the two groups. P a r e n t a l f a n n i n g , however, reached a h i g h i n t e n s i t y of 4 0 f o , f o u r to seven days a f t e r the eggs were f e r t i l i z e d , i n the normal male which had spawned. T h i s was d i f f e r e n t from t h a t i n the t r e a t e d c a s t r a t e d male which showed o n l y an i n i t i a l i n c r e a s e t o approximately 2 0 % , two t o three days a f t e r spawning. The t r e a t e d c a s t r a t e d male showed the same movement of"spawning" and " f e r t i l i z i n g " the eggs as the normal f i s h except t h a t no sperm were emitted. The presence of the l i v i n g eggs i n the nest i n the f i r s t two days s t i m u l a t e d the p a r e n t a l f a n n i n g i n these male c a s t r a t e s r e s u l t i n g i n the i n i t i a l r i s e i n f a n n i n g - 46 -i n t e n s i t y . However, since they were not f e r t i l i z e d , the eggs would die. The l e v e l of carton dioxide concentration i n the nest produced by the r e s p i r a t i o n of the eggs i s one of the external s t i m u l i for releasing and; regulating the i n t e n s i t y of the parental fanning as shown by Van I e r s a l ( l Q 5 3 ) . Thus, when the u n f e r t i l i z e d eggs died, t h i s external stimulus was e l i -minated, and the parental fanning shown by these castrates dropped. Gonadectomized females treated with methyl testosterone showed the nest-building behaviour. The same basic movements of the normal or castrated male were also performed by these treated females which showed sand-digging, c o l l e c t i n g material, gluing, pushing the nest material, as well as a low l e v e l of fanning, probably that associated with the nest-building. This indicated that the a b i l i t y to perform these behaviour elements was present i n both males and females and was not linked g e n e t i c a l l y by the sex chromosomes. I t was released normally only i n the sexually mature male. Probably the high l e v e l of androgen secreted by the t e s t i s was the i n t e r n a l con-t r o l l i n g f actor which would stimulate the neuromechanism i n the central nervous system to t r i g g e r the response. Differences existed between the nest-building behaviour performed by the treated castrated male and female. In the f i r s t place, a much lower percentage of castrated female showed the nest-building (5-8%) as compared to castrated males treated with the i d e n t i c a l dosage of methyl testosterone (50 -60%). Moreover, although the same basic movements were performed i n the males and females, except that the l a t t e r lacked the f i n a l - 47 -movement of boring through the nest, q u a l i t a t i v e differences existed between the nest-building by a normal or castrated male and that of the female. The l a t t e r was never seen to b u i l d a complete tunnel-shape nest. It was merely a mass of algae p i l e d loosely together and not pushed or glued together neatly as i n that of the male. It appeared that the female lacked the a b i l i t y to co-ordinate and d i r e c t these movements i n the proper manner to b u i l d a complete nest, although the d i f f e r e n t movements were performed. The sexual behaviour patterns were also completely absent i n the treated females. They showed no interest i n the gravid female and did not show the zigzag dance or lead the mature females which were introduced. No spawning was observed and fanning was only at a very low l e v e l . Thus, i t appeared that either the female was incapable of recognizing the normal external stimulus f o r a l l these behaviour patterns, namely the gravid female, or there was some genetic difference between the male and female as f a r as the performance of these sexual move-ments were concerned. A l l the above mentioned differences suggested that the treated castrated female was i n some way d i f f e r e n t from the male. This