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Endocrine control of sexual development in the male guppy Pandey, Satyendra 1968

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THE ENDOCRINE CONTROL OF SEXUAL DEVELOPMENT IN THE MALE GUPPY POECILIA RETICULATA PETERS by SATYENDRA PANDEY B.Sc. (Distn.) Bihar U n i v e r s i t y , 1955 M.Sc. Bihar U n i v e r s i t y , 1957 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia November, 1968 i In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h C olumbia, I agr e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department of ^ O C» L.0 GrY The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8 , Canada Date J>^OaAvv|cxi2A , 13. 6 t ABSTRACT The r o l e of p i t u i t a r y and gonadal s t e r o i d s i n the development and maintenance of the t e s t i s and secondary sex characters of the guppy P o e c i l i a r e t i c u l a t a Peters has been s t u d i e d by the technique of s u r g i c a l hypophysectomy and chemical i n h i b i t i o n of gonadotropic , a c t i o n using 'methallibur Hypophysectomy of j u v e n i l e or a d u l t guppies completely blocks m i t o s i s i n the spermatogonia and t h e i r transformation i n t o spermatocytes. However, spermatocytes, spermatids and sperm already present i n the a d u l t t e s t i s at the time of o p e r a t i o n transform i n t o spermatophores. In the absence of the p i t u i t a r y , the spermatophores rupture a f t e r e i g h t weeks and the r e s u l t i n g sperm are phagocytosed w i t h i n the sperm ducts. S e r t o l i c e l l s , i n t e r s t i t i a l c e l l s and the e p i t h e l i a l c e l l s l i n i n g the sperm ducts regress i n hypophysectomized f i s h Testosterone treatment of the hypophysectomized a d u l t guppy i n i t i a t e s spermatogonia1 m u l t i p l i c a t i o n and the transformation of spermatogonia i n t o spermatocytes; the regressed S e r t o l i c e l l s , i n t e r s t i t i a l c e l l s and the e p i t h e l i a l c e l l s l i n i n g the sperm ducts resume t h e i r normal appearance. Testosterone treatment of hypophysectomized j u v e n i l e guppies does not i n i t i a t e spermatogenesis but the sperm ducts become w e l l d i f f e r e n t i a t e d . Of two p a r t i c u l a r l y w e l l d i f f e r e n t i a t e d secondary sex characters of the a d u l t male guppy, the gonopodium (modified anal f i n ) remains u n a f f e c t e d a f t e r hypophysectomy whereas the lipophores (yellow and red pigments) present on the sides of body become obscure or e n t i r e l y disappear i n the absence of p i t u i t a r y ; the lipophores reappear a f t e r testosterone treatment. Secondary sex characters never appear i n guppies hypophysectomized as j u v e n i l e s . When hypophysectomized j u v e n i l e s are t r e a t e d w i t h testosterone, secondary sex characters (gonopodium and lipophores) become evident. The r e g r e s s i o n of the gametogenetic and the s t e r o i d o g e n e t i c t i s s u e s i n the t e s t i s of 'methallibure'- t r e a t e d ( l : l 0 ^ p a r t s ) a d u l t guppy i s not as complete as i n the hypophysectomized f i s h . This i n d i c a t e s that the r e l e a s e of p i t u i t a r y gonadotropins i s not completely blocked. With the same dose of 'methallibure', however, the gonadotropin r e l e a s e i n the j u v e n i l e s i s apparently blocked. In both a d u l t and j u v e n i l e guppies 'methallibure' b r i n g s about a c l e a r decrease i n both the number and mean c e l l diameter of gonadotrophs. The gonadotropic hormone b l o c k i n g a c t i v i t y of the compound seems to occur a t the l e v e l of hormone s y n t h e s i s . From these s t u d i e s i t has been concluded that m i t o t i c d i v i s i o n of spermatogonia and t h e i r transformation i n t o spermatocytes are dependent on p i t u i t a r y , but the transformation of spermatocytes, spermatids and sperm i n t o spermatophores are p i t u i t a r y - i n d e p e n d e n t . The r e l e a s e of spermatophores i s under the c o n t r o l of p i t u i t a r y . The regressed S e r t o l i c e l l s , i n t e r s t i t i a l c e l l s and the e p i t h e l i a l c e l l s l i n i n g the sperm ducts of hypophysectomized f i s h assume i v normal appearance w i t h testosterone treatment. The appearance of secondary sex characters i n hypophysectomized j u v e n i l e s t r e a t e d w i t h testosterone i n d i c a t e s that secondary sex characters are d i r e c t l y c o n t r o l l e d by t e s t o s t e r o n e . 'Methallibure' completely blocks the synthesis of gonadotropic hormones i n the j u v e n i l e guppies but not i n a d u l t s . V TABLE OF CONTENTS Page LIST OF TABLES v i i i LIST OF FIGURES x i ACKNOWLEDGMENTS x i v INTRODUCTION 1 SECTION I : HYPOPHYSECTOMY AND METHYL TESTOSTERONE TREATMENT OF THE ADULT MALES 5 INTRODUCTION 5 MATERIALS AND METHODS 5 Maintenance of f i s h i n breeding aquaria. 5 Maintenance of hypophysectomized f i s h . 6 procedure of hypophysectomy. 6 Methyl testosterone treatment. 8 H i s t o l o g y . 8 Measurements. 9 RESULTS 10 St r u c t u r e of the a d u l t t e s t i s . 10 Secondary sex characters of the male guppy. 13 St r u c t u r e of the t e s t i s and secondary sex characters of sham-operated c o n t r o l guppy. 14 St r u c t u r e of the t e s t i s of hypophysectomized guppy. 14 Secondary sex characters of hypophysectomized guppy. 21 Str u c t u r e of the t e s t i s and secondary sex characters of hypophysectomized c o n t r o l guppy (testosterone experiment). 21 S t r u c t u r e of the t e s t i s and secondary sex characters of sham-operated c o n t r o l guppy (testosterone experiment). 24 E f f e c t of methyl testosterone on the a d u l t t e s t i s of hypophysectomized guppy. 24 E f f e c t of methyl testosterone on secondary sex characters of hypophysectomized guppy. 28 v i Page SECTION I I : HYPOPHYSECTOMY AND METHYL TESTOSTERONE TREATMENT OF THE JUVENILE GUPPIES 3 7 INTRODUCTION 3 7 MATERIALS AND METHODS Maintenance of hypophysectomized j u v e n i l e f i s h . 37 Procedure of hypophysectomy. 38 Methyl testosterone treatment. 38 Hi s t o l o g y . 39 Measurements. 39 RESULTS S t r u c t u r e of the j u v e n i l e t e s t i s ( i n i t i a l c o n t r o l ) . 40 St r u c t u r e of the t e s t i s of hypophysectomized j u v e n i l e . 42 St r u c t u r e of the t e s t i s arid secondary sex characters of the sham-operated j u v e n i l e . 45 St r u c t u r e of the t e s t i s of hypophysectomized j u v e n i l e (Control of testosterone experiment). 46 St r u c t u r e of the t e s t i s and secondary sex characters of the sham-operated j u v e n i l e (Control of testosterone experiment). 46 E f f e c t of methyl testosterone on the t e s t i s of hypophysectomized j u v e n i l e . 50 E f f e c t of methyl testosterone on secondary sex c h a r c t e r s of hypophysectomized juvenile.. , 51 SECTION I I I : 'METHALLIBURE' TREATMENT OF THE JUVENILE AND ADULT MALES 59 INTRODUCTION 59 MATERIALS AND METHODS 59 Maintenance of 'methallibure' t r e a t e d f i s h . 59 Hi s t o l o g y . 60 Measurements. 60 RESULTS 61 St r u c t u r e of the t e s t i s and secondary sex characters of a d u l t control;.... 61 E f f e c t of 'methallibure' on the t e s t i s and secondary sex characters of the a d u l t guppy. 61 V l l Page Comparison of the e f f e c t s of hypophysectomy and 'methallibure' treatment on the a d u l t t e s t e s and secondary sex characters. 65 S t r u c t u r e of the j u v e n i l e t e s t i s ( I n i t i a l c o n t r o l ) . 67 S t r u c t u r e of the t e s t i s and secondary sex characters of the j u v e n i l e guppy not t r e a t e d w i t h 'methallibure'. 67 E f f e c t of 'methallibure' on the t e s t i s of the j u v e n i l e f i s h . 67 Comparison of the e f f e c t s of hypophysectomy and 'methallibure' treatment on the t e s t e s of the j u v e n i l e s . 71 Morphology of the p i t u i t a r y complex. 71 E f f e c t of "methallibure' on the meso- adenohypophysis of the a d u l t male guppy. 75 E f f e c t of 'methallibure' on the meso- adenohypophysis of the j u v e n i l e guppy. 77 GENERAL DISCUSSION 86 The r o l e of the p i t u i t a r y and the androgens i n the c o n t r o l of spermatogenesis i n c l u d i n g spermatophores. 88 The endocrine c o n t r o l of the r e l e a s e of spermatophores, the development and maintenance of i n t e r s t i t i a l c e l l s , e p i t h e l i a l c e l l s l i n i n g the sperm ducts and S e r t o l i c e l l s . 96 The r o l e of the p i t u i t a r y and the androgens i n the c o n t r o l of secondary sex chara c t e r s . 102 The b l o c k i n g a c t i o n of 'methallibure' on the e f f e c t s of the gonadotropins and the s i t e of a c t i o n of 'methallibure'. 105 SUMMARY 113 LITERATURE CITED 117 v i i i LIST OF TABLES TABLE PAGE I G.S.I, of hypophysectomized and sham-operated guppies a f t e r 8 weeks. 15 I I Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized and sham- operated guppies a f t e r 8 weeks. 16 I I I E p i t h e l i a l c e l l heights of the e f f e r e n t ducts of hypophysectomized and sham-operated guppies a f t e r 8 weeks. 17 IV G.S.I, of hypophysectomized and sham-operated guppies a f t e r 16 weeks. 18 V Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized and .sham- operated guppies a f t e r 16 weeks. 19 VI Number and percentage of spermatogonia and spermatophores i n hypophysectomized guppies a f t e r 8 weeks and 16 weeks. 22 V I I Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized, t e s t o s t e r o n e - t r e a t e d and sham-operated guppies. 23 V I I I E p i t h e l i a l c e l l heights of the e f f e r e n t ducts of hypophysectomized, t e s t o s t e r o n e - t r e a t e d and sham- operated guppies. 25 IX G.S.I, of hypophysectomized, t e s t o s t e r o n e - t r e a t e d and sham-operated guppies. 26 X Number and percentage of d i f f e r e n t stages of spermatogenesis i n j u v e n i l e c o n t r o l ; j u v e n i l e hypophysectomized and j u v e n i l e sham-operated guppies. 41 XI T o t a l number, width and lumen of e f f e r e n t ducts of j u v e n i l e c o n t r o l , j u v e n i l e hypophysectomized and j u v e n i l e sham-operated guppies. 43 X I I E p i t h e l i a l c e l l heights and lumen of main sperm duct of j u v e n i l e c o n t r o l ; j u v e n i l e hypophysectomized and j u v e n i l e sham-operated guppies. 44 i x TABLE X I I I PAGE XIV XV XVI XVII XVIII XIX XX XXI Number and percentage of d i f f e r e n t stages of spermatogenesis i n j u v e n i l e hypophysectomized; j u v e n i l e hypophysectomized and testosterone tr e a t e d ; j u v e n i l e sham-operated guppies. T o t a l number, width and lumen of e f f e r e n t ducts of j u v e n i l e hypophysectomized; j u v e n i l e hypophysectomized and testosterone t r e a t e d ; j u v e n i l e sham-operated guppies. E p i t h e l i a l c e l l heights and lumen of main sperm duct of j u v e n i l e hypophysectomized; j u v e n i l e hypophysectomized and t e s t o s t e r o n e - t r e a t e d ; j u v e n i l e sham-operated guppies. Gonosomatic index (G.S.I.) of 'methallibure' t r e a t e d and c o n t r o l guppies a f t e r 8 weeks. Number and percentage of d i f f e r e n t stages of spermatogenesis i n 'methallibure' t r e a t e d and c o n t r o l guppies. E p i t h e l i a l c e l l heights of e f f e r e n t ducts of •methallibure' t r e a t e d and c o n t r o l guppies. Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized and 'methallibure' t r e a t e d guppies a f t e r 8 weeks. Number and percentage of d i f f e r e n t stages of spermatogenesis i n j u v e n i l e c o n t r o l , j u v e n i l e 'methallibure' t r e a t e d and j u v e n i l e non- 1 m e t h a l l i b u r e ' t r e a t e d guppies. T o t a l number, width and lumen of e f f e r e n t ducts of j u v e n i l e c o n t r o l , j u v e n i l e 'methallibure' t r e a t e d and j u v e n i l e non-'methallibure' t r e a t e d guppies. 47 48 49 62 63 64 66 68 69 XXII E p i t h e l i a l c e l l heights and lumen of main sperm duct of j u v e n i l e c o n t r o l , j u v e n i l e 'methallibure' t r e a t e d , and j u v e n i l e non-'methallibure' t r e a t e d guppies. 7 0 XXII I Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized j u v e n i l e guppy a f t e r 6 weeks and 'methallibure' t r e a t e d j u v e n i l e guppy a f t e r 8 weeks. 72 X TABLE PAGE XXIV E f f e c t of 'met h a l l i b u r e 1 (ICI 33,828) on the mean c e l l diameter of the mesoadenohypophysial gonadotrophs, somatotrophs, and thyrotrophs of the a d u l t and j u v e n i l e guppy, P o e c i l i a r e t i c u l a t a , P e t e r s . 76 LIST OF FIGURES FIGURE PAGE l a S e c t i o n of whole t e s t i s of a d u l t guppy 29 l b D e t a i l of a p o r t i o n of t e s t i s i n d i c a t e d by arrow i n l a . 29 2a. S a g i t t a l s e c t i o n of a d u l t t e s t i s showing d i f f e r e n t stages of spermatogenesis 3 0 2b Magnified view of a p o r t i o n of 2a. 3 0 3a S a g i t t a l s e c t i o n of t e s t i s e i g h t weeks a f t e r hypophysectomy showing only spermatophores 3 0 3b Magnifi e d view of a p o r t i o n of 3a. 3 0 4 E f f e r e n t ducts of a d u l t t e s t i s c o n t a i n i n g spermatophores. 31 5 Ruptured spermatophores i n the e f f e r e n t ducts of t e s t i s e i g h t weeks a f t e r hypophysectomy 31 6 Main sperm duct of a d u l t t e s t i s c o n t a i n i n g spermatophores. 31 7 Ruptured spermatophores i n the main sperm duct of t e s t i s e i g h t weeks a f t e r hypophysectomy. 31 8 S e r t o l i c e l l s of a d u l t t e s t i s w i t h sperm heads attached to them. 3 2 9 Regressed S e r t o l i c e l l s of t e s t i s e i g h t weeks a f t e r hypophysectomy. 32 10 Spermatophores w i t h surrounding S e r t o l i c e l l s of a d u l t t e s t i s opening i n t o e f f e r e n t ducts. 3 2 11 T e s t i s at the end of t h i r d week a f t e r hypophy- sectomy c o n t a i n i n g o n l y sperm-cysts and spermatophores. 3 2 12 I n t e r s t i t i a l c e l l s of a d u l t t e s t i s . 33 13 Regressed i n t e r s t i t i a l c e l l s of t e s t i s e i g h t weeks a f t e r hypophysectomy. 33 14 I n t e r s t i t i a l c e l l s and e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts of a d u l t t e s t i s s t a i n e d w i t h Sudan black B. 33 x i i FIGURE PAGE 15 T e s t i s showing remnant sperm being phagocytosed i n e f f e r e n t ducts 16 weeks a f t e r hypophysectomy. 33 16 T e s t i s of hypophysectomized guppy t r e a t e d w i t h t estosterone f o r f i v e weeks. 34 17 Magnified view of a p o r t i o n of f i g u r e 16. 34 18 Remnant sperm i n main sperm duct of hypophysectomized guppy a f t e r two weeks of testosterone treatment. 34 19 T a l l e p i t h e l i a l c e l l s reappear i n sperm duct of hypophysectomized guppy a f t e r f i v e weeks of testosterone treatment. 34 20 Percentage of d i f f e r e n t stages of spermato- genesis i n hypophysectomized and sham- operated a d u l t guppies a f t e r e i g h t weeks. 35 21 Percentage of d i f f e r e n t stages of spermato- genesis i n hypophysectomized; tes t o s t e r o n e - t r e a t e d and sham-operated a d u l t guppies. 3 6 22 S a g i t t a l s e c t i o n of t e s t i s of j u v e n i l e guppy showing only spermatogonia1 c y s t s . 53 23 S a g i t t a l s e c t i o n of t e s t i s of j u v e n i l e guppy showing spermatogonia and e f f e r e n t ducts. 53 24 E f f e r e n t ducts of hypophysectomized j u v e n i l e t r e a t e d w i t h testosterone. 53 25 S a g i t t a l s e c t i o n of t e s t i s of j u v e n i l e guppy showing main sperm duct l i n e d by squamous e p i t h e l i a l c e l l s . 54 26 Main sperm duct of t e s t i s of hypophysectomized j u v e n i l e t r e a t e d w i t h testosterone. 54 27 Percentage of d i f f e r e n t stages of spermato- genesis i n j u v e n i l e c o n t r o l ; j u v e n i l e hypophysectomized and j u v e n i l e sham-operated guppies. 55 28 Percentage of d i f f e r e n t stages of spermato- genesis i n j u v e n i l e hypophysectomized; j u v e n i l e hypophysectomized and t e s t o s t e r o n e - t r e a t e d ; j u v e n i l e sham-operated guppies. 56 29 Anal f i n of j u v e n i l e guppy. 57 3 0 A n a l f i n of hypophysectomized j u v e n i l e . 57 x i i i FIGURE PAGE 31 Gonopodium (modified anal f i n ) of a d u l t male. 58 3 2 A n a l f i n of hypophysectomized j u v e n i l e t r e a t e d w i t h t e s t o s t e r o n e . 58 33 S a g i t t a l s e c t i o n of t e s t i s of a d u l t guppy t r e a t e d w i t h 'methallibure' showing few cysts of e a r l i e r stages of spermatogenesis. 79 34 Same as f i g u r e 33 showing e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts. 79 35 S a g i t t a l s e c t i o n of t e s t i s of j u v e n i l e guppy not t r e a t e d w i t h ' m e t h a l l i b u r e 1 . 79 3 6 Percentage of d i f f e r e n t stages of spermatogenesis i n ' m e t h a l l i b u r e ' - t r e a t e d and c o n t r o l a d u l t guppies a f t e r e i g h t weeks. 80 3 7 Percentage of d i f f e r e n t stages of spermatogenesis i n j u v e n i l e c o n t r o l ; j u v e n i l e 'methallibure'- t r e a t e d and j u v e n i l e non-'methallibure' t r e a t e d guppies. 81 38a S a g i t t a l s e c t i o n of p i t u i t a r y gland of a d u l t c o n t r o l guppy. 82 38b Magnified view of a p o r t i o n of 38a. 82 39a S a g i t t a l s e c t i o n of p i t u i t a r y gland of a d u l t ' m e t h a l l i b u r e ' - t r e a t e d guppy. 83 39b Magnified view of a p o r t i o n of 39a. 83 40 S a g i t t a l s e c t i o n of p i t u i t a r y gland of j u v e n i l e c o n t r o l guppy (Group I I I ) . 84 41 S a g i t t a l s e c t i o n of p i t u i t a r y gland of j u v e n i l e ' m e t h a l l i b u r e ' - t r e a t e d guppy. 84 42a S a g i t t a l s e c t i o n of p i t u i t a r y gland of j u v e n i l e c o n t r o l guppy (group vi) not t r e a t e d w i t h 'methallibure'. 85 42b Magnif i e d view of a p o r t i o n of 42a. 85 x i v ACKNOWLEDGMENTS I wish to express my s i n c e r e a p p r e c i a t i o n to Dr. W.S. Hoar f o r h i s guidance, e n t h u s i a s t i c support and c r i t i c a l s u p e r v i s i o n throughout the present i n v e s t i g a t i o n . Thanks are due to Drs. J.R. Adams, P. Ford, N.R. L i l e y and J.M. Taylor f o r t h e i r h e l p f u l suggestions during the pr e p a r a t i o n of the manuscript. I f u r t h e r wish to thank Dr. J.R. Adams f o r the use of h i s equipment f o r photographic work. For h i s i n t e r e s t i n my work and help extended during the p r e p a r a t i o n of the manuscript, I would l i k e to thank Dr. J.F. Leatherland. I am a l s o t h a n k f u l to Dr. M. Wiesbart, Mr. J.C. Fenwick and Mr. T.J. Lam f o r many f r u i t f u l d i s c u s s i o n s . I wish to acknowledge Mrs. J . Fenwick and Mrs. M. Jenson f o r drawing some f i g u r e s ; Mr. F. McConnell and Mr. L. Sharman f o r t e c h n i c a l help and Miss L. Bogstie f o r t y p i n g the manuscript. I am g r a t e f u l to the a u t h o r i t i e s of patna U n i v e r s i t y f o r g r a n t i n g me study leave which made t h i s study p o s s i b l e . F i n a l l y , I would l i k e to express my a p p r e c i a t i o n to my wi f e f o r her encouragement and my fa t h e r f o r h i s keen i n t e r e s t i n my education. INTRODUCTION The guppy P o e c i l i a r e t i c u l a t a peters i s a h i g h l y s p e c i a l i z e d monthly breeding ovoviviparous t e l e o s t belonging to the f a m i l y P o e c i l i i d a e . Male and female guppies are s e x u a l l y dimorphic; the female i s considerably l a r g e r than the male and of a pale g r e y i s h green c o l o r , while the male i s adorned w i t h s e v e r a l b r i g h t patches on the sides of the body. The d i f f e r e n c e i n pigmentation and c o l o r i n g i n the males belonging to d i f f e r e n t races i s considerable and depends on genes l o c a t e d i n sex chromosomes (Winge 1922; Winge and D i t l e v s o n 1947). The most conspicuous c o l o r patches are the lipophores which contain both y e l l o w pigments (Xanthophores) and r e d pigments (erythrophores). Another w e l l d i f f e r e n t i a t e d secondary sex character found i n the male guppy i s the h i g h l y modified a n a l f i n or gonopodium, w i t h which i n t e r n a l f e r t i l i z a t i o n i s e f f e c t e d . As an adaptation to i n t e r n a l f e r t i l i z a t i o n , the male guppy produces sperm-balls or spermatophores which probably prevent the l o s s of sperm by leakage i n t o the water during t r a n s f e r to the g e n i t a l t r a c t of female. In the guppy, development of the gonad and secondary sex characters has been described i n d e t a i l . D i l d i n e (1936) and Goodrich e_t a_l (1934) discussed the morphogenesis of gonads. Comparable s t u d i e s of the ovary were reported i n d e t a i l by Antennius (1959). vaupel (1929) described spermatogenesis and discussed the method of t r a n s f e r of the 2 sperm to the female. The morphogenesis of the gonopodium i n the male has been described by Hopper (1949a). Secondary sex characters have been shown to be i n v o l v e d i n complex c o u r t s h i p behavior (Clark and Aronson 1951; Kadow 1954; Baerends e t a l 19 55; L i l e y 1966). Although c o u r t s h i p behavior has been s t u d i e d i n d e t a i l , few analyses are a s s o c i a t e d w i t h the endocrinology of reproduction (Kadow 19 54; L i l e y 1965, 1968). Although the i n f l u e n c e of exogenous s t e r o i d s on the gonad and secondary sex characters of the guppy has been t e s t e d many times (Eversole 1939, 1941; Regnier 1941; Okada and Yamashita 1944b; Hopper 1949b, 1951), e f f o r t s have never been made to e l i m i n a t e the p o s s i b l e e f f e c t s of endogenous gonadotropins by the use of hypophysectomized animals. The p o s s i b i l i t y of a d i r e c t a c t i o n of the p i t u i t a r y i n the development of t e s t i s and the d i f f e r e n t i a t i o n of secondary sex characters of the guppy or any other p o e c i l i i d has not been i n v e s t i g a t e d . S i m i l a r l y , the endocrine c o n t r o l of spermatophore formation, the r e t e n t i o n of spermatophores w i t h i n the t e s t i s and t h e i r eventual r e l e a s e from the t e s t i s have not been s t u d i e d i n any f i s h . The technique of hypophysectomy was found to be r e l a t i v e l y s u c c e s s f u l w i t h both the a d u l t and j u v e n i l e guppies thus making p o s s i b l e c r i t i c a l experiments concerning p i t u i t a r y - g o n a d r e l a t i o n s . The r o l e of gonadal s t e r o i d s i n the maintenance or development of the t e s t i s and secondary sex characters may be r e a d i l y analyzed by t r e a t i n g the 3 hypophysectomized j u v e n i l e and a d u l t guppies w i t h testosterone preparations which have been repeatedly shown to be p h y s i o l o g i c a l l y e f f e c t i v e i n replacement of n a t u r a l androgens. In a d d i t i o n , a technique of chemically i n h i b i t i n g gonadotropic a c t i o n has r e c e n t l y been made a v a i l a b l e . 