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

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THE ENDOCRINE CONTROL OF SEXUAL DEVELOPMENT IN THE MALE GUPPY POECILIA RETICULATA  PETERS  by  B.Sc. M.Sc.  SATYENDRA PANDEY ( D i s t n . ) B i h a r U n i v e r s i t y , 1955 B i h a r 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 t h e Department of Zoology  We a c c e p t t h i s t h e s i s as conforming t o t h e required standard  The  U n i v e r s i t y o f B r i t i s h Columbia November, 1968  i  In p r e s e n t i n g an  this  thesis  in partial  advanced degree a t the U n i v e r s i t y  the  Library  I further for  shall  make i t f r e e l y  agree that  permission  h i s representatives.  of  this  written  thesis  ^  O C» L.0  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8 , Canada  Date  gain  J>^OaAvv|cxi2A  GrY Columbia  , 13. 6 t  I agree  that  copying of this  thesis  b y t h e Head o f my D e p a r t m e n t o r  It i s understood  for financial  Columbia,  f o r r e f e r e n c e and S t u d y .  f o rextensive  permission.  D e p a r t m e n t of  of B r i t i s h  available  s c h o l a r l y p u r p o s e s may be g r a n t e d  by  f u l f i l m e n t of the requirements f o r  shall  that  copying or p u b l i c a t i o n  n o t be a l l o w e d w i t h o u t  my  ABSTRACT  The r o l e o f 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  o f the t e s t i s and secondary sex  c h a r a c t e r s o f the 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 has been s t u d i e d by the t e c h n i q u e o f s u r g i c a l hypophysectomy and chemical i n h i b i t i o n of gonadotropic , action using 'methallibur Hypophysectomy o f j u v e n i l e o r a d u l t guppies c o m p l e t e l y b l o c k s m i t o s i s i n the spermatogonia and t h e i r t r a n s f o r m a t i o n i n t o spermatocytes.  However, s p e r m a t o c y t e s , s p e r m a t i d s and  sperm a l r e a d y p r e s e n t i n the a d u l t t e s t i s a t the time o f o p e r a t i o n t r a n s f o r m i n t o spermatophores. the p i t u i t a r y , the spermatophores  I n the absence o f  r u p t u r e a f t e r e i g h t weeks  and the r e s u l t i n g sperm a r e 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 d u c t s r e g r e s s i n hypophysectomized T e s t o s t e r o n e t r e a t m e n t o f the hypophysectomized  fish  adult  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 t r a n s f o r m a t i o n o f spermatogonia i n t o s p e r m a t o c y t e s ; the r e g r e s s e d 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 t h e sperm d u c t s resume t h e i r appearance.  Testosterone treatment of  normal  hypophysectomized  j u v e n i l e guppies does n o t i n i t i a t e spermatogenesis b u t the sperm d u c t s become w e l l  differentiated.  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 c h a r a c t e r s o f the a d u l t male guppy, the gonopodium ( m o d i f i e d a n a l f i n ) remains u n a f f e c t e d a f t e r hypophysectomy whereas  the l i p o p h o r e s  ( y e l l o w and r e d pigments) p r e s e n t on t h e s i d e s  o f body become obscure o r e n t i r e l y d i s a p p e a r o f p i t u i t a r y ; the l i p o p h o r e s reappear a f t e r treatment.  i n the absence testosterone  Secondary sex c h a r a c t e r s 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 a r e t r e a t e d w i t h t e s t o s t e r o n e , secondary sex characters  (gonopodium and l i p o p h o r e s ) become e v i d e n t .  The r e g r e s s i o n o f t h e gametogenetic and t h e 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 o f ' m e t h a l l i b u r e ' t r e a t e d ( l : l 0 ^ p a r t s ) a d u l t guppy i s n o t as complete as i n t h e 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. same dose o f ' m e t h a l l i b u r e ' , however, t h e  With 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 g u p p i e s ' m e t h a l l i b u r e ' b r i n g s about a c l e a r d e c r e a s e i n b o t h t h e number and mean c e l l diameter o f gonadotrophs.  The g o n a d o t r o p i c  hormone b l o c k i n g a c t i v i t y o f  the compound seems t o occur a t t h e l e v e l o f 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 o f spermatogonia and t h e i r t r a n s f o r m a t i o n  into  s p e r m a t o c y t e s a r e dependent on p i t u i t a r y , b u t the t r a n s f o r m a t i o n o f s p e r m a t o c y t e s , spermatids spermatophores a r e p i t u i t a r y - i n d e p e n d e n t .  and sperm i n t o The r e l e a s e o f  spermatophores i s under t h e c o n t r o l o f p i t u i t a r y .  The  r e g r e s s e d 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 t h e e p i t h e l i a l c e l l s l i n i n g the sperm ducts o f hypophysectomized f i s h assume  iv  normal appearance w i t h t e s t o s t e r o n e treatment.  The  appearance  o f secondary sex c h a r a c t e r s i n hypophysectomized j u v e n i l e s t r e a t e d w i t h t e s t o s t e r o n e i n d i c a t e s t h a t secondary sex c h a r a c t e r s are d i r e c t l y c o n t r o l l e d by  testosterone.  ' M e t h a l l i b u r e ' c o m p l e t e l y b l o c k s the s y n t h e s i s o f  gonadotropic  hormones i n the j u v e n i l e guppies b u t not i n a d u l t s .  V  TABLE OF CONTENTS Page LIST OF TABLES  v  LIST OF FIGURES ACKNOWLEDGMENTS  x  i  i  x  i  i  v  i  INTRODUCTION  1  SECTION I : HYPOPHYSECTOMY AND METHYL TESTOSTERONE TREATMENT OF THE ADULT MALES  5  INTRODUCTION  5  MATERIALS AND METHODS Maintenance o f f i s h i n b r e e d i n g a q u a r i a . Maintenance o f hypophysectomized f i s h . p r o c e d u r e o f hypophysectomy.  5 5 6 6  Methyl testosterone treatment.  8  Histology. Measurements.  8 9  RESULTS Structure o f the a d u l t t e s t i s . Secondary s e x c h a r a c t e r s o f t h e male guppy. S t r u c t u r e o f t h e t e s t i s and secondary sex c h a r a c t e r s o f sham-operated c o n t r o l guppy. S t r u c t u r e o f t h e t e s t i s o f hypophysectomized guppy. Secondary s e x c h a r a c t e r s o f hypophysectomized guppy. S t r u c t u r e o f t h e t e s t i s and secondary s e x c h a r a c t e r s o f hypophysectomized c o n t r o l guppy (testosterone experiment). S t r u c t u r e o f t h e t e s t i s and secondary s e x c h a r a c t e r s o f sham-operated c o n t r o l guppy (testosterone experiment). E f f e c t o f m e t h y l t e s t o s t e r o n e on t h e a d u l t t e s t i s o f hypophysectomized guppy. E f f e c t o f m e t h y l t e s t o s t e r o n e on secondary sex c h a r a c t e r s o f hypophysectomized guppy.  10 10 13 14 14 21 21 24 24 28  vi Page SECTION I I :  HYPOPHYSECTOMY AND METHYL TESTOSTERONE TREATMENT OF THE JUVENILE GUPPIES INTRODUCTION MATERIALS AND METHODS Maintenance o f hypophysectomized j u v e n i l e f i s h . P r o c e d u r e o f hypophysectomy. Methyl testosterone treatment. Histology. Measurements. RESULTS Structure of the juvenile t e s t i s ( i n i t i a l control) . S t r u c t u r e o f t h e t e s t i s o f hypophysectomized juvenile. S t r u c t u r e o f t h e t e s t i s arid secondary s e x c h a r a c t e r s o f t h e sham-operated j u v e n i l e . S t r u c t u r e o f t h e t e s t i s o f hypophysectomized j u v e n i l e (Control of testosterone experiment). S t r u c t u r e o f t h e t e s t i s and secondary s e x c h a r a c t e r s o f t h e sham-operated j u v e n i l e (Control of testosterone experiment).  37 37 37 38 38 39 39  40 42 45 46 46  E f f e c t o f m e t h y l t e s t o s t e r o n e on t h e t e s t i s o f hypophysectomized j u v e n i l e .  50  E f f e c t o f m e t h y l t e s t o s t e r o n e on secondary sex c h a r c t e r s o f hypophysectomized j u v e n i l e . . ,  51  SECTION I I I :  'METHALLIBURE' TREATMENT OF THE JUVENILE AND ADULT MALES  59  INTRODUCTION  59  MATERIALS AND METHODS Maintenance o f ' m e t h a l l i b u r e ' t r e a t e d f i s h . Histology. Measurements.  59 59 60 60  RESULTS S t r u c t u r e o f t h e t e s t i s and secondary s e x c h a r a c t e r s o f a d u l t control;.... E f f e c t o f ' m e t h a l l i b u r e ' on t h e t e s t i s and secondary s e x c h a r a c t e r s o f t h e a d u l t guppy.  61 61 61  Vll  Page Comparison o f the e f f e c t s o f hypophysectomy and ' m e t h a l l i b u r e ' t r e a t m e n t 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 . S t r u c t u r e o f the j u v e n i l e t e s t i s ( I n i t i a l control). S t r u c t u r e o f the t e s t i s and secondary sex c h a r a c t e r s o f the j u v e n i l e guppy n o t t r e a t e d with 'methallibure'. E f f e c t o f ' m e t h a l l i b u r e ' on the t e s t i s o f the juvenile fish. Comparison o f the e f f e c t s o f hypophysectomy and ' m e t h a l l i b u r e ' t r e a t m e n t on the t e s t e s o f the j u v e n i l e s .  65 67 67 67 71  Morphology o f the p i t u i t a r y complex. E f f e c t o f " m e t h a l l i b u r e ' on the meso-  71  adenohypophysis  75  o f the a d u l t male guppy.  E f f e c t o f ' m e t h a l l i b u r e ' on the mesoadenohypophysis o f the j u v e n i l e guppy. GENERAL DISCUSSION The r o l e o f the p i t u i t a r y and the androgens i n the c o n t r o l o f spermatogenesis i n c l u d i n g spermatophores.  77 86 88  The e n d o c r i n e c o n t r o l o f the r e l e a s e o f 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 d u c t s and S e r t o l i c e l l s .  96  The r o l e o f the p i t u i t a r y and the androgens i n the c o n t r o l o f secondary sex c h a r a c t e r s .  102  The b l o c k i n g a c t i o n o f ' m e t h a l l i b u r e ' on the e f f e c t s o f the g o n a d o t r o p i n s and the s i t e o f a c t i o n of 'methallibure'.  105  SUMMARY  113  LITERATURE CITED  117  viii  LIST OF TABLES TABLE I II  III  IV V  VI  VII  VIII  IX X  XI  XII  PAGE G.S.I, o f hypophysectomized and guppies a f t e r 8 weeks.  sham-operated 15  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized and shamo p e r a t e d guppies a f t e r 8 weeks.  16  E p i t h e l i a l c e l l h e i g h t s o f the e f f e r e n t d u c t s o f hypophysectomized and sham-operated guppies a f t e r 8 weeks.  17  G.S.I, o f hypophysectomized and g u p p i e s a f t e r 16 weeks.  18  sham-operated  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized and .shamo p e r a t e d guppies a f t e r 16 weeks.  19  Number and p e r c e n t a g e o f spermatogonia and spermatophores i n hypophysectomized guppies a f t e r 8 weeks and 16 weeks.  22  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized, testosterone-treated and sham-operated g u p p i e s .  23  E p i t h e l i a l c e l l h e i g h t s o f the e f f e r e n t d u c t s o f hypophysectomized, t e s t o s t e r o n e - t r e a t e d and shamoperated guppies.  25  G.S.I, o f hypophysectomized, and sham-operated g u p p i e s .  26  testosterone-treated  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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  T o t a l number, w i d t h and lumen o f e f f e r e n t d u c t s o f 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 g u p p i e s .  43  E p i t h e l i a l c e l l h e i g h t s and lumen o f main sperm d u c t o f 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 g u p p i e s . 44  ix TABLE  PAGE Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis 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 g u p p i e s .  47  T o t a l number, w i d t h and lumen o f e f f e r e n t d u c t s o f 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 g u p p i e s .  48  E p i t h e l i a l c e l l h e i g h t s and lumen o f main sperm d u c t o f 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 g u p p i e s .  49  Gonosomatic i n d e x (G.S.I.) o f ' 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 guppies a f t e r 8 weeks.  62  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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 guppies.  63  XVIII  E p i t h e l i a l c e l l heights o f e f f e r e n t ducts o f • 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 g u p p i e s .  64  XIX  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized and ' m e t h a l l i b u r e ' t r e a t e d guppies a f t e r 8 weeks.  66  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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 ' 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 nonm e t h a l l i b u r e ' t r e a t e d guppies.  68  T o t a l number, w i d t h and lumen o f e f f e r e n t d u c t s 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 n o n - ' m e t h a l l i b u r e ' t r e a t e d guppies.  69  E p i t h e l i a l c e l l h e i g h t s and lumen o f main sperm duct o f 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 n o n - ' m e t h a l l i b u r e ' t r e a t e d guppies.  70  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized j u v e n i l e guppy a f t e r 6 weeks and ' m e t h a l l i b u r e ' t r e a t e d j u v e n i l e guppy a f t e r 8 weeks.  72  XIII  XIV  XV  XVI XVII  XX  1  XXI  XXII  XXIII  X  TABLE XXIV  PAGE Effect of 'methallibure ( I C I 33,828) on t h e mean c e l l diameter o f t h e mesoadenohypophysial gonadotrophs, somatotrophs, and t h y r o t r o p h s o f 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 , Peters. 1  76  LIST OF FIGURES FIGURE  PAGE  la  S e c t i o n o f whole t e s t i s o f a d u l t guppy  29  lb  D e t a i l o f a p o r t i o n o f t e s t i s i n d i c a t e d by arrow i n l a .  29  2a.  Sagittal  section of adult testis  showing  d i f f e r e n t s t a g e s o f spermatogenesis  30  2b  Magnified  v i e w o f a p o r t i o n o f 2a.  30  3a  S a g i t t a l s e c t i o n o f t e s t i s e i g h t weeks a f t e r hypophysectomy showing o n l y spermatophores M a g n i f i e d v i e w o f a p o r t i o n o f 3a.  30 30  4  E f f e r e n t ducts o f a d u l t t e s t i s spermatophores.  31  5  Ruptured spermatophores i n t h e e f f e r e n t d u c t s of t e s t i s e i g h t weeks a f t e r hypophysectomy  31  6  Main sperm d u c t o f a d u l t t e s t i s spermatophores.  31  7  R u p t u r e d spermatophores i n t h e main sperm d u c t o f 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 o f a d u l t t e s t i s w i t h sperm heads a t t a c h e d t o them.  32  9  R e g r e s s e d S e r t o l i c e l l s o f 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 s u r r o u n d i n g S e r t o l i c e l l s o f a d u l t t e s t i s opening i n t o e f f e r e n t d u c t s .  32  11  T e s t i s a t the end o f t h i r d week a f t e r hypophysectomy c o n t a i n i n g o n l y sperm-cysts and spermatophores.  32  12  I n t e r s t i t i a l c e l l s of adult 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 o f t e s t i s weeks a f t e r hypophysectomy.  3b  14  containing  containing  eight  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 d u c t s o f a d u l t t e s t i s s t a i n e d w i t h Sudan b l a c k B.  33  33  xii FIGURE 15  PAGE T e s t i s showing remnant sperm b e i n g phagocytosed i n e f f e r e n t d u c t s 16 weeks a f t e r hypophysectomy.  33  16  T e s t i s o f hypophysectomized guppy t r e a t e d t e s t o s t e r o n e f o r f i v e weeks.  34  17  M a g n i f i e d v i e w o f a p o r t i o n o f f i g u r e 16.  34  18  Remnant sperm i n main sperm duct o f hypophysectomized guppy a f t e r two weeks o f testosterone treatment.  34  T a l l e p i t h e l i a l c e l l s reappear i n sperm d u c t o f hypophysectomized guppy a f t e r f i v e weeks o f testosterone treatment.  34  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatog e n e s i s i n hypophysectomized and shamo p e r a t e d a d u l t guppies a f t e r e i g h t weeks.  35  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatog e n e s i s 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 g u p p i e s .  36  22  S a g i t t a l s e c t i o n o f t e s t i s o f j u v e n i l e guppy showing o n l y spermatogonia1 c y s t s .  53  23  S a g i t t a l s e c t i o n o f t e s t i s o f j u v e n i l e guppy showing spermatogonia and e f f e r e n t d u c t s .  53  24  E f f e r e n t d u c t s o f hypophysectomized treated with testosterone.  53  25  S a g i t t a l s e c t i o n o f t e s t i s o f j u v e n i l e guppy showing main sperm d u c t l i n e d by squamous epithelial cells.  54  26  Main sperm d u c t o f t e s t i s o f hypophysectomized juvenile treated with testosterone.  54  27  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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.  55  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatog e n e s i s i n j u v e n i l e hypophysectomized; j u v e n i l e hypophysectomized and testosterone-treated; j u v e n i l e sham-operated g u p p i e s .  56  29  A n a l f i n o f j u v e n i l e guppy.  57  30  A n a l f i n o f hypophysectomized j u v e n i l e .  57  19  20  21  28  with  juvenile  xiii FIGURE  PAGE  31  Gonopodium  32  A n a l f i n o f hypophysectomized j u v e n i l e t r e a t e d with testosterone.  58  S a g i t t a l s e c t i o n o f t e s t i s o f 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 ' showing few c y s t s o f e a r l i e r stages o f spermatogenesis.  79  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 the e f f e r e n t d u c t s .  79  33  34 35  ( m o d i f i e d a n a l f i n ) o f a d u l t male.  lining  S a g i t t a l s e c t i o n o f t e s t i s o f 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 .  79  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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 g u p p i e s a f t e r e i g h t weeks.  80  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n juvenile control; juvenile 'methallibure't r e a t e d and j u v e n i l e n o n - ' m e t h a l l i b u r e ' treated guppies.  81  1  36  37  38a  58  S a g i t t a l section of p i t u i t a r y gland o f adult c o n t r o l guppy.  82  38b  Magnified  82  39a  S a g i t t a l s e c t i o n o f p i t u i t a r y gland o f 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. M a g n i f i e d v i e w o f a p o r t i o n o f 39a.  83 83  40  S a g i t t a l s e c t i o n o f 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 o f p i t u i t a r y gland o f 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 o f p i t u i t a r y gland o f j u v e n i l e c o n t r o l guppy (group v i ) n o t t r e a t e d w i t h 'methallibure'.  