might be due to some genetic difference linked i n the male sex chromosome which was responsible for the complete expression of the nest-building and sexual behaviour. The' neuromechanism required to co-ordinate the nest-building move-ments to a well organized pattern or the neuromuscular move-ments to perform the sexual act might be absent i n the females. However, i t was also possible that these females had the - 48 -complete p o t e n t i a l i t y to perform both the nest-building and the sexual behaviour, but that previous effectoof the n a t u r a l l y occurring estrogen might have desensitized the f i s h i n some 1 way so that these movements were not induced with such ease as i n the male, and a much more exact physiological condition was required to bring about the behaviour pattern. Thus, genetic, neuromechanism, or previous priming effect of the estrogen might be responsible f o r the difference i n these reproductive behaviour observed i n the gonadectomized females. Homosexuality has been reported i n a number of vertebrates. Beach (1948) described pseudomale and pseudofemale behaviour i n the r a t s . Morris (1952, 1955) also reported the perfor-mance of male or female reproductive behaviour i n the ten-spined sticklebacks. He observed fanning as well as the male courtship dance by the gravid female to other ripe females. These observation indicated that the p o t e n t i a l i t y for the reproductive behaviour was present i n both sexes and was t r i g -gered by the sudden inbalance of the hormonal l e v e l . Aronson (1957) also showed that the neuromuscular mechanism f o r per-forming the reproductive behaviour of the opposite sex was present i n the species. Others also demonstrated the possible separate controls of the d i f f e r e n t phases of the reproductive behaviour, as for instance, Laskowski (1954) showed that only the preliminary male courtship behaviour, but not the pre-copulatory or copulatory a c t i v i t y , could be induced i n the female Platypoecilus variatus by treatment with methyl testos-terone. The present data on the reproductive behaviour of the stickleback was i n l i n e with this phenomenon of the genetic - 49 -similarity between the sexes as far as the nest-building move-ments were concerned which were triggered in castrates of either sexes by the administration of methyl testosterone. This gonadal steroid was also the controlling factor for the sexual behaviour in the normal and castrated male, although i t s importance in triggering the sexual behaviour was not apparent either due to the non-physiological dosage of the hormone administered or to the genetic differences which exists between the two sexes. - 50 -V . SUMMARY AND CONCLUSIONS The s p e c i f i c e f f e c t o f t h e p i t u i t a r y hormone and t h a t o f t h e m a l e g o n a d a l s t e r o i d s , m e t h y l t e s t o s t e r o n e , i n e l i c i t i n g t h e s e c o n d a r y s e x u a l c h a r a c t e r i s t i c s and t h e male r e p r o -d u c t i v e "behaviour o f t h e t h r e e - s p i n e d s t i c k l e b a c k was i n -v e s t i g a t e d and s u m m a r i z e d as f o l l o w s : 1. T r e a t m e n t w i t h m e t h y l t e s t o s t e r o n e i n d u c e d t h e k i d n e y t u b u l e s Of b o t h g o n a d e c t o m i z e d m a l e and f e m a l e a d u l t f i s h and u n o p e r a t e d j u v e n i l e f i s h t o d e v e l o p i n t o g r a n u l a r and m u c u s - s e c r e t i n g c e l l s a c c o m p a n i e d b y an i n c r e a s e i n c e l l h e i g h t . Up t o a l i m i t , t h e i n c r e a s e i n c e l l h e i g h t i s a f u n c t i o n o f t h e l e n g t h o f hormone t r e a t m e n t . 