'Methallibure' ( l - a - m e t h y l a l l y l t h i o c a r b o m o y l - 2 - m e t h y l t h i o - carbamyl hydrazine; I.C.I. 33,828) blocks the s y n t h e s i s , r e l e a s e or a c t i o n of gonadotropic hormones i n t e l e o s t s (Hoar e t a l 1967; Wiebe 1968). This agent provides a u s e f u l a d d i t i o n a l technique i n the a n a l y s i s of p i t u i t a r y - g o n a d r e l a t i o n s i n f i s h e s . The s i t e of a c t i o n of 'methallibure' i n b l o c k i n g gonadotropic a c t i o n i s not known. With these techniques as a b a s i s f o r the experimental a n a l y s i s , the purposes of the present i n v e s t i g a t i o n of the male guppy are as f o l l o w s : 1. To examine the r o l e of the p i t u i t a r y and the gonadal hormones i n spermatogenesis w i t h p a r t i c u l a r reference to the i n i t i a t i o n of spermatocyte formation and the formation of spermatophores. 2. To i n v e s t i g a t e the p o s s i b i l i t y of a d i r e c t p i t u i t a r y involvement i n the expression of secondary sex characters of a d u l t males. 3. To describe the e f f e c t s of hypophysectomy and methyl testosterone treatment on the t e s t i s of the j u v e n i l e guppy p r i o r to i t s d i f f e r e n t i a t i o n and before the appearance of secondary sex char a c t e r s . 4 4. To compare 'methallibure' e f f e c t s on the t e s t e s and secondary sex characters of the a d u l t and j u v e n i l e guppies and to l o c a l i z e the s i t e of a c t i o n of 'methallibure' i n b l o c k i n g gonadotropic a c t i o n . 5 SECTION I HYPOPHYSECTOMY AND METHYL TESTOSTERONE TREATMENT OF THE ADULT ' MALES. INTRODUCTION The r o l e of the p i t u i t a r y and the gonadal hormones i n the r e p r o d u c t i v e endocrinology of the a d u l t male guppy has been s t u d i e d by an a l y z i n g the e f f e c t s of hypophysectomy on the te s t e s and secondary sex characters (gonopodium and lipophores) and subsequently t r e a t i n g such hypophysectomized animals w i t h methyl testosterone. MATERIALS AND METHODS Maintenance of f i s h i n breeding aquaria. The i n i t i a l sample of guppies was purchased from a l o c a l aquarium dealer (Northwest-Aquatics, Vancouver, Canada). These were allowed to breed i n f i v e g l a s s aquaria of 20- to 6 0 - l i t r e c a p a c i t y . The bottoms of these aquaria were covered to a depth of 2 cm w i t h coarse sand; water was continuously r e c i r c u l a t e d and f i l t e r e d through a pad of glass-wool. Each day feces and uneaten food were removed from the bottom of the aquaria by a fine-meshed net and f i l t e r s were cleaned a t l e a s t once a week. About 50% of water i n each of the aquaria was renewed once i n every two or three weeks. Aquatic p l a n t s (water s p r i t e , C e r a t o p t e r i s t h a l i c t r o i d e s ) were put on the surface of water i n aquaria to provide cover 6 f o r the young thus a v o i d i n g t h e i r being eaten by the a d u l t s . There was no s p e c i a l i l l u m i n a t i o n d i r e c t l y above the aquaria but the room was w e l l - l i g h t e d w i t h overhead f l u o r e s c e n t tubes and the photoperiod r e g u l a t e d a t n a t u r a l day l e n g t h by a p h o t o c e l l exposed i n a large window f a c i n g south. The temperature of a l l aquaria was maintained a t 25 ± 0.5°C. F i s h were fe d d a i l y w i t h f i n e l y ground commercial t r o u t f i s h food (J.R. C l a r k Co.) Maintenance of hypophysectomized f i s h . One week p r i o r to the operation b r i g h t l y c o l o r e d males (16-22 mm standard length, 9 0-250 mg i n weight) were taken from the breeding aquaria and put i n aquaria of 2 0 - l i t r e c a p a c i t y which contained 1 6 - l i t r e of f i s h s a l i n e jjSTaCl-5.5 gm, KCl-0.14 gm and cacl2~0.12 gm i n 1 l i t r e of d e c h l o r i n a t e d water (Young 1933JJ. The males were kept i n the s a l i n e u n t i l the end of the experiment. No p l a n t s were put i n these experimental aquaria. L i g h t was provided by 2 0-watt f l u o r e s c e n t tubes about 15 cm above the aquaria. The photoperiod of 16-hr d a i l y i l l u m i n a t i o n was c o n t r o l l e d by Intermatic model T101 c l o c k s . The temperature was maintained a t 25 1 0.5°C. F i s h were fed d a i l y w i t h f i n e l y ground commercial t r o u t f i s h food. Sham-operated a d u l t males which served as c o n t r o l s were a l s o kept i n the s a l i n e u n t i l the end of the experiment. Procedure of hypophysectomy. Males conditioned to f i s h - s a l i n e f o r one week, were ane s t h e t i z e d w i t h 1:600 MS 222 ( T r i c a i n e Methane Sulphonate-Sandoz). The f i s h was 7 then placed v e n t r a l s i d e uppermost on a p l a s t i c p l a t e f i x e d a t an angle on the surface of wax i n a rect a n g u l a r p l a s t i c t r a y (15x7^x4^ em). The f i s h was secured i n place by a p a i r of movable p l a s t i c hooks present on the p l a s t i c p l a t e . A f i n e g l a s s tube placed i n the bu c c a l c a v i t y of f i s h served to perfuse the g i l l s w i t h f i s h - s a l i n e . A l l subsequent procedures were c a r r i e d out under a b i n o c u l a r microscope (xlO). A small i n c i s i o n was made i n the gular r e g i o n i n f r o n t of b r a n c h i o s t e g a l membrane on the r i g h t s i d e and extended to the lower l i p . The mucous membrane covering the p a l a t e was to r n away g e n t l y w i t h a p a i r of f i n e forceps. The p i t u i t a r y gland could then be seen beneath the parasphenoid bone as a sm a l l white body j u s t p o s t e r i o r to the o p t i c chiasma. The parasphenoid bone was now broken by a p a i r of b l u n t forceps. The p i t u i t a r y was picked up w i t h a p a i r of f i n e forceps. I t was a s c e r t a i n e d under the microscope that a l l three lobes of p i t u i t a r y were always removed. Sham- operated f i s h r e c e i v e d the same treatment without p i t u i t a r y removal. The blood and t i s s u e fragments r e s u l t i n g from the opera t i o n were r a p i d l y f l u s h e d w i t h f i s h s a l i n e . A f t e r the oper a t i o n , the f i s h was l e f t i n the f i s h s a l i n e accumulated i n s i d e the t r a y d u r i n g the operation u n t i l i t s t a r t e d swimming a c t i v e l y again. I t was then t r a n s f e r r e d to an aquarium c o n t a i n i n g f i s h - s a l i n e . The wound healed i n ten to twelve days and no i n f e c t i o n was ever n o t i c e d . M o r t a l i t y 8 was high.,amounting to 2 0-25% during the operation; a f u r t h e r m o r t a l i t y of 25-30% occurred i n two weeks f o l l o w i n g the o p e r a t i o n . Methyl testosterone treatment. Hypophysectomized a d u l t males were t r a n s f e r r e d e i g h t weeks a f t e r the operation to aquaria c o n t a i n i n g 1 6 - l i t r e of f i s h s a l i n e . To avoid the changes of concentration of testosterone i n f i s h - s a l i n e , no sand or f i l t e r or p l a n t s were used; the aquarium was 7 continuously aerated. A concentration of 1:10 p a r t s (1.6 mgm i n 16 l i t r e s of f i s h s a l i n e ) of methyl testosterone ( C a l i f o r n i a Corporation f o r Biochemical Research, Los Angeles) was added to each aquarium every week (5 a p p l i c a t i o n s during 5 weeks). Methyl testosterone was added as a suspension (1.6 mgm i n 50 ml. of f i s h s a l i n e ) to the aquaria. Four l i t r e s of f i s h s a l i n e was renewed i n each aquarium every week. Controls were of two k i n d s : 1. the hypophysectomized males which d i d not r e c e i v e any methyl testosterone treatment and 2. the sham-operated males. The c o n t r o l s were always kept i n s a l i n e . H i s t o l o g y . The t e s t e s were f i x e d i n Bouins f l u i d ( p i c r i c a c i d - f o r m o l - a c e t i c acid) f o r r o u t i n e h i s t o l o g y . To check on the completeness of p i t u i t a r y removal, s u i t a b l e p o r t i o n s of the heads were f i x e d i n formic acid-Bouin f i x a t i v e and l e f t i n the f i x a t i v e to d e c a l c i f y f o r a week. Some of the t e s t e s were f i x e d i n Baker's formol-calcium f o r l i p i d s t a i n i n g . 9 The Bouin and Bouin-formic a c i d f i x e d t e s t e s and head p o r t i o n s were dehydrated i n a l c o h o l , c l e a r e d i n X y l o l and embedded i n paraffin-wax. S e r i a l l o n g i t u d i n a l s e c t i o n s were cut f o r t e s t e s at 5 u and of head p o r t i o n s a t 7 M and s t a i n e d w i t h E h r l i c h ' s haematoxylin and eo s i n . The t e s t i s f i x e d i n formol-calcium was embedded i n g e l a t i n (Gurr 1962) and sectioned a t 10-15 ;u on a f r e e z i n g microtome. Sections were f l o a t e d on to s l i d e s i n i c e d water. Sections were s t a i n e d w i t h Sudan black B (0.7% i n Propylene g l y c o l ) , d i f f e r e n t i a t e d i n 70% propylene g l y c o l and mounted i n Hydramount ( C h i f f e l l e & P u t t 1951). Measurements. The standard length was measured as the d i s t a n c e between the snout and the l a s t row of s c a l e s on the caudal peduncle. The o v e r a l l body weight before the removal of the gonad was measured to the nearest 1 mg and the gonad weight was measured to the nearest O.i mg. The gonad weight was expressed as a percentage of t o t a l body weight to give the gonosomatic index (GSI) GSI = G o n a < 3- weight i n grams X -̂ QQ T o t a l body weight i n grams To compare the h i s t o l o g i c a l p i c t u r e of the t e s t e s of sham-operated, hypophysectomized and t e s t o s t e r o n e - t r e a t e d guppies, the t o t a l number of a c i n i or cysts c o n t a i n i n g germ-cells of d i f f e r e n t stages of spermatogenesis i n the median s a g i t t a l s e c t i o n were counted and percentage of each c e l l type c a l c u l a t e d (one acinus u s u a l l y contains germ-cells 10 i n the same stage of maturation). The s i n g l e s e c t i o n thus s e l e c t e d had cy s t s c o n t a i n i n g a l l stages of spermatogenesis and gave e s s e n t i a l l y the same percentage composition as was obtained a f t e r counting every 20th s e c t i o n of the whole t e s t i s . The e p i t h e l i a l c e l l heights of e f f e r e n t ducts (branches of main sperm duct) of t e s t e s of sham-operated, hypophysectomized and t e s t o s t e r o n e - t r e a t e d f i s h were measured w i t h an ocu l a r micrometer. F i v e e f f e r e n t ducts from one s e c t i o n ( j u s t before the e f f e r e n t ducts j o i n to form the main sperm duct) were s e l e c t e d at random and f i v e measurements of e p i t h e l i a l c e l l heights were taken from each e f f e r e n t duct. Thus 25 e p i t h e l i a l c e l l heights were measured from one s e c t i o n of each t e s t i s . RESULTS St r u c t u r e of the a d u l t t e s t i s . The t e s t i s i s a two- lobed body fused i n the middle and l o c a t e d i n the p o s t e r i o r p a r t of the body-cavity, v e n t r a l to the swimbladder. I t i s covered by a t h i n , unpigmented p e r i t o n e a l membrane. The two branched main sperm ducts (one occupying the centre of each lobe of the t e s t i s ) u n i t e a t the p o s t e r i o r and v e n t r a l margins of t e s t i s to form a short vas deferens which opens almost immediately i n t o the u r o g e n i t a l sinus ( F i g . l a ) . The main sperm duct and i t s branches, the e f f e r e n t ducts are l i n e d by a cuboida l or columnar e p i t h e l i u m (Figs. 4 & 6). 11 The e f f e r e n t ducts are surrounded by cysts or a c i n i (the t e s t i s i s the acinus-type i n which no tubules have developed). Each cy s t or acinus u s u a l l y contains germ-cells i n the same stage of maturation. Cysts c o n t a i n i n g spermatogonia and spermatocytes are a t the periphery; i n t e r n a l to these are spermatid- and sperm-cysts and f i n a l l y c l o s e to the centre l y i n g adjacent to the e f f e r e n t ducts are cysts c o n t a i n i n g 'sperm-balls' or spermatophores ( F i g s , l b & 2a,b). S e r t o l i c e l l s are arranged around the periphery of the c y s t s . S e r t o l i c e l l s l i n i n g the cysts of spermatocytes and spermatids are t h i n and f l a t t e n e d . When the spermatids w i t h i n the cys t transform i n t o sperm, the S e r t o l i c e l l s become s t r i k i n g l y enlarged; t h e i r n u c l e i become l a r g e r and more rounded; and the sperm heads become attached to the inner margin of S e r t o l i c e l l s ( F i g . 8 ). Thus a sperm-ball or spermatophore i s formed w i t h a compact r i n g of sperm-heads around the periphery of cys t and the t a i l s i n the centre. According to Vaupel (1929), i n the guppy the sperm-heads of spermatophores now withdraw from the c y s t - w a l l , the t a i l s being entwined i n the process; a connection of the spermatophore w i t h the main canal i s e s t a b l i s h e d ; the spermatophores then pass i n t o the c e n t r a l c a n a l . He d i d not describe the f a t e of S e r t o l i c e l l s a f t e r the spermatophore withdraws from them and reaches the c e n t r a l c a n a l . Medlen (19 50) and Chavin v& Gordon (1951) were a l s o unable to e x p l a i n the f a t e of S e r t o l i c e l l s a f t e r the spermatophores 12 pass i n t o the lumen of the t e s t i c u l a r canal i n Gambusia a f f i n i s and Xiphophorus maculatus r e s p e c t i v e l y . In the present i n v e s t i g a t i o n i t was noted th a t the w a l l of the mature c y s t c o n t a i n i n g the spermatophore fuses w i t h the w a l l of the e f f e r e n t duct; the spermatophore passes i n t o the lumen of the e f f e r e n t duct and the S e r t o l i c e l l s become a p a r t of the e p i t h e l i a l l i n i n g of the e f f e r e n t duct (F i g . 10). Both S e r t o l i c e l l s and the e p i t h e l i a l c e l l s of the e f f e r e n t ducts have l a r g e o v a l n u c l e i w i t h d i s t i n c t n u c l e o l i . I t may be suggested th a t the spermatophores w i t h i n the germinal p o r t i o n of the t e s t i s are surrounded by S e r t o l i c e l l s ; and a f t e r the spermatophores pass i n t o the e f f e r e n t duct, the S e r t o l i c e l l s become continuous w i t h the e p i t h e l i a l l i n i n g of the e f f e r e n t duct. Wiebe (1967) has r e f e r r e d to e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts as S e r t o l i c e l l s i n the Shiner seaperch (Cymatogaster aggregata). The spermatophores congregate i n groups i n the e f f e r e n t ducts and the main sperm duct. A c o l l o i d a l m a t e r i a l i s n o t i c e d around and i n the spaces between the spermatophores (F i g . 6). The source of c o l l o i d a l m a t e r i a l i s presumed to be e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts and main sperm duct, because these appear cuboid a l and depleted where spermatophores are present, while columnar and f i l l e d w i t h s e c r e t o r y d r o p l e t s where spermatophores are absent. Medlen (1950) has suggested that the c o l l o i d a l m a t e r i a l contains a n u t r i t i v e substance f o r the maintenance of sperm and perhaps a mucilaginous substance to cause the spermatophores to adhere to one another. 13 There i s no prominent c o n n e c t i v e - t i s s u e core i n the t e s t i s . The t e s t i s c o n s i s t s of a tenuous, d i f f u s e , connective t i s s u e stroma w i t h c l o s e l y packed cyst s i n the i n t e r s t i c e s . The i n t e r s t i t i a l c e l l s (Leydig c e l l s ) w i t h rounded n u c l e i are found i n the c e n t r a l p a r t of the t e s t i s d i s persed i n the space between the branches of the e f f e r e n t ducts ( F i g . 12). Secondary sex characters of the male guppy. The a d u l t guppy has two p a r t i c u l a r l y w e l l - d i f f e r e n t i a t e d secondary sex c h a r a c t e r s : (a) s e v e r a l patches of b r i g h t c o l o r s on the s i d e s of the body and (b) the gonopodium. The most conspicuous c o l o r patches are the l i p o p h o r e s . The b r i g h t n e s s of the lipophores may be q u a n t i f i e d by a s s i g n i n g + signs — (++++) very b r i g h t ; (+++) b r i g h t ; (++) d u l l and (+) t r a c e . The v a l i d i t y of t h i s method i s open to question, s i n c e i t i s not known whether the a r b i t r a r y signs are i n l i n e a r r e l a t i o n to the content of y e l l o w and red pigments i n the area under observation. Since a l l a d u l t males u s u a l l y have b r i g h t l i p o p h o r e s , they may be assigned ++++ or +++ s i g n s . The morphological d e t a i l s of the transformation of the male a n a l f i n i n t o the gonopodium have been described by Hopper (1949a). There are 10 rays present i n the a n a l f i n of the guppy; i n the male, rays 3, 4 and 5 become elongated. Ray 3 becomes t h i c k (bone deposition) and develops a hood and v e n t r a l spines. Ray 4 a l s o develops spines but ray 5 has only a hook a t i t s t i p and no spines ( F i g . 31). 14 S t r u c t u r e of the t e s t i s and secondary sex characters of sham-operated c o n t r o l guppy. Sham-operation does not b r i n g about any changes i n the s t r u c t u r e of the a d u l t t e s t i s . The spermatogonial cyst s are at the periphery and the spermatophores are i n the centre; the area between the two i s f i l l e d w i t h the cyst s c o n t a i n i n g the various developmental stages of spermatocytes, spermatids and sperm (F i g s . 2a, 2b, 20 and t a b l e s II...arid V) . The e f f e r e n t ducts and the main sperm duct are f i l l e d w i t h i n t a c t spermatophores ( F i g s . 4 & 6). The e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts are t a l l and columnar ( F i g . 4 and Table I I I ) and contain l i p i d d r o p l e t s ( F i g . 14). The i n t e r s t i t i a l c e l l s have o v a l n u c l e i ( F i g . 12) and give a p o s i t i v e t e s t f o r l i p i d s ( F i g . 