85  Magnified  85  39b  42b  v i e w o f a p o r t i o n o f 38a.  v i e w o f a p o r t i o n o f 42a.  xiv  ACKNOWLEDGMENTS  I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o Dr. W.S. Hoar f o r h i s guidance, e n t h u s i a s t i c s u p p o r t and c r i t i c a l s u p e r v i s i o n throughout t h e p r e s e n t i n v e s t i g a t i o n . Thanks a r e due t o D r s . J.R. Adams, P. F o r d , N.R. L i l e y and J.M. T a y l o r f o r t h e i r h e l p f u l s u g g e s t i o n s during the p r e p a r a t i o n of the manuscript.  I further wish  to thank Dr. J.R. Adams f o r t h e use o f h i s equipment f o r p h o t o g r a p h i c work. For h i s i n t e r e s t i n my work and h e l p extended d u r i n g the p r e p a r a t i o n o f t h e m a n u s c r i p t , I would l i k e t o thank Dr. J . F . L e a t h e r l a n d .  I am a l s o t h a n k f u l t o Dr. M. W i e s b a r t ,  Mr. J.C. Fenwick and Mr. T . J . Lam f o r many f r u i t f u l discussions. I w i s h t o acknowledge Mrs. J . Fenwick and Mrs. M. Jenson for  drawing some f i g u r e s ; Mr. F. M c C o n n e l l and Mr. L. Sharman  for  t e c h n i c a l h e l p and M i s s L. B o g s t i e f o r t y p i n g t h e  manuscript. I am g r a t e f u l t o t h e a u t h o r i t i e s o f patna U n i v e r s i t y f o r g r a n t i n g me s t u d y l e a v e which made t h i s s t u d y p o s s i b l e . F i n a l l y , I would l i k e t o e x p r e s s my a p p r e c i a t i o n t o my w i f e f o r her encouragement and my f a t h e r f o r h i s keen i n t e r e s t i n my e d u c a t i o n .  INTRODUCTION  The 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 i s a h i g h l y s p e c i a l i z e d monthly b r e e d i n g o v o v i v i p a r o u s to  the f a m i l y P o e c i l i i d a e .  s e x u a l l y dimorphic;  t e l e o s t belonging  Male and female guppies a r e  t h e female i s c o n s i d e r a b l y l a r g e r than  the male and o f a p a l e g r e y i s h green c o l o r , w h i l e t h e male i s adorned w i t h s e v e r a l b r i g h t p a t c h e s on t h e s i d e s o f t h e body.  The d i f f e r e n c e i n p i g m e n t a t i o n  males b e l o n g i n g  and c o l o r i n g i n t h e  t o d i f f e r e n t r a c e s i s c o n s i d e r a b l e and  depends on genes l o c a t e d i n s e x chromosomes (Winge 1922; Winge and D i t l e v s o n 1947).  The most conspicuous c o l o r  p a t c h e s a r e t h e l i p o p h o r e s which c o n t a i n b o t h y e l l o w pigments (Xanthophores) and r e d pigments  (erythrophores).  A n o t h e r w e l l d i f f e r e n t i a t e d secondary s e x c h a r a c t e r found i n t h e male guppy i s t h e h i g h l y m o d i f i e d a n a l f i n o r gonopodium, w i t h w h i c h i n t e r n a l f e r t i l i z a t i o n As an a d a p t a t i o n t o i n t e r n a l f e r t i l i z a t i o n , produces s p e r m - b a l l s prevent  o r spermatophores w h i c h  t h e l o s s o f sperm by leakage  i seffected.  t h e male guppy probably  i n t o t h e water d u r i n g  t r a n s f e r t o t h e g e n i t a l t r a c t o f female. In t h e guppy, development o f t h e gonad and secondary sex c h a r a c t e r s has been d e s c r i b e d i n d e t a i l . and G o o d r i c h gonads.  D i l d i n e (1936)  e_t a_l (1934) d i s c u s s e d t h e morphogenesis o f  Comparable s t u d i e s o f t h e o v a r y were r e p o r t e d i n  d e t a i l by A n t e n n i u s (1959).  vaupel  (1929) d e s c r i b e d  spermatogenesis and d i s c u s s e d t h e method o f t r a n s f e r o f t h e  2  sperm t o t h e female.  The morphogenesis o f the gonopodium i n  the male has been d e s c r i b e d by Hopper  (1949a).  Secondary s e x c h a r a c t e r s have been shown t o be i n v o l v e d i n complex c o u r t s h i p b e h a v i o r ( C l a r k and A r o n s o n 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 b e h a v i o r has been s t u d i e d i n d e t a i l , few a n a l y s e s are a s s o c i a t e d w i t h the endocrinology of r e p r o d u c t i o n (Kadow 19 54; L i l e y 1965, 1968). A l t h o u g h t h e i n f l u e n c e o f exogenous s t e r o i d s on t h e gonad and secondary s e x c h a r a c t e r s o f t h e guppy has been t e s t e d many times ( E v e r s o l e 1939, 1941; R e g n i e r 1941; Okada and Y a m a s h i t a 1944b; Hopper 1949b, 1951), e f f o r t s have never been made t o e l i m i n a t e t h e p o s s i b l e e f f e c t s o f endogenous g o n a d o t r o p i n s by t h e use o f hypophysectomized a n i m a l s .  The  p o s s i b i l i t y o f a d i r e c t a c t i o n o f the p i t u i t a r y i n the development o f t e s t i s and t h e d i f f e r e n t i a t i o n o f secondary sex c h a r a c t e r s o f t h e guppy o r any o t h e r p o e c i l i i d has n o t 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 o f  spermatophore f o r m a t i o n , t h e r e t e n t i o n o f spermatophores w i t h i n t h e t e s t i s and t h e i r e v e n t u a l r e l e a s e from t h e t e s t i s have n o t been s t u d i e d i n any f i s h . The t e c h n i q u e o f hypophysectomy was found t o 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 b o t h t h e 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 e x p e r i m e n t s c o n c e r n i n g pituitary-gonad relations.  The r o l e o f g o n a d a l s t e r o i d s  i n t h e maintenance o r development o f t h e t e s t i s and secondary sex c h a r a c t e r s may be r e a d i l y a n a l y z e d 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 t e s t o s t e r o n e  p r e p a r a t i o n s which have been r e p e a t e d l y shown t o 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 o f n a t u r a l In a d d i t i o n , a t e c h n i q u e o f c h e m i c a l l y  androgens.  inhibiting  g o n a d o t r o p i c 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-methylallylthiocarbomoyl-2-methylthio-  carbamyl h y d r a z i n e ; I . C . I . 33,828) b l o c k s the s y n t h e s i s , r e l e a s e or a c t i o n o f g o n a d o t r o p i c hormones i n t e l e o s t s e t a l 1967; Wiebe 1968).  (Hoar  T h i s agent p r o v i d e s a u s e f u l  a d d i t i o n a l t e c h n i q u e i n the a n a l y s i s o f p i t u i t a r y - g o n a d relations i n fishes.  The s i t e o f a c t i o n o f  'methallibure'  i n b l o c k i n g g o n a d o t r o p i c a c t i o n i s n o t known. W i t h t h e s e t e c h n i q u e s as a b a s i s f o r the e x p e r i m e n t a l a n a l y s i s , the purposes o f the p r e s e n t i n v e s t i g a t i o n o f the male guppy a r e as f o l l o w s : 1.  To examine the r o l e o f 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 r e f e r e n c e t o the i n i t i a t i o n o f spermatocyte f o r m a t i o n and the f o r m a t i o n 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 o f a d i r e c t  p i t u i t a r y i n v o l v e m e n t i n the e x p r e s s i o n o f secondary sex c h a r a c t e r s o f a d u l t males. 3.  To d e s c r i b e the e f f e c t s o f hypophysectomy and  m e t h y l t e s t o s t e r o n e t r e a t m e n t on the t e s t i s o f the j u v e n i l e guppy p r i o r t o i t s d i f f e r e n t i a t i o n and b e f o r e the appearance o f secondary sex c h a r a c t e r s .  4  4.  To compare ' m e t h a l l i b u r e ' e f f e c t s on the t e s t e s  and secondary sex c h a r a c t e r s o f the a d u l t and j u v e n i l e guppies and t o l o c a l i z e  the s i t e o f a c t i o n o f ' m e t h a l l i b u r e ' i n  blocking 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 o f t h e p i t u i t a r y and t h e g o n a d a l hormones i n the r e p r o d u c t i v e e n d o c r i n o l o g y o f t h e a d u l t male guppy has been s t u d i e d by a n a l y z i n g t h e e f f e c t s o f hypophysectomy on t h e t e s t e s and secondary s e x c h a r a c t e r s (gonopodium and l i p o p h o r e s ) and s u b s e q u e n t l y  t r e a t i n g s u c h hypophysectomized  animals  with methyl testosterone. MATERIALS AND METHODS Maintenance o f f i s h i n b r e e d i n g a q u a r i a .  The i n i t i a l  sample o f g u p p i e s was p u r c h a s e d from a l o c a l aquarium d e a l e r (Northwest-Aquatics,  Vancouver, Canada).  These were a l l o w e d  to b r e e d i n f i v e g l a s s a q u a r i a o f 20- t o 6 0 - l i t r e c a p a c i t y . The bottoms o f these a q u a r i a were covered  t o a depth o f  2 cm w i t h coarse sand; water was c o n t i n u o u s l y r e c i r c u l a t e d and f i l t e r e d through a pad o f g l a s s - w o o l .  Each day f e c e s  and uneaten f o o d were removed from t h e bottom o f t h e a q u a r i a by a fine-meshed n e t and f i l t e r s were c l e a n e d a t l e a s t once a week.  About 50% o f water i n each o f t h e a q u a r i a was  renewed once i n e v e r y two o r t h r e e weeks. A q u a t i c 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 p u t on t h e s u r f a c e o f water i n a q u a r i a t o p r o v i d e  cover  6  for  the young thus a v o i d i n g t h e i r b e i n g e a t e n 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  b u t the room was tubes and  aquaria  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  the p h o t o p e r i o d  r e g u l a t e d a t n a t u r a l day  length  by a p h o t o c e l l exposed i n a l a r g e window f a c i n g s o u t h . temperature o f a l l a q u a r i a was  maintained  The  a t 25 ± 0.5°C.  F i s h were f e 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.)  M a i n t e n a n c e o f hypophysectomized f i s h . to  One  week p r i o r  the o p e r a t i o n b r i g h t l y c o l o r e d males (16-22 mm  l e n g t h , 9 0-250 mg  i n w e i g h t ) were taken from the  standard breeding  a q u a r i a and put i n a q u a r i a o f 2 0 - l i t r e c a p a c i t y w h i c h contained  1 6 - l i t r e o f 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 o f d e c h l o r i n a t e d water (Young 1933JJ.  The males were k e p t i n the s a l i n e u n t i l  end o f the e x p e r i m e n t . experimental  the  No p l a n t s were put i n these  aquaria.  L i g h t was  p r o v i d e d by 2 0-watt f l u o r e s c e n t tubes about  15 cm above the a q u a r i a . i l l u m i n a t i o n was  The  photoperiod  o f 16-hr  daily  c o n t r o l l e d by I n t e r m a t i c model T101 maintained  a t 25 1 0.5°C.  The  temperature was  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  clocks.  F i s h were food.  Sham-operated a d u l t males w h i c h s e r v e d as c o n t r o l s were a l s o k e p t i n the s a l i n e u n t i l the end o f the e x p e r i m e n t . P r o c e d u r e o f hypophysectomy.  Males c o n d i t i o n e d t o  f i s h - s a l i n e f o r one week, were a n e s t h e t i z e d w i t h  1:600  MS  fish  222  ( T r i c a i n e Methane S u l p h o n a t e - S a n d o z ) .  The  was  7  then p l a c e d 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 a n g l e on t h e s u r f a c e o f wax i n a r e c t a n g u l a r tray  (15x7^x4^ em).  The f i s h was s e c u r e d  plastic  i n p l a c e by a  p a i r o f movable p l a s t i c hooks p r e s e n t on t h e p l a s t i c p l a t e . A f i n e g l a s s tube p l a c e d i n t h e b u c c a l c a v i t y o f f i s h to perfuse  the g i l l s w i t h f i s h - s a l i n e .  served  A l l subsequent  p r o c e d u r e s were c a r r i e d o u t under a b i n o c u l a r m i c r o s c o p e ( x l O ) . A s m a l l i n c i s i o n was made i n t h e g u l a r r e g i o n i n f r o n t o f b r a n c h i o s t e g a l membrane on t h e r i g h t s i d e and extended t o t h e lower l i p . The mucous membrane c o v e r i n g t h e p a l a t e was t o r n away g e n t l y w i t h a p a i r o f f i n e f o r c e p s .  The  p i t u i t a r y g l a n d c o u l d then be seen b e n e a t h the p a r a s p h e n o i d bone as a s m a l l w h i t e body j u s t p o s t e r i o r t o t h e o p t i c chiasma.  The p a r a s p h e n o i d bone was now broken by a p a i r  of blunt forceps. of f i n e forceps.  The p i t u i t a r y was p i c k e d up w i t h a p a i r I t was a s c e r t a i n e d under t h e m i c r o s c o p e  t h a t a l l t h r e e l o b e s o f p i t u i t a r y were always removed. o p e r a t e d f i s h r e c e i v e d t h e same t r e a t m e n t w i t h o u t  Sham-  pituitary  removal. The b l o o d and t i s s u e fragments r e s u l t i n g from t h e o p e r a 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  o p e r a t i o n , t h e f i s h was l e f t i n t h e 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 a g a i n .  I t was then t r a n s f e r r e d t o an  aquarium c o n t a i n i n g f i s h - s a l i n e .  The wound h e a l e d i n t e n  t o twelve days and no i n f e c t i o n was ever n o t i c e d .  Mortality  8  was high.,amounting t o 2 0-25% d u r i n g the o p e r a t i o n ; a f u r t h e r m o r t a l i t y o f 25-30% o c c u r r e d i n two weeks f o l l o w i n g the operation. 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 o p e r a t i o n to a q u a r i a c o n t a i n i n g 1 6 - l i t r e o f f i s h s a l i n e .  To a v o i d  the changes o f c o n c e n t r a t i o n o f t e s t o s t e r o n e 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 7 continuously aerated. (1.6 mgm  A c o n c e n t r a t i o n o f 1:10  was  parts  i n 16 l i t r e s o f f i s h s a l i n e ) o f m e t h y l t e s t o s t e r o n e  ( C a l i f o r n i a C o r p o r a t i o n f o r B i o c h e m i c a l R e s e a r c h , Los A n g e l e s ) was added to each aquarium e v e r y week (5 a p p l i c a t i o n s d u r i n g 5 weeks). (1.6 mgm  M e t h y l t e s t o s t e r o n e was added as a s u s p e n s i o n i n 50 m l . o f f i s h s a l i n e ) t o the a q u a r i a .  Four  l i t r e s o f f i s h s a l i n e was renewed i n each aquarium e v e r y week. C o n t r o l s were o f two k i n d s :  1.  the hypophysectomized  males w h i c h d i d n o t r e c e i v e any m e t h y l t e s t o s t e r o n e t r e a t m e n t 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 . Histology.  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 routine h i s t o l o g y .  To  check on the completeness o f p i t u i t a r y r e m o v a l , s u i t a b l e p o r t i o n s o f the heads were f i x e d i n f o r m i c  acid-Bouin  f i x a t i v e and l e f t i n t h e f i x a t i v e t o d e c a l c i f y f o r a week. Some o f the t e s t e s were f i x e d i n Baker's f o r m o l - c a l c i u m f o r lipid  staining.  9  The B o u i n and B o u i n - f o r m i c 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 d e h y d r a t e d 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 p a r a f f i n - w a x .  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  c u t f o r t e s t e s a t 5 u and o f 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 h a e m a t o x y l i n and e o s i n . The t e s t i s f i x e d i n f o r m o l - c a l c i u m was embedded i n gelatin  (Gurr 1962) and s e c t i o n e d a t 10-15 ;u on a f r e e z i n g  microtome.  S e c t i o n s were f l o a t e d on t o s l i d e s i n i c e d w a t e r .  S e c t i o n s were s t a i n e d w i t h Sudan b l a c k B ( 0 . 7 % i n P r o p y l e n e g l y c o l ) , d i f f e r e n t i a t e d i n 70% p r o p y l e n e 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 s t a n d a r d l e n g t h was measured as  the d i s t a n c e between the snout and t h e l a s t row o f s c a l e s on the c a u d a l p e d u n c l e .  The o v e r a l l body w e i g h t b e f o r e the  removal o f the gonad was measured t o the n e a r e s t 1 mg and the gonad w e i g h t was measured t o t h e n e a r e s t O.i mg.  The  gonad w e i g h t was e x p r e s s e d as a p e r c e n t a g e o f t o t a l body w e i g h t t o g i v e the gonosomatic GSI =  i n d e x (GSI)  - w e i g h t i n grams T o t a l body w e i g h t i n grams  G o n a < 3  X  -^QQ  To compare the h i s t o l o g i c a l p i c t u r e o f the t e s t e s o f sham-operated, hypophysectomized  and t e s t o s t e r o n e - t r e a t e d  g u p p i e s , the t o t a l number o f a c i n i or c y s t s c o n t a i n i n g g e r m - c e l l s o f d i f f e r e n t s t a g e s o f spermatogenesis  i n the  median s a g i t t a l s e c t i o n were counted and p e r c e n t a g e o f each c e l l type c a l c u l a t e d (one a c i n u s u s u a l l y c o n t a i n s g e r m - c e l l s  10  i n t h e same s t a g e o f m a t u r a t i o n ) .  The s i n g l e s e c t i o n thus  s e l e c t e d had c y s t s c o n t a i n i n g a l l s t a g e s o f spermatogenesis and gave e s s e n t i a l l y t h e same p e r c e n t a g e c o m p o s i t i o n as was obtained  a f t e r counting  e v e r y 20th s e c t i o n o f t h e whole  testis. The  e p i t h e l i a l c e l l heights o f e f f e r e n t ducts  (branches  o f main sperm duct) o f t e s t e s o f sham-operated, hypophysectomized and t e s t o s t e r o n e - t r e a t e d w i t h an o c u l a r micrometer.  f i s h were measured  F i v e e f f e r e n t d u c t s from one  s e c t i o n ( j u s t b e f o r e t h e e f f e r e n t d u c t s j o i n t o form t h e main sperm duct) were s e l e c t e d a t random and f i v e measurements o f e p i t h e l i a l c e l l h e i g h t s were t a k e n from each e f f e r e n t duct. one  Thus 25 e p i t h e l i a l c e l l h e i g h t s were measured from  s e c t i o n o f each t e s t i s .  RESULTS Structure of the a d u l t t e s t i s .  The t e s t i s i s a two-  l o b e d body f u s e d i n t h e m i d d l e and l o c a t e d i n t h e p o s t e r i o r p a r t o f the body-cavity,  v e n t r a l t o t h e swimbladder.  c o v e r e d by a t h i n , unpigmented p e r i t o n e a l membrane.  It is The  two b r a n c h e d main sperm d u c t s (one o c c u p y i n g t h e c e n t r e o f each l o b e o f t h e t e s t i s ) u n i t e a t t h e p o s t e r i o r and v e n t r a l margins o f t e s t i s t o form a s h o r t vas d e f e r e n s w h i c h opens a l m o s t i m m e d i a t e l y i n t o t h e u r o g e n i t a l s i n u s  (Fig. l a ) .  The main sperm d u c t and i t s b r a n c h e s , t h e e f f e r e n t d u c t s a r e l i n e d by a c u b o i d a l o r columnar e p i t h e l i u m  (Figs. 4 & 6 ) .  11  The e f f e r e n t d u c t s a r e surrounded by c y s t s o r a c i n i (the t e s t i s i s t h e a c i n u s - t y p e i n w h i c h no t u b u l e s have developed).  Each c y s t o r a c i n u s u s u a l l y c o n t a i n s g e r m - c e l l s  i n t h e same s t a g e o f m a t u r a t i o n .  C y s t s c o n t a i n i n g spermatogonia  and s p e r m a t o c y t e s a r e a t t h e p e r i p h e r y ; i n t e r n a l t o t h e s e a r e s p e r m a t i d - and sperm-cysts and f i n a l l y c l o s e t o t h e c e n t r e l y i n g a d j a c e n t t o the e f f e r e n t d u c t s a r e c y s t s c o n t a i n i n g ' s p e r m - b a l l s ' or spermatophores  ( F i g s , l b & 2a,b).  