2. L o n g p h o t o p e r i o d s t i m u l a t e d t h e p r e - s p a w n i n g a g g r e s -s i v e n e s s i n g o n a d e c t o m i z e d m a l e s and f e m a l e s . T h e x i n c r e a s e i n a g g r e s s i v e b e h a v i o u r i s m a n i f e s t e d as a g r a d u a l i n c r e a s e i n t h e number o f a t t a c k s among t h e m s e l v e s l e a d i n g e v e n t u a l l y t o t h e d e v e l o p m e n t o f t e r r i t o r y by t h e d o m i n a n t f i s h . S h o r t p h o t o p e r i o d has no p a r a l l e l e f f e c t . 3 . A n d r o g e n t r e a t m e n t h a s no a p p a r e n t e f f e c t i n m o d i f y i n g t h e p r e - s p a w n i n g a g g r e s s i v e n e s s a l r e a d y s t i m u l a t e d by t h e l o n g p h o t o p e r i o d . 4. M e t h y l t e s t o s t e r o n e a d m i n i s t r a t i o n t o g e t h e r w i t h l o n g p h o t o p e r i o d s t i m u l a t e d t h e n e s t - b u i l d i n g b e h a v i o u r i n g o n a d e c t o m i z e d f i s h o f b o t h s e x e s . The r e s p o n s e was a t a much h i g h e r l e v e l i n t h e m a l e c a s t r a t e s t h a n i n t h e g o n a d e c t o m i z e d f e m a l e s . 5. The n e s t - b u i l d i n g movements p e r f o r m e d b y t h e a n d r o g e n - 51 -treated male castrates were similar to those of normal males matured i n nature. Gonadectomized androgen treated females show s l i g h t v a r i a t i o n i n t h e i r nest-building elements. 6. Hale castrates treated with androgen showed sexual behaviour s i m i l a r to that of normal male. Nest-building females, however, treated i n exactly the same way, never show any sexual behaviour. - 52 -VI. BIBLIOGRAPHY A l l e e , W. C , N. E. C o l l i n and G.Z. Lutherman 1939. Modification of s o c i a l order i n flo c k s of hens by-in j e c t i o n of testosterone propionate. Physiol. Zool. 12: 412 - 440. Al l e e , W. C , D. Foreman and C. H. Holabrid 1953. E f f e c t s of an androgen on dominance and subordination i n s i x common birds of Gallus g a l l u s . Physiol. Zool. 28: 89 - 115. Aronson, L. R. 1957. Reproductive and parental behaviour. In "The Physiology of Fishes", edited by M«E. Brown, pp. 272 - 296. Academic Press, New York. Aronson, W. R. 1959. Hormones and reproductive behaviour: some phylogenetic considerations. In "Comparative Endocrinology" edited by A. Gorbman. pp. 98 - 120. Wiley, New York. Baggerman, B. 1957. An experimental study on the timing of breeding and migration i n the three-spined sticklebacks. Arch. Neerl. Zool. 12: 105 - 317-B a l l , J . 1940. E f f e c t s of testosterone on the sex behaviour of female rat s . J . Comp. Psychol. 29: 151 - 165. Beach, F. A. 1942. Analysis of factors involved i n the arousal, maintenance and manifestation of sexual excitement i n male animals. Psychosomat. Med. 4: 173 Beach, F. A. 1948. Hormones and Behaviour. Hoeber, New York and London. Beach, F. A. 1952. Mechanism of hormonal action upon behaviour. Ciba Found. C o l l . Endocrinol. 3: 209 - 220. Beach, F. A. and G. Levinson 1950. E f f e c t s of androgen on the glans penis and mating behaviour of castrated male r a t s . J . E x p t l . Zool. 114: 159 - 171. Bishop, J". E. 1959. A h i s t o l o g i c a l and histoehemical study of the kidney tubule of the common garter snake, Thamnophis s i s t a l i s . with s p e c i a l reference to the sexual segment i n the male. J . Morph. 104: 307 - 357. Bock, F. 1928. Kastration und sehundare Geschlechtsmerkmale bei Teleostiern. Z.wiss. Zool. 130: 455 - 4-68. Carpenter, C. R. 195§. T e r r i t o r i a l i t y : A review of concepts and problems, i n "Behaviour and Evolution" edited by Roe and Simpson.- -p. 224. Yale University Press. - 53 -Cohen, H. 1.946. E f f e c t s of sex hormones on the development of the p l a t y f i s h , Platypoeoilus maculatus. Zoologica 3 1 : i21. Gourrier, R. 1922. Etude preliminaire du determinisme des caracteres sexuels secondaires chez les poissons. Arch. Anat. H i s t o l . et Embryol. 1: 118 - 144. Evans, L. T. 1952. Endocrine rel a t i o n s h i p i n t u r t l e s . I I I . Some effects of male hormones i n t u r t l e . Herpatologica 8 : 11 - 14. Eversole, W. J . 