14). The secondary sex characters are unchanged i n the sham-operated males. S t r u c t u r e of the t e s t i s of hypophysectomized guppy. A d u l t male guppies were hypophysectomized and two to three f i s h were k i l l e d a t weekly i n t e r v a l s up to e i g h t weeks i n one experiment. In another experiment the hypophysectomized f i s h were k i l l e d a f t e r 16 weeks. In both the experiments there i s a considerable decrease i n the gonosomatic i n d i c e s of the operated f i s h (Tables I and I V ) . A f t e r e i g h t weeks the t e s t i s of a hypophysectomized guppy i s completely packed w i t h spermatophores except f o r a few spermatogonial cyst s a t the periphery (Figs. 3a, 3b, 20 and Table I I ) . I t seems that the cyst s c o n t a i n i n g spermatocytes, spermatids and sperm present at the time of o p e r a t i o n transform TABLE I G.S.I, of hypophysectomized and sham-operated guppies a f t e r 8 weeks. G. S. I. F i s h No Hypophysectomized Sham-operated 1 0.84 - 3.53 2 0.86 3.80 3 0.93 2.75 4 0.91 2.94 5 0.97 4.39 Mean 1 + + *** -±e= 0.9 0 * 0 . 0 2 3 . 4 8 * 0 . 3 0 -'-Standard 'Error * * * P < o . 001 TABLE I I Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized and sham-operated guppies a f t e r 8 weeks. Stages of spermatogenesis SPG SPC SPD SPM SPR No % No % No % No % No % Mean 1 1.6 1.82 0 0 0 97.6 98.18 Hypophysectomized ^ x ±0.6 +0.48 ±16.3 ±0.75 Mean-1 2.6 1.11 23.0 10.04 16.0 6.84 23.8 11.07 185.2 70.93 Sham-operated ±Sx ±0.3 ±0.13 ±3.5 ±2.10 ±1.5 ±0.77 ±3.5 ±2.65 ±42.6 +±5.47 •l-Mean of 5 observations P < 0.001, contingency t a b l e (row x column) SPG-^-Spermatogonia; SPC - spermaocytes; SPD - spermatids, SPM-sperm; SPR-spermatophores. 17 TABLE I I I E p i t h e l i a l c e l l heights of the e f f e r e n t ducts of hypophysectomized and sham-operated guppies a f t e r 8 weeks. Mean e p i t h e l i a l c e l l height"*" (u) F i s h No Hypophysectomized Sham-operated 1 4.3 33.2 2 3.3 20.5 3 4.0 27.1 4 4.2 13,1 5 4.6 22.6 Grand Mean 4.1 23.3 "'"Mean of 25 5 e f f e r e n t counts (5 e p i t h e l i a l ducts i n one s e c t i o n c e l l heights i n each of of each t e s t i s . ) P < 0.005 - A n a l y s i s of variance based on 5 median counts, 1 i n each of 5 e f f e r e n t ducts of each t e s t i s . TABLE TV G-S.I. of hypophysectomized and sham-operated guppies a f t e r 16 weeks. F i s h No Hypophysectomized Sham-operated 1 0.63 3.61 2 0.38 4.12 3 0.49 3.73 4 0.46 3.41 5 0.31 3.84 Mean ±Sx 0.45+0.05 3.7410.12 *** p < 0.001 TABLE V Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized and sham-operated guppies a f t e r 16 weeks. Stages of spermatogenesis SPG SPC SPD SPM SPR No % No No % No % No % Hypophysectomi zed Mean 1 ±Sx- = 2.2 ±0.8 4.68 ±0.92 0 0 0 47.4 +13.6 95.32 ±0.92 Sham-operated Mean 1 ±Sx 3.4 ±0.4 1.88 ±0.29 23.6 ±3.7 12. ±3. 45 02 14.6 ±2.1 7.53 "±1.25 22.2 "±3.9 10.73 ±0.27 137.4 ±22.9 67.41 ±4.95 Mean of 5 observations P < 0.001, contingency t a b l e (row x column) 20 i n t o spermatophores. During the second week a f t e r the ope r a t i o n few spermatocytes, many spermatids and sperm are evident i n the t e s t i s but by the end of the t h i r d week only sperm-cysts are n o t i c e d ( F i g . 11). From the f o u r t h week onwards a f t e r the ope r a t i o n the t e s t i s contains only spermatogonia and spermatophores. This i n d i c a t e s t h a t spermatogonia are not transformed i n t o spermatocytes i n the absence of the p i t u i t a r y but spermatocytes, spermatids and sperm are transformed i n t o spermatophores during the f i r s t three weeks. The presence of spermatophores at the periphery of the t e s t i s of a hypophysectomized guppy ( F i g s . 3a,b) w i t h no t r a c e of d i s i n t e g r a t i n g cysts c o n t a i n i n g e a r l i e r stages of spermatogenesis suggests t h a t spermatocytes, spermatids and sperm do not d i s i n t e g r a t e but transform i n t o spermatophores. In the hypophysectomized guppy no m i t o t i c d i v i s i o n of spermatogonia i s evident; many spermatophores are found ruptured i n the e f f e r e n t ducts and the main sperm duct (Figs. 5 & 7). The e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts decrease i n height to become squamous ( F i g . 5 and Table I I I ) . The e p i t h e l i a l c e l l s of the e f f e r e n t ducts do not c o n t a i n any l i p i d d r o p l e t s . S e r t o l i c e l l s a l s o regress i n the hypophysectomized animal; t h e i r n u c l e i change from rounded c o n d i t i o n to f l a t t e n e d shape ( F i g . 9 ). There i s an apparent increase i n the number of f i b r o b l a s t s throughout the t e s t i s ( F i g . 5). The i n t e r s t i t i a l c e l l s are regressed w i t h shrunken n u c l e i ( F i g . 13) and no l i p i d d r o p l e t s were evident i n frozen s e c t i o n s s t a i n e d w i t h Sudan black B. The s t r u c t u r e of the t e s t i s of a hypophysectomized guppy a f t e r 16 weeks i s s i m i l a r to the t e s t i s a f t e r e i g h t weeks except f o r the f a c t that most of the spermatophores are ruptured (Table V and V I ) . The d i f f e r e n c e s i n percentag compositions of spermatogonia and spermatophores i n the t e s t e s of hypophysectomized guppies a f t e r e i g h t and 16 weeks are 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 (Table V I ) . Thus the trend of d e t e r i o r a t i n g spermatophores which i s n o t i c e a b l e a f t e r e i g h t weeks of hypophysectomy i s much more pronounced a f t e r 16 weeks. There i s a strong i n d i c a t i o n t h a t the sperm r e s u l t i n g from ruptured spermatophores are being phagocytosed w i t h i n the e f f e r e n t ducts and the main sperm duct ( F i g . 15). The e n t i r e t e s t i s appears as a mass of f i b r o u s connective t i s s u e ( F i g . 15) because of rupture and disappearance of spermatophores and pronounced increase i n the number of f i b r o b l a s t s . Secondary sex characters of hypophysectomized guppy. In both the experiments (eight and 16 weeks a f t e r hypophy- . sectomy) the s t r u c t u r e of the gonopodium i s unaltered. The lipophores become f a i n t or e n t i r e l y disappear both on the sides of the body and on the t a i l and were r a t e d as 0 to + i n the a r b i t r a r y s c a l e described e a r l i e r . S t r u c t u r e of the t e s t i s and secondary sex characters of hypophysectomized c o n t r o l guppy (testosterone experiment) The t e s t i s has few spermatogonial cyst s a t the periphery; the r e s t of the t e s t i s i s f i l l e d w i t h spermatophores, many of which are ruptured (Figs . 3, 21 and Table V I I ) . S e r t o l i TABLE VI Number and percentage of spermatogonia and spermatophores i n hypophysectomized guppies a f t e r 8 weeks and 16 weeks Stages of spermatogenesis SPC SPD SPM SPR No % No % No % No % No % 8 weeks a f t e r Mean 1 1.6 1.82 0 0 0 97.6 98.18 hypophy- ±Sx ±0.48 ±0.75 sectomy ±0.6 ±16.3 16 weeks a f t e r Mean 1 2.2 4.68 0 0 0 47.4 95.32 Hypophy- ±Sx ±0.8 ±0.92 ±13.6 ±0.92 sectomy Mean of 5 observations p<0.5, contingency t a b l e (row x column) TABLE V I I Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized, t e s t o s t e r o n e - t r e a t e d and sham-operated guppies. Stages of spermatogenesis SPG SPC SPD SPM SPR No r No % No % No % No % (A) 1 Mean 1.8 3. 52 0 0 0 55.8 96.48 Hypo phy s e c tomi zed ±S it- ±0.4 ±0. 99 ±11.0 ±0.99 (B) Testosterone- t r e a t e d Mean ±Sx 9.8 ±1.9 46. ±2. 23 16 4.4 22.16 ±0.5 ±1.19 0 0 6.8 ±1.5 31.58 ±1.87 (C) Mean 3.8 1. 70 23.4 11.08 14.4 6.94 20.2 9. 70 152.4 70.57 S ham-opera ted -Sx ±1.1 ±0. 35 ±2.2 ±1.16 ±3.2 ±1.59 ±6.7 ±3. 24 ±1:9.4 ±5.52 Mean of 5 observations P < 0.001 between A & B; A & C; B & C: Contingency t a b l e (row x column) 24 c e l l s and i n t e r s t i t i a l c e l l s regress and t h e i r n u c l e i are shrunken (Fig s . 9 & 13). The e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts are regressed ( F i g . 5 and Table V I I I ) . The lipophores become f a i n t or e n t i r e l y disappear. S t r u c t u r e of the t e s t i s and secondary sex characters of sham-operated c o n t r o l guppy (testosterone experiment). The s t r u c t u r e of the t e s t i s of sham-operated guppy i s e x a c t l y s i m i l a r to the a d u l t t e s t i s p r e v i o u s l y described. A l l stages of spermatogenesis are present ( F i g s . 2 & 21 and Table V I I ) . The e f f e r e n t ducts and the main sperm duct are f i l l e d w i t h i n t a c t spermatophores (Fig s . 4 & 6). The e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts are t a l l and columnar ( F i g . 4 and Table VIXI)- The secondary sex characters are unchanged i n sham-operated males. E f f e c t of methyl testosterone on the a d u l t t e s t i s of hypophysectomized guppy. Hypophysectomized male guppies were t r e a t e d w i t h methyl testosterone f o r f i v e weeks. During t h i s treatment two to three f i s h were k i l l e d a t weekly i n t e r v a l s ; the remaining ( f i v e ) were k i l l e d a t the end of the f i f t h week. There i s a s i g n i f i c a n t increase i n the gonosomatic index of hypophysectomized guppy f o l l o w i n g methyl testosterone treatment (Table IX) a s s o c i a t e d w i t h a r a p i d m u l t i p l i c a t i o n of both spermatogonial and spermatocyte-cysts ( F i g s . 16, 21 and Table V I I ) . Cysts c o n t a i n i n g spermatocytes are evident by the end of second week of testosterone treatment but no l a t e r stages of spermatogenesis appear during the next three 25 TABLE V I I I E p i t h e l i a l c e l l heights of the e f f e r e n t ducts of hypophysectomized, t e s t o s t e r o n e - t r e a t e d and sham- operated guppies. Mean e p i t h e l i a l c e l l height (u) i s h < A ) <B> < C ) No Hypophysectomized Testosterone- Sham-operated t r e a t e d 1 4.4 34.0 25.5 2 4.8 31.5 21.2 3 3.8 30.2 18.6 4 4.3 38.9 22.4 5 3.5 39.4 20.1 £ r * n d 4.2 34.8 21.6 Mean 1Mean of 25 counts (5 e p i t h e l i a l c e l l heights i n each of 5 e f f e r e n t ducts i n one s e c t i o n of each t e s t i s ) . P<0.01 between A & B; A & C; B & C. Tukey's t e s t - W Q 1 = 5.05. Test based on 5 median counts, 1 i n each of 5 e f f e r e n t ducts of each t e s t i s . 26 TABLE IX G.S.I, of hypophysectomized, testosterone - - t r e a t e d and sham-operated guppies. G. S. I . (A) (B) (C) F i s h Testosterone- No Hypophysectomized t r e a t e d Sham-operated 1 0.69 1.37 3. 04 2 0.67 1.39 3.10 3 0.48 1.18 2.76 4 0.77 1.34 3.41 5 0.71 1.12 3.27 +?- n 0.66*0.04 1.28±0.05 3.12*0.11** ** P<0.01 between A & B; A & C; B & C. Tukey's t e s t - W Q 1 = 0.40 27 weeks. Thus testosterone treatment does not l e a d to complete r e s t o r a t i o n of spermatogenesis i n hypophysectomized guppies. Occasional m i t o t i c d i v i s i o n i n the spermatogonial cy s t s can be n o t i c e d ( F i g . 17). The r e s o r p t i o n of sperm, r e s u l t i n g from the rupture of spermatophores, i s complete w i t h i n the e f f e r e n t ducts and the main sperm duct during the f i r s t two weeks of testosterone treatment ( F i g . 18). This i n d i c a t e s t h a t the exogenous testosterone enhances the rupture of spermatophores and the phagocytosis (resorption) of sperm. Only few spermatophores are l e f t i n t a c t at the periphery ( F i g . 16). Methyl testosterone treatment s t i m u l a t e s the e p i t h e l i a l c e l l s of both e f f e r e n t ducts and main sperm duct so that they assume the t a l l columnar appearance of the normal animal ( F i g . 19 and Table V I I I ) . S e r t o l i c e l l s and i n t e r s t i t i a l c e l l s are a l s o r e s t o r e d to normal and t h e i r n u c l e i become s p h e r i c a l i n shape w i t h conspicuous n u c l e o l i ( F i g . 17). The e p i t h e l i a l c e l l s l i n i n g the sperm ducts, S e r t o l i c e l l s and i n t e r s t i t i a l c e l l s respond to the testosterone treatment i n a s i m i l a r way. They regress i n the absence of the p i t u i t a r y and a t t a i n t h e i r normal appearance f o l l o w i n g testosterone treatment. Except f o r the c y s t s c o n t a i n i n g spermatogonia, spermatocytes and spermatophores, the whole t e s t i s i s f i l l e d w i t h f i b r o u s connective t i s s u e ( F i g . 16). 28 E f f e c t of methyl testosterone on secondary sex characters of hypophysectomized guppy. The gonopodium which was unaffected by hypophysectomy remains unchanged when the animals are t r e a t e d w i t h methyl testosterone. There i s moderate recovery i n the content of lipophores present on the s i d e s of body and the t a i l . The lipophores were r a t e d as ++ i n the a r b i t r a r y q u a n t i t a t i v e r a t i n g . This suggests that the lipophores are d i r e c t l y c o n t r o l l e d by the androgens. 29 F i g u r e l a : S e c t i o n of whole t e s t i s of a d u l t guppy. Fi g u r e l b : D e t a i l of a p o r t i o n of t e s t i s i n d i c a t e d by arrow i n l a . lobe of testis spermatogonium spermatocyte 30 Fi g u r e 2a: S a g i t t a l s e c t i o n of a d u l t t e s t i s showing d i f f e r e n t stages of spermatogenesis (x 200) Figure 2b: Magnified view of a p o r t i o n of 2a (x 9 00) Figur e 3a: S a g i t t a l s e c t i o n of t e s t i s e i g h t weeks a f t e r hypophysectomy showing only spermatophores (x 200) Fi g u r e 3b: Magnified view of a p o r t i o n of 3a (x 9 00) SG - Spermatogia; SC - Spermatocytes; SD - Spermatids; SM - Sperm; SR - Spermatophores  31 Fi g u r e 4: E f f e r e n t ducts of a d u l t t e s t i s c o n t a i n i n g spermatophores (x 2 00) . Fi g u r e 5: Ruptured spermatophores i n the e f f e r e n t ducts of t e s t i s e i g h t weeks a f t e r hypophysectomy (x 200) Fi g u r e 6: Main sperm duct of a d u l t t e s t i s c o n t a i n i n g spermatophores (x 2 00). Note the c o l l o i d a l m a t e r i a l i n main sperm duct. Fi g u r e 7: Ruptured spermatophores i n the main sperm duct of t e s t i s e i g h t weeks a f t e r hypophysectomy (x 200) E.D. - E f f e r e n t ducts; M.D.- Main sperm duct; E.C. - E p i t h e l i a l c e l l s ; C L . - C o l l o i d a l m a t e r i a l .  32 Figu r e 8: S e r t o l i c e l l s of a d u l t t e s t i s w i t h sperm heads attached to them (x 900). Fi g u r e 9: Regressed S e r t o l i c e l l s of t e s t i s e i g h t weeks a f t e r hypophysectomy (x 9 00) . Fi g u r e 10: Spermatophores w i t h surrounding S e r t o l i c e l l s of a d u l t t e s t i s opening i n t o e f f e r e n t ducts ( i n d i c a t e d by arrows) (x 200). Figure 11: T e s t i s a t the end of t h i r d week a f t e r hypophy- sectomy c o n t a i n i n g o n l y sperm-cysts and spermatophores (x 200). S.C. - S e r t o l i c e l l s .  33 Figur e 12: I n t e r s t i t i a l c e l l s of a d u l t t e s t i s (x 900). Figur e 13: Regressed i n t e r s t i t i a l c e l l s of t e s t i s e i g h t weeks a f t e r hypophysectomy (x 9 00). F i g u r e 14: I n t e r s t i t i a l c e l l s and e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts of a d u l t t e s t i s s t a i n e d w i t h Sudan black B (x 350). F i g u r e 15: T e s t i s showing remnant sperm being phagocytosed i n e f f e r e n t ducts 16 weeks a f t e r hypophysectomy (x 200) . I.C - I n t e r s t i t i a l c e l l s ; R.S - Remnant sperm  34 F i g u r e 16: T e s t i s of hypophysectomized guppy t r e a t e d w i t h testosterone f o r f i v e weeks (x 2 00). F i g u r e 17: Magnified view of a p o r t i o n of f i g u r e 16 (x 900). Arrow i n d i c a t e s m i t o t i c d i v i s i o n i n spermatogonia. S e r t o l i c e l l s assume normal appearance. Fi g u r e 18: Remnant sperm i n main sperm duct of hypophy- sectomized guppy a f t e r two weeks of tes t o s t e r o n e - treatment (x 200). Note regressed e p i t h e l i a l c e l l s l i n i n g main sperm duct. Fi g u r e 19: T a l l e p i t h e l i a l c e l l s reappear i n sperm duct of hypophysectomized guppy a f t e r f i v e weeks of testosterone treatment (x 200).  35 Fi g u r e 20: Percentage of d i f f e r e n t stages of spermato- genesis i n hypophysectomized and sham- operated a d u l t guppies a f t e r e i g h t weeks. The v e r t i c a l l i n e on top of each bar represents Standard E r r o r . SPG - Spermatogonia; SPC - Spermatocytes; SPD - Spermatids; SPM - Sperm; SPR - Spermatophores. SPG' SPC SPD SPM S P R 36 F i g u r e 21: Percentage of d i f f e r e n t stages of spermato- genesis i n hypophysectomized; t e s t o s t e r o n e - t r e a t e d and sham-operated a d u l t guppies. 1 0 0 S P G S P C S P D S P M S P R 37 SECTION I I HYPOPHYSECTOMY AND METHYL TESTOSTERONE TREATMENT OF THE JUVENILE GUPPIES. INTRODUCTION Although gonadal d i f f e r e n t i a t i o n occurs s h o r t l y before b i r t h , the two sexes are e x t e r n a l l y i n d i s t i n g u i s h a b l e i n the newly born young. In male embryos, the germ c e l l s are r e l a t i v e l y uniformly d i s t r i b u t e d among stroma c e l l s but at b i r t h the spermatogonia tend to migrate to the periphery. Later (21 days a f t e r b i r t h ) the sperm ducts appear as narrow s l i t s surrounded by stroma c e l l s (Goodrich et a l 1934). In t h i s s e c t i o n , the e f f e c t s of hypophysectomy on such j u v e n i l e males without secondary sex characters and w i t h gonads con t a i n i n g only spermatogonia, are described. The r e s u l t s of t r e a t i n g such hypophysectomized j u v e n i l e males w i t h methyl testosterone should i n d i c a t e whether secondary sex characters are d i r e c t l y c o n t r o l l e d by p i t u i t a r y hormones or se c o n d a r i l y by the androgens. MATERIALS AND METHODS Maintenance of hypophysectomized j u v e n i l e f i s h . One week p r i o r to the opera t i o n j u v e n i l e guppies (9-11 mm standard length; 14-3 0 mg i n weight) i n c l u d i n g both males and females (since sex i s i n d i s t i n g u i s h a b l e at t h i s stage) 38 were taken from the breeding aquaria and put i n aquaria w i t h 1 6 - l i t r e of f i s h s a l i n e . The j u v e n i l e s (both hypophysectomized and sham-operated) were kept i n t h i s medium u n t i l the end of the experiment. A group of j u v e n i l e s was k i l l e d as i n i t i a l c o n t r o l s immediately a f t e r removal from the breeding aquaria. The maintenance of j u v e n i l e s was s i m i l a r to that described e a r l i e r f o r hypophysectomized a d u l t males. Procedure of hypophysectomy. J u v e n i l e guppies co n d i t i o n e d to f i s h s a l i n e f o r one week were an e s t h e t i z e d w i t h 1:800 MS 222 ( T r i c a i n e Methane Sulphonate-Sandoz). The f i s h was placed v e n t r a l s i d e uppermost on a(6 x 3 x ^ cm) piece of s o f t p l a s t i c sponge f i x e d i n the middle of a w a x - f i l l e d r e c t a n g u l a r p l a s t i c t r a y (16 x 8 x 3^ cm). The f i s h was secured i n place by a t h i n . s t r i p of s o f t p l a s t i c sponge s t r e t c h e d across the v e n t r a l surface j u s t p o s t e r i o r to the g i l l s and was immersed i n f i s h s a l i n e during the operation. The op e r a t i o n of the j u v e n i l e guppy f o r the removal of the p i t u i t a r y was s i m i l a r to t h a t p r e v i o u s l y described f o r the a d u l t male. M o r t a l i t y was high, amounting to 30-35% during the operation; a f u r t h e r m o r t a l i t y of 25-30% occurred i n two weeks f o l l o w i n g the o p e r a t i o n . Methyl testosterone treatment; The aquaria w i t h 1 6 - l i t r e of f i s h s a l i n e were used f o r testosterone experiment. Two d i f f e r e n t concentrations of methyl testosterone were used; 1:2x10^ (8 mgm of methyl testosterone i n 16 l i t r e s of 7 f i s h s a l i n e per week) and 1:10 (1.6 mgm of methyl testosterone i n 16 l i t r e s of f i s h s a l i n e every week). The treatment of hypophysectomized j u v e n i l e guppies w i t h methyl 39 testosterone was begun one week a f t e r the ope r a t i o n . The treatments continued f o r e i g h t weeks. The maintenance of j u v e n i l e s was s i m i l a r to that described e a r l i e r f o r methyl testosterone t r e a t e d hypophysectomized a d u l t males. Hypophysectomized j u v e n i l e guppies which d i d not r e c e i v e any testosterone treatment and sham-operated j u v e n i l e s were the two types of c o n t r o l s and were a l s o kept i n f i s h s a l i n e . H i s t o l o g y . Because of the small s i z e , the e n t i r e body of the j u v e n i l e guppy except f o r i t s caudal peduncle and the t a i l was f i x e d i n formic acid-Bouin f i x a t i v e and l e f t f o r seven days i n f i x a t i v e to d e c a l c i f y before dehydration i n e t h y l a l c o h o l and embedding i n p a r a f f i n wax. S e r i a l l o n g i t u d i n a l s e c t i o n s were cut a t 5 u and s t a i n e d w i t h E h r l i c h ' s haematoxylin and eos i n . Measurements. The percentage of d i f f e r e n t stages of spermatogenesis present i n the te s t e s of the j u v e n i l e or the a d u l t guppies (since sham-operated j u v e n i l e s became a d u l t s during the experimental period) was c a l c u l a t e d i n the same way as expl a i n e d p r e v i o u s l y f o r the a d u l t males. The t o t a l number of e f f e r e n t ducts i n the median s a g i t t a l s e c t i o n was counted. The width and lumen of f i v e randomly chosen e f f e r e n t ducts (before they j o i n to form main sperm duct) were measured w i t h an ocular micrometer i n one s e c t i o n of each t e s t i s of hypophysectomized, testosterone t r e a t e d or sham-operated guppies. The height of e p i t h e l i a l c e l l s and 40 width of lumen (space between e p i t h e l i a l c e l l l i n i n g s ) of main sperm duct i n the median s a g i t t a l s e c t i o n of each t e s t i s were measured a t f i v e d i f f e r e n t places which covered the e n t i r e l e n g t h of main sperm duct. RESULTS S t r u c t u r e of the j u v e n i l e t e s t i s ( i n i t i a l c o n t r o l ) . The j u v e n i l e t e s t i s c o n s i s t s of two lobes connected by a bridge of mesodermal or stroma c e l l s . I t i s surrounded by a t h i n , squamous, p e r i t o n e a l membrane. The t e s t i s i s s i t u a t e d i n the p o s t e r i o r p a r t of the body c a v i t y , v e n t r a l to the a i r b l a d d e r and d o r s a l to the pancreas and the c o i l s of i n t e s t i n e . No other stages of spermatogenesis except spermatogonia are found i n the t e s t i s . Spermatogonia are present i n s m a l l cy s t s at the periphery of t e s t i s ( F i g s . 