S e r t o l i c e l l s a r e a r r a n g e d around t h e p e r i p h e r y o f the c y s t s .  S e r t o l i c e l l s l i n i n g t h e c y s t s o f spermatocytes  and s p e r m a t i d s a r e t h i n and f l a t t e n e d .  When the s p e r m a t i d s  w i t h i n t h e c y s t t r a n s f o r m i n t o sperm, t h e S e r t o l i  cells  become s t r i k i n g l y e n l a r g e d ; t h e i r n u c l e i become l a r g e r and more rounded; and t h e sperm heads become a t t a c h e d t o t h e i n n e r margin o f S e r t o l i c e l l s spermatophore  ( F i g . 8 ) . Thus a s p e r m - b a l l o r  i s formed w i t h a compact r i n g o f sperm-heads  around t h e p e r i p h e r y o f c y s t and t h e t a i l s i n the c e n t r e . A c c o r d i n g t o V a u p e l (1929), i n t h e guppy the sperm-heads o f spermatophores now withdraw from the c y s t - w a l l , the t a i l s b e i n g e n t w i n e d i n the p r o c e s s ; a c o n n e c t i o n o f the spermatophore w i t h t h e main c a n a l 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  n o t d e s c r i b e the f a t e o f S e r t o l i c e l l s a f t e r t h e spermatophore withdraws from them and r e a c h e s t h e c e n t r a l c a n a l . (19 50) and Chavin v&  Medlen  Gordon (1951) were a l s o unable t o  e x p l a i n the f a t e o f 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 o f the t e s t i c u l a r  c a n a l 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 p r e s e n t i n v e s t i g a t i o n i t was n o t e d t h a t the w a l l o f the mature c y s t c o n t a i n i n g the spermatophore f u s e s w i t h the w a l l o f the e f f e r e n t duct; the spermatophore passes i n t o the lumen o f the e f f e r e n t d u c t and the S e r t o l i  cells  become a p a r t o f the e p i t h e l i a l l i n i n g o f the e f f e r e n t d u c t ( F i g . 10).  Both S e r t o l i c e l l s and t h e e p i t h e l i a l c e l l s o f  the e f f e r e n t d u c t s have l a r g e o v a l n u c l e i w i t h nucleoli.  distinct  I t may be s u g g e s t e d t h a t the spermatophores w i t h i n  the g e r m i n a l p o r t i o n o f t h e t e s t i s a r e 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 d u c t , the S e r t o l i c e l l s become c o n t i n u o u s w i t h t h e 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 t o 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 d u c t s as S e r t o l i c e l l s i n the S h i n e r s e a p e r c h (Cymatogaster a g g r e g a t a ) . The spermatophores congregate i n groups i n the e f f e r e n t d u c t s and the main sperm d u c t .  A colloidal material i s  n o t i c e d around and i n the spaces between the spermatophores (Fig. 6).  The s o u r c e o f c o l l o i d a l m a t e r i a l i s presumed t o  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 d u c t s and main sperm d u c t , because t h e s e appear c u b o i d a l and d e p l e t e d where spermatophores a r e p r e s e n t , w h i l e columnar and  filled  w i t h s e c r e t o r y d r o p l e t s where spermatophores a r e a b s e n t . Medlen  (1950) has s u g g e s t e d t h a t the c o l l o i d a l m a t e r i a l  c o n t a i n s a n u t r i t i v e s u b s t a n c e f o r t h e maintenance o f sperm and perhaps a m u c i l a g i n o u s s u b s t a n c e t o cause the spermatophores to adhere t o one a n o t h e r .  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 testis.  The  connective  t e s t i s c o n s i s t s o f a tenuous, d i f f u s e ,  t i s s u e stroma w i t h c l o s e l y packed c y s t s i n the  interstices.  The  interstitial  cells  (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 o f the  testis  d i s p e r s e d i n the space between the branches of the e f f e r e n t ducts  ( F i g . 12). Secondary sex c h a r a c t e r s o f the male guppy.  The  adult  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 characters:  (a) s e v e r a l p a t c h e s o f b r i g h t c o l o r s on  s i d e s o f the body and  (b) the gonopodium.  c o l o r p a t c h e s are the l i p o p h o r e s . l i p o p h o r e s may  b r i g h t ; (++)  The b r i g h t n e s s o f  d u l l and  the  The most c o n s p i c u o u s  be q u a n t i f i e d by a s s i g n i n g + s i g n s —  v e r y b r i g h t ; (+++)  sex  (+) t r a c e .  the (++++) The  v a l i d i t y o f t h i s method i s open t o q u e s t i o n , s i n c e i t i s not known whether the a r b i t r a r y s i g n s are i n l i n e a r r e l a t i o n t o the c o n t e n t o f y e l l o w and r e d pigments i n the a r e a under observation.  S i n c e 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 a s s i g n e d ++++ or +++  The m o r p h o l o g i c a l  signs.  d e t a i l s o f the t r a n s f o r m a t i o n o f  the male a n a l f i n i n t o the gonopodium have been d e s c r i b e d Hopper (1949a).  There a r e 10 r a y s p r e s e n t i n the a n a l f i n  o f the guppy; i n the male, r a y s 3, 4 and Ray  by  5 become e l o n g a t e d .  3 becomes t h i c k (bone d e p o s i t i o n ) and develops a hood  and v e n t r a l s p i n e s .  Ray  4 a l s o develops spines but ray 5  has o n l y a hook a t i t s t i p and no s p i n e s  ( F i g . 31).  14  S t r u c t u r e o f the t e s t i s and secondary sex c h a r a c t e r s of sham-operated  c o n t r o l guppy.  Sham-operation  does n o t  b r i n g about any changes i n the s t r u c t u r e o f the a d u l t t e s t i s . The s p e r m a t o g o n i a l c y s t s a r e a t the p e r i p h e r y and the spermatophores  a r e i n the c e n t r e ; the a r e a between the two  i s f i l l e d w i t h the c y s t s c o n t a i n i n g the v a r i o u s developmental s t a g e s o f s p e r m a t o c y t e s , s p e r m a t i d s 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 d u c t s and the main  sperm d u c t a r e f i l l e d w i t h i n t a c t spermatophores 6).  (Figs. 4 &  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 d u c t s a r e t a l l  and columnar (Fig.  14).  ( F i g . 4 and T a b l e I I I ) and c o n t a i n l i p i d  droplets  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 g i v e 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 c h a r a c t e r s a r e unchanged i n the  sham-operated  males. S t r u c t u r e o f the t e s t i s o f hypophysectomized  guppy.  A d u l t male guppies were hypophysectomized and two t o t h r e e f i s h were k i l l e d a t weekly i n t e r v a l s up t o e i g h t weeks i n one experiment.  I n another experiment the  f i s h were k i l l e d a f t e r 16 weeks.  hypophysectomized  I n b o t h the experiments  t h e r e i s a c o n s i d e r a b l e decrease i n the  gonosomatic  i n d i c e s o f the o p e r a t e d 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 o f a  hypophysectomized  guppy i s c o m p l e t e l y packed w i t h spermatophores  except f o r a  few s p e r m a t o g o n i a l c y s t s a t the p e r i p h e r y ( F i g s . 3a, 3b, and T a b l e I I ) .  20  I t seems t h a t the c y s t s c o n t a i n i n g s p e r m a t o c y t e s ,  s p e r m a t i d s and sperm p r e s e n t a t the time o f o p e r a t i o n t r a n s f o r m  TABLE I  G.S.I, o f hypophysectomized and sham-operated g u p p i e s a f t e r 8 weeks.  G. S. I . Fish 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  -'-Standard 'Error ***  P  < o . 001  -  +  3.48*0.3 0  ***  TABLE I I Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized and sham-operated g u p p i e s a f t e r 8 weeks.  Stages o f s p e r m a t o g e n e s i s SPG No  Hypophysectomized  Mean  %  No  SPD %  0  No  SPM %  0  No  SPR %  0  No  %  97.6  98.18  ±16.3  ±0.75  70.93  1.6  1.82  ±0.6  +0.48  Mean- 2.6  1.11  23.0  10.04  16.0  6.84  23.8  11.07  185.2  ±Sx ±0.3  ±0.13  ±3.5  ±2.10  ±1.5  ±0.77  ±3.5  ±2.65  ±42.6 +±5.47  1  ^x  1  Sham-operated  SPC  •l-Mean o f 5 o b s e r v a t i o n s P < 0.001, c o n t i n g e n c y t a b l e (row x column) SPG-^-Spermatogonia; SPC - spermaocytes; SPD - s p e r m a t i d s , SPM-sperm; SPR-spermatophores.  17  TABLE I I I E p i t h e l i a l c e l l h e i g h t s o f t h e e f f e r e n t d u c t s o f 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) Fish 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  4.1  23.3  Grand Mean  "'"Mean o f 25 counts (5 e p i t h e l i a l c e l l h e i g h t s i n each o f 5 e f f e r e n t d u c t s i n one s e c t i o n o f each t e s t i s . ) P < 0.005 - A n a l y s i s o f v a r i a n c e based on 5 median c o u n t s , 1 i n each o f 5 e f f e r e n t d u c t s o f each t e s t i s .  TABLE TV  Fish No  G-S.I. o f hypophysectomized and sham-operated g u p p i e s a f t e r 16 weeks.  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 *** p < 0.001  3.7410.12  TABLE V  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized and sham-operated guppies a f t e r 16 weeks. Stages o f spermatogenesis SPG  No Hypophysectomi zed  Sham-operated  SPC  %  2.2  4.68  ±Sx- = ±0.8  ±0.92  Mean  Mean ±Sx  1  1  3.4  1.88  ±0.4  ±0.29  SPD  No  No  0  0  SPM  %  No  %  0  23.6  12. 45  14.6  7.53  22.2  ±3.7  ±3. 02  ±2.1 "±1.25  "±3.9  Mean o f 5 o b s e r v a t i o n s P < 0.001, c o n t i n g e n c y t a b l e  SPR  (row x column)  10.73  No  %  47.4  95.32  +13.6  ±0.92  137.4  67.41  ±0.27 ±22.9  ±4.95  20 i n t o spermatophores.  D u r i n g t h e second week a f t e r the  o p e r a t i o n few s p e r m a t o c y t e s , many s p e r m a t i d s and sperm a r e e v i d e n t i n t h e t e s t i s b u t by the end o f t h e t h i r d week o n l y sperm-cysts a r e n o t i c e d  ( F i g . 1 1 ) . From t h e f o u r t h week  onwards a f t e r the o p e r a t i o n the t e s t i s c o n t a i n s o n l y spermatogonia and spermatophores.  This indicates  that  spermatogonia a r e n o t t r a n s f o r m e d i n t o spermatocytes i n the  absence o f t h e p i t u i t a r y b u t s p e r m a t o c y t e s , s p e r m a t i d s  and sperm a r e t r a n s f o r m e d i n t o spermatophores t h r e e weeks. of  The p r e s e n c e o f spermatophores  t h e t e s t i s o f a hypophysectomized  during the f i r s t a t the p e r i p h e r y  guppy ( F i g s . 3a,b) w i t h  no t r a c e o f d i s i n t e g r a t i n g c y s t s c o n t a i n i n g e a r l i e r s t a g e s o f spermatogenesis suggests t h a t s p e r m a t o c y t e s , s p e r m a t i d s and sperm do n o t d i s i n t e g r a t e b u t t r a n s f o r m i n t o In of  spermatophores.  t h e hypophysectomized guppy no m i t o t i c  spermatogonia i s e v i d e n t ; many spermatophores  division a r e found  r u p t u r e d i n t h e e f f e r e n t d u c t s and t h e main sperm d u c t (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  d u c t s decrease i n h e i g h t t o 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 o f t h e e f f e r e n t d u c t s do  n o t c o n t a i n any l i p i d d r o p l e t s . i n the hypophysectomized  S e r t o l i c e l l s also regress  a n i m a l ; t h e i r n u c l e i change from  rounded c o n d i t i o n t o f l a t t e n e d shape ( F i g . 9 ) . There i s an a p p a r e n t i n c r e a s e i n t h e number o f f i b r o b l a s t s the  testis  ( F i g . 5 ) . The i n t e r s t i t i a l  w i t h shrunken n u c l e i  throughout  c e l l s are regressed  ( F i g . 13) and no l i p i d d r o p l e t s were  e v i d e n t i n f r o z e n s e c t i o n s s t a i n e d w i t h Sudan b l a c k B.  The s t r u c t u r e o f the t e s t i s o f a  hypophysectomized  guppy a f t e r 16 weeks i s s i m i l a r t o the t e s t i s a f t e r e i g h t weeks e x c e p t f o r the f a c t t h a t most o f the are  r u p t u r e d (Table V and V I ) .  spermatophores  The d i f f e r e n c e s i n p e r c e n t a g  c o m p o s i t i o n s o f spermatogonia and spermatophores i n the t e s t e s o f 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  t r e n d o f 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 o f hypophysectomy a f t e r 16 weeks.  i s much more pronounced  There i s a s t r o n g 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 r u p t u r e d spermatophores a r e b e i n g phagocytosed w i t h i n the e f f e r e n t d u c t s 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 o f  fibrous connective tissue  ( F i g . 15) because o f r u p t u r e and  d i s a p p e a r a n c e o f spermatophores and pronounced i n c r e a s e i n the  number o f f i b r o b l a s t s . Secondary sex c h a r a c t e r s o f hypophysectomized guppy.  In b o t h the e x p e r i m e n t s ( e i g h t and 16 weeks a f t e r hypophy- . sectomy)  the s t r u c t u r e o f the gonopodium i s u n a l t e r e d .  The  l i p o p h o r e s become f a i n t o r e n t i r e l y d i s a p p e a r b o t h on the s i d e s o f 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 d e s c r i b e d e a r l i e r . S t r u c t u r e o f the t e s t i s and secondary sex c h a r a c t e r s of  hypophysectomized c o n t r o l guppy ( t e s t o s t e r o n e experiment)  The t e s t i s has few s p e r m a t o g o n i a l c y s t s a t the p e r i p h e r y ; the  r e s t o f the t e s t i s i s f i l l e d w i t h spermatophores, many  of w h i c h a r e r u p t u r e d ( F i g s . 3, 21 and T a b l e V I I ) .  Sertoli  TABLE V I  Number and p e r c e n t a g e o f spermatogonia and spermatophores i n hypophysectomized guppies a f t e r 8 weeks and 16 weeks  Stages of  spermatogenesis  SPC No 8 weeks a f t e r hypophysectomy  Mean  16 weeks a f t e r Hypophysectomy  Mean  1  ±Sx  ±Sx  1  %  1.6  1.82  ±0.6  ±0.48  2.2  4.68  ±0.8  ±0.92  No 0  0  SPD %  No  SPM %  No  0  0  Mean o f 5 o b s e r v a t i o n s p < 0 . 5 , c o n t i n g e n c y t a b l e (row x column)  0  0  SPR %  No  %  97.6  98.18  ±16.3  ±0.75  47.4  95.32  ±13.6  ±0.92  TABLE V I I Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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 o f spermatogenesis SPG No 1 (A) Mean Hypo phy s e c tomi zed ±S it-  SPC r  1.8  3. 52  ±0.4  ±0. 99  No  SPD %  0  No  SPM %  0  %  0  (B) Testosteronetreated  Mean  9.8  46. 23  4.4  22.16  ±Sx  ±1.9  ±2. 16  ±0.5  ±1.19  (C) S ham-opera t e d  Mean  3.8  1. 70  23.4  11.08  14.4  ±1.1  ±0. 35  ±2.2  ±1.16  ±3.2 ±1.59  -Sx  No  SPR  0  0  6.94  Mean o f 5 o b s e r v a t i o n s P < 0.001 between A & B; A & C; B & C: Contingency t a b l e (row x column)  20.2  9. 70  ±6.7 ±3. 24  No  %  55.8  96.48  ±11.0  ±0.99  6.8  31.58  ±1.5  ±1.87  152.4  70.57  ±1:9.4  ±5.52  24  c e l l s and i n t e r s t i t i a l c e l l s r e g r e s s and t h e i r n u c l e i a r e shrunken  ( F i g 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 d u c t s a r e r e g r e s s e d ( F i g . 5 and T a b l e V I I I ) .  The  l i p o p h o r e s become f a i n t o r e n t i r e l y d i s a p p e a r . S t r u c t u r e o f the t e s t i s and secondary sex c h a r a c t e r s o f sham-operated c o n t r o l guppy ( t e s t o s t e r o n e e x p e r i m e n t ) .  The  s t r u c t u r e o f the t e s t i s o f sham-operated guppy i s e x a c t l y s i m i l a r t o the a d u l t t e s t i s p r e v i o u s l y d e s c r i b e d . A l l s t a g e s o f spermatogenesis a r e p r e s e n t ( F i g s . 2 & 21 and T a b l e V I I ) . The e f f e r e n t d u c t s and t h e main sperm d u c t a r e 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  l i n i n g t h e e f f e r e n t d u c t s a r e t a l l and columnar Table VIXI)-  cells  ( F i g . 4 and  The secondary s e x c h a r a c t e r s a r e unchanged i n  sham-operated males. E f f e c t o f m e t h y l t e s t o s t e r o n e on the a d u l t t e s t i s o f hypophysectomized  guppy.  Hypophysectomized  male guppies were  t r e a t e d w i t h m e t h y l t e s t o s t e r o n e f o r f i v e weeks.  During t h i s  t r e a t m e n t two t o t h r e e f i s h were k i l l e d a t weekly  intervals;  the r e m a i n i n g ( f i v e ) were k i l l e d a t t h e end o f the f i f t h week.  There i s a s i g n i f i c a n t i n c r e a s e i n the gonosomatic  i n d e x o f hypophysectomized guppy f o l l o w i n g m e t h y l t e s t o s t e r o n e t r e a t m e n t (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 o f b o t h s p e r m a t o g o n i a l and s p e r m a t o c y t e - c y s t s ( F i g s . 16, 21 and Table V I I ) .  C y s t s c o n t a i n i n g spermatocytes a r e e v i d e n t  by the end o f second week o f t e s t o s t e r o n e t r e a t m e n t b u t no l a t e r s t a g e s o f spermatogenesis appear d u r i n g t h e n e x t t h r e e  25  TABLE V I I I  E p i t h e l i a l c e l l heights o f the e f f e r e n t d u c t s o f hypophysectomized, t e s t o s t e r o n e - t r e a t e d and shamoperated guppies.  Mean e p i t h e l i a l c e l l h e i g h t (u) ish No  < Hypophysectomized A)  B  < Sham-operated C)  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  4.2  34.8  21.6  £ * Mean r  <> Testosteronetreated  n d  1  Mean o f 25 counts (5 e p i t h e l i a l c e l l h e i g h t s i n each o f 5 e f f e r e n t d u c t s i n one s e c t i o n o f each testis).  P < 0 . 0 1 between A & B; A & C; B & C. Tukey's t e s t - W = 5.05. T e s t based on 5 median c o u n t s , 1 i n each o f 5 e f f e r e n t d u c t s o f each t e s t i s . Q 1  26  TABLE I X  Fish No  G.S.I, o f hypophysectomized, t e s t o s t e r o n e - - t r e a t e d and sham-operated g u p p i e s .  (A) Hypophysectomized  G. S. I . (B) Testosteronetreated  (C) 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  0.66*0.04  1.28±0.05  +?-  n  ** P < 0 . 0 1  between A & B; A & C; B & C. Tukey's t e s t - W = 0.40 Q 1  3.12*0.11**  27  weeks.  Thus t e s t o s t e r o n e 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. O c c a s i o n a l m i t o t i c d i v i s i o n i n the c y s t s can be n o t i c e d ( F i g . 17).  spermatogonial  The r e s o r p t i o n o f sperm,  r e s u l t i n g from the r u p t u r e o f 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 d u r i n g f i r s t two weeks of t e s t o s t e r o n e treatment  ( F i g . 18).  the  This  i n d i c a t e s t h a t the exogenous t e s t o s t e r o n e enhances the r u p t u r e o f spermatophores and the p h a g o c y t o s i s o f sperm. periphery  (resorption)  Only few spermatophores a r e l e f t i n t a c t a t the ( 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 o f b o t h e f f e r e n t ducts main sperm duct so t h a t they assume the t a l l appearance o f the normal a n i m a l  and  columnar  ( F i g . 