@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Science, Faculty of"@en, "Zoology, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Pandey, Satyendra"@en ; dcterms:issued "2011-06-22T23:19:51Z"@en, "1968"@en ; vivo:relatedDegree "Doctor of Philosophy - PhD"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """The role of pituitary and gonadal steroids in the development and maintenance of the testis and secondary sex characters of the guppy Poecilia reticulata Peters has been studied by the technique of surgical hypophysectomy and chemical inhibition of gonadotropic action using 'methallibure’. Hypophysectomy of juvenile or adult guppies completely blocks mitosis in the spermatogonia and their transformation into spermatocytes. However, spermatocytes, spermatids and sperm already present in the adult testis at the time of operation transform into spermatophores. In the absence of the pituitary, the spermatophores rupture after eight weeks and the resulting sperm are phagocytosed within the sperm ducts. Sertoli cells, interstitial cells and the epithelial cells lining the sperm ducts regress in hypophysectomized fish. Testosterone treatment of the hypophysectomized adult guppy initiates spermatogonial multiplication and the transformation of spermatogonia into spermatocytes; the regressed Sertoli cells, interstitial cells and the epithelial cells lining the sperm ducts resume their normal appearance. Testosterone treatment of hypophysectomized juvenile guppies does not initiate spermatogenesis but the sperm ducts become well differentiated. Of two particularly well differentiated secondary sex characters of the adult male guppy, the gonopodium (modified anal fin) remains unaffected after hypophysectomy whereas the lipophores (yellow and red pigments) present on the sides of body become obscure or entirely disappear in the absence of pituitary; the lipophores reappear after testosterone treatment. Secondary sex characters never appear in guppies hypophysectomized as juveniles. When hypophysectomized juveniles are treated with testosterone, secondary sex characters (gonopodium and lipophores) become evident. The regression of the gametogenetic and the steroidogenetic tissues in the testis of 'methallibure'-treated (1:10⁶ parts) adult guppy is not as complete as in the hypophysectomized fish. This indicates that the release of pituitary gonadotropins is not completely blocked. With the same dose of 'methallibure', however, the gonadotropin release in the juveniles is apparently blocked. In both adult and juvenile guppies 'methallibure' brings about a clear decrease in both the number and mean cell diameter of gonadotrophs. The gonadotropic hormone blocking activity of the compound seems to occur at the level of hormone synthesis. From these studies it has been concluded that mitotic division of spermatogonia and their transformation into spermatocytes are dependent on pituitary, but the transformation of spermatocytes, spermatids and sperm into spermatophores are pituitary-independent. The release of spermatophores is under the control of pituitary. The regressed Sertoli cells, interstitial cells and the epithelial cells lining the sperm ducts of hypophysectomized fish assume normal appearance with testosterone treatment. The appearance of secondary sex characters in hypophysectomized juveniles treated with testosterone indicates that secondary sex characters are directly controlled by testosterone. 'Methallibure' completely blocks the synthesis of gonadotropic hormones in the juvenile guppies but not in adults."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/35665?expand=metadata"@en ; skos:note "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 ) < 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- Mean1 1.6 1. 82 0 0 0 97.6 98. 18 sectomized ±Sx ±0.6 ±0. 48 - ±16.3 ±0. 75 'Methallibure' Mean1 . 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 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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"@en ; edm:hasType "Thesis/Dissertation"@en ; edm:isShownAt "10.14288/1.0104198"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Zoology"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Endocrine control of sexual development in the male guppy"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/35665"@en .