22, 27 and Table X); the centre i s f i l l e d w i t h stroma c e l l s ( F i g . 23). M i t o t i c d i v i s i o n i s not evident i n the spermatogonia1 c y s t s . Spermatogonia are o v a l w i t h d i s t i n c t c e l l u l a r membranes and homogeneous cytoplasm except f o r the presence of o c c a s i o n a l f i n e b a s o p h i l i a . Their n u c l e i are almost round w i t h evenly d i s t r i b u t e d f i n e chromatin granules. There i s u s u a l l y a s i n g l e n u c l e o l u s . The stroma c e l l s , on the other hand, have no d i s t i n c t c e l l u l a r membranes. Their n u c l e i are e l l i p t i c a l and each contains an i r r e g u l a r and coarse r e t i c u l u m . The S e r t o l i c e l l s and i n t e r s t i t i a l c e l l s are not evident i n the t e s t i s . TABLE X Number and percentage of d i f f e r e n t stages of spermatogenesis i n j u v e n i l e c o n t r o l ; j u v e n i l e hypophysectomized and j u v e n i l e sham-operated guppies. Stages of spermatogenesis SPG SPC SPD SPM SPR No % No % No % No % No % (A) J u v e n i l e C o n t r o l Mean"1" ±Sx 13.4 100 ±1.28 P 0 0 0 (B) J u v e n i l e Hypophysectomized 1 Mean ±Sx 12.4 100 ±0.68 0 0 0 0 (C) J u v e n i l e S ham-operated Mean"*" ±Sx 3.8 2.17 +0.8 ±0.45 34.8 20.03 ±4.6 ±2.83 23.0 13.07 ±1.1 ±0.29 36.4 ±2.6 20. ±1. 92 96 78 143. ±10.9 ±4. 81 31 Mean of 5 observations. P < 0.001 - A & C; B & C. Contingency t a b l e (row x column) 42 During development the stroma c e l l s i n the centre of the t e s t i s are rearranged to form s m a l l e f f e r e n t ducts, although the lumen i n the e f f e r e n t duct i s hardly d i s t i n c t ( F i g . 23 and Table X i ) . The e f f e r e n t ducts from the two lobes of the t e s t i s open at the p o s t e r i o r end i n t o a main sperm duct w i t h a narrow lumen ( F i g . 25 and Table X I I ) . The e p i t h e l i a l c e l l s l i n i n g the main sperm 'duct' are squamous" and are deri v e d from stroma c e l l s (the main sperm duct appears as a r e s u l t of rearrangement of stroma c e l l s ) . The main sperm duct opens i n t o the u r o g e n i t a l s i n u s . S t r u c t u r e of the t e s t i s of hypophysectomized j u v e n i l e . Since the c o n t r o l sham-operated j u v e n i l e s d i f f e r e n t i a t e d i n t o a d u l t s i n s i x weeks, the hypophysectomized j u v e n i l e guppies were a l s o k i l l e d a f t e r s i x weeks. No changes take place i n the s t r u c t u r e of j u v e n i l e t e s t i s i n the absence of the p i t u i t a r y except f o r an increase i n the number of connective t i s s u e c e l l s . The spermatogonia1 cysts are present at the periphery but show no m i t o t i c d i v i s i o n ; consequently there i s no increase i n t h e i r number (Figs. 22, 27 and Table X). The S e r t o l i c e l l s are not evident. The stroma c e l l s are not d i f f e r e n t i a t e d i n t o the i n t e r s t i t i a l c e l l s (stroma c e l l s have e l l i p t i c a l n u c l e i and contain an i r r e g u l a r r e t i c u l u m , whereas i n t e r s t i t i a l c e l l s have round n u c l e i with a d i s t i n c t n u c l e o l u s ) . There i s no increase i n the t o t a l number, width and the s i z e of lumina of the e f f e r e n t ducts ( F i g . 23 and Table X I ) . The e p i t h e l i a l TABLE XI T o t a l number, width and lumen of e f f e r e n t ducts of j u v e n i l e c o n t r o l , j u v e n i l e hypophysectomized and j u v e n i l e sham-operated guppies. E f f e r e n t ducts (A) J u v e n i l e c o n t r o l F i s h No (B) J u v e n i l e Hypophysectomized (C) J u v e n i l e Sham-operated No Width(ul Mean1±Sx Lumenfu) Mean^Sx No Width(u) Mean^Sx Lumen (u)_ Mean1±Sx No Width(ul Mean1±Sx Lumenful Meani^Sx 1 8 15.66 1. 08 7 13.50 1.08 19 122. 18 76. 26 ±1.90 ±0.18 ±1.30 ±0.18 ±7. 38 ±6. 30 2 9 16.38 1.26 7 14.22 1.44 17 114. 80 63. 96 ±1.52 ±0.22 ±0.77 ±0.22 ±8. 60 ±7. 95 3 7 18.18 1.26 8 10.98 1.62 19 127. 10 71. 34 ±1.12 ±0.22 ±0.52 ±0:34 ±15. 0 ±14. 87 4 6 10.62 1.08 6 10.80 1.26 18 121. 76 77. 08 ±0.34 ±0.18 ±0.80 ±0.22 ±14. 36 ±12. 12 5 9 12.96 1.44 8 16. 56 1.26 21 118. 90 64. 78 ±0.. 46 ±0.36 ±1.67 ±0.22 ±9. 44 ±7. 03 Mean 1 7.2 7.2 18.8 . ±Sx ±0.58 ±0.37 ±0.66 1Mean of 5 observations P < 0. 01 - A Sc C; B & C (for no., width and lumen) Not s i g n i f i c a n t - A & B (for no. , width and lumen) • Tukey 1 s t e s t - W.oi f o r no. - 2.77 W.oi f o r width - 11.98 w . 01 f o r lumen - 10.67 TABLE X I I E p i t h e l i a l c e l l heights and lumen of main sperm duct of j u v e n i l e c o n t r o l ; j u v e n i l e hypophysectomized and j u v e n i l e sham-operated guppies. Main sperm duct (u; Mean 1 + Sx ) (A) (B) (c) J u v e n i l e F i s h J u v e n i l e C o n t r o l Hypophysectomized J u v e n i l e Sham-operated No E p i t h e l i a l c e l l s Lumen E p i t h e l i a l c e l l s Lumen E p i t h e l i a l c e l l s Lumen 1 3.24 2.16 2.88 1.18 12.34 159.80 ±0.22 ±0.46 ±0.18 ±0.28 ±1.27 ±8.30 2 2.88 1.62 2.70 1.62 13.12 82.82 ±0.18 ±0.34 ±0.28 ±0.44 ±0.82 ±4.38 3 2.52 1.98 2.52 1.98 17.22 : .179-. 58 ±0.18 ±0.18 ±0.34 ±0.34 ±2.39 ±12.53 4 1.98 1.26 2.34 1.80 13.12 182.04 ±0.18 +0.22 ±0.22 ±0.28 ±0.82 ±7. 09 5 2.88 1.26 2.16 1.62 13.94 86.10 ±0.52 ±0.36 ±0.22 ±0.18 ±1.00 +±2.90 iMean of 5 observations P< 0.01 - A & C; B & C ( f o r e p i t h e l i a l c e l l s and lumen) Not s i g n i f i c a n t - A & B (for e p i t h e l i a l c e l l s and lumen)'. Tukey's t e s t - W > 01 f o r e p i t h e l i a l c e l l s - 1.71 w.01 f o r lumen - 23.59 45 c e l l s l i n i n g the main sperm duct remain squamous and the width of the lumen of the main sperm duct i s not increased ( F i g . 25 and Table X I I ) . Thus i t i s apparent that f u r t h e r development of the j u v e n i l e t e s t i s i n c l u d i n g the d i f f e r e n t i a t i o n of the i n t e r s t i t i a l c e l l s and S e r t o l i c e l l s i s stopped i n the absence of the p i t u i t a r y . No secondary sex characters are present i n the j u v e n i l e s a t the beginning of the experiment ( F i g . 29). Secondary sex characters do not develop i n animals hypophysectomized as j u v e n i l e s . Both lipophore pigments and the anal f i n remain i n the s e x u a l l y i n d i f f e r e n t c o n d i t i o n ( F i g . 3 0). Thus i t i s evident that the presence of the p i t u i t a r y i s e s s e n t i a l f o r the d i f f e r e n t i a t i o n of secondary sex characters i n the j u v e n i l e guppy. S t r u c t u r e of the t e s t i s and secondary sex characters of the sham-operated j u v e n i l e . By the end of the experiment ( s i x weeks) as was to be expected, the sham-operated j u v e n i l e s were d i f f e r e n t i a t e d i n t o a d u l t males and females. The t e s t i s contains a l l stages of spermatogenesis ( F i g . 2 and Table X). Spermatophores are a l s o present i n the e f f e r e n t ducts and the main sperm duct (Figs 4 & 6). There i s a considerable increase i n the t o t a l number, width and the s i z e of lumina of the e f f e r e n t ducts ( F i g . 4 and Table X I ) . The width of lumen of the main sperm duct a l s o increases considerably and the e p i t h e l i a l c e l l s l i n i n g i t are columnar ( F i g . 6 and Table X I I ) . 46 Secondary sex characters are w e l l d i f f e r e n t i a t e d i n the males. The anal f i n i s transformed i n t o the gonopodium (F i g . 31) w h i l e b r i g h t patches of lipophores appear on the sides of the body and on the t a i l . S t r u c t u r e of the t e s t i s of hypophysectomized j u v e n i l e ( c o n t r o l of testosterone experiment). The t e s t i s contains only spermatogonial cyst s a t the periphery and stroma c e l l s i n the centre ( F i g s . 22,28 and Table X I I I ) . The e f f e r e n t ducts w i t h narrow luminaare present i n the centre of t e s t i s ( F i g . 23 and Table XIV)- The main sperm duct has narrow lumen and i s l i n e d by squamous e p i t h e l i a l c e l l s ( F i g . 25 and Table XV). No secondary sex characters develop. St r u c t u r e of the t e s t i s and secondary sex characters of the sham-operated j u v e n i l e . (Control of testosterone experiment). As might be expected, the sham-operated j u v e n i l e s were d i f f e r e n t i a t e d i n t o a d u l t males and females by the end of the experiment (eight weeks). The t e s t i s contains a l l stages of spermatogeneis (Figs 2, 28 and Table X I I l ) . The e f f e r e n t ducts are w e l l d i f f e r e n t i a t e d and are f i l l e d w i t h spermatophores ( F i g . 4 and Table XIV). The main sperm duct i s a l s o f i l l e d w i t h spermatophores and has columnar e p i t h e l i a l c e l l s ( F i g . 6 and Table XV)~. • The males have d i s t i n c t secondary sex char a c t e r s . The anal f i n i s f u l l y transformed i n t o a t y p i c a l gonopodium (F i g . 31), while b r i g h t patches of lipophores appear on the sides of the body; the lipophore index f o r these f i s h was +++ and ++++. TABLE X I I I Number and percentage of d i f f e r e n t stages of spermatogenesis i n j u v e n i l e hypophysectomized; j u v e n i l e hypophysectomized and testosterone tre a t e d ; j u v e n i l e sham-operated guppies. Stages of spermatogenesis SPG SPC SPD SPM SPR No % No °A No % No- % No % (A) Mean 1 J u v e n i l e hypophysectomized + S x 12.8 ±0.4 100 0 0 0 0 T  ( B )., Meanl J u v e n i l e hypo p hy s e c tomi ze d - test o s t e r o n e t r e a t e d 14.2 ±2.1 100 0 0 0 0 (C) Mean 1 J u v e n i l e sham-operated ±Sx 4.2 ±1.0 2.63 ±0.65 34.2 21. ±5.1 ±5. 32 1 22.4 14.20 ±1.8 ±1.40 27.6 ±4.5 17.43 ±2.85 71.4 44.42 ±7.2 ±2.96 Mean of 5 observations P< 0.001 - A & C; B & C , contingency t a b l e (row x column) TABLE XIV T o t a l number, width and lumen of e f f e r e n t ducts of j u v e n i l e hypophysectomized; j u v e n i l e hypophysecto- mized and testosterone t r e a t e d ; j u v e n i l e sham- operated guppies E f f e r e n t ducts F i s h No ... J u v e n i l e --' •• Hypophysectomi zed (B) J u v e n i l e Hypox. & Testo. t r e a t e d (C) J u v e n i l e Sham-operated No Width(u) Mean1±Sx Lumen(u) Mean1±Sx No Width(u) Mean !±Sx Lumen (u) Mean!±Sx No Width(u) Mean!±Sx Lumen(u) Mean1±Sx 1 9 18.36 ±0.67 1.08 ±0.18 39 33.44 ±5.38 14.76 ±3.99 12 132.84 ±15.43 75.44 ±12.67 2 9 17.10 ±1.03 1.44 ±0.36 41 32.58 ±3.42 11.70 ±2.55 17 108.22 ±8.06 59. 04 ±8.36 3 8 12.06 ±0.73 1.62 ±0.18 31 28.44 ±2.95 9.36 ±1.38 19 117.26 ±7.05 63.14 ±8.16 4 7 12.24 1 ±1.26 1.62 ±0.34 28 27.36 ±2.44 8.28 ±1.04 18 111.52 ±8.44 7-2-. 16 ±4.78 5 7 11.16 ±0.61 1.44 ±0.36 26 30. 06 ±2.63 8.28 ±1.25 20 102.5 ±6.08 63.82 ±3.23 Meanl + S x 8 +0.1 33 ±3.0 19 ±0.71 xMean of 5 observations P<0.01 - A & B (for number and width only) P < 0.01 - A & G; B & C; (for no, width and lumen) Tukey's t e s t - W # 01 f o r n o ~ 9-07 w ! o i f° r width - 11.75 W. 01 f o r lumen - 9.16 P <0.05 - A & B (for lumen only) Tukey's t e s t - W.05 f o r lumen - 7.28 TABLE XV E p i t h e l i a l c e l l heights and lumen of main sperm duct of j u v e n i l e hypophysectomized; j u v e n i l e hypophysectomized and t e s t o s t e r o n e - t r e a t e d ; j u v e n i l e sham-operated guppies. Main sperm duct (u; Mean 1 ±Sx) F i s h (A) J u v e n i l e Hypop hy s e c tomi zed (B) J u v e n i l e Hypox. & Testesterone-treated (C) J u v e n i l e sham-operated No E p i t h e l i a l c e l l s Lumen E p i t h e l i a l c e l l s Lumen E p i t h e l i a l c e l l s Lumen 1 3 . 6 0 ±0 .28 1.08 ± 0 . 1 8 1 6 . 2 0 ±2 .36 26 .28 ±3 .26 10 .74 V : ± 0 . 9 5 136 .94 ±11 .12 2 3 .42 ± 0 . 3 4 1.26 ± 0 . 2 2 9 .18 ± 1 . 0 0 21 .96 ± 4 . 0 8 13 .94 ±4 .02 9 4 . 3 0 ±10 .37 3 2 .52 ± 0 . 3 4 2 .16 ± 0 . 3 6 1 1 . 8 8 ±0 .96 1 9 . 8 0 ± 3 . 1 4 11 .48 ±1.53 113 .16 ±13 .57 4 2 . 8 8 ± 0 . 3 4 1.62 ± 0 . 1 8 8 .46 ± 0 . 7 3 16 .56 ± 3 . 0 6 14 .72 ±2 .78 113 .16 ±14.53 5 2 .16 ± 0 . 2 2 1 .80 ± 0 . 2 8 7 .02 ± 0 . 6 0 10 .62 ± 1 . 7 4 13 .12 ± 2 . 0 1 130 .38 ±18 .89 'Mean of 5 observations P < 0 .01 - A & B; A & C; (f o r e p i t h e l i a l c e l l s and lumen) ;p < 0 . 0 1 - B & C (only lumen) Tukey's t e s t - W QI f o r e p i t h e l i a l c e l l s - 3 .39 W*oi f o r lumen - 16 .57 Not s i g n i f i c a n t - B & C ( e p i t h e l i a l c e l l s ) 50 E f f e c t of methyl testosterone on the t e s t i s of hypophysectomized j u v e n i l e . No d i f f e r e n c e s were evident i n animals exposed to the two d i f f e r e n t concentrations of 6 V methyl testosterone (1:2 x 10 and 1:10 ). Methyl testosterone treatment of f i s h hypophysectomized as j u v e n i l e s does not seem to s t i m u l a t e m i t o t i c d i v i s i o n i n the spermatogonial c y s t s , nor i s there any increase i n the number of the cysts (Figs. 22, 28 and Table X I I I ) . Spermatocytes or any other stages of spermatogenesis do not appear i n the t e s t i s . In c o n t r a s t , the methyl testosterone treatment of the hypophysectomized a d u l t guppy s t i m u l a t e s m i t o t i c d i v i s i o n s i n the spermatogonial c y s t s and spermatocytes a l s o appear. This i n d i c a t e s that exogenous testosterone cannot d i r e c t l y a c t on the j u v e n i l e t e s t i s and i n i t i a t e spermato- genesis. In the j u v e n i l e guppy the p i t u i t a r y was removed before the t e s t i s r e c e i v e d any gonadotropin s t i m u l a t i o n . I t seems that spermatogenesis i n the j u v e n i l e t e s t i s can be t r i g g e r e d o n l y by gonadotropins. The stroma c e l l s present i n the centre of the t e s t i s of the hypophysectomized j u v e n i l e guppy do not become d i f f e r e n t i a t e d i n t o i n t e r s t i t i a l c e l l s f o l l o w i n g testosterone treatment. The S e r t o l i c e l l s are not evident. Perhaps, gonadotropins r e g u l a t e the d i f f e r e n t i a t i o n of i n t e r s t i t i a l c e l l s and S e r t o l i c e l l s i n the j u v e n i l e t e s t i s . The most s i g n i f i c a n t change i n the s t r u c t u r e of the t e s t i s of testosterone t r e a t e d j u v e n i l e i s the d i f f e r e n t i a t i o n 51 of the e f f e r e n t ducts and the main sperm duct. The t o t a l number, width and the s i z e of lumina of the e f f e r e n t ducts increase s i g n i f i c a n t l y ( F i g . 24 and Table XIV). The lumen of the main sperm duct becomes very wide and the e p i t h e l i a l c e l l s l i n i n g sperm duct become t a l l and columnar and even exceed i n s i z e the e p i t h e l i a l c e l l s of the sperm duct of sham- operated j u v e n i l e s ( F i g . 26 and Table XV). This suggest that the d i f f e r e n t i a t i o n of the e f f e r e n t ducts and the main sperm duct i s under the c o n t r o l of the androgens and gonodotropins are not d i r e c t l y i n v o l v e d . Methyl testosterone treatment a l s o causes an increase i n the number of f i b r o b l a s t s i n the j u v e n i l e t e s t i s . E f f e c t of methyl testosterone on secondary sex characters of hypophysectomized j u v e n i l e . . A l l changes r e q u i r e d to transform an anal f i n i n t o a gonopodium are i n i t i a t e d a f t e r testosterone treatment of hypophysectomized j u v e n i l e s but the development of the f i n i s not complete. The ray 3 becomes t h i c k (bone deposition) and develops a small hood which does not extend beyond the ray 3 (as i n a normal gonopodium). The segments of rays 3 and 4 do not develop spines, which are w e l l developed i n a normal gonopodium. There i s a small hook developed a t the t i p of ray 5 but i t i s smaller when compared to the hook of a normal gonopodium ( F i g . 32). The lipophores appear as two narrow bands, one on the upper margin and the other on the lower margin of the caudal f i n . These two bands j o i n a t the p o s t e r i o r margin of 52 the caudal peduncle and extend onto the caudal peduncle as a s i n g l e band. I t i s not evident beyond the area of the caudal peduncle (as i s found i n the a d u l t males). Lipophores are a l s o found dispersed on the anal f i n . The brightness of lipophores i s l e s s than i n normal a d u l t males and was assigned ++ i n the a r b i t r a r y s c a l e described e a r l i e r . The secondary sex characters which developed f o l l o w i n g testosterone treatment are s i m i l a r i n the hypophysectomized male and female j u v e n i l e s . I t seems tha t the secondary sex characters i n the guppy are d i r e c t l y under the c o n t r o l of androgens. The incomplete d i f f e r e n t i a t i o n of the secondary sex characters may be due to degree of d i s s i m i l a r i t y between the s y n t h e t i c exogenous androgen and the natura.lly occurring endogenous androgen or that the exogenous androgen cannot lead to complete d i f f e r e n t i a t i o n of secondary sex characters i n ..the absence of the p i t u i t a r y . 53 Figu r e 22: S a g i t t a l s e c t i o n of t e s t i s of j u v e n i l e guppy showing only spermatogonia! cysts (x 900). Figu r e 23: S a g i t t a l s e c t i o n of t e s t i s of j u v e n i l e guppy showing spermatogonia and e f f e r e n t ducts (x 9 00). F i g u r e 24: E f f e r e n t ducts of hypophysectomized j u v e n i l e t r e a t e d w i t h testosterone (x 9 00).  54 Figu r e 25: S a g i t t a l s e c t i o n of t e s t i s of j u v e n i l e guppy showing main sperm duct l i n e d by squamous e p i t h e l i a l c e l l s (x 900). Figu r e 26: Main sperm duct of t e s t i s of hypophysectomized j u v e n i l e t r e a t e d w i t h testosterone (x 900). Note the columnar e p i t h e l i a l c e l l s l i n i n g main sperm duct.  55 F i g u r e 27:. Percentage of d i f f e r e n t stages of spermatogenesis i n j u v e n i l e c o n t r o l ; j u v e n i l e hypophysectomized and j u v e n i l e sham-operated guppies. 0 SPG SPC SPD S P M SPR 56 Fig u r e 28: Percentage of d i f f e r e n t stages of spermatogenesis in.' j u v e n i l e hypophysectomized; j u v e n i l e hypophysectomized and t e s t o s t e r o n e - t r e a t e d ; j u v e n i l e sham-operated guppies.  57 F i g u r e 29: Anal f i n of j u v e n i l e guppy. F i n - r a y s are numberd from c r a n i a l (ventral) to caudal (dorsal) border of f i n . The l i n e below the diagram represents 1 mm. Figure 3 0: Anal f i n of hypophysectomized j u v e n i l e .  58 F i g u r e 31: Gonopodium (modified anal f i n ) of a d u l t male. Fi g u r e 32: Anal f i n of hypophysectomized j u v e n i l e t r e a t e d w i t h t e s t o s t e r o n e .  