19 and T a b l e 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 a r e 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  epithelial cells  the sperm d u c t s , 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 r e s p o n d t o the t e s t o s t e r o n e treatment  cells  i n a s i m i l a r way.  r e g r e s s i n the absence o f the p i t u i t a r y and a t t a i n  They  their  normal appearance f o l l o w i n g t e s t o s t e r o n e t r e a t m e n t . for  lining  the c y s t s c o n t a i n i n g spermatogonia, spermatocytes  Except 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 o f m e t h y l t e s t o s t e r o n e on secondary sex c h a r a c t e r s o f hypophysectomized guppy.  The gonopodium w h i c h  was u n a f f e c t e d 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 r e c o v e r y i n t h e c o n t e n t o f l i p o p h o r e s p r e s e n t the s i d e s o f body and the t a i l .  on  The l i p o p h o r e s 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 .  T h i s suggests  t h a t the l i p o p h o r e s a r e d i r e c t l y c o n t r o l l e d by the androgens.  29  Figure l a :  S e c t i o n o f whole t e s t i s o f a d u l t guppy.  F i g u r e l b : D e t a i l o f a p o r t i o n o f 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  F i g u r e 2a:  S a g i t t a l s e c t i o n o f a d u l t t e s t i s showing d i f f e r e n t s t a g e s o f spermatogenesis (x 200)  F i g u r e 2b:  M a g n i f i e d v i e w o f a p o r t i o n o f 2a (x 9 00)  F i g u r e 3a:  S a g i t t a l s e c t i o n o f t e s t i s e i g h t weeks a f t e r hypophysectomy showing o n l y  spermatophores  (x 200)  F i g u r e 3b:  M a g n i f i e d v i e w o f a p o r t i o n o f 3a (x 9 00)  SG - Spermatogia;  SC - Spermatocytes;  SD - S p e r m a t i d s ; SM - Sperm; SR -  Spermatophores  31  F i g u r e 4:  E f f e r e n t ducts o f a d u l t t e s t i s  containing  spermatophores (x 2 00) .  F i g u r e 5:  R u p t u r e d spermatophores i n t h e e f f e r e n t d u c t s o f t e s t i s e i g h t weeks a f t e r  F i g u r e 6:  hypophysectomy (x 200)  Main sperm d u c t o f a d u l t t e s t i s  containing  spermatophores (x 2 00). Note t h e c o l l o i d a l m a t e r i a l i n main sperm d u c t .  F i g u r e 7:  R u p t u r e d spermatophores i n the main sperm duct o f t e s t i s e i g h t weeks a f t e r  E.D. - E f f e r e n t d u c t s ; M.D.-  hypophysectomy (x 200)  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  material.  32  F i g u r e 8:  S e r t o l i c e l l s o f a d u l t t e s t i s w i t h sperm heads a t t a c h e d t o them (x 9 0 0 ) .  F i g u r e 9:  R e g r e s s e d S e r t o l i c e l l s o f 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 10: Spermatophores w i t h s u r r o u n d i n g S e r t o l i c e l l s o f 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).  F i g u r e 11: T e s t i s a t t h e end o f 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  cells.  33  F i g u r e 12:  I n t e r s t i t i a l c e l l s of adult testis  F i g u r 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 weeks a f t e r hypophysectomy  F i g u r e 14:  (x 9 0 0 ) .  eight  (x 9 00).  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  cells  lining  the e f f e r e n t d u c t s o f a d u l t t e s t i s s t a i n e d w i t h Sudan b l a c k B (x 350).  F i g u r e 15:  T e s t i s showing remnant sperm b e i n g phagocytosed i n e f f e r e n t d u c t s 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 o f hypophysectomized guppy t r e a t e d w i t h t e s t o s t e r o n e f o r f i v e weeks (x 2 00).  F i g u r e 17:  M a g n i f i e d v i e w o f a p o r t i o n o f 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. Sertoli  F i g u r e 18:  c e l l s assume normal appearance.  Remnant sperm i n main sperm d u c t o f hypophys e c t o m i z e d guppy a f t e r two weeks o f t e s t o s t e r o n e - t r e a t m e n t (x 200). regressed e p i t h e l i a l  Note  c e l l s l i n i n g main sperm  duct.  F i g u r e 19:  Tall epithelial  c e l l s r e a p p e a r i n sperm d u c t o f  hypophysectomized guppy a f t e r f i v e weeks o f t e s t o s t e r o n e t r e a t m e n t (x 200).  35  F i g u r e 20:  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatog e n e s i s i n hypophysectomized and shamo p e r a t e d 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 o f each b a r represents Standard E r r o r .  SPG - Spermatogonia;  SPC - Spermatocytes;  SPD - S p e r m a t i d s ; SPM - Sperm; SPR -  Spermatophores.  SPG'  SPC  SPD  SPM  SPR  36  F i g u r e 21:  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatog e n e s i s 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.  100  S P G  S P C  S P D  S P M  SPR  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 b e f o r e b i r t h , t h e two sexes a r e e x t e r n a l l y i n t h e newly b o r n young.  indistinguishable  I n male embryos, t h e germ c e l l s  a r e r e l a t i v e l y u n i f o r m l y d i s t r i b u t e d among stroma c e l l s b u t a t b i r t h t h e spermatogonia tend t o m i g r a t e t o t h e p e r i p h e r y . Later  (21 days a f t e r b i r t h ) t h e sperm d u c t s appear as narrow  s l i t s surrounded by stroma c e l l s  ( G o o d r i c h e t a l 1934).  t h i s s e c t i o n , t h e e f f e c t s o f hypophysectomy  In  on such j u v e n i l e  males w i t h o u t secondary s e x c h a r a c t e r s and w i t h gonads containing  o n l y spermatogonia, a r e d e s c r i b e d .  The r e s u l t s  o f t r e a t i n g such hypophysectomized j u v e n i l e males w i t h m e t h y l t e s t o s t e r o n e s h o u l d i n d i c a t e whether secondary s e x c h a r a c t e r s 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 o r secondarily  by t h e androgens.  MATERIALS AND METHODS Maintenance o f hypophysectomized j u v e n i l e f i s h .  One  week p r i o r t o t h e o p e r a t i o n j u v e n i l e guppies (9-11 mm standard length;  14-3 0 mg i n w e i g h t ) i n c l u d i n g b o t h males  and females ( s i n c e s e x i s i n d i s t i n g u i s h a b l e a t t h i s stage)  38  were t a k e n from the b r e e d i n g a q u a r i a and p u t i n a q u a r i a w i t h 16-litre  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 k e p t i n t h i s medium u n t i l the end o f the experiment. A group o f j u v e n i l e s was k i l l e d as  initial  c o n t r o l s i m m e d i a t e l y a f t e r removal from the b r e e d i n g a q u a r i a . The maintenance  o f j u v e n i l e s was s i m i l a r t o t h a t d e s c r i b e d  e a r l i e r f o r hypophysectomized  a d u l t males.  P r o c e d u r e o f hypophysectomy.  J u v e n i l e guppies  c o n d i t i o n e d t o f i s h s a l i n e f o r one week were a n 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).  f i s h was p l a c e d v e n t r a l s i d e uppermost on a(6 x 3 x ^  The cm)  p i e c e o f s o f t p l a s t i c sponge f i x e d i n the m i d d l e o f 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 s e c u r e d i n p l a c e by a t h i n . s t r i p o f s o f t p l a s t i c sponge s t r e t c h e d a c r o s s the v e n t r a l s u r f a c e j u s t p o s t e r i o r t o the g i l l s and was immersed i n f i s h s a l i n e d u r i n g the o p e r a t i o n . The o p e r a t i o n o f the j u v e n i l e guppy f o r the removal o f the p i t u i t a r y was s i m i l a r t o t h a t p r e v i o u s l y d e s c r i b e d f o r the a d u l t male.  M o r t a l i t y was h i g h , amounting  t o 30-35%  d u r i n g the o p e r a t i o n ; a f u r t h e r m o r t a l i t y o f 25-30% o c c u r r e d 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 a q u a r i a w i t h  1 6 - l i t r e o f f i s h s a l i n e were used f o r t e s t o s t e r o n e experiment. Two  d i f f e r e n t c o n c e n t r a t i o n s o f m e t h y l t e s t o s t e r o n e were  used; 1:2x10^ (8 mgm  o f m e t h y l t e s t o s t e r o n e i n 16 l i t r e s o f 7  f i s h s a l i n e per week) and 1:10  (1.6 mgm  of methyl  t e s t o s t e r o n e i n 16 l i t r e s o f f i s h s a l i n e e v e r y week). t r e a t m e n t o f hypophysectomized  The  j u v e n i l e guppies w i t h m e t h y l  39  t e s t o s t e r o n e was begun one week a f t e r t h e o p e r a t i o n . treatments continued  f o r e i g h t weeks.  The  The maintenance o f  j u v e n i l e s was s i m i l a r t o t h a t d e s c r i b e d e a r l i e r f o r m e t h y l 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 w h i c h d i d n o t r e c e i v e any  testosterone  t r e a t m e n t and sham-operated j u v e n i l e s were  the two types o f c o n t r o l s and were a l s o k e p t i n f i s h s a l i n e . Histology.  Because o f the s m a l l s i z e , the e n t i r e body  o f t h e j u v e n i l e guppy e x c e p t f o r i t s c a u d a l p e d u n c l e and t h e t a i l was f i x e d i n f o r m i c a c i d - B o u i n  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 t o d e c a l c i f y b e f o r e  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.  Serial  l o n g i t u d i n a l s e c t i o n s were c u t a t 5 u and s t a i n e d w i t h E h r l i c h ' s h a e m a t o x y l i n and e o s i n . Measurements.  The p e r c e n t a g e o f d i f f e r e n t s t a g e s o f  spermatogenesis p r e s e n t i n the t e s t e s o f the j u v e n i l e o r t h e a d u l t g u p p i e s ( s i n c e sham-operated j u v e n i l e s became a d u l t s during the experimental  p e r i o d ) was c a l c u l a t e d i n the same  way as e x p l a i n e d p r e v i o u s l y f o r t h e a d u l t males.  The t o t a l  number o f e f f e r e n t d u c t s i n the median s a g i t t a l s e c t i o n was counted. The w i d t h and lumen o f f i v e randomly chosen e f f e r e n t d u c t s ( b e f o r e they j o i n t o form main sperm duct) were measured w i t h an o c u l a r micrometer i n one s e c t i o n o f each t e s t i s o f hypophysectomized, t e s t o s t e r o n e sham-operated g u p p i e s .  treated or  The h e i g h t o f e p i t h e l i a l c e l l s and  40  w i d t h o f lumen (space between e p i t h e l i a l c e l l l i n i n g s ) o f main sperm d u c t i n the median s a g i t t a l s e c t i o n o f each t e s t i s were measured a t f i v e d i f f e r e n t p l a c e s which c o v e r e d the e n t i r e l e n g t h o f main sperm d u c t . RESULTS S t r u c t u r e o f the j u v e n i l e t e s t i s  (initial  control).  The j u v e n i l e t e s t i s c o n s i s t s o f two l o b e s connected by a b r i d g e o f mesodermal or stroma c e l l s . t h i n , squamous, p e r i t o n e a l membrane.  I t i s surrounded by a The t e s t i s i s  s i t u a t e d i n t h e p o s t e r i o r p a r t o f the body c a v i t y ,  ventral  t o the a i r b l a d d e r and d o r s a l t o the pancreas and the c o i l s of i n t e s t i n e . No o t h e r s t a g e s o f spermatogenesis e x c e p t spermatogonia a r e found i n the t e s t i s .  Spermatogonia a r e p r e s e n t i n s m a l l  c y s t s a t the p e r i p h e r y o f t e s t i s  ( F i g s . 22, 27 and T a b l e X ) ;  the c e n t r e i s f i l l e d w i t h stroma c e l l s  ( F i g . 23).  d i v i s i o n i s n o t e v i d e n t i n the spermatogonia1  Mitotic  cysts.  Spermatogonia a r e 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 c y t o p l a s m e x c e p t f o r the p r e s e n c e o f o c c a s i o n a l fine basophilia.  T h e i r n u c l e i a r e a l m o s t round w i t h e v e n l y  d i s t r i b u t e d f i n e chromatin granules. single nucleolus.  There i s u s u a l l y a  The stroma c e l l s , on the o t h e r 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 c o n t a i n s an i r r e g u l a r and c o a r s e 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 a r e not e v i d e n t i n the testis.  TABLE X  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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 o f SPG No (A) Juvenile Control (B) Juvenile Hypophysectomized (C) Juvenile S ham-operated  13.4  ±Sx  ±1.28  1  1 Mean ±Sx Mean"*" ±Sx  SPC %  Mean" "  12.4  spermatogenesis  No  SPD %  No  SPM %  No  SPR  %  No  100  P  0  0  0  100  0  0  0  0  %  ±0.68 2.17  34.8  20.03  23.0  13.07  36.4  20. 92  +0.8 ±0.45  ±4.6  ±2.83  ±1.1  ±0.29  ±2.6  ±1. 96  3.8  Mean o f 5 o b s e r v a t i o n s . P < 0.001 - A & C; B & C. C o n t i n g e n c y t a b l e (row x column)  78  143.81  ±10.9 ±4. 31  42  D u r i n g development  the stroma c e l l s i n the c e n t r e o f  the t e s t i s a r e r e a r r a n g e d to form s m a l l e f f e r e n t d u c t s , a l t h o u g h the lumen i n the e f f e r e n t d u c t i s h a r d l y d i s t i n c t ( F i g . 23 and T a b l e X i ) .  The e f f e r e n t d u c t s from the two  l o b e s o f the t e s t i s open a t 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 T a b l e 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' a r e squamous" and a r e d e r i v e d from stroma c e l l s  (the main sperm duct appears as a  r e s u l t o f rearrangement o f 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 o f the t e s t i s o f hypophysectomized S i n c e the c o n t r o l sham-operated  juvenile.  juveniles differentiated into  a d u l t s i n s i x weeks, the hypophysectomized were a l s o k i l l e d a f t e r s i x weeks.  j u v e n i l e guppies  No changes take p l a c e i n  the s t r u c t u r e o f j u v e n i l e t e s t i s i n the absence o f the p i t u i t a r y e x c e p t f o r an i n c r e a s e i n the number o f c o n n e c t i v e tissue cells.  The spermatogonia1 c y s t s a r e p r e s e n t a t the  p e r i p h e r y b u t show no m i t o t i c d i v i s i o n ; c o n s e q u e n t l y t h e r e i s no i n c r e a s e i n t h e i r number ( F i g s . 22, 27 and T a b l e X ) . The S e r t o l i c e l l s a r e n o t e v i d e n t . The stroma c e l l s a r e n o t d i f f e r e n t i a t e d i n t o the interstitial cells  (stroma c e l l s have e l l i p t i c a l n u c l e i and  c o n t a i n 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 have round n u c l e i w i t h a d i s t i n c t n u c l e o l u s ) .  cells  There i s no  i n c r e a s e i n the t o t a l number, w i d t h and the s i z e o f lumina o f the e f f e r e n t d u c t s ( F i g . 23 and T a b l e X I ) .  The  epithelial  TABLE X I  T o t a l number, w i d t h and lumen o f e f f e r e n t d u c t s o f 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 g u p p i e s . Efferent  (A) Juvenile  Fish No  ducts  (B) Juvenile Hypophysectomized  control  (C) Juvenile Sham-operated  No  Width(ul Mean ±Sx  Lumenfu) Mean^Sx  No  Width(u) Mean^Sx  Lumen (u)_ Mean ±Sx  No  Width(ul Mean ±Sx  Lumenful Meani^Sx  1  8  15.66 ±1.90  1. 08 ±0.18  7  13.50 ±1.30  1.08 ±0.18  19  122. 18 ±7. 38  76. 26 ±6. 30  2  9  16.38 ±1.52  1.26 ±0.22  7  14.22 ±0.77  1.44 ±0.22  17  114. 80 ±8. 60  63. 96 ±7. 95  3  7  18.18 ±1.12  1.26 ±0.22  8  10.98 ±0.52  1.62 ±0:34  19  127. 10 ±15. 0  71. 34 ±14. 87  4  6  10.62 ±0.34  1.08 ±0.18  6  10.80 ±0.80  1.26 ±0.22  18  121. 76 ±14. 36  77. 08 ±12. 12  5  9  12.96 46  1.44 ±0.36  8  16. 56 ±1.67  1.26 ±0.22  21  118. 90 ±9. 44  64. 78 ±7. 03  1  ±0..  Mean . ±Sx  1  7.2 ±0.58  1  7.2 ±0.37 1  18.8 ±0.66  Mean o f 5 o b s e r v a t i o n s  P < 0. 01 - A Sc C; B & C ( f o r no., w i d t h and lumen) Not s i g n i f i c a n t - A & B ( f o r no. , w i d t h and lumen) • Tukey s t e s t - W.oi f o r no. - 2.77 W.oi f o r w i d t h - 11.98 . 01 f o r lumen - 10.67 1  w  1  TABLE X I I E p i t h e l i a l c e l l h e i g h t s and lumen o f main sperm d u c t o f 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 g u p p i e s . M a i n sperm d u c t (u; Mean Juvenile Control Epithelial  cells  + Sx )  (B)  (A)  Fish No  1  Lumen  (c)  Juvenile Hypophysectomized Epithelial  cells  J u v e n i l e Sham-operated Lumen  Epithelial  cells  Lumen  1  3.24 ±0.22  2.16 ±0.46  2.88 ±0.18  1.18 ±0.28  12.34 ±1.27  159.80 ±8.30  2  2.88 ±0.18  1.62 ±0.34  2.70 ±0.28  1.62 ±0.44  13.12 ±0.82  82.82 ±4.38  3  2.52 ±0.18  1.98 ±0.18  2.52 ±0.34  1.98 ±0.34  17.22 ±2.39  : .179-. 58 ±12.53  4  1.98 ±0.18  1.26 +0.22  2.34 ±0.22  1.80 ±0.28  13.12 ±0.82  182.04 ±7. 09  5  2.88 ±0.52  1.26 ±0.36  2.16 ±0.22  1.62 ±0.18  13.94 ±1.00  86.10 +±2.90  iMean o f 5 o b s e r v a t i o n s 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 ( f o r e p i t h e l i a l c e l l s and lumen)'. Tukey's t e s t - W 1 e p i t h e l i a l c e l l s - 1.71 . 0 1 f o r lumen - 23.59 f o r  >0  w  45  c e l l s l i n i n g the main sperm duct remain squamous and the w i d t h o f the lumen o f the main sperm d u c t i s n o t i n c r e a s e d (Fig.  25 and T a b l e X I I ) . Thus i t i s apparent t h a t f u r t h e r development o f 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 o f 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 o f the p i t u i t a r y . No secondary sex c h a r a c t e r s a r e p r e s e n t i n the j u v e n i l e s a t the b e g i n n i n g o f the experiment Secondary  ( F i g . 29).  sex c h a r a c t e r s do not develop i n a n i m a l s  hypophysectomized  as j u v e n i l e s .  Both l i p o p h o r e pigments  the a n a l 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 (Fig.  3 0).  and  condition  Thus i t i s e v i d e n t t h a t the presence o f 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 o f secondary sex c h a r a c t e r s i n the j u v e n i l e guppy. S t r u c t u r e o f the t e s t i s and secondary sex c h a r a c t e r s of  the sham-operated j u v e n i l e .  ( s i x weeks) as was  By the end o f the experiment  to be e x p e c t e d , 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 f e m a l e s . t e s t i s c o n t a i n s a l l s t a g e s o f spermatogenesis Table X ) .  The  ( F i g . 2 and  Spermatophores a r e a l s o p r e s e n t i n the e f f e r e n t  d u c t s and the main sperm duct ( F i g s 4 & 6 ) .  There i s a  c o n s i d e r a b l e i n c r e a s e i n the t o t a l number, w i d t h and the s i z e o f lumina o f the e f f e r e n t d u c t s ( F i g . 4 and Table X I ) . The w i d t h o f lumen o f the main sperm duct a l s o i n c r e a s e s c o n s i d e r a b l y 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 a r e (Fig.  6 and Table X I I ) .  columnar  46  Secondary sex c h a r a c t e r s a r e w e l l i n the males. (Fig.  differentiated  The a n a l f i n i s t r a n s f o r m e d i n t o the gonopodium  31) w h i l e b r i g h t patches o f l i p o p h o r e s appear on the  s i d e s o f the body and on the t a i l . S t r u c t u r e o f the t e s t i s o f hypophysectomized (control of testosterone experiment).  juvenile  The t e s t i s c o n t a i n s o n l y  s p e r m a t o g o n i a l c y s t s a t the p e r i p h e r y and stroma c e l l s i n the c e n t r e ( F i g s . 22,28 and T a b l e X I I I ) .  The e f f e r e n t d u c t s  w i t h narrow l u m i n a a r e p r e s e n t i n the c e n t r e o f t e s t i s ( F i g . 