1941. The effects of pregneninolone and re-lated steroids on sexual development of f i s h (Libertes  r e t i c u l a t u s ) . Endocrinology 2 8 : 6 0 3 . Fontaine, M. and J . Leloup 1 9 5 9 . Influence de l a nage a contra-courant sur l e metabolisms de l'iode et l e fonctionnement. C. R. Acad. S c i . (Paris) 249: 343 - 3 4 7 . Harris, G. W., R. P. Michael and P.^« Scott 1958. Neurological s i t e of action of s t i l b o e s t r o l i n e l i c i t i n g sexual behaviour. Ciba Foundation, Symbosia on neurological basis of behaviour. C h u r c h i l l , London, pp. 236 - 2 3 4 . Heuts, M. J. 1947. Experimental studies on adaptive evolution i n Gasterosteus aculeatus L. Evolution 1 ( 1 - 2 ) : 8 9 - 1 0 2 . Hoar, W. S. 1935 . Reproduction i n teleost f i s h . Mem. Soc. , Endocrinol. 4 : 5 - 24. Hoar, W. S. 1961 . Hormones and the reproductive behaviour of poikilothermos vertebrates. 22nd Ann. B i o l . Colloq., C o r v a l l i s , Oregon. Hoar, W. S., M. H. A. Keenleyside and R. C. Goodall 1955- The e f f e c t s of thyroxine and gonadal steroids on the a c t i v i t y of salmon and go l d f i s h . Can. J". Zool. 3 3 : 428 - 4 3 9 . Hoar, W. S., D. MacKinnon and A. Redlich 1952. E f f e c t s of some hormones on the behaviour of salmon f r y . Can. J. Zool. 3 0 : 2 7 3 . Van I e r s e l , J. J. A. 1953 The parental behaviour of the male three-spined stickleback. Behaviour Suppl. I I I . Ikeda, K. 1 9 3 3 . E f f e c t of castration on the secondary sexual characters of anadromous three-spined sticklebacks, Gasterosteus aculeatus aculeatus L. Jap. J . Zool. 5 : 135 - 157. Jones, J. W. and G. M. King 1 9 5 2 . The spawning of the male salmon parr .(Salmo salar Linn. Juv.). Proc. Zool. Soc. London 122 - 615 . - 54 -Laskowski, W. 1954. Einige Verhaltensstudien an Platypoecilus  variatus. B i o l . Zentr. 73: 429. Lehrman, D. S. 1959. Hormonal response to i n t e r n a l s t i m u l i i n birds. I b i s 104: 478 -495. Morris, D. 1952. Homosexuality i n the ten-spined stickleback. Behaviour 4: 233 - 261. Morris, D. 1955. The causation of pseudomale and pseudofemale behaviour: A further comment. Behaviour 7: 46 - 36. Noble, G. K. and K. F. Kumpf 1936. The sexual behaviour and secondary sexual c h a r a c t e r i s t i c of gonadectomized f i s h . Anat. Rec. 67(Suppl. 1): 113. Noble, G. K. and M. Wurm 1940, The effect of testosterone propionate on the black-crowned night heron. Endocrin. 26: 837 - 850. Oguro, C. 1958. E f f e c t s of sex hormones on the kidney of the three-spined stickleback. J. Faculty Science. Hokkaido University 14 No. 1, 45. Pickford, G. E. 1952. Induction of a spawning r e f l e x i n hypophysectomized k i l l i f i s h . Nature 170: 807. Pickford, G. E. 1954a. The response of hypophysectomized male k i l l i f i s h to p u r i f i e d f i s h growth hormone, as compared with the response to p u r i f i e d beef growth hormone. Endocrinol. 55: 274 - 287. Pickford, G. E. and J . W. Atz 1957- The Physiology of the P i t u i t a r y Gland of Fishes. New York Zool. S o c , New York. Ri s s , W., E. S. Valenstein, J. Sink and W. 0. Young 1955. Development of sexual behaviour i n male guinea pig from ge n e t i c a l l y d i f f e r e n t stock under controlled condition of androgen treatment and caging. Endocrinology 57* 139 - 146. Tavolga, W. N. 1955. E f f e c t s of gonadectomy and hypophysectomy on pre-spawning behaviour i n males of the gobiid f i s h , Bathygobius soporator. Physiol. Zool. 28: 218 - 233. Tinb/ergen, N. 1953. S o c i a l Behaviour i n Animals. Methuen, London. Wilkelmi, A.E., G. E. Pickford and W. H. Sawyer. 1955. I n i t i a t i o n of the spawning response i n Fundulus by administration of f i s h and mammalian neurohypophysial preparation and synthetic oxytocin. Endocrinology 57: 243 -253. 1 

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