59 SECTION I I I •METHALLIBURE1 TREATMENT OF THE JUVENILE AND ADULT MALES INTRODUCTION Hoar et a l (1967) and Wiebe (1968) concluded that 'methallibure' e f f e c t i v e l y blocks the p i t u i t a r y gonadotropic a c t i o n . This p o i n t may be f u r t h e r s u b s t a n t i a t e d by comparing the e f f e c t s of 'methallibure' treatment w i t h that of hypophysectomy. In t h i s s e c t i o n , an attempt i s made to compare the e f f e c t s of 'methallibure' treatment (pharmacological hypophysectomy) on the t e s t e s and secondary sex characters of the a d u l t and j u v e n i l e guppies w i t h that of s u r g i c a l hypophysectomy described i n e a r l i e r s e c t i o n s . The s i t e of a c t i o n of 'methallibure' i n e l i m i n a t i n g gonadotropic hormone a c t i v i t y i s not known. An a c t i o n at the l e v e l of the p i t u i t a r y would be r e f l e c t e d i n the cytology of the gland. The e f f e c t s of 'methallibure' on the appearance of the p i t u i t a r y complex, i n p a r t i c u l a r of the mesoadenohypophysis are described. MATERIALS AND METHODS Maintenance of 'methallibure' t r e a t e d f i s h . Mature a d u l t males (17-22 mm standard length; 100-230 mg i n weight) and immature j u v e n i l e guppies (8-12 mm standard length; 8-28 mg i n weight) were taken from the breeding aquaria and put i n four aquaria (two f o r ad u l t s and two f o r j u v e n i l e s ) , each of which contained 16 l i t r e s of de c h l o r i n a t e d tap water. 60 A group of j u v e n i l e f i s h was k i l l e d immediately a f t e r removal from the breeding aquaria to serve as i n i t i a l c o n t r o l s . The maintenance was s i m i l a r to that p r e v i o u s l y described f o r methyl testosterone t r e a t e d hypophysectomized a d u l t and j u v e n i l e guppies. 'Methallibure' treatment was i n i t i a t e d a f t e r the f i s h were acclimated f o r one week. Since two aquaria contained a d u l t s and two contained j u v e n i l e s , 'methallibure' treatment was l i m i t e d to one aquarium i n each case; the other served as c o n t r o l . A concentration of 1:10^ p a r t s (16 mgm i n 16 l i t r e s of water) of 'methallibure' was added to the aquaria every a l t e r n a t e day (28 a p p l i c a t i o n s during e i g h t weeks). H i s t o l o g y . The f i x a t i o n and s t a i n i n g of the t e s t e s of the a d u l t and j u v e n i l e guppies were s i m i l a r to that described e a r l i e r . The heads were f i x e d i n formic acid-Bouins a f t e r removal of the roof of the s k u l l and l e f t f o r seven days i n the f i x a t i v e to d e c a l c i f y . A f t e r dehydration i n e t h y l a l c o h o l and embedding i n p a r a f f i n , the heads were sectioned l o n g i t u d i n a l l y or t r a n s v e r s e l y a t 5 u, the s e c t i o n s were s t a i n e d w i t h combinations of a l c i a n blue-PAS-orange G (AB - PAS - OG), aldehyde t h i o n i n e - PAS - napthol y e l l o w (AT - PAS - NY), Gabe's aldehyde f u c h s i n (AF) or Cleveland- Wolfe trichrome (Herlant 19 56) . Measurements. Measurements of the t e s t e s of 'methallibure' t r e a t e d a d u l t and j u v e n i l e guppies were s i m i l a r to the t e s t i s of hypophysectomized a d u l t and j u v e n i l e guppies described e a r l i e r . By means of an ocular micrometer, 61 the diameters of ten c e l l s of each of gonadotrophs, somatotrophs and thyrotrophs from the s a g i t t a l s e c t i o n s of f i v e p i t u i t a r y glands of both a d u l t and j u v e n i l e guppies : were measured (a t o t a l of 50 c e l l s of each type i n each group). RESULTS St r u c t u r e of the t e s t i s and secondary sex characters of a d u l t c o n t r o l . The s t r u c t u r e of the t e s t i s of the a d u l t guppy has been described i n d e t a i l i n the e a r l i e r s e c t i o n s . In short, a l l stages of spermatogenesis are present ( F i g s . 2, 36 and Table X V I I ) . The e f f e r e n t ducts and the main sperm duct are f i l l e d w i t h i n t a c t spermatophores (Figs. 4 & 6). The e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts are t a l l and columnar ( F i g . 4 and Table X V I I I ) . The secondary sex characters have a l s o been described e a r l i e r . The lipophore index was r a t e d +++ or ++++. E f f e c t of 'methallibure' on the t e s t i s and secondary sex characters of the a d u l t guppy. The 'methallibure' treatment was continued f o r e i g h t weeks. The gonosomatic index of 'methallibure' t r e a t e d f i s h i s s i g n i f i c a n t l y decreased (Table XVI). The t e s t i s contains a few cysts of spermatogonia, spermatocytes, spermatids and sperm but spermatophores are present i n abundance (Figs . 33, 36 and Table X V I I ) . The spermatophores present i n the e f f e r e n t ducts and the main sperm duct are u s u a l l y i n t a c t ( F i g . 34). The e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts become considerably reduced ( F i g . 34 and Table XVIII) but S e r t o l i c e l l s and i n t e r s t i t i a l c e l l s do not seem to be regressed. TABLE XVI Gonosomatic index (G.S-I.) of 'methallibure' t r e a t e d and c o n t r o l guppies a f t e r 8 weeks. G. S. I . F i s h No 'Methallibure' t r e a t e d C o n t r o l 1 1.30 2.92 2 1.89 3.53 3 1.91 3.40 4 1.37 3.28 5 1.18 3.63 Mean J. _ L *** +r- 1.53+0.12 3.35+0.12 ID *** p < 0.001 TABLE XVII Number and percentage of d i f f e r e n t stages of spermatogenesis i n 'methallibure' t r e a t e d and c o n t r o l guppies. SPG No % Stages of spermatogenesis SPC SPD SPM No % No % No % SPR No % 1 M e t h a l l i b u r e 1 t r e a t e d Mean" ±Sx 2.4 ±0.6 1.39 3.4 2.02 6.6 3.91 +0.38 +0.8 +0.54 +2.7 +1.53 7.4 4.43 159 88.26 ±219 ±1.82 ±19.1 ±3.66 Con t r o l Mean 2.6 ± Sx ±0.7 1.18 33.6 14.74 14.2 6.99 18.4 8.79 159.8 68.29 ±0.37 ±5.9 ±2.47 ±3.6 ±2.00 ±3.7 ±2.20 ±26.9 ±5.34 xMean of 5 observations P ^ 0.001, contingency t a b l e (row x column) 64 TABLE XVIII E p i t h e l i a l c e l l heights of e f f e r e n t ducts of 'methallibure' t r e a t e d and c o n t r o l guppies. F i s h No Mean e p i t h e l i a l c e l l " M e t h a l l i b u r e 1 t r e a t e d h e i g h t ( u ) x C o n t r o l 1 7.3 17.7 2 9.9 22.9 3 6.9 16.4 4 6.8 15.1 5 7.9 14.1 Grand Mean 7.8 17.2 xMean of 2 5 counts (5 e p i t h e l i a l c e l l heights i n each of 5 e f f e r e n t ducts i n one s e c t i o n of each t e s t i s ) P-^0.025 - a n a l y s i s of variance based on 5 median counts, 1 i n each of 5 e f f e r e n t ducts i n 1 s e c t i o n of each t e s t i s . 65 No changes are evident i n the s t r u c t u r e of the gonopodium but there i s a marked decrease i n the lipophore pigmentation both on the si d e s of the body and on the t a i l . The lipophore index was r a t e d + or ++. Comparison of the e f f e c t s of hypophysectomy and 'methallibure' treatment on the a d u l t t e s t e s and secondary sex c h a r a c t e r s . The gonosomatic index i n both cases i s s i g n i f i c a n t l y decreased. The e f f e c t s of hypophysectomy on spermatogenetic stages of t e s t i s are more apparent than i n 'methallibure' treatment. The t e s t i s of a hypophysectomized guppy contains o n l y spermatogonial cyst s and spermatophores whereas the t e s t i s of 'methallibure' t r e a t e d guppy contains a l l stages of spermatogenesis, although the e a r l i e r stages are few (Figs . 3, 33 and Table XIX). Since hypophysectomy completely blocks the transformation of spermatogonia i n t o spermatocytes the presence of few spermatocytes i n the 'methallibure' t r e a t e d t e s t i s suggests t h a t a complete blockage of gonadotropin s e c r e t i o n was not a t t a i n e d (Table XIX). A f t e r hypophysectomy many spermatophores are found ruptured i n the e f f e r e n t ducts and the main sperm duct; by co n t r a s t , f o l l o w i n g 'methallibure' treatment the spermatophores are u s u a l l y i n t a c t . The e p i t h e l i a l c e l l heights of the e f f e r e n t ducts are more reduced f o l l o w i n g hypophysectomy than 'methallibure' treatment. Hypophysectomy b r i n g s about the r e g r e s s i o n of S e r t o l i c e l l s and i n t e r s t i t i a l c e l l s but •methallibure' treatment does not. Perhaps, even a small r e l e a s e of gonadotropins prevents the r e g r e s s i o n of S e r t o l i c e l l s and i n t e r s t i t i a l c e l l s . TABLE XIX Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized and 'methallibure' t r e a t e d guppies a f t e r 8 weeks. Stages of spermatogenesis SPG SPC SPD SPM SPR No % No % No % No °A No 7< Hypophy- Mean 1 1.6 1. 82 0 0 0 97.6 98. 18 sectomized ±Sx ±0.6 ±0. 48 - ±16.3 ±0. 75 'Methallibure' Mean 1 . 2.4 1. 39 3.4 2.02 6.6 3.91 7.4 4. 43 159 88. 26 t r e a t e d *Sx ±0.6 ±0. 38 ±0.8 ±0.54 ±2.7 ±1.53 ±2.9 ±1. 82 ±19 .1 ±3. 66 Mean of 5 observations P <0.001, contingency t a b l e (row x column) f o r a l l stages of spermatogenesis P <C 0.005, contingency t a b l e (row x column) f o r only 2 stages, spermatogonia and spermatocytes of hypophysectomized and 'methallibure'- t r e a t e d guppies. 67 No changes are evident i n the s t r u c t u r e of the gonopodium of an a d u l t f i s h e i t h e r a f t e r hypophysectomy or 'methallibure' treatment. The decrease i n lipophore pigmentation i s more pronounced a f t e r hypophysectomy than 'methallibure' treatment. S t r u c t u r e of the j u v e n i l e t e s t i s ( I n i t i a l c o n t r o l ) The s t r u c t u r e of the j u v e n i l e t e s t i s has been already described i n S e c t i o n I I . Spermatogonial cyst s are present a t the periphery (Figs. 22, 37 and Table XX); e f f e r e n t ducts w i t h narrow lumira are present i n the centre ( F i g . 23 and Table XXI). The main sperm duct has narrow lumen and squamous e p i t h e l i a l c e l l s ( F i g . 25 and Table X X I I ) . S t r u c t u r e of the t e s t i s and secondary sex characters of the j u v e n i l e guppy not t r e a t e d w i t h 'methallibure'. As might be expected, the j u v e n i l e s not t r e a t e d w i t h 'methallibure', were d i f f e r e n t i a t e d i n t o a d u l t males and females by the end of the experiment (eight weeks). A l l stages of spermatogenesis are present i n the t e s t i s (Figs. 2, 37 and Table XX) . Although the e f f e r e n t ducts ( F i g . 3 5 and Table XXI) and the main sperm duct ( F i g . 3 5 and Table XXII) are w e l l d i f f e r e n t i a t e d , they do not contain spermatophores. Secondary sex characters are very d i s t i n c t i n the males. E f f e c t of 'methallibure' on the t e s t i s of the j u v e n i l e f i s h . The j u v e n i l e f i s h were t r e a t e d w i t h 'methallibure' f o r e i g h t weeks. No developmental changes occurred i n the s t r u c t u r e of the j u v e n i l e t e s t i s f o l l o w i n g 'methallibure' treatment. M i t o t i c d i v i s i o n i n spermatogonial cysts was never evident and the number of spermatogonial cyst s remains TABLE XX Number and percentage of d i f f e r e n t stages of spermatogenesis i n j u v e n i l e c o n t r o l , j u v e n i l e •methallibure' t r e a t e d and j u v e n i l e non-"methallibure' t r e a t e d guppies. Stages of spermatogenesis SPG SPC SPD SPM SPR No °/° No % No % No °A No Vo (A) J u v e n i l e C o n t r o l Mean 1 ±Sx 15.8 ±1.3 100 0 0 0 0 (B) J u v e n i l e Meth. t r e a t e d Mean 1 + S x 17.8 +0.6 100 0 P 0 0 (c) J u v e n i l e non- meth. t r e a t e d Mean 1 ±Sx 1.8 -0.2 1.38 ±0.19 35.2 26. ±6.3 ±3. 14 42 24.0 18. ±2.5 ±1. 13 85 43.6 31. ±10.9-±5. 37 04 29.6 22 ±2.3 ±3 .98 .31 Mean of 5 observations P < 0.001 - A & C; B & C: Econtingency t a b l e (row x column) TABLE XXI T o t a l number, width and lumen of e f f e r e n t ducts of j u v e n i l e c o n t r o l , j u v e n i l e ' m e t h a l l i b u r e 1 t r e a t e d and j u v e n i l e non-'methallibure 1 t r e a t e d guppies E f f e r e n t ducts J u v e n i l e J u v e n i l e Non- J u v e n i l e C o n t r o l ' M e t h a l l i b u r e ' t r e a t e d (B) ' M e t h a l l i b u r e ' t r e a t e d (C) NO No Width(u) Mean 1 : tSx Lumen(u) Mean1±Sx No Width(u) Mean1±Sx Lumen(u) Mean1±Sx No Width(u) Mean1±Sx Lumen(u) Mean1±Sx 1 18 19.80 ±1.51 1.44 ±0.36 21 19.08 ±0.77 1.08 ±0.18 41 64.78 ±7.27 33 .62 ±8.14 2 16 15.66 ±0.54 1.26 ±0.22 20 19.44 ±0.46 1.62 ±0.18 29 •61.50 ±4.67 28.70 ±3.43 3 14 20.16 ±0.93 1.80 ±0.28 15 19.62 ±1.98 1.62 ±0.34 28 71.34 ±5.28 38.54 ±6.34 4 18 18. 0 ±0.57 1.44 ±0.22 22 17.82 ±0.72 1.80 ±0.28 29 74.62 ±8.54 35.26 ±6.16 5 16 17.28 ±0.52 1.62 ±0.34 18 17.64 ±0.73 1.80 ±0.28 31 68.06 ±4.60 36.90 ±5.50 Mean 1 ±Sx 16.4 ±0.7 19.2 ±1.2 31.6 ±2.4 Mean of 5 observations P < 0.01 - A & C, B & C (for t o t a l na, width and lumen) Not s i g n i f i c a n t - A & B (for t o t a l no., width and lumen) Tukey's t e s t - W > 01 f°r no. - 8.16 w] Ql f ° r width - 6.96 w[Ql f ° r lumen - 6.42 TABLE XXII E p i t h e l i a l c e l l heights and lumen of main sperm duct of j u v e n i l e c o n t r o l , j u v e n i l e 'methallibure' t r e a t e d , and j u v e n i l e non-'methallibure t r e a t e d guppies. Main sperm duct (u; Mean 1 ±Sx) F i s h No (A) J u v e n i l e C o n t r o l (B) J u v e n i l e •Methallibure' 'treated (c) J u v e n i l e non- 'Methallibure' t r e a t e d E p i t h e l i a l c e l l s Lumen E p i t h e l i a l c e l l s Lumen E p i t h e l i a l c e l l s Lumen 1 3.06 -0.22 1.62 ±0.34 3.60 ±0.40 1.44 ±0.36 15.98 ±1.53 54.12 ±5.98 2 2.88 ±0.34 1.80 ±0.28 3.42 ±0.34 2.34 ±0.46 15.98 ±2.39 40.18 ±2.91 3 3.42 ±0.34 2.70 ±0.40 3.06 ±0.61 1.98 ±0.18 9.84 ±1.00 85.90 ±5.27 4 3.78 ±0.34 3.22 ±0.36 3.42 ±0.44 2.34 ±0.36 10.66 ±1.00 86.10 ±9.52 5 3.60 ±0.40 2.52 ±0.66 3.78 ±0.18 3.06 ±0.46 10.66 ±1.64 76.26 ±6.81 Mean of 5 observations P 4i0.01 - A & C; B & C (for e p i t h e l i a l c e l l s and lumen) Not s i g n i f i c a n t - A & B (for e p i t h e l i a l c e l l s and lumen) Tukey's t e s t - W Q-̂  f o r e p i t h e l i a l c e l l s - 2.14 W*Qi f° r lumen - 11.34 71 the same (Figs. 22, 37 and Table XX). There i s no increase i n the t o t a l number, width and the s i z e of lumina of the e f f e r e n t ducts ( F i g . 23;and Table XXI). The e p i t h e l i a l c e l l s l i n i n g main sperm duct remain squamous and the width of lumen of main sperm duct i s not increased ( F i g . 25 and Table XXII):. . Thus i t seems tha t the f u r t h e r development of the t e s t i s i s stopped when the j u v e n i l e s are t r e a t e d w i t h ' m e t h a l l i b u r e 1 . Secondary sex characters are absent i n the j u v e n i l e s . They do not develop i n j u v e n i l e s t r e a t e d w i t h 'methallibure'. Thus i t i s apparent that 'methallibure' prevents the appearance of secondary sex characters i n j u v e n i l e s . Comparison of the e f f e c t s of hypophysectomy and 'methallibure' treatment on the t e s t e s of the j u v e n i l e s . In the j u v e n i l e guppy, treatment w i t h ' m e t h a l l i b u r e 1 produced i d e n t i c a l e f f e c t s to hypophysectomy. The t e s t i s contains only p e r i p h e r a l spermatogonial cyst s (Table X X I I I ) . Both the treatments i n h i b i t the appearance of secondary sex characters i n the j u v e n i l e f i s h . These p o i n t s i n d i c a t e that gonadotropin s e c r e t i o n i s completely stopped when the j u v e n i l e s are t r e a t e d w i t h 'methallibure'. Morphology of the p i t u i t a r y complex. The s t r u c t u r e of the adenohypophysis (AH) of the guppy has been described i n p a r t by Sokol (1961); a b r i e f d e s c r i p t i o n of the p i t u i t a r y complex w i t h s u f f i c e here. The p i t u i t a r y complex i n the guppy i s d i v i d e d , as i n most t e l e o s t s , i n t o 4 r e g i o n s , the pro-, meso T, and meta-AH, and the neurohypophysis (NH) ( F i g . 38a). TABLE X X I I I Number and percentage of d i f f e r e n t stages of spermatogenesis i n hypophysectomized j u v e n i l e guppy a f t e r 6 weeks and 'methallibure* t r e a t e d j u v e n i l e guppy a f t e r 8 weeks. Stages of spermatogenesis SPG SPC SPD SPM SPR No % No % No % No % No % Hypophy- sectomized Mean 1 -Sx 12.4 -0.68 100 0 0 0 0 'Methallibure' t r e a t e d Mean 1 ±Sx 17.8 +0.6 100 0 0 0 0 Mean of 5 observations. 73 The pro-AH/ (Figs. 38a, 39a & 40-42) which composes 3 0-40% of the p i t u i t a r y gland i s l a r g e l y formed of c l o s e l y packed a c i d o p h i l s of diameter 4.7 + 0.5 u; these are probably homologous w i t h the c e l l s i n the pro-AH of Fundulus h e t e r o c l i t u s which are capable of b i n d i n g a n t i - o v i n e p r o l a c t i n serum (Emmart, p i c k f o r d & Wilhelmi 1966), and are thought to be ' p r o l a c t i n c e l l s ' . Bordering the r a m i f i c a t i o n s of the NH w i t h the pro-AH i s found a f u r t h e r c e l l type, l e s s organe G p o s i t i v e (OG +ve) than the ' p r o l a c t i n c e l l s ' , of diameter 4.0 1" 0.3 u which probably represent the adrenocorticotrophs described by Ol i v e r e a u (1964). The meso-AH i s composed of 3 c e l l types recognizable by t h e i r s t a i n i n g c h a r a c t e r i s t i c s . One i s a c i d o p h i l i c , more or l e s s rounded, of diameter ranging from 3.9 to 5.2 u; the c e l l s are s c a t t e r e d throughout the c e n t r a l zone of the meso-AH (Figs. 38b & 39b) and are considered to have a somatotrophic f u n c t i o n (Olivereau & Ridgeway 1962; Levenstein 1939; 1 Fontaine & O l i v e r e a u 1949). The two remaining c e l l types are ba s o p h i l s which, i n the guppy, are arranged i n two d i s t i n c t r e g i o n s . One r e g i o n i s i n the v e n t r a l h a l f of the meso-AH ( F i g . 38a); i n mature a d u l t s these c e l l s are of angular o u t l i n e w i t h mean diameter 4.8 to 5.9 u; and s t a i n r e a d i l y w i t h AB, AT, AF, and PAS and are commonly d i s t r i b u t e d around l a r g e sinuses or c a p i l l a r i e s which are prevale n t i n t h i s v e n t r a l r e g i o n of the gland ( F i g s . 38 & 39). Sokol (1961) considered these b a s o p h i l s to have a gonadotrophic f u n c t i o n because of t h e i r changing appearance 74 during sexual maturation, although she was unable to separate them t i n c t o r i a l l y from the second group of meso-AH ba s o p h i l s found c l o s e to the r a m i f i c a t i o n s of the NH i n t o the meso-AH. The c e l l s of t h i s second group (Figs. 38b & 39b) are o v a l i n s e c t i o n and g e n e r a l l y l a r g e r (6.7 to 8.3 u) w i t h the cytoplasm more PAS +ve than the gonadotrophs; a f t e r s t a i n i n g w i t h AT-PAS-NY, the cytoplasm appears grey i n colour while that of the gonadotrophs i s dark blu e . They are thought to have a t h y r o t r o p h i c a c t i v i t y (Sokol 1961; O l i v e r e a u 1963). Two c e l l types are r e a d i l y recognised i n the meta-AH (F i g . 38a) one of which i s r e a d i l y s t a i n a b l e w i t h PAS ( c e l l diameter 6.9 t 0.2 u). The f u n c t i o n s of these two c e l l types are not known; one i s thought to produce melanophore s t i m u l a t i n g hormone. The NH i s formed of nerve f i b r e s which have two o r i g i n s . One type of f i b r e o r i g i n a t e s i n the p r e - o p t i c nucleus (PON) and contains m a t e r i a l r e a d i l y s t a i n a b l e w i t h the s o - c a l l e d •neurosecretory' s t a i n s such as AB, AF, and AT+NY (Leatherland, Budtz & Dodd 1966)(Figs. 38a,b). The s t a i n a b l e m a t e r i a l i n P_. r e t i c u l a t a i s found i n g r e a t e s t amounts i n the i n t e r d i g i t a t i o n s of the NH w i t h the meta-AH, and l e s s so i n the r a m i f i c a t i o n s i n t o the meso- and pro-AH. In these regions the s t a i n a b l e m a t e r i a l appears f i n e l y granulated, whereas i n the d o r s a l r e g i o n of the NH i t takes the form of l a r g e e x t r a c e l l u l a r accumulations (Fi g s . 41 & 42b). The second type of nerve f i b r e t e r m i n a t i n g i n the NH o r i g i n a t e s i n the 75 nucleus l a t e r a l i s t u b e r i s (NLT)(Fig. 38b) and i s not s t a i n a b l e w i t h these 'neurosecretory' s t a i n s . The nucleus i s composed of neurones of diameter 6.0 to 7.0 u and i s found i n the hypothalamus immediately d o r s o - l a t e r a l to the p i t u i t a r y gland, as w e l l as i n the d o r s a l r e g i o n of the NH i t s e l f . Neither the neurones nor the axons of the NLT s t a i n w i t h the combinations used here, although the AT-PAS-NY combination s t a i n s the cytoplasm s l i g h t l y grey and the nucleus yellow. In the experimental procedures described below, no changes are found i n e i t h e r the s i z e or appearance of the i n t r i n s i c endocrine c e l l s of the pro- or meta-AH, nor of the neurones of NLT or PON; although there -was considerable v a r i a t i o n i n the amount of s t a i n a b l e m a t e r i a l i n the NH, i t was not c o n s i s t e n t w i t h experimental c o n d i t i o n s . Only the c e l l types of the meso-AH show changes and w i l l be considered below. E f f e c t of 'methallibure' on the meso-adenohypophysis of the a d u l t male guppy. There appears to be more gonadotroph p i t u i t a r y c e l l s i n the v e n t r a l meso-AH of the c o n t r o l f i s h (group I ) ( F i g s . 