23 and T a b l e X I V ) -  The main sperm d u c t 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 T a b l e XV).  No secondary sex c h a r a c t e r s d e v e l o p . S t r u c t u r e o f the t e s t i s and secondary sex c h a r a c t e r s of  the sham-operated  experiment).  juvenile.  (Control of testosterone  As might be e x p e c t e d , 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 o f the experiment  ( e i g h t weeks).  a l l s t a g e s o f spermatogeneis  The t e s t i s c o n t a i n s  ( F i g s 2, 28 and Table X I I l ) .  The e f f e r e n t d u c t s a r e w e l l d i f f e r e n t i a t e d and a r e w i t h spermatophores  ( F i g . 4 and T a b l e X I V ) .  duct i s a l s o f i l l e d w i t h spermatophores epithelial cells  filled  The main sperm  and has  columnar  ( F i g . 6 and Table XV)~. •  The males have d i s t i n c t secondary sex c h a r a c t e r s .  The  a n a l f i n i s f u l l y t r a n s f o r m e d i n t o a t y p i c a l gonopodium (Fig.  31), w h i l e b r i g h t p a t c h e s o f l i p o p h o r e s appear on the  s i d e s o f the body; the l i p o p h o r e i n d e x f o r these f i s h +++  and ++++.  was  TABLE X I I I  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis 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 g u p p i e s .  Stages o f SPG  (A) Juvenile hypophysectomized  Mean +  1  ±Sx  °A  %  No-  No  %  12.8  100  0  0  0  0  100  0  0  0  0  No  No  SPR  SPM  No  14.2  ( B )  T  Mean  SPD  SPC  %  %  ±0.4  Sx  Meanl J u v e .n i,l e hypo p hy s e c tomi ze d testosterone treated (C) Juvenile sham-operated  spermatogenesis  ±2.1  1  4.2  2.63  34.2  21. 32  22.4 14.20  27.6  17.43  71.4  44.42  ±1.0  ±0.65  ±5.1  ±5. 1  ±1.8 ±1.40  ±4.5  ±2.85  ±7.2  ±2.96  Mean o f 5 o b s e r v a t i o n s P < 0.001 - A & C; B & C , c o n t i n g e n c y t a b l e  (row x column)  TABLE XIV  T o t a l number, w i d t h and lumen o f e f f e r e n t ducts o f 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 shamo p e r a t e d guppies Efferent  Fish No  1  1  (B) J u v e n i l e Hypox. & Testo. t r e a t e d Width(u) Lumen (u) No Mean!±Sx Mean !±Sx  No  (C) Juvenile Sham-operated Lumen(u) Width(u) Mean!±Sx Mean ±Sx 1  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 ±1.26  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 Sx +  No  ... J u v e n i l e --' •• Hypophysectomi zed Width(u) Lumen(u) Mean ±Sx Mean ±Sx  ducts  1  8 +0.1  33 ±3.0  19 ±0.71  Mean o f 5 o b s e r v a t i o n s P < 0 . 0 1 - A & B ( f o r number and w i d t h o n l y ) P < 0.01 - A & G; B & C; ( f o r no, w i d t h and lumen) Tukey's t e s t - W 1 ~ 9-07 ! o i f ° w i d t h - 11.75 W. 01 f o r lumen - 9.16 P <0.05 - A & B ( f o r lumen o n l y ) Tukey's t e s t - W.05 f o r lumen - 7.28 x  f o r  n  #0  w  r  o  TABLE XV  E p i t h e l i a l c e l l h e i g h t s and lumen o f main sperm d u c t o f 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 g u p p i e s . Main sperm d u c t (u; Mean  Fish No  (A) Juvenile Hypop hy s e c tomi zed Epithelial  cells  Lumen  1  ±Sx) (C)  (B) J u v e n i l e Hypox. & Testesterone-treated Epithelial  cells  Juvenile Lumen  sham-operated  Epithelial  cells  Lumen  1  3.60 ±0.28  1.08 ±0.18  16.20 ±2.36  26.28 ±3.26  10.74 V:±0.95  136.94 ±11.12  2  3.42 ±0.34  1.26 ±0.22  9.18 ±1.00  21.96 ±4.08  13.94 ±4.02  94.30 ±10.37  3  2.52 ±0.34  2.16 ±0.36  11.88 ±0.96  19.80 ±3.14  11.48 ±1.53  113.16 ±13.57  4  2.88 ±0.34  1.62 ±0.18  8.46 ±0.73  16.56 ±3.06  14.72 ±2.78  113.16 ±14.53  5  2.16 ±0.22  1.80 ±0.28  7.02 ±0.60  10.62 ±1.74  13.12 ±2.01  130.38 ±18.89  'Mean o f 5 o b s e r v a t i o n s P < 0 . 0 1 - 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 ( o n l y 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 . 3 9 W*oi f o r lumen - 1 6 . 5 7 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 o f m e t h y l t e s t o s t e r o n e on the t e s t i s o f hypophysectomized j u v e n i l e . i n animals  No d i f f e r e n c e s were e v i d e n t  exposed t o the two d i f f e r e n t c o n c e n t r a t i o n s o f 6 V  methyl testosterone  (1:2 x 10  t e s t o s t e r o n e treatment  and 1:10 ) .  Methyl  o f f i s h hypophysectomized as j u v e n i l e s  does n o t seem t o 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 t h e r e any i n c r e a s e i n the number  o f the c y s t s ( F i g s . 22, 28 and T a b l e X I I I ) .  Spermatocytes  or any o t h e r stages o f spermatogenesis do n o t appear i n the t e s t i s .  I n c o n t r a s t , the m e t h y l t e s t o s t e r o n e  treatment  o f 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 s p e r m a t o g o n i a l  c y s t s and spermatocytes a l s o  appear. T h i s i n d i c a t e s t h a t exogenous t e s t o s t e r o n e 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 spermatogenesis.  I n the j u v e n i l e guppy the p i t u i t a r y was removed  b e f o r e the t e s t i s r e c e i v e d any g o n a d o t r o p i n  stimulation.  I t seems t h a t 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 p r e s e n t i n the c e n t r e o f the t e s t i s o f the hypophysectomized j u v e n i l e guppy do n o t become d i f f e r e n t i a t e d into i n t e r s t i t i a l c e l l s following testosterone treatment.  The S e r t o l i c e l l s a r e n o t e v i d e n t .  Perhaps,  g o n a d o t r o p i n s r e g u l a t e the d i f f e r e n t i a t i o n o f 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 o f the t e s t i s o f 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 i s the d i f f e r e n t i a t i o n  51  o f the e f f e r e n t d u c t s and the main sperm d u c t .  The  total  number, w i d t h and the s i z e o f lumina o f the e f f e r e n t d u c t s increase s i g n i f i c a n t l y  ( F i g . 24 and T a b l e X I V ) .  The lumen o f  the main sperm d u c t becomes v e r y wide and the e p i t h e l i a l  cells  l i n i n g sperm d u c t 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 o f the sperm d u c t o f shamoperated juveniles  ( F i g . 26 and T a b l e XV).  T h i s suggest  t h a t the d i f f e r e n t i a t i o n o f the e f f e r e n t d u c t s and the main sperm d u c t i s under the c o n t r o l o f the androgens and gonodotropins are not d i r e c t l y i n v o l v e d .  Methyl testosterone  t r e a t m e n t a l s o causes an i n c r e a s e i n the number o f f i b r o b l a s t s i n the j u v e n i l e  testis.  E f f e c t o f m e t h y l t e s t o s t e r o n e on secondary s e x c h a r a c t e r s o f hypophysectomized j u v e n i l e . .  A l l changes  r e q u i r e d t o t r a n s f o r m an a n a l f i n i n t o a gonopodium a r e i n i t i a t e d a f t e r t e s t o s t e r o n e t r e a t m e n t o f hypophysectomized j u v e n i l e s b u t the development o f the f i n i s n o t complete. The r a y 3 becomes t h i c k  (bone d e p o s i t i o n ) and d e v e l o p s a  s m a l l hood w h i c h does n o t e x t e n d beyond the r a y 3 (as i n a normal gonopodium).  The segments o f r a y s 3 and 4 do n o t  d e v e l o p s p i n e s , w h i c h a r e w e l l d e v e l o p e d i n a normal gonopodium.  There i s a s m a l l hook developed a t the t i p o f  r a y 5 b u t i t i s s m a l l e r when compared t o the hook o f a normal gonopodium ( F i g . 3 2 ) . The l i p o p h o r e s appear as two narrow bands, one on the upper m a r g i n and the o t h e r on the lower m a r g i n o f 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 o f  52  the c a u d a l peduncle and e x t e n d onto the c a u d a l peduncle as a s i n g l e band.  I t i s n o t e v i d e n t beyond the a r e a o f the c a u d a l  peduncle (as i s found i n the a d u l t m a l e s ) . a l s o found d i s p e r s e d on the a n a l f i n .  Lipophores are  The b r i g h t n e s s o f  l i p o p h o r e s i s l e s s than i n normal a d u l t males and  was  a s s i g n e d ++ i n the a r b i t r a r y s c a l e d e s c r i b e d e a r l i e r . The secondary sex c h a r a c t e r s w h i c h d e v e l o p e d f o l l o w i n g t e s t o s t e r o n e t r e a t m e n t a r e 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 t h a t  the secondary sex c h a r a c t e r s i n the guppy a r e d i r e c t l y under the c o n t r o l o f androgens.  The i n c o m p l e t e d i f f e r e n t i a t i o n  o f the secondary sex c h a r a c t e r s may be due t o degree o f 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 occurring endogenous androgen or t h a t the exogenous androgen cannot l e a d t o complete  differentiation  o f secondary sex c h a r a c t e r s i n ..the absence o f the p i t u i t a r y .  53  F i g u r e 22:  S a g i t t a l s e c t i o n o f t e s t i s o f j u v e n i l e guppy showing o n l y s p e r m a t o g o n i a ! c y s t s (x 9 0 0 ) .  F i g u r e 23:  S a g i t t a l s e c t i o n o f t e s t i s o f j u v e n i l e guppy showing spermatogonia and e f f e r e n t d u c t s (x 9 00).  F i g u r e 24:  E f f e r e n t d u c t s o f hypophysectomized j u v e n i l e treated with testosterone  (x 9 00).  54  F i g u r e 25:  Sagittal  s e c t i o n o f t e s t i s o f j u v e n i l e guppy  showing main sperm duct l i n e d by squamous epithelial cells  F i g u r e 26:  (x 900).  Main sperm d u c t o f t e s t i s o f hypophysectomized juvenile treated with testosterone  (x 900).  Note t h e 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:. P e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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 sham-operated g u p p i e s .  0  juvenile  SPG  SPC  SPD  SPM  SPR  56  F i g u r e 28:  P e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis in.' j u v e n i l e hypophysectomized; hypophysectomized and j u v e n i l e sham-operated  juvenile  testosterone-treated; guppies.  57  F i g u r e 29:  A n a l f i n o f j u v e n i l e guppy. numberd from c r a n i a l  (ventral) to caudal  (dorsal) border of f i n . diagram r e p r e s e n t s 1  F i g u r e 3 0:  F i n - r a y s are  The  l i n e b e l o w the  mm.  A n a l f i n o f hypophysectomized  juvenile.  58  F i g u r e 31:  Gonopodium ( m o d i f i e d a n a l f i n ) o f a d u l t male.  F i g u r e 32:  A n a l f i n o f hypophysectomized j u v e n i l e t r e a t e d with  testosterone.  59 SECTION I I I •METHALLIBURE  1  TREATMENT OF  THE JUVENILE AND ADULT MALES  INTRODUCTION Hoar e t a l (1967) and Wiebe (1968) c o n c l u d e d t h a t 'methallibure' e f f e c t i v e l y blocks the p i t u i t a r y action.  gonadotropic  T h i s 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 o f ' m e t h a l l i b u r e ' t r e a t m e n t w i t h t h a t o f hypophysectomy.  I n t h i s s e c t i o n , an attempt i s made t o  compare t h e e f f e c t s o f ' m e t h a l l i b u r e ' t r e a t m e n t  (pharmacological  hypophysectomy) on t h e t e s t e s and secondary s e x c h a r a c t e r s o f t h e a d u l t and j u v e n i l e guppies w i t h t h a t o f s u r g i c a l hypophysectomy d e s c r i b e d i n e a r l i e r s e c t i o n s . action of 'methallibure' i n e l i m i n a t i n g hormone a c t i v i t y i s n o t known.  The s i t e o f  gonadotropic  An a c t i o n a t t h e l e v e l o f  the p i t u i t a r y would be r e f l e c t e d i n t h e c y t o l o g y o f t h e g l a n d . The  e f f e c t s o f ' m e t h a l l i b u r e ' on t h e appearance o f t h e  p i t u i t a r y complex, i n p a r t i c u l a r o f t h e mesoadenohypophysis are  described.  MATERIALS AND METHODS Maintenance o f ' m e t h a l l i b u r e ' t r e a t e d f i s h . a d u l t males (17-22 mm s t a n d a r d  Mature  l e n g t h ; 100-230 mg i n weight)  and immature j u v e n i l e guppies (8-12 mm s t a n d a r d  length;  8-28 mg i n weight) were t a k e n from t h e b r e e d i n g  a q u a r i a and  put i n four aquaria  (two f o r a d u l t s and two f o r j u v e n i l e s ) ,  each o f w h i c h c o n t a i n e d  16 l i t r e s o f d e c h l o r i n a t e d t a p w a t e r .  60  A group o f j u v e n i l e f i s h was from the b r e e d i n g a q u a r i a maintenance was  k i l l e d i m m e d i a t e l y a f t e r removal  to s e r v e as i n i t i a l c o n t r o l s .  s i m i l a r to that p r e v i o u s l y described  methyl testosterone  t r e a t e d hypophysectomized a d u l t  The  for and  j u v e n i l e guppies. ' M e t h a l l i b u r e ' t r e a t m e n t was were a c c l i m a t e d a d u l t s and was  two  f o r one week. contained  l i m i t e d t o one  control.  i n i t i a t e d a f t e r the  S i n c e two a q u a r i a  contained  j u v e n i l e s , 'methallibure'  treatment  aquarium i n each case; the o t h e r  A concentration  o f 1:10^  parts  fish  (16 mgm  served  i n 16  l i t r e s o f water) of  ' m e t h a l l i b u r e ' was  e v e r y a l t e r n a t e day  (28 a p p l i c a t i o n s d u r i n g e i g h t weeks).  Histology. o f the a d u l t and  The  f i x a t i o n and  as  added t o the  aquaria  s t a i n i n g of the  testes  j u v e n i l e g u p p i e s were s i m i l a r to t h a t  described e a r l i e r .  The  heads were f i x e d i n f o r m i c  a f t e r r e m o v a l of the r o o f of the s k u l l and i n the f i x a t i v e t o d e c a l c i f y .  acid-Bouins  l e f t f o r seven days  A f t e r dehydration  in ethyl  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 c o m b i n a t i o n s o f a l c i a n blue-PAS-orange G (AB - PAS  - OG),  (AT - PAS  - NY), Gabe's aldehyde f u c h s i n (AF) or  Wolfe t r i c h r o m e  aldehyde t h i o n i n e - PAS  yellow  Cleveland-  ( H e r l a n t 19 56) .  Measurements. 'methallibure'  - napthol  Measurements o f the t e s t e s of  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 o f hypophysectomized a d u l t and guppies d e s c r i b e d e a r l i e r .  juvenile  By means o f an o c u l a r micrometer,  61 the d i a m e t e r s o f t e n c e l l s o f each o f gonadotrophs, somatotrophs and t h y r o t r o p h s five pituitary  from t h e s a g i t t a l s e c t i o n s o f  g l a n d s o f b o t h a d u l t and j u v e n i l e guppies : were  measured (a t o t a l o f 50 c e l l s o f each type i n each  group).  RESULTS S t r u c t u r e o f t h e t e s t i s and secondary sex c h a r a c t e r s of a d u l t c o n t r o l .  The s t r u c t u r e o f t h e t e s t i s o f t h e a d u l t  guppy has been d e s c r i b e d i n d e t a i l i n t h e e a r l i e r  sections.  I n s h o r t , a l l s t a g e s o f spermatogenesis a r e p r e s e n t 36 and T a b l e X V I I ) .  ( F i g s . 2,  The e f f e r e n t ducts and t h e main sperm  d u c t a r e 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 t h e e f f e r e n t ducts a r e t a l l and columnar  ( F i g . 4 and T a b l e X V I I I ) .  The secondary s e x  c h a r a c t e r s have a l s o been d e s c r i b e d e a r l i e r .  The l i p o p h o r e  i n d e x was r a t e d +++ o r ++++. E f f e c t o f ' m e t h a l l i b u r e ' on t h e t e s t i s and secondary sex c h a r a c t e r s o f t h e a d u l t guppy. t r e a t m e n t was c o n t i n u e d  The  f o r e i g h t weeks.  'methallibure' The gonosomatic  index o f '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 (Table X V I ) .  decreased  The t e s t i s c o n t a i n s a few c y s t s o f spermatogonia,  s p e r m a t o c y t e s , s p e r m a t i d s and sperm b u t spermatophores a r e present  i n abundance ( F i g s . 33, 36 and T a b l e X V I I ) .  spermatophores p r e s e n t  The  i n t h e e f f e r e n t d u c t s and the main sperm  d u c t a r e u s u a l l y i n t a c t ( F i g . 3 4 ) . The e p i t h e l i a l c e l l s  lining  the e f f e r e n t ducts become c o n s i d e r a b l y r e d u c e d ( F i g . 34 and T a b l e X V I I I ) b u t 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 n o t seem t o be r e g r e s s e d .  TABLE XVI  Gonosomatic i n d e x (G.S-I.) o f ' 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 guppies a f t e r 8 weeks.  G. S. I . Fish No  'Methallibure' treated  Control  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 +r-  J. 1.53 0.12 +  ID  *** p < 0.001  _ L *** 3.35+0.12  TABLE X V I I  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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 guppies. Stages o f spermatogenesis SPC  SPG  1  Methallibure treated  1  Mean" ±Sx Mean  Control  ± Sx  %  No  SPM  %  2.4  1.39  3.4  2.02  ±0.6  +0.38  +0.8  +0.54  +2.7 +1.53  ±219 ±1.82  ±19.1 ±3.66  2.6  1.18  33.6  14.74  14.2  18.4  8.79  159.8 68.29  ±0.7  ±0.37  ±5.9  ±2.47  ±3.6 ±2.00  ±3.7 ±2.20  ±26.9 ±5.34  6.6  % 3.91  6.99  Mean o f 5 o b s e r v a t i o n s  P ^ 0.001, c o n t i n g e n c y t a b l e  (row x column)  No  SPR  No  x  No  SPD  7.4  %  No  %  4.43  159  88.26  64  TABLE X V I I I  E p i t h e l i a l c e l l heights of efferent d u c t s o f ' 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 guppies.  Mean e p i t h e l i a l Fish No  "Methallibure  1  c e l l height(u)  treated  x  Control  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  x  Mean o f 2 5 counts (5 e p i t h e l i a l c e l l h e i g h t s i n each o f 5 e f f e r e n t d u c t s i n one s e c t i o n o f each testis)  P-^0.025 - a n a l y s i s o f v a r i a n c e based on 5 median c o u n t s , 1 i n each o f 5 e f f e r e n t d u c t s i n 1 s e c t i o n o f each t e s t i s .  65  No changes a r e e v i d e n t i n the s t r u c t u r e o f the gonopodium b u t t h e r e i s a marked decrease i n the l i p o p h o r e p i g m e n t a t i o n b o t h on t h e s i d e s o f t h e body and on t h e t a i l . The l i p o p h o r e i n d e x was r a t e d + o r ++. Comparison  o f t h e e f f e c t s o f hypophysectomy and  ' m e t h a l l i b u r e ' t r e a t m e n t 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 significantly  decreased.  i n d e x i n b o t h cases i s  The e f f e c t s o f hypophysectomy on  s p e r m a t o g e n e t i c s t a g e s o f t e s t i s a r e more apparent than i n 'methallibure' treatment.  The t e s t i s o f a  hypophysectomized  guppy c o n t a i n s o n l y s p e r m a t o g o n i a l c y s t s and  spermatophores  whereas t h e t e s t i s o f ' m e t h a l l i b u r e ' t r e a t e d guppy c o n t a i n s a l l stages o f spermatogenesis, although the e a r l i e r are  few ( F i g s . 3, 33 and T a b l e X I X ) .  stages  S i n c e hypophysectomy  c o m p l e t e l y b l o c k s t h e t r a n s f o r m a t i o n o f spermatogonia  into  spermatocytes t h e presence o f few spermatocytes i n the ' m e t h a l l i b u r e ' t r e a t e d t e s t i s s u g g e s t s t h a t a complete b l o c k a g e o f g o n a d o t r o p i n s e c r e t i o n was n o t a t t a i n e d XIX).  