38a,b) compared w i t h the 'methallibure' t r e a t e d f i s h (group I I ) ( F i g s . 39a,b). The mean diameter of these c e l l s i s s i m i l a r l y s i g n i f i c a n t l y higher (p ^ 0.001) and the amount of AF+ve m a t e r i a l i n t h e i r cytoplasm i s n o t i c e a b l y greater i n the c o n t r o l f i s h (Table XXIV). As already noted, the v e n t r a l p a r t of the meso-AH i s o f t e n r i c h l y s u p p l i e d w i t h TABLE XXIV E f f e c t of 'methallibure' (ICI 33.828) on the mean c e l l diameter of the mesoadenohypophysial gonadotrophs, somatotrophs, and thyrotrophs of the a d u l t and j u v e n i l e guppy, P o e c i l i a r e t i c u l a t a , P e t e rs. + Experimental No. Weight range No. of Condition of (mg) glands F i s h I n i t i a l F i n a l measured Gonadotroph Somatotroph Thyrotroph Mean diameter of c e l l s (u) I Standard Error Group I A d u l t Controls 13 100-230 120-275 5.5*0.2 4.3+0.1 7.4+0.2 Group I I Ad u l t 'Methallibure' 12 100-190 130-200 4.2+0.1 4.3+0.1* 8.5+0.1 Group I I I J u v e n i l e Controls 12 8-20 4.5+0.1 4.5+0.2 5.5^.1 Group IV J u v e n i l e Controls 10 8-20 90-180 5.7-0.1 4.6+0.1 8.9+0.3 Group V J u v e n i l e ' M e t h a l l i b u r e 1 16 9-28 11-55 3.7+0.2 + + *** 7.5+0.2 + gonadotrophs not recognised i n 1 case; ++ only recognised i n 1 case; +++ thyrotrophs only recognised i n 2 cases. * somatotrophs not recognised i n 2 cases; ** only recognised i n 2 cases; *** not recognised i n any case. p< 0.001 between mean diameter of gonadotrophs of group I V and v , I and I I , I I I and I V and p = 0.01-0.02 between groups I I I and V; p ^ 0.001 between mean diameter of thyrotrophs of groups I V and V, I and I I , I and I V , I I and I I I , I and I I I , I I I and V and I I I and I V . Di f f e r e n c e s between mean diameters of gonadotrophs of groups I and I V , I I and I I I , I and I I I , and of somatotrophs of groups I and I I , I and I V , I I and I I I , I and I I I , I I I and I V not s i g n f i c a n t . 77 blood sinuses or c a p i l l a r i e s which appear to be i n t i m a t e l y connected w i t h the gonadotroph c e l l s ; a l a r g e r number of the •methallibure' t r e a t e d f i s h are found to have, these l a r g e sinuses; i n c o n t r o l f i s h they tend to be l e s s numerous. Few somatotrophs are evident i n e i t h e r the c o n t r o l f i s h , ( i n which the c e l l s were absent or not rec o g n i s a b l e i n 2 cases) or the experimental f i s h ; no d i f f e r e n c e s are found between the mean c e l l diameter of the two groups (Table XXIV). Few thyrotrophs are found i n the meso-AH of the f i s h i n group I compared w i t h the experimental group I I ( F i g s . 38b & 39b) i n which the c e l l diameter i s a l s o s i g n i f i c a n t l y (p< 0.001) l a r g e r (8.5 ± 0.1 u i n the 'methallibure' t r e a t e d f i s h compared w i t h 7.4 t 0.2 u i n the c o n t r o l group). The thyrotrophs of the 'methallibure' t r e a t e d f i s h are a l s o more PAS+ve than those of the c o n t r o l f i s h . E f f e c t of 'methallibure' on the meso-adenohypophysis of the j u v e n i l e guppy. The meso-AH of the young f i s h k i l l e d a t the beginning of the experiment (group I I I ) i s only p a r t i a l l y d i f f e r e n t i a t e d ( F i g . 40). The gonadotrophs are very s i g n i f i c a n t l y smaller (p < 0.001) i n diameter and fewer i n number than i n the a d u l t c o n t r o l s (group I ) , and have very l i t t l e AF+ve cytoplasm. Blood sinuses are evident i n only one of the p i t u i t a r y glands examined. The thyrotrophs and somatotrophs are not c l e a r l y d i f f e r e n t i a t e d i n t h i s group; they were recognised i n only two cases. The somatotrophs are s i m i l a r i n s i z e and appearance to those of the a d u l t , but 78 the thyrotrophs are very s i g n i f i c a n t l y (p ^ 0.001) smaller than those of e i t h e r of the a d u l t groups (I or I I ) . The c o n t r o l f i s h K i l l e d 8 weeks a f t e r commencement of the experiment (group IV) have a w e l l d i f f e r e n t i a t e d meso-AH ( F i g . 42a) w i t h more numerous gonadotrophs of a s i g n i f i c a n t l y l a r g e r mean diameter (p <10.001) than those of e i t h e r of the f i s h of Group I I I , or of the 'methallibure' t r e a t e d a d u l t s (group I I ) . Blood sinuses a s s o c i a t e d w i t h the gonadotrophs are more numerous i n t h i s group than i n the younger c o n t r o l f i s h of group I I I . Thyrotrophs i n t h i s group (IV) are f a i r l y numerous ( F i g . 42b) and of s i g n i f i c a n t l y greater diameter (p £ 0.001) than i n groups I, I I I , or V. Gonadotrophs are evident i n o n l y two of the p i t u i t a r y glands of the j u v e n i l e 'methallibure' t r e a t e d f i s h (group V) (F i g . 41), and where present they contain l i t t l e AF+ve s t a i n a b l e m a t e r i a l . They are of s i g n i f i c a n t l y smaller diameter (p <, 0.001) than those of a l l the other groups. Blood sinuses are commonly found but are g e n e r a l l y s m a l l . Somatotrophs were not i d e n t i f i e d i n any of the glands examined i n t h i s group (V), although t h i s may be because the poor d i f f e r e n t i a t i o n of the gonadotrophs d i d not a l l o w p o s i t i v e i d e n t i f i c a t i o n of these c e l l s . The meso-AH of t h i s group (V) contains numerous thyrotrophs, although not as many as the comparable c o n t r o l s of the same age (group IV); they are, however, s i g n i f i c a n t l y l a r g e r (p<0.001) than those of the c o n t r o l f i s h of s i m i l a r s i (group I I I ) . 79 F i g u r e 33: S a g i t t a l s e c t i o n of t e s t i s of a d u l t guppy t r e a t e d w i t h 'methallibure 1 showing few cysts of e a r l i e r stages of spermatogenesis (x 200). Figu r e 34: Same as f i g u r e 33 showing e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts (x 200). Figure 35: S a g i t t a l s e c t i o n of t e s t i s of j u v e n i l e guppy not t r e a t e d w i t h 'methallibure' (which became adult; during the p e r i o d of experiment). Note the e f f e r e n t ducts and main sperm duct do not contain spermatophores (x 200) .  80 F i g u r e 36: Percentage of d i f f e r e n t stages of spermatogenesis i n 'methallibure'-treated and c o n t r o l a d u l t guppies a f t e r e i g h t weeks. 1 0 0 S P G S P C SPD S P M %PR 81 F i g u r e 37: Percentage of d i f f e r e n t stages of spermato- genesis i n j u v e n i l e c o n t r o l ; j u v e n i l e • m e t h a l l i b u r e ' - t r e a t e d and j u v e n i l e non- 'methallibure* t r e a t e d guppies. 1 0 0 8 0 CONTROL METHALLIBURE NON-TREATED UJ 6 0 2 Ul u or ui o_ 4 0 2 0 - SPG SPC SPD SPM SPR 82 F i g u r e 38a: S a g i t t a l s e c t i o n of p i t u i t a r y gland of a d u l t c o n t r o l guppy. Note numerous gonadotrophs i n v e n t r a l r e g i o n and blood sinuses a s s o c i a t e d w i t h these c e l l s . F i g u r e 38b: Magnified view of a p o r t i o n of 38a. BS - Blood Sinuses; BV - Blood Vess e l s ; GN - Gonadotrophs; MS - Meso-adenohypophysis; MT - Meta-adenohypophysis; NH - Neurohypophysis; NT - Nucleus l a t e r a l i s t u b e r i s ; NS - Neurosecretion; PR - Pro-adenohypophysis; SM - Somatotrophs; TH - Thyrotrophs; TV - T h i r d v e n t r i c l e .  83 Figu r e 39a: S a g i t t a l s e c t i o n of p i t u i t a r y gland of a d u l t ' m e t h a l l i b u r e 1 - t r e a t e d guppy. Note few gonadotrophs around l a r g e blood sinuses, l a r g e thyrotrophs c l o s e to neurohypophysis. Figu r e 39b: Magnified view of a p o r t i o n of 39a.  84 F i g u r e 40: S a g i t t a l s e c t i o n of p i t u i t a r y gland of j u v e n i l e c o n t r o l guppy (group I I I ) . Note poor development of meso-adenohypophysisv. few gonadotrophs, other c e l l types not rec o g n i s a b l e . F i g u r e 41: S a g i t t a l s e c t i o n of p i t u i t a r y gland of j u v e n i l e • m e t h a l l i b u r e ' - t r e a t e d guppy. Note almost complete absence of gonadotrophs ( S i m i l a r to f i g . 40) and developed thyrotrophs adjacent to neurohypophysis.  85 F i g u r e 42a: S a g i t t a l s e c t i o n of p i t u i t a r y gland of j u v e n i l e c o n t r o l guppy (group VI) not t r e a t e d w i t h 'methallibure' (which became a d u l t during the p e r i o d of experiment). Note numerous gonadotrophs i n v e n t r a l margin and thyrotrophs adjacent to neurohypophysis. Fig u r e 42b: Magnified view of a p o r t i o n of 42a.  86 GENERAL DISCUSSION The i n v e s t i g a t i o n was i n i t i a t e d w i t h the premise that a study of the hypophysectomized male guppy would f u r t h e r the understanding of p i t u i t a r y - g o n a d r e l a t i o n s h i p s i n f i s h e s , c l a r i f y some of the c o n t r o v e r s i a l i s s u e s concerning the d e t a i l s of gonadotropic and androgenic a c t i o n on the t e s t i s and male secondary sex characters and provide s p e c i f i c i n f o r m a t i o n on p h y s i o l o g i c a l r e g u l a t i o n of endocrine c o n t r o l s i n the male of a l i v e - b e a r i n g cyprinodont. The study has d i f f e r e d from t h a t of previous workers i n t h i s f i e l d not only i n i t s o r i e n t a t i o n toward a male ovoviviparous species but a l s o i n the comparative study of a d u l t s w i t h j u v e n i l e s hypophysectomized p r i o r to the d i f f e r e n t i a t i o n of the gonad and secondary sex charac t e r s . V i v i e n (1941) was the f i r s t and o n l y i n v e s t i g a t o r to demonstrate t h a t hypophysectomy prevents the development of the gonad i n a j u v e n i l e t e l e o s t (Gobius paganellus). V i v i e n ' s i n v e s t i g a t i o n , however, was a general one and provided no d e t a i l s of the e f f e c t s on the cytology of the gonad. An added feat u r e i n the present a n a l y s i s of p i t u i t a r y - g o n a d r e l a t i o n s has been the use of the gonadotropic b l o c k i n g agent 'methallibure'. The i n i t i a l premise has been j u s t i f i e d and p e r t i n e n t a d d i t i o n a l data have been obtained w i t h respect to (a) the locus of p i t u i t a r y r e g u l a t i o n i n spermatogenesis (b) the p i t u i t a r y involvement i n spermiation (c) the r o l e of androgens i n spermatogenesis and i n the c o n t r o l of 87 secondary sex characters and (d) the physiology and hormonal r e g u l a t i o n of the S e r t o l i c e l l s and the e p i t h e l i a l c e l l s l i n i n g the sperm ducts. In a d d i t i o n , s e v e r a l areas of gonadal physiology have been i n v e s t i g a t e d f o r the f i r s t time i n f i s h e s w i t h s i g n i f i c a n t data p e r t a i n i n g to (a) the development and endocrine c o n t r o l of spermatophore formation (b) the hormonal involvement i n d i f f e r e n t i a t i o n of the gonad and secondary sex characters of j u v e n i l e f i s h e s and (c) the comparative study of 'methallibure' e f f e c t s on j u v e n i l e s and a d u l t s . The f o l l o w i n g t o p i c s are considered s i g n i f i c a n t c o n t r i b u t i o n s of t h i s t h e s i s and have been s e l e c t e d f o r d i s c u s s i o n : a. The r o l e of the p i t u i t a r y and the androgens i n the c o n t r o l of spermatogenesis i n c l u d i n g spermatophores. b. The endocrine c o n t r o l of the r e l e a s e of spermatophores, the development and maintenance of i n t e r s t i t i a l c e l l s , e p i t h e l i a l c e l l s l i n i n g the sperm ducts and S e r t o l i c e l l s . c The r o l e of the p i t u i t a r y and the androgens i n the c o n t r o l of secondary sex characters, d. The b l o c k i n g a c t i o n of 'methallibure' on the e f f e c t s of the gonadotropins and the s i t e of a c t i o n of 'methallibure'. 88 a. The r o l e of the p i t u i t a r y and the androgens i n the c o n t r o l of spermatogenesis i n c l u d i n g spermatophores. Role of the p i t u i t a r y While i t i s w e l l e s t a b l i s h e d that t h t t e s t e s of t e l e o s t s show a suppression of spermatogenesis a f t e r hypophysectomy, there are comparatively few d e t a i l e d s t u d i e s of the c y t o l o g i c a l changes. The o l d e r l i t e r a t u r e i n c l u d e s the work of V i v i e n (1938, 1941) on Gobius paqanellus, and that of Matthews (1939) and Burger (1941) on Fundulus h e t e r o c l i t u s . More recent i n v e s t i g a t i o n s are those of Barr (1963) on Pleuronectes p l a t e s s a , Roy (1964) and Belsare (1965) on Ophicephalus punctatus, Ahsan (1966) on Couesius plumbeus, L o f t s et aJL (1966) on Fundulus h e t e r o c l i t u s , Donaldson and McBride " (1967) on Salmo g a i r d n e r i i , Sundararaj and Nayyar (1967) on Heteropneustes f o s s i l i s , and Yamazaki and Donaldson (1968) on Carassius auratus. From a review of t h i s l i t e r a t u r e , i t i s evident that the e f f e c t of hypophysectomy on the t e s t e s v a r i e s considerably a t d i f f e r e n t timesof the year (Matthews 1939; V i v i e n 1941; Barr 1963). There i s considerable disagreement i n the l i t e r a t u r e concerning the p r e c i s e stage i n spermatogenesis which i s a f f e c t e d by hypophysectomy. In Fundulus h e t e r o c l i t u s , spermatogonial d i v i s i o n s continue but the l a t e r stages of spermatogenesis are suppressed (Matthews 1939; Burger 1941; P i c k f o r d 1953; L o f t s e_t a l 1966) . L i k e w i s e , Sundararaj and Nayyar (1967) using Heteropneustes f o s s i l i s found that spermatogonial d i v i s i o n continues and the only c e l l types 89 present i n the t e s t i s of hypophysectomized f i s h are the spermatogonia and the sperm. On the contrary, however, Barr (1963) working on p l a i c e , pleuronectes p l a t e s s a and Ahsan (1966) on lake chub, Couesius plumbeus described the suppression of spermatogonial d i v i s i o n s i n the absence of the p i t u i t a r y ; the conversion of spermatogonia i n t o spermatocytes ceases but spermatogenesis, i f w e l l underway, continues and sperm are formed. Dodd et al_ (1960) i n t h e i r s t u d i e s on S c y l i o r h i n u s c a n i c u i u s , l i k e w i s e , found that hypophysectomy stops the transformation of a spermatogonium i n t o a spermatocyte, but spermatocytes and a l l succeeding stages of spermatogenesis already e s t a b l i s h e d a t the time of operation apparently develop i n normal f a s h i o n i n t o sperm. Dodd e_t aJL (1960) d i d not observe m i t o s i s i n spermatogonia. Yamazaki and Donaldson (1968) noted that i n Carassius auratus, the m i t o t i c d i v i s i o n of spermatogonia i s completely suppressed by hypophysectomy and spermatocytes, spermatids and sperm disappear. The t e l e o s t s s t u d i e d i n a l l these previous i n v e s t i g a t i o n s are oviparous, seasonal breeders. The guppy P o e c i l i a r e t i c u l a t a d i f f e r s from the t e l e o s t s p r e v i o u s l y s t u d i e d i n being an ovoviviparous, monthly breeder. As an adaptation to i n t e r n a l f e r t i l i z a t i o n the male guppy produces sperm-balls or spermatophores. The h i s t o l o g y of spermatophore formation has been described i n a number of p o e c i l i i d s : P h a l l o c e r o s caudo-maculatus and Cnesterodon decem-maculatus ( P h i l i p p i 1908); P o e c i l i a r e t i c u l a t a (vaupel 1929; Goodrich 90 et_ al_ 1934); Gambusia a f f i n i s (Self 1940; Medlen 1950); Xiphophorus h e l l e r i (Essenberg 1924; Vallowe 1957) and Xiphophorus maculatus (Wolf 1931; Chavin and Gordon 19 51). However, the p o s s i b l e endocrine c o n t r o l of spermatophore formation, the physiology of the spermatophores while i n the t e s t i s and t h e i r eventual discharge have not been p r e v i o u s l y described. In the t e s t i s of a c o n t r o l guppy, the spermatogonial cysts are p e r i p h e r a l l y l o c a t e d while the spermatophores are i n the centre; the area between the two i s f i l l e d w i t h cysts c o n t a i n i n g the various developmental stages of spermatocytes, spermatids and sperm ( F i g . 2). By c o n t r a s t , the t e s t i s of a hypophysectomized guppy i s completely packed w i t h spermatophores except f o r a few spermatogonial cysts near the periphery ( F i g . 3). This suggests that hypophysectomy (presumably the l a c k of gonadotropin) b l o c k s the transformation of spermatogonia i n t o spermatocytes, but does not prevent the transformation of spermatocytes, spermatids, and sperm i n t o spermatophores. This f i n d i n g i s s i m i l a r to that described i n Pleuronectes p l a t e s s a (Barr 1963), Couesius plumbeus (Ahsan 1966) and S c y l i o r h i n u s c a n i c u l u s (Dodd et a l 1960). A s i m i l a r s i t u a t i o n has been noted i n the Amphibia; van Oordt (1956) concluded from study of the f r o g , Rana temporaria that the l a t e r stages of spermatogenesis beyond spermatogonia are not under the p i t u i t a r y c o n t r o l . The presence of spermatophores at the periphery of the t e s t i s of a hypophysectomized guppy w i t h no t r a c e of 91 d i s i n t e g r a t i n g cysts c o n t a i n i n g e a r l i e r stages of spermatogenesis i n d i c a t e s that spermatocytes, spermatids and sperm do not degenerate but transform i n t o spermatophores. This observation i s i n c o n t r a d i c t i o n to the f i n d i n g s i n oviparous t e l e o s t s such as Gobius paganellus ( V i v i e n 1941), Fundulus h e t e r o c l i t u s (Matthews 1939; Burger 1941; P i c k f o r d 1953; L o f t s et a l 1966), pleuronectes p l a t e s s a (Barr 1963), Couesius plumbeus (Ahsan 1966), Heteropneustes f o s s i l i s (Sundararaj and Nayyar 1967) and Carassius auratus (Yamazaki and Donaldson 1968). I n v e s t i g a t o r s who have s t u d i e d these oviparous species s t a t e that the spermatocytes and spermatids degenerate or disappear i n the absence of the p i t u i t a r y . This d i f f e r e n c e i s d i f f i c u l t to e x p l a i n but may be due to the f a c t that the guppy i s an ovoviviparous t e l e o s t and produces r e l a t i v e l y few spermatophores, thus conservation of the e a r l i e r stages i s e s s e n t i a l ; whereas the r e s t are oviparous and produce enormous number of sperm. Since m i t o t i c d i v i s i o n was never noted i n the spermato- g o n i a l c y s t s of the hypophysectomized guppy, it.seems l i k e l y t h a t spermatogonial m u l t i p l i c a t i o n ceases i n the absence of the p i t u i t a r y . This i s f u r t h e r evidenced by the f a c t that percentage composition of spermatogonial cyst s of c o n t r o l and experimental guppies has not changed during the eight-week experimental p e r i o d (Table I I ) . This f i n d i n g i s i n accord w i t h that of Yamazaki and Donaldson (1968) on g o l d f i s h , Carassius auratus and that of van Oordt (1960) on f r o g , Rana temporaria. 92 Testes of a newly born guppy contain only spermatogonial c y s t s . The primary spermatogonia begin to d i v i d e to form nests of c e l l s (beginning of spermatogenesis) a t about 36 days a f t e r b i r t h (Goodrich et a l 1934). Sokol (1961) found th a t gonadotrophs i n the v e n t r a l r e g i o n of meso-adenohypophysis become granulated ( i n i t i a t i o n of gonadotropin se c r e t i o n ) during the f i f t h week a f t e r b i r t h . I t i s thus evident t h a t there i s a d i r e c t c o r r e l a t i o n between the s e c r e t i o n of gonadotropins and the i n i t i a t i o n of spermatogenesis. The r o l e of the p i t u i t a r y i n the development of t e s t i s has been analyzed by removing the p i t u i t a r y of the j u v e n i l e guppy before the gonadotrophs were d i f f e r e n t i a t e d . No changes take place i n the s t r u c t u r e of the j u v e n i l e t e s t i s f o l l o w i n g hypophysectomy. Mitoses were never observed and there was no evidence of an increase i n the number of spermatogonial cyst s (Table X). The l a t e r stages of spermatogenesis do not appear. During the course of the above experiment sham-operated j u v e n i l e s became a d u l t s and t h e i r t e s t e s contained a l l stages of spermatogenesis. I t i n d i c a t e s that hypophysectomy prevents the m i t o t i c d i v i s i o n of spermatogonia and t h e i r transformation i n t o spermatocytes. This f i n d i n g i s s i m i l a r to that of Chang and W i t s c h i (1955) on urodeles ( T r i t u r u s ) and anurans (Rana, Bufo). They noted t h a t even a f t e r a prolonged p e r i o d , the t e s t e s of hypophysectomized l a r v a l amphibians contain only spermatogonia. 