A f t e r hypophysectomy many spermatophores  (Table  a r e found  r u p t u r e d i n t h e e f f e r e n t d u c t s and t h e main sperm duct; by c o n t r a s t , f o l l o w i n g ' m e t h a l l i b u r e ' t r e a t m e n t the spermatophores are  usually intact.  The e p i t h e l i a l c e l l h e i g h t s o f the  e f f e r e n t d u c t s a r e more reduced f o l l o w i n g hypophysectomy than 'methallibure' treatment.  Hypophysectomy b r i n g s about t h e  r e g r e s s i o n o f 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  • m e t h a l l i b u r e ' t r e a t m e n t does n o t . Perhaps, even a s m a l l r e l e a s e o f 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  cells.  TABLE X I X  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized and ' m e t h a l l i b u r e ' t r e a t e d guppies a f t e r 8 weeks. Stages o f  SPG No Hypophysectomized  'Methallibure' treated  Mean  1  ±Sx Mean *Sx  1  spermatogenesis  SPC %  No  SPM  SPD %  0  No  %  1. 82  ±0.6  ±0. 48  . 2.4  1. 39  3.4  2.02  ±0.6  ±0. 38  ±0.8  ±0.54  °A  0  0  1.6  No  SPR  -  6.6  3.91  ±2.7 ±1.53  7.4  4. 43  ±2.9 ±1. 82  Mean o f 5 o b s e r v a t i o n s P <0.001, c o n t i n g e n c y t a b l e (row x column) f o r a l l s t a g e s o f spermatogenesis P <C 0.005, c o n t i n g e n c y t a b l e (row x column) f o r o n l y 2 s t a g e s , spermatogonia and spermatocytes o f hypophysectomized and ' m e t h a l l i b u r e ' t r e a t e d guppies.  No  7<  97.6  98. 18  ±16.3  ±0. 75  159  88. 26  ±19 .1  ±3. 66  67  No changes a r e e v i d e n t i n the s t r u c t u r e o f the gonopodium o f an a d u l t f i s h e i t h e r a f t e r hypophysectomy o r treatment.  'methallibure'  The decrease i n l i p o p h o r e p i g m e n t a t i o n  i s more  pronounced a f t e r hypophysectomy than ' m e t h a l l i b u r e ' S t r u c t u r e o f the j u v e n i l e t e s t i s  treatment.  ( I n i t i a l control)  The s t r u c t u r e o f the j u v e n i l e t e s t i s has been a l r e a d y i n Section I I . periphery  described  S p e r m a t o g o n i a l c y s t s a r e p r e s e n t a t the  ( F i g s . 22, 37 and T a b l e XX); e f f e r e n t ducts  narrow lumira a r e p r e s e n t  i n the c e n t r e  with  ( F i g . 23 and T a b l e X X I ) .  The main sperm d u c t has narrow lumen and squamous e p i t h e l i a l cells  ( F i g . 25 and T a b l e X X I I ) . S t r u c t u r e o f the t e s t i s and secondary sex c h a r a c t e r s  o f t h e j u v e n i l e guppy n o t t r e a t e d w i t h ' m e t h a l l i b u r e ' . might be e x p e c t e d , the j u v e n i l e s n o t t r e a t e d w i t h  As  'methallibure',  were d i f f e r e n t i a t e d i n t o a d u l t males and females by the end o f the e x p e r i m e n t ( e i g h t weeks). are present  i n the t e s t i s  A l l s t a g e s o f spermatogenesis  ( F i g s . 2, 37 and T a b l e XX) .  A l t h o u g h the e f f e r e n t d u c t s  ( F i g . 3 5 and T a b l e XXI) and the  main sperm d u c t ( F i g . 3 5 and T a b l e X X I I ) a r e w e l l d i f f e r e n t i a t e d , they do n o t c o n t a i n spermatophores. Secondary sex c h a r a c t e r s a r e v e r y d i s t i n c t i n the males. E f f e c t o f ' m e t h a l l i b u r e ' on the t e s t i s o f the j u v e n i l e fish.  The j u v e n i l e f i s h were t r e a t e d w i t h ' m e t h a l l i b u r e ' f o r  e i g h t weeks.  No d e v e l o p m e n t a l changes o c c u r r e d  s t r u c t u r e o f the j u v e n i l e t e s t i s f o l l o w i n g treatment.  i n the  'methallibure'  M i t o t i c d i v i s i o n i n s p e r m a t o g o n i a l c y s t s was  never e v i d e n t and the number o f s p e r m a t o g o n i a l c y s t s remains  TABLE XX  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f 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 • 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.  Stages o f spermatogenesis SPG  (A) Juvenile Control  Mean  (B) Juvenile Meth. t r e a t e d  Mean  (c)  J u v e n i l e nonmeth. t r e a t e d  1  ±Sx  +  ±Sx  SPD  SPM  SPR  No  °/°  No  15.8  100  0  0  0  0  100  0  P  0  0  %  No  %  No  °A  No  Vo  ±1.3 1  Sx  Mean  SPC  17.8 +  1  0.6 1.8  1.38  35.2  26. 14  24.0 18. 13  43.6 31. 37  29.6  22 .98  -0.2  ±0.19  ±6.3  ±3. 42  ±2.5 ±1. 85  ±10.9-±5. 04  ±2.3  ±3 .31  Mean o f 5 o b s e r v a t i o n s P < 0.001 - A & C; B & C:  Econtingency table  (row x column)  TABLE X X I  T o t a l number, w i d t h and lumen o f e f f e r e n t ducts o f juvenile control, juvenile 'methallibure treated and j u v e n i l e n o n - ' m e t h a l l i b u r e t r e a t e d guppies 1  1  E f f e r e n t ducts Juvenile 'Methallibure'treated  Juvenile Control  (B)  J u v e n i l e Non' M e t h a l l i b u r e ' t r e a t e d (C)  NO  No  Width(u) Mean Sx  Lumen(u) Mean ±Sx  No  Width(u) Mean ±Sx  Lumen(u) Mean ±Sx  No  Width(u) Mean ±Sx  Lumen(u) Mean ±Sx  1  18  19.80 ±1.51  1.44 ±0.36  21  19.08 ±0.77  1.08 ±0.18  41  64.78 ±7.27  33 .62 ±8.14  2  16  15.66 ±0.54  1.26 ±0.22  20  19.44 ±0.46  1.62 ±0.18  29  •61.50 ±4.67  28.70 ±3.43  3  14  20.16 ±0.93  1.80 ±0.28  15  19.62 ±1.98  1.62 ±0.34  28  71.34 ±5.28  38.54 ±6.34  4  18  18. 0 ±0.57  1.44 ±0.22  22  17.82 ±0.72  1.80 ±0.28  29  74.62 ±8.54  35.26 ±6.16  5  16  17.28 ±0.52  1.62 ±0.34  18  17.64 ±0.73  1.80 ±0.28  31  68.06 ±4.60  36.90 ±5.50  Mean ±Sx  1  16.4 ±0.7  1:t  1  1  1  19.2 ±1.2  1  1  31.6 ±2.4  Mean o f 5 o b s e r v a t i o n s P < 0.01 - A & C, B & C ( f o r t o t a l na, w i d t h and lumen) Not s i g n i f i c a n t - A & B ( f o r t o t a l no., w i d t h and lumen) Tukey's t e s t - W 1 ° r no. - 8.16 w] Ql f ° w i d t h - 6.96 w[Ql f ° lumen - 6.42 f  >0  r  r  TABLE X X I I  E p i t h e l i a l c e l l h e i g h t s and lumen o f main sperm d u c t 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 n o n - ' m e t h a l l i b u r e t r e a t e d guppies. Main sperm d u c t (u; M e a n  (A) Fish No  Juvenile Epithelial  Control  cells  Lumen  1  ±Sx)  (B) Juvenile • M e t h a l l i b u r e ' 'treated Epithelial  cells  Lumen  (c) J u v e n i l e non'Methallibure' treated Epithelial  cells  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 o f 5 o b s e r v a t i o n s P 4i0.01  - 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 ( f o r 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 ° lumen - 11.34 r  71  the same ( F i g s . 22, 37 and T a b l e XX). i n the t o t a l number, w i d t h and e f f e r e n t ducts  There i s no  the s i z e o f lumina o f  ( F i g . 23;and T a b l e X X I ) .  The  l i n i n g main sperm duct remain squamous and o f main sperm duct  increase  i s not i n c r e a s e d  the  epithelial  cells  the w i d t h of lumen  ( F i g . 25 and T a b l e XXII):. .  Thus i t seems t h a t the f u r t h e r development o f the t e s t i s i s s t o p p e d when the j u v e n i l e s a r e t r e a t e d w i t h  'methallibure . 1  Secondary sex c h a r a c t e r s a r e absent i n the j u v e n i l e s . They do not d e v e l o p 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 a p p a r e n t t h a t ' m e t h a l l i b u r e ' p r e v e n t s  the  appearance o f secondary sex c h a r a c t e r s i n j u v e n i l e s . Comparison o f the e f f e c t s o f hypophysectomy  and  ' m e t h a l l i b u r e ' t r e a t m e n t on the t e s t e s o f the j u v e n i l e s . the j u v e n i l e guppy, t r e a t m e n t w i t h ' m e t h a l l i b u r e i d e n t i c a l e f f e c t s t o hypophysectomy.  The  produced  1  t e s t i s contains  p e r i p h e r a l s p e r m a t o g o n i a l c y s t s (Table X X I I I ) .  stopped when the j u v e n i l e s a r e  'methallibure'.  Morphology o f the p i t u i t a r y complex. the adenohypophysis (AH) p a r t by S o k o l  characters  These p o i n t s i n d i c a t e t h a t g o n a d o t r o p i n  s e c r e t i o n i s completely treated with  only  Both the  t r e a t m e n t s i n h i b i t the appearance o f secondary sex i n the j u v e n i l e f i s h .  In  (1961);  The  s t r u c t u r e of  o f the guppy has been d e s c r i b e d i n  a b r i e f d e s c r i p t i o n o f the  pituitary  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 p r o - , meso , and meta-AH, T  and the neurohypophysis (NH)  ( F i g . 38a).  TABLE X X I I I  Number and p e r c e n t a g e o f d i f f e r e n t s t a g e s o f spermatogenesis i n hypophysectomized j u v e n i l e guppy a f t e r 6 weeks and ' m e t h a l l i b u r e * 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 SPG  Hypophysectomized  Mean  'Methallibure' treated  Mean  1  -Sx  ±Sx  spermatogenesis  SPC No  SPD %  SPM  SPR  No  %  12.4  100  0  0  0  0  100  0  0  0  0  No  %  No  %  No  -0.68 1  17.8 +  0.6 Mean o f 5 o b s e r v a t i o n s .  %  73  The pro-AH/ 3 0-40%  ( F i g s . 38a, 39a & 40-42) w h i c h composes  o f t h e p i t u i t a r y g l a n d i s l a r g e l y formed o f c l o s e l y  packed a c i d o p h i l s probably  o f diameter 4.7  +  0.5 u; these a r e  homologous w i t h the c e l l s i n t h e pro-AH o f Fundulus  h e t e r o c l i t u s w h i c h a r e capable o f b i n d i n g  anti-ovine  p r o l a c t i n serum (Emmart, p i c k f o r d & W i l h e l m i 1966), and a r e thought t o 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  o f t h e NH w i t h t h e pro-AH i s found a f u r t h e r organe G p o s i t i v e  c e l l type,  less  (OG +ve) than t h e ' p r o l a c t i n c e l l s ' , o f  diameter 4.0 1" 0.3 u w h i c h p r o b a b l y r e p r e s e n t t h e adrenocorticotrophs  d e s c r i b e d by O l i v e r e a u  (1964).  The meso-AH i s composed o f 3 c e l l types 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 .  recognizable  One i s a c i d o p h i l i c , more  or l e s s rounded, o f diameter r a n g i n g from 3.9 t o 5.2 u; t h e c e l l s a r e s c a t t e r e d throughout t h e c e n t r a l zone o f t h e meso-AH ( F i g s . 38b & 39b) and a r e c o n s i d e r e d t o have a somatotrophic 1939;  f u n c t i o n ( O l i v e r e a u & Ridgeway 1962; L e v e n s t e i n  1 F o n t a i n e & O l i v e r e a u 1949).  The two r e m a i n i n g  types a r e b a s o p h i l s which, i n t h e guppy, a r e a r r a n g e d d i s t i n c t regions.  cell i n two  One r e g i o n i s i n t h e v e n t r a l h a l f o f t h e  meso-AH ( F i g . 3 8 a ) ; i n mature a d u l t s these c e l l s a r e o f a n g u l a r o u t l i n e w i t h mean diameter 4.8 t o 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 a r e commonly d i s t r i b u t e d around l a r g e s i n u s e s o r c a p i l l a r i e s w h i c h a r e prevalent i n t h i s v e n t r a l region of the gland Sokol  ( F i g s . 38 & 3 9 ) .  (1961) c o n s i d e r e d these b a s o p h i l s t o have a  gonadotrophic  f u n c t i o n because o f t h e i r changing appearance  74  during sexual maturation, separate  a l t h o u g h she was  unable t o  them t i n c t o r i a l l y from the second group o f meso-AH  b a 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 o f the NH the meso-AH. 39b)  The  into  c e l l s o f t h i s second group ( F i g s . 38b  &  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 t o 8.3  w i t h the c y t o p l a s m more PAS  +ve  u)  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  c o l o u r w h i l e t h a t o f the gonadotrophs i s dark b l u e . 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  They a r e  1961;  O l i v e r e a u 1963). Two (Fig.  38a)  c e l l types are r e a d i l y r e c o g n i s e d i n the meta-AH one o f which i s r e a d i l y s t a i n a b l e w i t h PAS  diameter 6.9  t 0.2  u).  types a r e n o t known; one  The  f u n c t i o n s o f these two  (cell  cell  i s thought to produce melanophore  s t i m u l a t i n g hormone. The One  NH i s formed o f nerve f i b r e s w h i c h have two  type o f 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 n u c l e u s  origins. (PON)  and c o n t a i n s 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 ( L e a t h e r l a n d ,  Budtz & Dodd 1 9 6 6 ) ( F i g s . 38a,b).  The  stainable material 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 o f 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 g r a n u l a t e d , whereas i n the d o r s a l r e g i o n o f the NH i t takes the form o f l a r g e e x t r a c e l l u l a r accumulations  ( F i g s . 41 & 42b).  The  second  type o f 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  ( N L T ) ( F i g . 38b) and i s not s t a i n a b l e  w i t h these ' n e u r o s e c r e t o r y ' s t a i n s . o f neurones o f diameter 6.0  The n u c l e u s i s composed  to 7.0 u and i s found i n the  hypothalamus i m m e d i a t e l y d o r s o - l a t e r a l t o the p i t u i t a r y g l a n d , as w e l l as i n the d o r s a l r e g i o n o f the NH i t s e l f . the neurones nor the axons o f the NLT  Neither  s t a i n w i t h the  c o m b i n a t i o n s used here, a l t h o u g h the AT-PAS-NY c o m b i n a t i o n s t a i n s the c y t o p l a s m s l i g h t l y grey and the n u c l e u s y e l l o w . In the e x p e r i m e n t a l procedures d e s c r i b e d below, no changes a r e found i n e i t h e r the s i z e or appearance o f the i n t r i n s i c e n d o c r i n e c e l l s o f the p r o - or meta-AH, nor o f the neurones o f NLT or PON;  a l t h o u g h t h e r e -was c o n s i d e r a b l e  v a r i a t i o n i n the amount o f s t a i n a b l e m a t e r i a l i n the  NH,  i t was n o t c o n s i s t e n t w i t h e x p e r i m e n t a l c o n d i t i o n s .  Only  the c e l l types o f the meso-AH show changes and w i l l  be  c o n s i d e r e d below. E f f e c t o f ' m e t h a l l i b u r e ' on the meso-adenohypophysis o f the a d u l t male guppy.  There appears t o 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 o f the c o n t r o l  fish  (group I ) ( F i g s . 38a,b) compared w i t h the ' m e t h a l l i b u r e ' t r e a t e d fish  (group I I ) ( F i g s . 39a,b).  The mean diameter o f 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 h i g h e r (p ^ 0.001) and the amount o f AF+ve m a t e r i a l i n t h e i r c y t o p l a s m i s n o t i c e a b l y g r e a t e r i n the c o n t r o l f i s h  (Table XXIV).  As a l r e a d y noted,  the v e n t r a l p a r t o f 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  Experimental Condition  E f f e c t o f ' m e t h a l l i b u r e ' ( I C I 33.828) on t h e mean c e l l diameter o f the mesoadenohypophysial gonadotrophs, somatotrophs, and t h y r o t r o p h s o f 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 . No. of Fish  Weight range (mg) Initial Final  No. o f glands measured  Mean diameter o f c e l l s (u) +I Standard E r r o r Gonadotroph Somatotroph T h y r o t r o p h  Group I Adult Controls  13  100-230  120-275  5.5*0.2  4.3+0.1  7.4+0.2  Group I I Adult 'Methallibure'  12  100-190  130-200  4.2+0.1  4.3+0.1*  8.5+0.1  Group I I I Juvenile Controls  12  8-20  4.5+0.1  4.5+0.2  5.5^.1  Group IV Juvenile Controls  10  8-20  90-180  5.7-0.1  4.6+0.1  8.9+0.3  16  9-28  11-55  3.7+0.2  ***  7.5+0.2  Group V Juvenile 'Methallibure  ++  1  + gonadotrophs n o t r e c o g n i s e d i n 1 case; ++ o n l y r e c o g n i s e d i n 1 case; +++ t h y r o t r o p h s o n l y r e c o g n i s e d i n 2 c a s e s . * somatotrophs n o t r e c o g n i s e d i n 2 c a s e s ; ** o n l y r e c o g n i s e d i n 2 cases; *** n o t r e c o g n i s e d i n any case. p < 0.001 between mean diameter o f gonadotrophs o f 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 d i a m e t e r o f t h y r o t r o p h s o f 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 . D i f f e r e n c e s between mean d i a m e t e r s o f gonadotrophs o f groups I and I V , I I and I I I , I and I I I , and o f somatotrophs o f 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 n o t signficant.  77  b l o o d s i n u s e s o r c a p i l l a r i e s which appear t o be i n t i m a t e l y connected  w i t h t h e gonadotroph c e l l s ; a l a r g e r number o f t h e  • m e t h a l l i b u r e ' t r e a t e d f i s h a r e found t o have, these l a r g e s i n u s e s ; i n c o n t r o l f i s h they tend t o be l e s s numerous. Few somatotrophs a r e e v i d e n t i n e i t h e r the c o n t r o l fish,  ( i n w h i c h t h e c e l l s were absent o r n o t r e c o g n i s a b l e  i n 2 cases) o r t h e e x p e r i m e n t a l f i s h ; no d i f f e r e n c e s a r e found between t h e mean c e l l diameter o f the two groups (Table XXIV). Few t h y r o t r o p h s a r e found i n the meso-AH o f t h e f i s h i n group I compared w i t h t h e e x p e r i m e n t a l group I I ( F i g s . 38b & 39b) i n w h i c h t h e c e l l diameter (p< 0.001) l a r g e r  i s also s i g n i f i c a n t l y  (8.5 ± 0.1 u i n the ' m e t h a l l i b u r e ' t r e a t e d  f i s h compared w i t h 7.4 t 0.2 u i n t h e c o n t r o l g r o u p ) .  The  t h y r o t r o p h s o f the ' m e t h a l l i b u r e ' t r e a t e d f i s h a r e a l s o more PAS+ve than those o f the c o n t r o l  fish.  E f f e c t o f ' m e t h a l l i b u r e ' on t h e meso-adenohypophysis o f t h e j u v e n i l e guppy.  The meso-AH o f t h e young f i s h  a t t h e b e g i n n i n g o f t h e experiment partially differentiated  killed  (group I I I ) i s o n l y  ( F i g . 4 0 ) . The gonadotrophs a r e v e r y  s i g n i f i c a n t l y s m a l l e r (p < 0.001) i n diameter and fewer i n number than i n t h e a d u l t c o n t r o l s (group I ) , and have v e r y l i t t l e AF+ve cytoplasm.  Blood sinuses are evident i n only  one o f the p i t u i t a r y g l a n d s examined.  The t h y r o t r o p h s and  somatotrophs a r e n o t 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 r e c o g n i s e d i n o n l y two c a s e s .  The somatotrophs  a r e s i m i l a r i n s i z e and appearance t o those o f the a d u l t , b u t  78  the t h y r o t r o p h s a r e v e r y s i g n i f i c a n t l y  (p ^ 0.001) s m a l l e r  than those o f e i t h e r o f 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  differentiated  meso-AH ( F i g . 42a) w i t h more numerous gonadotrophs s i g n i f i c a n t l y l a r g e r mean diameter  of a  (p <10.001) than those o f  e i t h e r o f the f i s h o f Group I I I , or o f the ' m e t h a l l i b u r e ' t r e a t e d a d u l t s (group I I ) . the gonadotrophs  Blood sinuses a s s o c i a t e d w i t h  a r e 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 o f group I I I . T h y r o t r o p h s i n t h i s group (IV) a r e f a i r l y numerous ( F i g . 42b) and o f s i g n i f i c a n t l y g r e a t e r diameter  (p £ 0.001)  than i n groups I , I I I , or V. Gonadotrophs a r e e v i d e n t i n o n l y two o f the  pituitary  g l a n d s o f the 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 f i s h (group V) ( F i g . 