93 I t i s concluded that hypophysectomy of both a d u l t and j u v e n i l e guppies prevents m i t o s i s i n the spermatogonia and blocks t h e i r transformation i n t o spermatocytes. The hypophysectomy of the a d u l t guppy does not prevent the transformation of spermatocytes, spermatids and sperm i n t o spermatophores. This i n d i c a t e s that the l a t e r stages of spermatogenesis are p i t u i t a r y - i n d e p e n d e n t . Role of the androgens In a study of e f f e c t s of various s t e r o i d s i n sexual development of i n t a c t guppy, Eversole (1939, 1941) suggested that testosterone propionate hastens germ c e l l maturation. There have been s e v e r a l other r e p o r t s of s t i m u l a t i o n of t e s t e s of i n t a c t t e l e o s t f i s h by androgens ( p i c k f o r d and A t z 1957; Dodd 1960). The r o l e of androgens i n spermatogenesis may be r e a d i l y analyzed by t r e a t i n g hypophysectomized f i s h w i t h androgens. There are, however, very few observations on the e f f e c t s of androgens on the t e s t e s of hypophysectomized f i s h (Burger 1942; L o f t s et a l 1966; Sundararaj and Nayyar 1967). L o f t s et a l (1966) working on Fundulus h e t e r o c l i t u s and Sundararaj and Nayyar (1967) on Heteropneustes f o s s i l i s demonstrated that spermatogenesis i s completely r e s t o r e d w i t h testosterone treatment. On the contrary, however, Burger (1942) a l s o working w i t h the hypophysectomized Fundulus s t a t e d t h a t spermatogenesis i s p o o r l y maintained by androgens. Methyl testosterone treatment of hypophysectomized a d u l t guppies s i g n i f i c a n t l y increases t h e i r gonosomatic i n d i c e s . 94 This f i n d i n g i s similar, to that described i n Fundulus (Loft s e t a l 1966) and Heteropneustes (Sundararaj and Nayyar 1967). Exogenous methyl testosterone appears to have a d i r e c t spermatokinetic e f f e c t on the t e s t i s of the hypophysectomized guppy. In c o n t r a s t to the hypophysectomized c o n t r o l s , a c t i v e m i t o t i c d i v i s i o n s are evident w i t h i n spermatogonial c y s t s ; the number of spermatogonial c y s t s increases and cysts c o n t a i n i n g spermatocytes are d i f f e r e n t i a t e d (Table V I I ) . T e s t i c u l a r s t i m u l a t i o n reaches only the spermatocytal stage. This observation i s i n c o n t r a s t to the f i n d i n g s of L o f t s et a l (1966) i n Fundulus and Sundararaj and Nayyar (1967) i n Heteropneustes. They reported complete r e s t o r a t i o n of spermatogenesis w i t h testosterone. The d i f f e r e n c e s i n a c t i v a t i o n of the regressed t e s t i s i s d i f f i c u l t to e x p l a i n but may be a t t r i b u t e d to the f a c t t h a t P o e c i l i a i s an ovoviviparous, monthly breeder whereas Fundulus and Heteropneustes are oviparous, seasonal breeders. In higher vertebrates the e f f e c t of testosterone on the regressed t e s t i s v a r i e s . Testosterone i s known to exert a s t i m u l a t o r y e f f e c t on spermatogenesis i n hypophysectomized mammals (Boccabella 1963; Clermont and Harvey 1966). In c o n t r a s t to the f i n d i n g s noted above, Basu and Nandi (1965) reporte d that treatment of hypophysectomized frogs w i t h testosterone r e s u l t s i n greater suppression of spermatogenesis than that observed a f t e r hypophysectomy alone. 95 There i s no pu b l i s h e d account of the e f f e c t s of testosterone treatment on hypophysectomized j u v e n i l e f i s h . U n l i k e the e f f e c t found i n a d u l t s , methyl testosterone treatment of hypophysectomized j u v e n i l e guppies does not st i m u l a t e m i t o t i c d i v i s i o n s i n the spermatogonial c y s t s ; l a t e r stages of spermatogenesis do not appear. The d i f f e r e n c e s i n a c t i v a t i o n of the t e s t e s of hypophysectomized a d u l t and j u v e n i l e guppies by methyl testosterone may be explained i n the f o l l o w i n g way: the a d u l t t e s t i s had w e l l d i f f e r e n t i a t e d i n t e r s t i t i a l c e l l s which became regressed i n the absence of p i t u i t a r y and stopped producing androgen. The methyl testosterone treatment of the hypophysectomized a d u l t compensates f o r t h i s l o s s i n androgen production and that i s why the wave of spermatogenesis i n i n i t i a t e d . I t seems l i k e l y that the production of androgen by i n t e r s t i t i a l c e l l s i n a d u l t t e s t i s has s y n e r g i s t i c a c t i o n w i t h gonadotropins i n spermatogenesis. On the other hand i n the j u v e n i l e s , the i n t e r s t i t i a l c e l l s were not d i f f e r e n t i a t e d and the p i t u i t a r y was removed before the t e s t i s had ever r e c e i v e d any gonadotropin s t i m u l a t i o n . I t may be suggested th a t exogenous testosterone cannot a c t d i r e c t l y on the j u v e n i l e t e s t i s and b r i n g about spermatogenesis and d i f f e r e n t i a t i o n of i n t e r s t i t i a l c e l l s u n t i l the t i s s u e has been primed or t r i g g e r e d by gonadotropins. 96 b. The endocrine c o n t r o l of the r e l e a s e of spermatophores, the development and maintenance of i n t e r s t i t i a l c e l l s , e p i t h e l i a l c e l l s l i n i n g the sperm ducts and S e r t o l i c e l l s . Release of spermatophores There i s considerable disagreement i n l i t e r a t u r e concerning the endocrine c o n t r o l of spermiation i n oviparous t e l e o s t s . This c o n t r o l e v i d e n t l y v a r i e s i n the same f i s h at d i f f e r e n t times of the year. When the p i t u i t a r y of Gobius paganellus was removed during winter, sperm disappeared from the t e s t i s , whereas 7 0 percent of operated f i s h r e t a i n e d sperm when the p i t u i t a r y was removed s h o r t l y before the n a t u r a l r e p r o d u c t i v e climax ( V i v i e n 1938, 1941). S i m i l a r r e s u l t s were obtained i n Fundulus h e t e r o c l i t u s during the f a l l and s p r i n g (Matthews 1939). P i c k f o r d (19 53) found no sperm i n Fundulus f i v e months a f t e r the operation. L o f t s e t a l (1966) a l s o working w i t h Fundulus described empty lo b u l e s without sperm i n the t e s t i s a f t e r hypophysectomy. In pleuronectes p l a t e s s a , sperm are shed normally i n the absence of the p i t u i t a r y (Barr 1963). Ahsan (1966) noted th a t Couesius plumbeus spermiates normally i n the absence of t h e . p i t u i t a r y i n both the prespawning and spawning phases of i t s annual c y c l e . Likewise, Yamazaki and Donaldson (1968) found t h a t sperm disappear from the t e s t i s of hypophysectomized Carassius auratus. On the contrary, however, Sundararaj and Nayyar (1967) working on Heteropneustes f o s s i l i s noted that sperm p e r s i s t as long as 337 days a f t e r hypophysectomy. 97 The endocrine c o n t r o l of the r e l e a s e of spermatophores from the t e s t i s has not been p r e v i o u s l y s t u d i e d . E i ght weeks a f t e r hypophysectomy, many spermatophores i n the t e s t i s of the guppy are found ruptured but the sperm are not r e l e a s e d . The rupture of the spermatophores may be due to the f a c t that the e p i t h e l i a l c e l l s of the sperm ducts surrounding the spermatophores cease to produce the n u t r i t i v e or c o l l o i d a l m a t e r i a l i n the absence of the p i t u i t a r y . There i s a strong i n d i c a t i o n that the sperm r e s u l t i n g from ruptured spermato- phores are being phagocytosed w i t h i n the e f f e r e n t ducts and the main sperm duct (F i g . 15). The rupture of the spermatophores and the r e s o r p t i o n of the sperm w i t h i n the sperm ducts become a c c e l e r a t e d when the hypophysectomized guppy i s t r e a t e d w i t h t e s t o s t e r o n e . No sperm are n o t i c e d i n the sperm ducts a f t e r two weeks of testosterone treatment (Fig 18), whereas remnant sperm are present i n the hypophysectomized c o n t r o l . I t may be suggested that the exogenous testosterone a c c e l e r a t e s the phagocytosis of sperm. I n t e r s t i t i a l c e l l s . Buser-Lahaye (1953), L o f t s e t a l (1966), Sundararaj and Nayyar (1967) and Yamazaki and Donaldson (1968) found signs of atrophy i n the i n t e r s t i t i a l c e l l s of d i f f e r e n t t e l e o s t s f o l l o w i n g hypophysectomy. Ahsan (1966) noted that p i t u i t a r y removal suppresses the s e c r e t o r y a c t i v i t y of lobule-boundary c e l l s i n Couesius plumbeus. Likewise, hypophysectomy causes the r e g r e s s i o n of the i n t e r s t i t i a l 98 c e l l s i n the a d u l t guppy. The i n t e r s t i t i a l c e l l s do not contain any l i p i d d r o p l e t s and t h e i r n u c l e i become shrunken. Regressed i n t e r s t i t i a l c e l l s of hypophysectomized guppy are s t i m u l a t e d f o l l o w i n g testosterone a d m i n i s t r a t i o n . Their n u c l e i assume a more rounded shape w i t h a d i s t i n c t n u c l e o l u s . This f i n d i n g i s i n accord w i t h that of L o f t s et a l (1966) i n Fundulus. On the contrary, however, Sundararaj and Nayyar (1967) found that the i n t e r s t i t i a l c e l l s remained a t r o p h i e d i n Heteropneustes. The stroma c e l l s of the t e s t i s of hypophysectomized j u v e n i l e guppy are not d i f f e r e n t i a t e d i n t o i n t e r s t i t i a l c e l l s . Testosterone treatment of the hypophysectomized j u v e n i l e does not cause the d i f f e r e n t i a t i o n of i n t e r s t i t i a l c e l l s e i t h e r . I t may be concluded from the data that the d i f f e r e n t i a t i o n of i n t e r s t i t i a l c e l l s from the stroma c e l l s r e q u i r e s priming or t r i g g e r i n g by gonadotropins as i n spermatogenesis. The testosterone can s t i m u l a t e the regressed i n t e r s t i t i a l c e l l s of a d u l t t e s t i s which produced androgens before hypophysectomy but cannot lead to the p r o l i f e r a t i o n of the i n t e r s t i t i a l c e l l s i n the j u v e n i l e s . E p i t h e l i a l c e l l s l i n i n g the sperm ducts. The e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts of an i n t a c t a d u l t guppy are of the t a l l columnar type and contain l i p i d d r o p l e t s . The presence of l i p i d d r o p l e t s i n d i c a t e s that these e p i t h e l i a l c e l l s may be r e s p o n s i b l e f o r s e c r e t i n g androgens. Moser (1967), l i k e w i s e , noted that s u d a n o p h i l i c d r o p l e t s are p r e v a l e n t i n the c e l l s of the e f f e r e n t ducts 99 of the r o c k f i s h , Sebastodes p a u c i n s p i n i s . Wiebe (1967) a l s o rep o r t e d the r.presence of s t e r o i d dehydrogenases i n e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts of the seaperch, Cymatogaster aggregata. The spermatophores i n an e f f e r e n t duct of an i n t a c t guppy are surrounded by a c o l l o i d a l m a t e r i a l which seems to be secreted by the e p i t h e l i a l c e l l s . The c o l l o i d a l m a t e r i a l might contain a n u t r i t i v e substance as has been suggested by Medlen (1950) i n h i s study on Gambusia a f f i n i s . The e p i t h e l i a l l i n i n g of the e f f e r e n t ducts becomes reduced and does not contain any l i p i d d r o p l e t s a f t e r hypophysectomy. No c o l l o i d a l m a t e r i a l i s secreted by the e p i t h e l i a l c e l l s of the e f f e r e n t ducts i n the absence of the p i t u i t a r y and i t i s thought t h a t the rupture of spermatophores i n the e f f e r e n t ducts and the main sperm duct might be due to the f a c t that the e p i t h e l i a l c e l l s cease to produce androgens and the c o l l o i d a l m a t e r i a l . When the hypophysectomized guppy i s t r e a t e d w i t h t e s t o s t e r o n e , the regressed e p i t h e l i a l c e l l s of the e f f e r e n t ducts and the main sperm duct hypertrophy and assume the t a l l columnar appearance of the normal animal. This i n d i c a t e s that the e p i t h e l i a l l i n i n g of the e f f e r e n t ducts i s under the s t e r o i d c o n t r o l . Likewise, Burger (1942) and L o f t s et a l (1966) noted a hypertrophy of the e f f e r e n t ducts i n Fundulus h e t e r o c l i t u s . There i s no increase i n the number, width and the s i z e of the lumina of the e f f e r e n t ducts i n the t e s t i s of j u v e n i l e 100 guppy f o l l o w i n g hypophysectomy. Methyl testosterone treatment of the hypophysectomized j u v e n i l e s b r i n g s about complete d i f f e r e n t i a t i o n of the e f f e r e n t ducts and the main sperm duct. The t o t a l number, width and the s i z e of lumina of the e f f e r e n t ducts increase s i g n i f i c a n t l y . The lumen of the main sperm duct becomes very wide and the l i n i n g e p i t h e l i a l c e l l s a t t a i n t a l l columnar appearance of the a d u l t guppy. I t i s concluded that the d i f f e r e n t i a t i o n and maintenance of the e f f e r e n t ducts and the main sperm duct of both the j u v e n i l e and a d u l t t e s t e s depend on androgens and gonadotropin s e c r e t i o n i s not d i r e c t l y i n v o l v e d . U n l i k e the game.togenetic and endocrine t i s s u e s of the t e s t i s , an i n i t i a l priming w i t h gonadotropin i s e v i d e n t l y unnecessary f o r the a c t i o n of androgen. S e r t o l i c e l l s Among the f i s h e s , S e r t o l i c e l l s have been reported i n Gasterosteus (Craig-Bennet 1931), P o e c i l i a ( F o l l e n i u s 1953, Fundulus ( L o f t s et. a l . 1966) and i n Elasmobranchs (Matthews 1950; Stanley 1962), but these workers do not describe the f u n c t i o n of S e r t o l i c e l l s . In the t e s t i s of an i n t a c t guppy, S e r t o l i c e l l s p l a y an important p a r t i n the formation of spermatophores. These c e l l s become s t r i k i n g l y enlarged as the sperm become more mature and the sperm heads become attached to t h e i r inner margin. Considering the enormous change i n the s i z e of S e r t o l i c e l l s and sperm heads becoming attached to them, i t may be suggested that S e r t o l i c e l l s serve as n u t r i e n t c e l l s as they are b e l i e v e d to be i n higher 101 v e r t e b r a t e s . S e r t o l i c e l l s i n mammals contain considerable glycogen i n t h e i r cytoplasm and t h i s i s considered evidence that they serve as n u t r i e n t c e l l s f o r the germinal e p i t h a l i u m (Paulsen 1968). Burgos (1955) reported that glycogen i s present i n the S e r t o l i c e l l s of f r o g , Rana pipiens.. - T i n c t o r i a l response of carbohydrates i s a l s o detectable i n S e r t o l i c e l l s of c a r a s s i u s auratus (Yamamoto and Yamazaki 1967). Moser (1967) reported that the tubule boundary c e l l s (corresponding to S e r t o l i c e l l s ) of Sebastodes p a u c i s p i n i s contain carbohydrate granules • and a c t as a s u b s t r a t e f o r the n u t r i t i o n of gametes. However, t h i s may not be the only f u n c t i o n of these c e l l s . According to Vaupel (1929) the S e r t o l i c e l l s of the guppy are phagocytic and i n g e s t the cytoplasm (co n t a i n i n g the G o l g i remnant) discarded when spermatids are transformed i n t o sperm. S i m i l a r f u n c t i o n has been a s c r i b e d to S e r t o l i c e l l s i n mammals (Lacy 1968). There i s no p u b l i s h e d account of the e f f e c t of hypophysectomy on S e r t o l i c e l l s i n any t e l e o s t . In the present study, i t was evident t h a t the S e r t o l i c e l l s of the hypophysectomized guppy regress and t h e i r n u c l e i change from rounded c o n d i t i o n to f l a t t e n e d shape ( F i g . 9 ) . Burgos (1955) found that S e r t o l i c e l l s of Rana p i p i e n s atrophy and t h e i r glycogen content disappears f o l l o w i n g hypophysectomy. Regressed S e r t o l i c e l l s of the hypophysectomized guppy are r e s t o r e d to normal f o l l o w i n g testosterone a d m i n i s t r a t i o n ( F i g . 17). Their n u c l e i become rounded i n shape once more w i t h a conspicuous n u c l e o l u s . This i n d i c a t e s t h a t S e r t o l i 102 c e l l s are under s t e r o i d c o n t r o l . The S e r t o l i c e l l s are not d i f f e r e n t i a t e d i n the j u v e n i l e t e s t i s a f t e r hypophysectomy. Even testosterone treatment of hypophysectomized j u v e n i l e s does not b r i n g about the p r o l i f e r a t i o n of S e r t o l i c e l l s . Perhaps S e r t o l i c e l l s can be d i f f e r e n t i a t e d o n l y a f t e r being primed w i t h gonadotropins. In the guppy, not only S e r t o l i c e l l s regress a f t e r hypophysectomy and a t t a i n t h e i r normal appearance w i t h t e s t o s t e r o n e treatment; both the i n t e r s t i t i a l c e l l s and the e p i t h e l i a l c e l l s l i n i n g the sperm ducts behave i n a s i m i l a r f a s h i o n . The i n t e r s t i t i a l c e l l s and the e p i t h e l i a l c e l l s l i n i n g the sperm ducts contain l i p i d d r o p l e t s and produce androgens. I t may be suggested t h a t S e r t o l i c e l l s a l s o produce androgens as they are b e l i e v e d to do i n mammals (Teilum 1950). c. The r o l e of the p i t u i t a r y and the androgens i n the c o n t r o l of secondary sex c h a r a c t e r s . A l l secondary sex characters i n t e l e o s t s are under t e s t i c u l a r c o n t r o l as shown by treatment w i t h exogenous sex s t e r o i d s and c a s t r a t i o n experiments ( P i c k f o r d and A t z 1957; Dodd 1960; Hoar 1965, 1966). The p i t u i t a r y d i r e c t l y r e g u l a t e s the androgen production of the t e s t i s and thus i n d i r e c t l y c o n t r o l s the secondary sex c h a r a c t e r s . In Gobius paganellus ( V i v i e n 1938, 1941) and Fundulus h e t e r o c l i t u s (Matthews 1939; Burger 1941; P i c k f o r d 1953; L o f t s e t a l 1966) the n u p t i a l c o l o r a t i o n or breeding dress (the most 103 apparent secondary sex character) disappeais i n the absence of the p i t u i t a r y . L ikewise, the b r i g h t lipophores present on the sides of the body of a d u l t guppy become very f a i n t or e n t i r e l y disappear f o l l o w i n g hypophysectomy. In c o n t r a s t , no changes are evident i n the s t r u c t u r e of the gonopodium of the hypophysectomized guppy. M o r p h o l o g i c a l l y the gonopodium i s a w e l l d i f f e r e n t i a t e d s t r u c t u r e and i t i s n a t u r a l to expect t h a t once morphogenesis i s complete, the s t r u c t u r e w i l l not reg r e s s . Although the i n f l u e n c e of exogenous s t e r o i d s on the sex acc e s s o r i e s and secondary sex characters of the t e l e o s t s has been t e s t e d many times ( p i c k f o r d and A t z 1957), e f f o r t s have r a r e l y been made to e l i m i n a t e the p o s s i b l e e f f e c t s of endogenous gonadotropins by the use of hypophysectomized animals. There are only two published accounts on the e f f e c t s of androgens on the secondary sex characters of hypophysectomized f i s h . Burger (1942) and L o f t s et a l (1966) noted t h a t the n u p t i a l c o l o r a t i o n reappeared i n hypophysectomized Fundulus f o l l o w i n g testosterone treatment. S i m i l a r l y the testosterone treatment of the a d u l t hypophysectomized guppy leads to moderate recovery i n the content of lipophores present on the sides of the body. The gonopodium of the guppy, which remains unaffected a f t e r hypophysectomy, does not change a f t e r testosterone treatment. The r e s p e c t i v e r o l e of the p i t u i t a r y and the androgens i n the d i f f e r e n t i a t i o n of secondary sex characters of the j u v e n i l e guppy has been analyzed by f i r s t removing the 104 p i t u i t a r y and subsequently t r e a t i n g such hypophysectomized j u v e n i l e s w i t h t e s t o s t e r o n e . Secondary sex characters i n c l u d i n g both the lipophore pigments and the gonopodium, are absent i n the j u v e n i l e guppy; the j u v e n i l e appearance i s maintained i n the hypophysectomized j u v e n i l e s u n t i l the end of experimental p e r i o d (eight weeks). During t h i s p e r i o d , the sham-operated j u v e n i l e s developed i n t o a d u l t males w i t h d i s t i n c t secondary sex c h a r a c t e r s . When the hypophysectomized j u v e n i l e s are t r e a t e d w i t h t e s t o s t e r o n e , the secondary sex characters (gonopodium and lipophores) become d i f f e r e n t i a t e d but the d i f f e r e n t i a t i o n i s not as complete