41), and where p r e s e n t they c o n t a i n l i t t l e AF+ve stainable material. diameter  They a r e o f s i g n i f i c a n t l y s m a l l e r  (p <, 0.001) than those o f a l l the o t h e r groups.  Blood  s i n u s e s are commonly found b u t a r e 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 o f the g l a n d s examined i n t h i s group ( V ) , a l t h o u g h t h i s may be because the poor d i f f e r e n t i a t i o n  o f 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 o f these c e l l s . The meso-AH o f t h i s group (V) c o n t a i n s numerous t h y r o t r o p h s , a l t h o u g h not as many as the comparable c o n t r o l s of the same age larger  (group I V ) ; they a r e , however, s i g n i f i c a n t l y  (p<0.001) than those o f the c o n t r o l f i s h o f s i m i l a r s i  (group I I I ) .  79  F i g u r e 33:  Sagittal  s e c t i o n o f t e s t i s o f a d u l t guppy  treated with  F i g u r e 34:  'methallibure  1  showing few c y s t s  o f e a r l i e r s t a g e s o f spermatogenesis  (x 200).  Same as f i g u r e 33 showing e p i t h e l i a l  cells  l i n i n g t h e e f f e r e n t d u c t s (x 200).  F i g u r e 35:  Sagittal  s e c t i o n o f t e s t i s o f j u v e n i l e guppy n o t  treated with  'methallibure'  (which became a d u l t ;  d u r i n g the p e r i o d o f e x p e r i m e n t ) .  Note the  e f f e r e n t d u c t s and main sperm duct do n o t c o n t a i n 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 ' 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.  1 0 0  S P G  SPC  SPD  SPM  %PR  81  F i g u r e 37:  Percentage of d i f f e r e n t stages of genesis i n j u v e n i l e c o n t r o l ;  spermato-  juvenile  •methallibure'-treated  and j u v e n i l e  'methallibure* treated  guppies.  non-  100 CONTROL METHALLIBURE 80  NON-TREATED  UJ 6 0 2 Ul  u  or ui o_ 4 0  20-  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 o f p i t u i t a r y g l a n d o f 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 b l o o d s i n u s e s 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: M a g n i f i e d v i e w o f a p o r t i o n o f 38a.  BS - B l o o d S i n u s e s ; BV - B l o o d V e s s e l s ; GN - Gonadotrophs; MS - Meso-adenohypophysis; MT - Meta-adenohypophysis; NH - Neurohypophysis; NT - N u c l e u s l a t e r a l i s t u b e r i s ; NS - N e u r o s e c r e t i o n ; PR - Pro-adenohypophysis; SM - Somatotrophs; TH - T h y r o t r o p h s ; TV - T h i r d v e n t r i c l e .  83  F i g u 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 adult ' m e t h a l l i b u r e - t r e a t e d guppy. 1  Note few  gonadotrophs around l a r g e b l o o d large thyrotrophs  Figure  39b:  Magnified  sinuses,  close to neurohypophysis.  v i e w o f a p o r t i o n o f 39a.  84  F i g u r e 40:  S a g i t t a l section 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 o f meso-adenohypophysisv.  few  gonadotrophs, o t h e r c e l l t y p e s n o t r e c 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 a l m o s t  complete absence o f gonadotrophs ( S i m i l a r t o f i g . 40) and d e v e l o p e d t h y r o t r o p h s a d j a c e n t 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 g l a n d 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 d u r i n g  p e r i o d of experiment).  Note numerous  gonadotrophs i n v e n t r a l margin and adjacent  Figure  42b:  the  to n e u r o h y p o p h y s i s .  Magnified view of a p o r t i o n of  42a.  thyrotrophs  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 t h e premise t h a t  a study o f t h e hypophysectomized male guppy would f u r t h e r the u n d e r s t a n d i n g  of pituitary-gonad relationships i nfishes,  c l a r i f y some o f t h e c o n t r o v e r s i a l i s s u e s c o n c e r n i n g t h e d e t a i l s o f gonadotropic  and a n d r o g e n i c a c t i o n on t h e t e s t i s  and male secondary s e x c h a r a c t e r s and p r o v i d e  specific  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 o f e n d o c r i n e c o n t r o l s i n t h e male o f a l i v e - b e a r i n g c y p r i n o d o n t .  The study has  d i f f e r e d from t h a t o f p r e v i o u s workers i n t h i s f i e l d n o t o n l y i n i t s o r i e n t a t i o n toward a male o v o v i v i p a r o u s  species but  a l s o i n t h e c o m p a r a t i v e study o f 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 t o the d i f f e r e n t i a t i o n o f t h e gonad and secondary s e x c h a r a c t e r s .  V i v i e n (1941) was t h e f i r s t  and o n l y i n v e s t i g a t o r t o demonstrate t h a t hypophysectomy prevents  t h e development o f t h e gonad i n a j u v e n i l e t e l e o s t  (Gobius p a g a n e l l u s ) .  V i v i e n ' s i n v e s t i g a t i o n , however, was a  g e n e r a l one and p r o v i d e d no d e t a i l s o f the e f f e c t s on t h e c y t o l o g y o f t h e gonad.  An added f e a t u r e i n t h e p r e s e n t  a n a l y s i s o f 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 t h e use o f t h e gonadotropic The  b l o c k i n g agent  'methallibure'.  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 o b t a i n e d w i t h r e s p e c t t o (a) t h e l o c u s o f p i t u i t a r y r e g u l a t i o n i n spermatogenesis (b) t h e p i t u i t a r y involvement i n spermiation  (c) t h e r o l e o f  androgens i n spermatogenesis and i n t h e c o n t r o l o f  87  secondary sex c h a r a c t e r s  and  (d) the p h y s i o l o g y and  r e g u l a t i o n o f the S e r t o l i c e l l s and l i n i n g the sperm d u c t s .  hormonal  the e p i t h e l i a l  cells  In a d d i t i o n , s e v e r a l areas o f  g o n a d a l p h y s i o l o g y 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 d a t a p e r t a i n i n g to development and  (a)  the  e n d o c r i n e c o n t r o l o f spermatophore f o r m a t i o n  (b) the hormonal i n v o l v e m e n t i n d i f f e r e n t i a t i o n o f the gonad and  secondary sex c h a r a c t e r s  comparative study of  o f j u v e n i l e f i s h e s and  'methallibure'  (c)  e f f e c t s on j u v e n i l e s  the and  adults. The  f o l l o w i n g t o p i c s are c o n s i d e r e d 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  for  discussion: a.  The  r o l e o f the p i t u i t a r y and  the androgens i n the  c o n t r o l o f spermatogenesis i n c l u d i n g spermatophores. b.  The  e n d o c r i n e c o n t r o l o f the r e l e a s e o f spermatophores,  the development and maintenance of i n t e r s t i t i a l e p i t h e l i a l c e l l s l i n i n g the sperm d u c t s and c  The  r o l e o f the p i t u i t a r y and  c o n t r o l o f secondary sex d.  The b l o c k i n g a c t i o n o f the g o n a d o t r o p i n s and  cells,  Sertoli  the androgens i n  cells.  the  characters,  'methallibure'  on the e f f e c t s o f  the s i t e of a c t i o n o f  'methallibure'.  88 a.  The r o l e o f the p i t u i t a r y and the androgens of  spermatogenesis i n c l u d i n g  i n the c o n t r o l  spermatophores.  R o l e o f 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 t h a t t h t t e s t e s o f t e l e o s t s show a s u p p r e s s i o n o f spermatogenesis a f t e r hypophysectomy, t h e r e a r e c o m p a r a t i v e l y few d e t a i l e d s t u d i e s o f 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 p a q a n e l l u s , and t h a t o f Matthews (1939) and Burger  (1941) on Fundulus  heteroclitus.  More r e c e n t i n v e s t i g a t i o n s a r e those o f B a r r (1963) P l e u r o n e c t e s p l a t e s s a , Roy  on  (1964) and B e l s a r e (1965)  on  O p h i c e p h a l u s p u n c t a t u s , Ahsan (1966) on Couesius plumbeus, L o f t s e t aJL (1966) on Fundulus h e t e r o c l i t u s , Donaldson McBride " (1967) on Salmo g a i r d n e r i i , S u n d a r a r a j and (1967) on Heteropneustes f o s s i l i s , Donaldson  and Yamazaki  (1968) on C a r a s s i u s a u r a t u s .  and  Nayyar  and  From a r e v i e w o f  t h i s l i t e r a t u r e , i t i s e v i d e n t t h a t the e f f e c t o f hypophysectomy on the t e s t e s v a r i e s c o n s i d e r a b l y a t d i f f e r e n t t i m e s o f the year (Matthews  1939; V i v i e n 1941; B a r r 1963).  There i s c o n s i d e r a b l e disagreement i n the l i t e r a t u r e c o n c e r n i n g the p r e c i s e s t a g e i n spermatogenesis w h i c h i s a f f e c t e d by hypophysectomy.  In Fundulus  heteroclitus,  s p e r m a t o g o n i a l d i v i s i o n s c o n t i n u e b u t the l a t e r s t a g e s o f spermatogenesis a r e s u p p r e s s e d (Matthews P i c k f o r d 1953; L o f t s e_t a l 1966) . Nayyar  1939; Burger  1941;  L i k e w i s e , S u n d a r a r a j and  (1967) u s i n g H e t e r o p n e u s t e s f o s s i l i s  found t h a t  s p e r m a t o g o n i a l d i v i s i o n c o n t i n u e s and the o n l y c e l l types  89  p r e s e n t i n the t e s t i s o f hypophysectomized spermatogonia and the sperm.  f i s h a r e the  On the c o n t r a r y , however, B a r r  (1963) w o r k i n g on p l a i c e , p l e u r o n e c t e s p l a t e s s a and Ahsan (1966) on l a k e chub, Couesius plumbeus d e s c r i b e d the s u p p r e s s i o n o f s p e r m a t o g o n i a l d i v i s i o n s i n the absence o f the p i t u i t a r y ; the c o n v e r s i o n o f spermatogonia i n t o  spermatocytes  ceases b u t s p e r m a t o g e n e s i s , i f w e l l underway, c o n t i n u e s and sperm a r e formed.  Dodd e t a l _ (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 t h a t hypophysectomy s t o p s the t r a n s f o r m a t i o n o f a spermatogonium i n t o a s p e r m a t o c y t e , b u t spermatocytes and a l l s u c c e e d i n g s t a g e s o f spermatogenesis a l r e a d y e s t a b l i s h e d a t the time o f o p e r a t i o n a p p a r e n t l y develop i n normal f a s h i o n i n t o sperm. (1960) d i d n o t observe m i t o s i s i n spermatogonia. and Donaldson  Dodd e_t aJL Yamazaki  (1968) n o t e d t h a t i n C a r a s s i u s a u r a t u s ,  the m i t o t i c d i v i s i o n o f spermatogonia i s c o m p l e t e l y s u p p r e s s e d by hypophysectomy and s p e r m a t o c y t e s , s p e r m a t i d s and sperm d i s a p p e a r . The t e l e o s t s s t u d i e d i n a l l t h e s e p r e v i o u s 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 b e i n g an o v o v i v i p a r o u s , monthly b r e e d e r .  As an  a d a p t a t i o n t o 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 s p e r m - b a l l s or spermatophores.  The h i s t o l o g y o f  spermatophore  f o r m a t i o n has been d e s c r i b e d i n a number o f 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  ( v a u p e l 1929;  Goodrich  90 et_ al_ 1934); Gambusia a f f i n i s Xiphophorus h e l l e r i  ( S e l f 1940; Medlen  1950);  (Essenberg 1924; V a l l o w e 1957)  Xiphophorus maculatus  and  (Wolf 1931; C h a v i n and Gordon 19 51).  However, the p o s s i b l e e n d o c r i n e c o n t r o l o f  spermatophore  f o r m a t i o n , the p h y s i o l o g y o f the spermatophores w h i l e i n the t e s t i s and t h e i r e v e n t u a l d i s c h a r g e have n o t been p r e v i o u s l y described. In the t e s t i s o f a c o n t r o l guppy, the s p e r m a t o g o n i a l c y s t s a r e p e r i p h e r a l l y l o c a t e d w h i l e the spermatophores a r e i n the c e n t r e ; the a r e a between the two i s f i l l e d w i t h c y s t s c o n t a i n i n g the v a r i o u s d e v e l o p m e n t a l s t a g e s o f s p e r m a t o c y t e s , s p e r m a t i d s and sperm ( F i g . 2 ) .  By c o n t r a s t , the t e s t i s o f  a hypophysectomized guppy i s c o m p l e t e l y packed w i t h spermatophores e x c e p t f o r a few s p e r m a t o g o n i a l c y s t s near the periphery (Fig. 3). T h i s s u g g e s t s t h a t hypophysectomy  (presumably the l a c k  o f g o n a d o t r o p i n ) b l o c k s the t r a n s f o r m a t i o n o f spermatogonia i n t o spermatocytes, but  does n o t p r e v e n t the t r a n s f o r m a t i o n  o f s p e r m a t o c y t e s , s p e r m a t i d s , 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  Scyliorhinus caniculus  situation  (Dodd e t a l 1960).  A similar  has been n o t e d i n the Amphibia; van Oordt (1956) c o n c l u d e d from s t u d y o f the f r o g , Rana t e m p o r a r i a t h a t the l a t e r s t a g e s o f spermatogenesis beyond spermatogonia a r e n o t under the pituitary  control.  The p r e s e n c e o f spermatophores a t the p e r i p h e r y o f the t e s t i s o f a hypophysectomized guppy w i t h no t r a c e o f  91  d i s i n t e g r a t i n g c y s t s c o n t a i n i n g e a r l i e r stages o f spermatogenesis i n d i c a t e s t h a t s p e r m a t o c y t e s , s p e r m a t i d s and sperm do n o t degenerate b u t t r a n s f o r m i n t o spermatophores.  This observation  i s i n c o n t r a d i c t i o n t o the f i n d i n g s i n o v i p a r o u s t e l e o s t s such as Gobius p a g a n e l l u s ( V i v i e n 1941), Fundulus  heteroclitus  (Matthews 1939; Burger 1941; P i c k f o r d 1953; L o f t s e t a l 1966), p l e u r o n e c t e s p l a t e s s a (Barr 1963), Couesius plumbeus 1966), H e t e r o p n e u s t e s f o s s i l i s  ( S u n d a r a r a j and Nayyar  and C a r a s s i u s a u r a t u s (Yamazaki and Donaldson I n v e s t i g a t o r s who  (Ahsan 1967)  1968).  have s t u d i e d these o v i p a r o u s s p e c i e s s t a t e t h a t  the  spermatocytes and s p e r m a t i d s degenerate or d i s a p p e a r i n  the  absence o f 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 b u t may be due t o the f a c t t h a t the guppy i s an o v o v i v i p a r o u s t e l e o s t and produces r e l a t i v e l y  few  spermatophores, thus c o n s e r v a t i o n o f the e a r l i e r s t a g e s i s e s s e n t i a l ; whereas the r e s t a r e o v i p a r o u s and produce enormous number o f sperm. S i n c e m i t o t i c d i v i s i o n was never n o t e d i n the spermatog o n i a l c y s t s o f the hypophysectomized guppy, i t . s e e m s  likely  t h a t s p e r m a t o g o n i a l m u l t i p l i c a t i o n ceases i n the absence o f the  pituitary.  T h i s i s f u r t h e r e v i d e n c e d by the f a c t t h a t  percentage composition of spermatogonial cysts of c o n t r o l and e x p e r i m e n t a l guppies has not changed d u r i n g the eight-week e x p e r i m e n t a l p e r i o d (Table I I ) .  This finding i s i n accord  w i t h t h a t o f Yamazaki and Donaldson  (1968) on g o l d f i s h ,  C a r a s s i u s a u r a t u s and t h a t o f van Oordt Rana t e m p o r a r i a .  (1960) on f r o g ,  92 T e s t e s o f a newly b o r n guppy c o n t a i n o n l y s p e r m a t o g o n i a l cysts.  The p r i m a r y spermatogonia b e g i n t o d i v i d e t o form  nests of c e l l s  ( b e g i n n i n g o f spermatogenesis) a t about  days a f t e r b i r t h  ( G o o d r i c h e t a l 1934).  t h a t gonadotrophs  36  S o k o l (1961) found  i n the v e n t r a l r e g i o n o f meso-adenohypophysis  become g r a n u l a t e d ( i n i t i a t i o n o f g o n a d o t r o p i n s e c r e t i o n ) d u r i n g the f i f t h week a f t e r b i r t h .  I t i s thus e v i d e n t t h a t  t h e r e 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 o f g o n a d o t r o p i n s and the i n i t i a t i o n o f spermatogenesis.  The  r o l e o f the p i t u i t a r y i n the development o f t e s t i s has been a n a l y z e d by removing the p i t u i t a r y o f the j u v e n i l e guppy b e f o r e the gonadotrophs were d i f f e r e n t i a t e d . No changes take p l a c e i n the s t r u c t u r e o f 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.  M i t o s e s were never  o b s e r v e d and t h e r e was no e v i d e n c e o f an i n c r e a s e i n the number o f s p e r m a t o g o n i a l c y s t s (Table X ) . o f spermatogenesis do n o t appear.  The l a t e r s t a g e s  D u r i n g the course o f  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 c o n t a i n e d a l l s t a g e s o f spermatogenesis. I t i n d i c a t e s t h a t hypophysectomy p r e v e n t s the m i t o t i c d i v i s i o n o f spermatogonia and t h e i r t r a n s f o r m a t i o n i n t o spermatocytes. and W i t s c h i Bufo).  T h i s f i n d i n g i s s i m i l a r t o t h a t o f Chang  (1955) on u r o d e l e s ( T r i t u r u s ) and anurans  (Rana,  They n o t e d t h a t even a f t e r a p r o l o n g e d p e r i o d , the  t e s t e s o f hypophysectomized spermatogonia.  l a r v a l amphibians  contain only  93  I t i s c o n c l u d e d t h a t hypophysectomy o f b o t h a d u l t and j u v e n i l e guppies p r e v e n t s m i t o s i s i n the spermatogonia b l o c k s t h e i r t r a n s f o r m a t i o n i n t o spermatocytes.  and  The  hypophysectomy o f the a d u l t guppy does n o t p r e v e n t the t r a n s f o r m a t i o n o f s p e r m a t o c y t e s , s p e r m a t i d s and sperm i n t o spermatophores.  T h i s i n d i c a t e s t h a t the l a t e r s t a g e s o f  spermatogenesis a r e p i t u i t a r y - i n d e p e n d e n t . R o l e o f the  androgens  In a s t u d y o f e f f e c t s o f v a r i o u s s t e r o i d s i n s e x u a l development  o f i n t a c t guppy, E v e r s o l e (1939, 1941)  suggested  t h a t t e s t o s t e r o n e p r o p i o n a t e hastens germ c e l l m a t u r a t i o n . There have been s e v e r a l o t h e r r e p o r t s o f s t i m u l a t i o n o f t e s t e s o f i n t a c t t e l e o s t f i s h by androgens Atz  ( p i c k f o r d and  1957; Dodd 1960). The r o l e o f androgens  i n spermatogenesis may be r e a d i l y  a n a l y z e d by t r e a t i n g hypophysectomized  f i s h with  androgens.  There a r e , however, v e r y few o b s e r v a t i o n s on the e f f e c t s o f androgens  on the t e s t e s o f hypophysectomized  1942; L o f t s e t a l 1966; S u n d a r a r a j and Nayyar  fish  (Burger  1967).  Lofts  e t a l (1966) w o r k i n g on Fundulus h e t e r o c l i t u s and S u n d a r a r a j and Nayyar  (1967) on H e t e r o p n e u s t e s f o s s i l i s demonstrated  that  spermatogenesis i s c o m p l e t e l y r e s t o r e d w i t h t e s t o s t e r o n e treatment.  On the c o n t r a r y , however, Burger  (1942)  also  w o r k i n g 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 m a i n t a i n e d by Methyl t e s t o s t e r o n e treatment of  androgens. hypophysectomized  a d u l t guppies s i g n i f i c a n t l y i n c r e a s e s t h e i r gonosomatic  indices.  94  T h i s f i n d i n g i s s i m i l a r , t o t h a t d e s c r i b e d i n Fundulus ( L o f t s e t a l 1966) and H e t e r o p n e u s t e s (Sundararaj and Nayyar 1967).  