as a d u l t c o n t r o l s . The incomplete d i f f e r e n t i a t i o n of secondary sex characters may be due to degree of d i s s i m i l a r i t y between the s y n t h e t i c exogenous androgen and the n a t u r a l l y o c c u r r i n g endogenous androgen or t h a t the exogenous androgen cannot lead to complete d i f f e r e n t i a t i o n of secondary sex characters i n the absence of p i t u i t a r y gonadotropins. I t might a l s o be suggested t h a t the dose used was inadequate. However, the conce n t r a t i o n of testosterone (I ::2xic£ par ts) used i n the experiment appears q u i t e s u f f i c i e n t when compared to the dosages s u c c e s s f u l l y used by previous workers f o r s t i m u l a t i n g t e s t i s and secondary sex characters of i n t a c t f i s h ( P i c k f o r d and A t z 1957) and i s probably not the main reason f o r the incomplete d i f f e r e n t i a t i o n of secondary sex characters. Data obtained from hypophysectomy and testosterone treatment of both a d u l t and j u v e n i l e guppies suggest t h a t 105 secondary sex characters are d i r e c t l y c o n t r o l l e d by the androgens and i n d i r e c t l y by the p i t u i t a r y gonadotropins. d. The b l o c k i n g a c t i o n of 'methallibure' on the e f f e c t s of the gonadotropins and the s i t e of a c t i o n of 'methallibure' B l o c k i n g a c t i o n of 'methallibure'. The e f f e c t of 'me t h a l l i b u r e 1 on j u v e n i l e f i s h has not been p r e v i o u s l y reported. No developmental changes occur i n the s t r u c t u r e of t e s t i s of j u v e n i l e guppy f o l l o w i n g •methallibure' treatment (Table XX). The number of spermatogonial cyst s remains the same and these are not transformed i n t o spermatocytes. The f u r t h e r d i f f e r e n t i a t i o n of the e f f e r e n t ducts and the main sperm duct i s checked. Secondary sex characters do not appear. A l l developmental changes of the j u v e n i l e t e s t i s are stopped i n the same fa s h i o n as i s evident a f t e r hypophysectomy. Since 'methallibure' treatment produces i d e n t i c a l e f f e c t s to hypophysectomy i t i s concluded that gonadotropin s e c r e t i o n of the j u v e n i l e guppy i s e n t i r e l y blocked w i t h 'methallibure'. In the j u v e n i l e s the gonadotropin s e c r e t i o n was completely blocked because the 'methallibure' treatment was begun before the gonadotrophs were d i f f e r e n t i a t e d , s i n c e according to Sokol (1961) i t i s not u n t i l the f i f t h week a f t e r the b i r t h t h a t l i g h t l y granulated b a s o p h i l s appear i n the v e n t r a l r e g i o n of meso-adenohypophysis. Thus chemical hypophysectomy ('methallibure' treatment) of j u v e n i l e s i s as 106 e f f e c t i v e as s u r g i c a l hypophysectomy as f a r as the development of gonad and appearance of secondary sex characters are concerned. The e f f e c t of 'methallibure' on the gonads and gonadal f u n c t i o n s has been s t u d i e d i n o n l y three species of t e l e o s t f i s h (Hoar et a_l 1967; Wiebe 1968) . The gonosomatic index of 'methallibure' t r e a t e d a d u l t guppy i s markedly reduced. This f i n d i n g i s i n accord w i t h Hoar e_t a_l (1967) and Wiebe (1968). In the a d u l t guppy, 'methallibure' treatment b r i n g s about s i g n i f i c a n t changes i n the percent composition of d i f f e r e n t stages of spermatogenesis. A f t e r e i g h t weeks of treatment', there are few c y s t s c o n t a i n i n g spermatogonia, spermatocytes, spermatids and sperm but spermatophores are present i n abundance (Table X V I I ) . Since hypophysectomy completely b l o c k s the transformation of spermatogonia i n t o spermatocytes, the presence of spermatocyte-cysts i n •methallibure' t r e a t e d t e s t i s suggests t h a t a complete p i t u i t a r y blockage of gonadotropin was not a t t a i n e d i n a d u l t s a t t h i s dose l e v e l (1:10^ parts) i n e i g h t weeks (Table XIX). Hoar et a l (1967) and Wiebe (1968) concluded t h a t •methallibure' e f f e c t i v e l y b locks the p i t u i t a r y gonadotropic a c t i o n . Their c o n c l u s i o n i s based on the f a c t t h a t the stages of r e d u c t i o n d i v i s i o n and subsequent spermiogenesis are blocked. Wiebe (1968) found that i n 'methallibure' t r e a t e d seaperch, Cymatogaster aggregata, the spermatogonia and spermatocytes comprise 96% of the l o b u l e area (10% of the 107 area i n c o n t r o l ) and the spermatophores disappear. Wiebe (1968) f u r t h e r noted t h a t i n t e r s t i t i a l c e l l s of Leydig and columnar e p i t h e l i a l ( S e r t o l i ) border c e l l s atrophy f o l l o w i n g • m e t h a l l i b u r e 1 treatment. The r e s u l t s of the present i n v e s t i g a t i o n are i n c o n t r a d i c t i o n to these f i n d i n g s . In the 'methallibure' t r e a t e d a d u l t guppy, spermatogonia and spermatocytes comprise 3.4% of the t o t a l t e s t i s area (16% of the area i n c o n t r o l ) and spermatophores are i n t a c t and cover 88% of the t e s t i s area (68% of the area i n c o n t r o l Table X V I I ) . The i n t e r s t i t i a l c e l l s and S e r t o l i c e l l s do not seem to be regressed, perhaps, even a small r e l e a s e of gonodotropins prevents t h e i r r e g r e s s i o n (complete r e g r e s s i o n ensues a f t e r hypophysectomy). I t seems l i k e l y t hat i n the guppy the 'methallibure' treatment suppresses the transformation of spermatogonia i n t o spermatocytes (Table XIX). Wiebe (1968) demonstrated t h a t the block f o l l o w i n g 'methallibure' treatment i s between primary and secondary spermatocytes. The d i f f e r e n c e s i n r e s u l t s might be a t t r i b u t e d to two species of f i s h - P o e c i l i a r e t i c u l a t a (ovoviviparous, monthly breeder) and Cymatogaster aggregata ( v i v i p a r o u s , seasonal breeder). In 'methallibure' t r e a t e d male guppy, there i s a marked decrease i n lipophore pigmentation both on the s i d e s of the body and on the t a i l . Hoar e t a l (1967) demonstrated that the height of kidney tubule of s t i c k l e b a c k (a secondary sex character) i s reduced f o l l o w i n g 'methallibure' treatment. 108 Wiebe (1968) found t h a t 'methallibure' causes the atrophy of secondary sex m o d i f i c a t i o n s on Cymatogaster male a n a l f i n . These r e s u l t s i n d i c a t e that 'methallibure' treatment causes r e d u c t i o n i n gonadal s t e r o i d o g e n e s i s . Since 'methallibure' treatment of the a d u l t guppy does not l e a d to complete r e g r e s s i o n of gametogenetic and s t e r o i d o g e n e t i c t i s s u e s of the t e s t i s , as i s evident i n the absence of the p i t u i t a r y , i t i s concluded t h a t 'methallibure' does not completely b l o c k the r e l e a s e of the p i t u i t a r y gonadotropins. The s i t e of a c t i o n of 'methallibure' Although i t i s recognised t h a t 'methallibure' blocks the a c t i o n of p i t u i t a r y gonadotropins on both the gameto- genet i c and endocrine t i s s u e s of t e s t i s (Hoar e t a l 1967; Wiebe 1968), the s i t e of a c t i o n of 'methallibure' i s not known. 'Methallibure' treatment of both a d u l t and j u v e n i l e guppies b r i n g s about a d e p l e t i o n of the aldehyde f u c h s i n p o s i t i v e granules from the cytoplasm of the p i t u i t a r y gonado tropic'ce l i s . As i n a l l s i m i l a r s t u d i e s , there i s a problem of i n t e r p r e t a t i o n ; i s a c e l l which i s depleted of granules i n a c t i v e , or i s the r a t e of s y n t h e s i s of the granules simply equal t o , or l e s s than the r a t e of release? Examination of the mean c e l l diameter of the gonadotrophs and of the t o t a l number of c e l l s i n d i c a t e s that there i s a very s i g n i f i c a n t (p< 0.001) r e d u c t i o n i n the s i z e of the gonadotrophs of the t r e a t e d f i s h compared w i t h those of the c o n t r o l s i n both the 109 a d u l t and j u v e n i l e stages which would tend to i n d i c a t e hypo- r a t h e r than h y p e r a c t i v i t y . On t h i s assumption, the gonadotropic b l o c k i n g a c t i o n of •methallibure' may be e f f e c t i v e i n one of two ways: (a) i t may block the hypothalamic c o n t r o l over gonadotropic hormone s y n t h e s i s , or, (b) i t may d i r e c t l y block the synthesis of the hormone w i t h i n the i n t r i n s i c p i t u i t a r y endocrine c e l l s . The hypothalamic nucleus l a t e r a l i s t u b e r i s (NLT) ( F l o r e n t i n 1934; H i l d 1950; Stahl 1957; Brehm 1958; B i l l e n s t e i n 1962; Oztan 1963) and p r e - o p t i c nucleus (PON) (see Dodd, Perks and Dodd 1966 f o r review) have been i m p l i c a t e d i n the r e g u l a t i o n of the sexual c y c l e . O l i v e r e a u and B a l l (1966) demonstrated by means of a u t o t r a n s p l a n t s of the p i t u i t a r y that an i n t i m a t e contact w i t h the hypothalamus i s necessary f o r the maintenance of gonadotrophs. I t i s p o s s i b l e that 'methallibure' blocks the s y n t h e s i s or r e l e a s e of f a c t o r s produced by these n u c l e i . However, most of the above authors describe changes i n the appearance of the neurons of the n u c l e i or changes i n the amount of s t a i n a b l e m a t e r i a l i n the neuro-hypophysis a s s o c i a t e d w i t h the sexual c y c l e . In the present i n v e s t i g a t i o n no such changes are apparent i n the n u c l e i f o l l o w i n g the b l o c k of gonadotrophic a c t i v i t y , o f f e r i n g no support f o r the f i r s t hypothesis. On the other hand, a decrease i n the number and s i z e of gonadotroph c e l l s as w e l l as a d e p l e t i o n of aldehyde f u c h s i n p o s i t i v e granules i n t h e i r cytoplasm, lend support to the second hypothesis. 110 Large blood sinuses are found i n the v e n t r a l r e g i o n of meso-adenohypophysis of 'methallibure' t r e a t e d f i s h (group I I ) and i n those c o n t r o l groups i n which the gonadotrophs are developed and presumably f u n c t i o n a l (groups I and I V ) . The r o l e of sinuses or c a p i l l a r i e s i s not c l e a r although t h e i r c l o s e s p a t i a l a s s o c i a t i o n w i t h the gonadotrophs suggests a r e l a t e d f u n c t i o n . An increasedblood supply to the gonadotrophs may f a c i l i t a t e a r i s e i n the r e l e a s e of gonadotropic hormones, and the c o n t r o l of hypothalamus over the i n t r i n s i c endocrine c e l l s of the p i t u i t a r y may be c o n t r o l l e d i n t h i s way (Leatherland 1967). Thus a negative feed-back mechanism f o l l o w i n g a d e c l i n e i n testosterone or estrogen production a f t e r treatment w i t h 'methallibure' would r e s u l t i n an enlargement of the blood sinuses c o n t r o l l e d by the hypothalamus. An enlargement of these v e s s e l s would a l s o be a n t i c i p a t e d i n those f i s h a c t i v e l y s e c r e t i n g gonadotropic hormones (groups I and I V ) . There i s l i t t l e d i f f e r e n c e i n the number or appearance of the somatotrophs i n the a d u l t groups'(I and I I ) , which may be expected i n more or l e s s f u l l y grown f i s h . There were, however, fewer of these c e l l s i n the j u v e n i l e f i s h R i l l e d a t the commencement of the experiment (group I I I ) compared wit h c o n t r o l s k i l l e d e i g h t weeks l a t e r (group IV). Somatotrophs were not i d e n t i f i e d i n any of the 'methallibure' t r e a t e d j u v e n i l e f i s h , which may i n d i c a t e a d i r e c t or i n d i r e c t i n f l u e n c e of the compound on the production of growth I l l hormone i n the j u v e n i l e f i s h . The very l i m i t e d growth of the • m e t h a l l i b u r e 1 t r e a t e d j u v e n i l e s compared w i t h the c o n t r o l s would s i m i l a r l y i n d i c a t e an e f f e c t on growth hormone synth e s i s and/or r e l e a s e . The thyrotrophs of the a d u l t 'methallibure' t r e a t e d f i s h are both s i g n i f i c a n t l y l a r g e r and more numerous than those of the c o n t r o l s . The thyrotrophs of the 'methallibure' t r e a t e d j u v e n i l e f i s h are s i m i l a r l y l a r g e r and more numerous compared w i t h the c o n t r o l s k i l l e d a t the beginning of experiment, but not when compared w i t h the c o n t r o l f i s h k i l l e d e i g h t weeks l a t e r . I t i s probably more c o n s i s t e n t to compare the 'methallibure' t r e a t e d j u v e n i l e s w i t h the group I I I c o n t r o l s which are of a s i m i l a r weight r a t h e r than group IV c o n t r o l s of a s i m i l a r age. 'Methallibure' has been shown to have an ' a n t i - t h y r o i d * e f f e c t i n mammals w i t h two d i s t i n c t a c t i o n s , one a t the l e v e l of the t h y r o i d b l o c k i n g the production of the t h y r o i d hormone, and simultaneously a t the l e v e l of the p i t u i t a r y and/or hypothalamus tending to cause t h y r o i d i n v o l u t i o n (Walpole 1965; T u l l o c h e t a l 1963). However, the c l e a r increase i n number and s i z e of the thyrotrophs i n 'methallibure' t r e a t e d a d u l t and j u v e n i l e f i s h i s i n d i c a t i v e of an i n c r e a s e d TSH production r a t h e r than a reduced a c t i v i t y . There i s a d i r e c t c o r r e l a t i o n between the t h y r o i d c e l l e p i t h e l i a l height (TEH) and the number and s i z e of the p i t u i t a r y thyrotrophs w i t h i n the f i v e groups, the TEH of the 'methallibure' t r e a t e d a d u l t group i s greater than t h a t of the c o n t r o l s . In the j u v e n i l e groups, the TEH i s l a r g e s t i n the f i s h of group TV which are r a p i d l y developing and which, during the course of the experiment, achieved sexual m a t u r i t y , and sm a l l i n the c o n t r o l group I I I k i l l e d a t the beginning of the experiment. The t h y r o i d f o l l i c l e s of the 'methallibure' t r e a t e d groups have a much reduced c o l l o i d content compared w i t h the c o n t r o l s , c o n s i s t e n t w i t h a decreased t h y r o i d hormone production. The apparent h y p e r a c t i v i t y of the p i t u i t a r y thyrotrophs i n the 'methallibure' t r e a t e d f i s h thus appears to r e s u l t from a f a l l i n t h y r o i d hormone production, and th a t of the c o n t r o l group (IV) from an increased t h y r o i d a c t i v i t y a t t h i s stage of the sexual c y c l e (Stolk 1959). I t i s concluded that 'methallibure' treatment of a d u l t and j u v e n i l e guppies causes a decrease i n both the number and mean c e l l diameter of gonadotrophs, an-, increase i n the number and mean c e l l diameter of the thyrotrophs and a decrease i n the number of somatotrophs of t r e a t e d j u v e n i l e s . 113 SUMMARY 1. Hypophysectomy of the a d u l t guppy causes marked r e g r e s s i o n i n the t e s t i s . The t e s t i s contains only spermatogonia and spermatophores because the spermatocytes, spermatids and sperm present a t the time of operation transform i n t o spermatophores. The m i t o t i c d i v i s i o n s of spermatogonia and t h e i r transformation i n t o spermatocytes cease. I t i s concluded that the d i v i s i o n of spermatogonia and t h e i r transformation i n t o spermatocytes are p i t u i t a r y - dependent but the transformation of spermatocytes, spermatids and sperm i n t o spermatophores are p i t u i t a r y - i n d e p e n d e n t . 2. In the absence of the p i t u i t a r y , the spermatophores rupture a f t e r e i g h t weeks and the r e s u l t i n g sperm i n s t e a d of being discharged, are phagocytosed w i t h i n the regressed e f f e r e n t ducts and the main sperm duct. I t seems that the r e l e a s e of spermatophores i s under the c o n t r o l of the p i t u i t a r y . 3. A f t e r hypophysectomy, S e r t o l i c e l l s , i n t e r s t i t i a l c e l l s and e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts and the main sperm duct regress. Methyl testosterone treatment of hypophysectomized animals causes the hypertrophy of the e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts and the main sperm duct; regressed S e r t o l i c e l l s and i n t e r s t i t i a l c e l l s are a l s o r e s t o r e d to normal. i t i s concluded that S e r t o l i c e l l s , i n t e r s t i t i a l c e l l s and e p i t h e l i a l c e l l s l i n i n g the duct are maintained by androgens and not by p i t u i t a r y gonadotropins. 114 4. Hypophysectomy and methyl testosterone treatment of the a d u l t guppy do not b r i n g about any change i n the s t r u c t u r e of the gonopodium. I t suggests that once morphogenesis of the gonopodium i s complete, i t becomes independent of the p i t u i t a r y and the androgens. The lipop h o r e s , on the other hand, become obscure or e n t i r e l y disappear i n the absence of the p i t u i t a r y but there i s moderate recovery i n the content of lipophores a f t e r t e stosterone treatment. I t i s concluded that the lipophores are r e g u l a t e d by androgens. 5. Exogenous methyl testosterone appears to have a d i r e c t spermatokinetic e f f e c t on the t e s t i s of hypophysectomized animal. Spermatogonial cyst s d i v i d e r a p i d l y and transform i n t o spermatocytes but l a t e r stages of spermatogenesis do not appear. 6. Hypophysectomy of the j u v e n i l e guppy prevents the m i t o t i c d i v i s i o n of spermatogonia i n the t e s t i s ; no other stages of spermatogenesis appear and the i n t e r s t i t i a l c e l l s and S e r t o l i c e l l s are not d i f f e r e n t i a t e d . Testosterone treatment of the hypophysectomized j u v e n i l e s do not i n i t i a t e spermatogenesis ( i n c o n t r a s t to the adult) and the i n t e r s t i t i a l c e l l s and S e r t o l i c e l l s are not evident. I t i s concluded that the spermatogenesis and the d i f f e r e n t i a t i o n of the i n t e r s t i t i a l c e l l s and S e r t o l i c e l l s are under the c o n t r o l of p i t u i t a r y gonadotropins only. 115 7. In the absence of the p i t u i t a r y , the sperm ducts do not d i f f e r e n t i a t e and the secondary sex characters do not appear i n the j u v e n i l e guppy. F o l l o w i n g testosterone treatment the sperm ducts are w e l l d i f f e r e n t i a t e d and the secondary sex characters become evident. I t i s concluded th a t the androgens cause the d i f f e r e n t i a t i o n and maintenance of the sperm ducts and the secondary sex characters ( s i m i l a r to the a d u l t ) . 8. The t e s t i s of the a d u l t guppy t r e a t e d w i t h ' m e t h a l l i b u r e 1 contains few c y s t s of e a r l i e r stages of spermatogenesis. The spermatophores remain i n t a c t . The e p i t h e l i a l c e l l s l i n i n g the e f f e r e n t ducts are reduced i n s i z e but there are no changes i n S e r t o l i c e l l s and i n t e r s t i t i a l c e l l s . There i s a marked decrease i n the amount of lipophore pigmentation. Since 'methallibure' treatment of the a d u l t guppy does not l e a d to complete regressions of the gametogenetic and s t e r o i d o g e n e t i c t i s s u e s of t e s t i s , as i s evident i n the absence of the p i t u i t a r y , i t seems tha t 'methallibure' does not completely block the r e l e a s e of p i t u i t a r y gonadotropins. 9. No developmental changes occur i n the t e s t i s of the j u v e n i l e guppy f o l l o w i n g 'methallibure' treatment. The secondary sex characters are not d i f f e r e n t i a t e d . Since 'methallibure' produces i d e n t i c a l e f f e c t s to hypophysectomy, i t i s concluded that gonadotropin s e c r e t i o n of the j u v e n i l e guppies i s e n t i r e l y blocked w i t h 'methallibure'. 116 10. 'Methallibure' treatment does not cause any change i n the s i z e or appearance of the c e l l s of pro- and meta-adenohypophysis of the a d u l t or j u v e n i l e guppies but the c e l l types of meso-adenohypophysis (gonadotrophs, thyrotrops and somatotrophs) show changes. A c l e a r decrease i n both the number and mean c e l l diameter of gonadotrophs, and an increase i n the number and mean c e l l diameter of the thyrotrophs i s evident i n 'methallibure' t r e a t e d a d u l t and j u v e n i l e f i s h , whereas a decrease i s n o t i c e d i n the number of somatotrophs of the j u v e n i l e s . The gonadotropic b l o c k i n g a c t i v i t y of the compound i s considered to occur a t the l e v e l of hormone s y n t h e s i s . 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