Exogenous m e t h y l t e s t o s t e r o n e appears t o  have a d i r e c t s p e r m a t o k i n e t i c e f f e c t on t h e t e s t i s o f the hypophysectomized guppy.  I n c o n t r a s t t o the hypophysectomized  controls, 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 ; t h e number o f s p e r m a t o g o n i a l  cysts  i n c r e a s e s and c y s t s c o n t a i n i n g spermatocytes a r e 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 r e a c h e s o n l y 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 o f L o f t s e t a l (1966) i n Fundulus and S u n d a r a r a j and Nayyar (1967) i n H e t e r o p n e u s t e s .  They r e p o r t e d complete  r e s t o r a t i o n o f spermatogenesis w i t h t e s t o s t e r o n e .  The  d i f f e r e n c e s i n a c t i v a t i o n o f the r e g r e s s e d t e s t i s i s d i f f i c u l t t o e x p l a i n b u t may be a t t r i b u t e d t o the f a c t t h a t P o e c i l i a i s an o v o v i v i p a r o u s , monthly b r e e d e r whereas Fundulus and H e t e r o p n e u s t e s a r e o v i p a r o u s , s e a s o n a l  breeders.  In h i g h e r v e r t e b r a t e s the e f f e c t o f t e s t o s t e r o n e on the r e g r e s s e d t e s t i s v a r i e s .  Testosterone  i s known t o  e x e r t a s t i m u l a t o r y e f f e c t on spermatogenesis i n hypophysectomized mammals ( B o c c a b e l l a 1963; Clermont and Harvey 1966).  I n c o n t r a s t t o t h e f i n d i n g s n o t e d above,  and Nandi (1965) r e p o r t e d t h a t treatment  Basu  o f hypophysectomized  frogs w i t h testosterone r e s u l t s i n greater suppression of spermatogenesis than t h a t o b s e r v e d a f t e r hypophysectomy a l o n e .  95  There i s no p u b l i s h e d account o f the e f f e c t s o f t e s t o s t e r o n e t r e a t m e n t on hypophysectomized Unlike  juvenile  fish.  the e f f e c t found i n a d u l t s , m e t h y l t e s t o s t e r o n e  t r e a t m e n t o f hypophysectomized stimulate  j u v e n i l e guppies does n o t  m i t o t i c d i v i s i o n s i n the s p e r m a t o g o n i a l c y s t s ;  l a t e r s t a g e s o f spermatogenesis do n o t appear. The d i f f e r e n c e s hypophysectomized  i n a c t i v a t i o n of the testes o f  a d u l t and j u v e n i l e guppies by m e t h y l  t e s t o s t e r o n e may be e x p l a i n e d i n t h e f o l l o w i n g way: 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  the  cells  w h i c h became r e g r e s s e d i n the absence o f p i t u i t a r y and stopped p r o d u c i n g androgen. The m e t h y l t e s t o s t e r o n e t r e a t m e n t o f t h e hypophysectomized  a d u l t compensates f o r t h i s l o s s i n  androgen p r o d u c t i o n and t h a t i s why t h e wave o f spermatogenesis in initiated.  I t seems l i k e l y t h a t t h e p r o d u c t i o n o f  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 t h e o t h e r hand i n the j u v e n i l e s , t h e i n t e r s t i t i a l c e l l s were n o t 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 b e f o r e t h e t e s t i s had ever r e c e i v e d stimulation.  any g o n a d o t r o p i n  I t may be s u g g e s t e d t h a t exogenous t e s t o s t e r o n e  cannot a c t d i r e c t l y on t h e j u v e n i l e t e s t i s and b r i n g spermatogenesis and d i f f e r e n t i a t i o n o f i n t e r s t i t i a l u n t i l the t i s s u e has been primed o r t r i g g e r e d by gonadotropins.  about cells  96  b.  The e n d o c r i n e c o n t r o l o f t h e r e l e a s e o f spermatophores, the development and maintenance o f i n t e r s t i t i a l  cells,  e p i t h e l i a l c e l l s l i n i n g t h e sperm d u c t s and S e r t o l i  cells.  R e l e a s e o f spermatophores There i s c o n s i d e r a b l e 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 teleosts.  T h i s c o n t r o l e v i d e n t l y v a r i e s i n t h e same f i s h a t  d i f f e r e n t times o f t h e y e a r .  When t h e p i t u i t a r y o f Gobius  p a g a n e l l u s was removed d u r i n g w i n t e r , sperm d i s a p p e a r e d from the t e s t i s , whereas  7 0 percent of operated f i s h retained  sperm when t h e p i t u i t a r y was removed s h o r t l y b e f o r e t h e n a t u r a l r e p r o d u c t i v e c l i m a x ( V i v i e n 1938, 1941).  Similar  r e s u l t s were o b t a i n e d i n Fundulus h e t e r o c l i t u s d u r i n g t h e f a l l and s p r i n g (Matthews 1939).  Pickford  (19 53) found no  sperm i n Fundulus f i v e months a f t e r t h e o p e r a t i o n .  Lofts  e t a l (1966) a l s o w o r k i n g w i t h Fundulus d e s c r i b e d empty l o b u l e s w i t h o u t sperm i n t h e t e s t i s a f t e r  hypophysectomy.  In p l e u r o n e c t e s p l a t e s s a , sperm a r e shed n o r m a l l y i n t h e absence o f t h e p i t u i t a r y  (Barr 1963).  Ahsan  (1966) n o t e d  t h a t Couesius plumbeus s p e r m i a t e s n o r m a l l y i n t h e absence o f t h e . p i t u i t a r y i n b o t h t h e prespawning and spawning phases o f i t s a n n u a l c y c l e . Donaldson  L i k e w i s e , Yamazaki and  (1968) found t h a t sperm d i s a p p e a r from t h e t e s t i s  o f hypophysectomized C a r a s s i u s a u r a t u s . however, S u n d a r a r a j and Nayyar  On t h e c o n t r a r y ,  (1967) w o r k i n g on H e t e r o p n e u s t e s  f o s s i l i s n o t e d t h a t sperm p e r s i s t as l o n g as 337 days a f t e r hypophysectomy.  97  The e n d o c r i n e c o n t r o l o f the r e l e a s e o f spermatophores from the t e s t i s has n o t been p r e v i o u s l y s t u d i e d .  E i g h t weeks  a f t e r hypophysectomy, many spermatophores i n the t e s t i s o f the  guppy a r e found r u p t u r e d b u t t h e sperm a r e n o t r e l e a s e d .  The r u p t u r e o f the spermatophores may be due t o the f a c t t h a t the  e p i t h e l i a l c e l l s o f the sperm d u c t s s u r r o u n d i n g the  spermatophores cease t o produce the n u t r i t i v e o r c o l l o i d a l m a t e r i a l i n t h e absence o f the p i t u i t a r y .  There i s a s t r o n g  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 r u p t u r e d spermatophores a r e b e i n g p h a g o c y t o s e d w i t h i n the e f f e r e n t d u c t s and the main sperm duct ( F i g .  15).  The r u p t u r e o f the  spermatophores and the r e s o r p t i o n o f t h e sperm w i t h i n the sperm d u c t s become a c c e l e r a t e d when t h e 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 a r e n o t i c e d  i n the sperm d u c t s a f t e r two weeks o f t e s t o s t e r o n e t r e a t m e n t ( F i g 1 8 ) , whereas remnant sperm a r e p r e s e n t i n the hypophysectomized c o n t r o l .  I t may be s u g g e s t e d t h a t the  exogenous t e s t o s t e r o n e a c c e l e r a t e s t h e p h a g o c y t o s i s o f sperm. Interstitial  cells.  Buser-Lahaye (1953), L o f t s e t a l (1966), S u n d a r a r a j and Nayyar (1967) and Yamazaki and Donaldson (1968) found s i g n s o f a t r o p h y i n the i n t e r s t i t i a l c e l l s o f 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. pituitary  Ahsan (1966) n o t e d t h a t  r e m o v a l s u p p r e s s e s the s e c r e t o r y a c t i v i t y o f  l o b u l e - b o u n d a r y c e l l s i n C o u e s i u s plumbeus.  Likewise,  hypophysectomy causes the r e g r e s s i o n o f 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. c o n t a i n any  The  interstitial  l i p i d d r o p l e t s and  Regressed i n t e r s t i t i a l  c e l l s do  t h e i r n u c l e i become shrunken. c e l l s o f hypophysectomized  guppy a r e s t i m u l a t e d f o l l o w i n g t e s t o s t e r o n e  administration.  T h e i r n u c l e i assume a more rounded shape w i t h a nucleolus.  not  distinct  This f i n d i n g i s i n accord with that of L o f t s et a l  (1966) i n F u n d u l u s .  On the c o n t r a r y , however, S u n d a r a r a j  and Nayyar (1967) found t h a t the i n t e r s t i t i a l  c e l l s remained  atrophied i n Heteropneustes. The  stroma c e l l s o f the t e s t i s o f hypophysectomized  j u v e n i l e guppy a r e 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  cells.  T e s t o s t e r o n e t r e a t m e n t o f the hypophysectomized j u v e n i l e does n o t cause the d i f f e r e n t i a t i o n either.  I t may  differentiation  of i n t e r s t i t i a l  be c o n c l u d e d from the d a t a t h a t of i n t e r s t i t i a l  cells  the  c e l l s from the stroma c e l l s  r e q u i r e s p r i m i n g or t r i g g e r i n g by g o n a d o t r o p i n s as i n spermatogenesis. regressed  proliferation  The  testosterone  interstitial  androgens b e f o r e  Epithelial  The  can s t i m u l a t e  the  c e l l s o f a d u l t t e s t i s which produced  hypophysectomy b u t cannot l e a d to  o f 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 .  c e l l s l i n i n g the sperm d u c t s . epithelial  c e l l s l i n i n g the e f f e r e n t d u c t s o f  i n t a c t a d u l t guppy a r e o f the t a l l columnar type and l i p i d droplets.  The  an  contain  presence of l i p i d d r o p l e t s i n d i c a t e s  t h a t these e p i t h e l i a l c e l l s may androgens.  the  be r e s p o n s i b l e f o r s e c r e t i n g  Moser (1967), l i k e w i s e , n o t e d t h a t  d r o p l e t s are p r e v a l e n t  sudanophilic  i n the c e l l s o f the e f f e r e n t d u c t s  99  o f the r o c k f i s h , Sebastodes also reported  paucinspinis.  Wiebe (1967)  the r.presence o f 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 d u c t s o f the seaperch, Cymatogaster  aggregata.  The spermatophores  i n an e f f e r e n t duct o f an i n t a c t  guppy a r e surrounded by a c o l l o i d a l m a t e r i a l which seems t o be s e c r e t e d 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  material  might c o n t a i n a n u t r i t i v e substance as has been suggested by Medlen (1950) i n h i s s t u d y 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 o f the e f f e r e n t d u c t s becomes r e d u c e d and does n o t c o n t a i n 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 s e c r e t e d by the  e p i t h e l i a l c e l l s o f the e f f e r e n t d u c t s i n the absence o f the p i t u i t a r y and i t i s thought t h a t the r u p t u r e o f  spermatophores  i n the e f f e r e n t d u c t s and the main sperm duct might be  due  t o the f a c t t h a t the e p i t h e l i a l c e l l s cease t o produce androgens and the c o l l o i d a l m a t e r i a l . When the hypophysectomized testosterone,  guppy i s t r e a t e d  with  the r e g r e s s e d e p i t h e l i a l c e l l s o f the e f f e r e n t  d u c t s and the main sperm d u c t h y p e r t r o p h y and assume the t a l l columnar appearance o f the normal a n i m a l .  This  i n d i c a t e s t h a t the e p i t h e l i a l l i n i n g o f the e f f e r e n t d u c t s 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 e t a l (1966) n o t e d a h y p e r t r o p h y o f the e f f e r e n t d u c t s i n Fundulus h e t e r o c l i t u s . There i s no i n c r e a s e  i n the number, w i d t h and the s i z e  o f the lumina o f the e f f e r e n t d u c t s i n the t e s t i s o f j u v e n i l e  100  guppy f o l l o w i n g hypophysectomy.  Methyl t e s t o s t e r o n e treatment  o f t h e hypophysectomized j u v e n i l e s b r i n g s about complete differentiation duct.  o f the e f f e r e n t d u c t s and the main sperm  The t o t a l number, w i d t h and the s i z e o f lumina o f 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 o f the main  sperm d u c t becomes v e r y wide and t h e l i n i n g e p i t h e l i a l attain  cells  t a l l columnar appearance o f t h e a d u l t guppy. I t i s c o n c l u d e d t h a t the d i f f e r e n t i a t i o n  and  maintenance o f the e f f e r e n t d u c t s and the main sperm duct o f b o t h 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 the  gonadotropin s e c r e t i o n i s not d i r e c t l y involved.  Unlike  game.togenetic and e n d o c r i n e t i s s u e s o f 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 o f androgen. Sertoli  cells Among the f i s h e s ,  S e r t o l i c e l l s have been r e p o r t e d i n  G a s t e r o s t e u s ( C r a i g - B e n n e t 1931), P o e c i l i a  ( F o l l e n i u s 1953,  F u n d u l u s ( L o f t s et. a l . 1966) and i n Elasmobranchs (Matthews 1950; S t a n l e y 1962), b u t these w o r k e r s do n o t d e s c r i b e t h e function of S e r t o l i c e l l s .  I n the t e s t i s o f an  intact  guppy, S e r t o l i c e l l s p l a y an i m p o r t a n t p a r t i n the f o r m a t i o n o f spermatophores. These c e l l s become s t r i k i n g l y as  enlarged  the sperm become more mature and the sperm heads become  attached to t h e i r  inner margin.  C o n s i d e r i n g the enormous  change i n the s i z e o f S e r t o l i c e l l s and sperm heads becoming a t t a c h e d t o them, i t may be suggested t h a t S e r t o l i  cells  s e r v e as n u t r i e n t c e l l s as they a r e b e l i e v e d t o be i n h i g h e r  101  vertebrates.  S e r t o l i c e l l s i n mammals c o n t a i n  g l y c o g e n i n t h e i r c y t o p l a s m and  considerable  t h i s i s considered  evidence  t h a t they s e r v e as n u t r i e n t c e l l s f o r the g e r m i n a l e p i t h a l i u m (Paulsen  1968).  Burgos (1955) r e p o r t e d t h a t g l y c o g e n i s  p r e s e n t i n the S e r t o l i c e l l s o f 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 S e r t o l i c e l l s o f c a r a s s i u s a u r a t u s (Yamamoto and 1967).  in  Yamazaki  Moser (1967) r e p o r t e d that the t u b u l e boundary c e l l s  (corresponding  to S e r t o l i c e l l s ) o f Sebastodes p a u c i s p i n i s  c o n t a i n c a r b o h y d r a t e g r a n u l e s • 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 o f gametes. f u n c t i o n o f these c e l l s .  However, t h i s may According  not be the  only  to V a u p e l (1929) the  S e r t o l i c e l l s o f the guppy are p h a g o c y t i c  and i n g e s t  the  c y t o p l a s m ( c o n t a i n i n g the G o l g i remnant) d i s c a r d e d when s p e r m a t i d s a r e t r a n s f o r m e d i n t o sperm.  Similar function  been a s c r i b e d t o S e r t o l i c e l l s i n mammals (Lacy  has  1968).  There i s no p u b l i s h e d a c c o u n t o f the e f f e c t o f hypophysectomy on S e r t o l i c e l l s i n any s t u d y , i t was  evident  teleost.  t h a t the S e r t o l i c e l l s o f  I n the p r e s e n t the  hypophysectomized guppy r e g r e s s and t h e i r n u c l e i change from rounded c o n d i t i o n t o f l a t t e n e d shape ( F i g . 9 ) .  Burgos (1955)  found t h a t S e r t o l i c e l l s o f Rana p i p i e n s a t r o p h y and  their  g l y c o g e n c o n t e n t d i s a p p e a r s f o l l o w i n g hypophysectomy. R e g r e s s e d S e r t o l i c e l l s o f the hypophysectomized guppy a r e r e s t o r e d t o normal f o l l o w i n g t e s t o s t e r o n e ( F i g . 17).  administration  T h e i r 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 that 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 t e s t o s t e r o n e t r e a t m e n t o f hypophysectomized  juveniles  does not b r i n g about the p r o l i f e r a t i o n o f 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 b e i n g primed w i t h  gonadotropins.  In the guppy, not o n l y S e r t o l i c e l l s r e g r e s s 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 t r e a t m e n t ; b o t h 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 d u c t s behave i n a s i m i l a r fashion.  The  interstitial  c e l l s and  l i n i n g the sperm d u c t s c o n t a i n androgens.  I t may  be  the e p i t h e l i a l c e l l s  l i p i d droplets  and  produce  suggested t h a t S e r t o l i c e l l s  produce androgens as t h e y a r e b e l i e v e d  also  to do i n mammals  (Teilum 1950). c.  The  r o l e o f the p i t u i t a r y and  o f secondary sex  the androgens i n the  control  characters.  A l l secondary sex c h a r a c t e r s 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 t r e a t m e n t w i t h exogenous s t e r o i d s and Dodd 1960; regulates  c a s t r a t i o n e x p e r i m e n t s ( P i c k f o r d and A t z  Hoar 1965,  1941)  Burger 1941;  1957;  pituitary directly  the secondary sex c h a r a c t e r s .  ( V i v i e n 1938,  (Matthews 1939; 1966)  The  the androgen p r o d u c t i o n o f the t e s t i s and  i n d i r e c t l y controls paganellus  1966).  sex  thus In Gobius  and Fundulus h e t e r o c l i t u s P i c k f o r d 1953;  Lofts et a l  the n u p t i a l c o l o r a t i o n or b r e e d i n g d r e s s (the most  103  apparent secondary s e x c h a r a c t e r ) of the p i t u i t a r y .  disappeais i n t h e absence  Likewise, the b r i g h t lipophores  on t h e s i d e s o f t h e body o f a d u l t guppy become v e r y or e n t i r e l y disappear  present faint  f o l l o w i n g hypophysectomy.  In c o n t r a s t , no changes a r e e v i d e n t i n t h e s t r u c t u r e o f t h e gonopodium o f t h e hypophysectomized guppy. M o r p h o l o g i c a l l y t h e 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 t o e x p e c t t h a t once morphogenesis i s complete, t h e s t r u c t u r e w i l l n o t r e g r e s s . A l t h o u g h t h e i n f l u e n c e o f exogenous s t e r o i d s on t h e s e x a c c e s s o r i e s and secondary s e x c h a r a c t e r s o f t h e 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 t o e l i m i n a t e t h e p o s s i b l e e f f e c t s o f endogenous g o n a d o t r o p i n s by t h e use o f hypophysectomized animals.  There a r e o n l y two p u b l i s h e d a c c o u n t s on t h e  e f f e c t s o f androgens on t h e secondary s e x c h a r a c t e r s o f hypophysectomized f i s h .  Burger (1942) and L o f t s e t a l (1966)  noted that the n u p t i a l c o l o r a t i o n reappeared i n hypophysectomized F u n d u l u s f o l l o w i n g t e s t o s t e r o n e  treatment.  S i m i l a r l y the testosterone treatment o f the a d u l t hypophysectomized guppy l e a d s t o moderate r e c o v e r y  i n the  c o n t e n t o f l i p o p h o r e s p r e s e n t on t h e s i d e s o f t h e body.  The  gonopodium o f t h e guppy, w h i c h remains u n a f f e c t e d a f t e r hypophysectomy, does n o t change a f t e r t e s t o s t e r o n e  treatment.  The r e s p e c t i v e r o l e o f t h e p i t u i t a r y and t h e androgens i n t h e d i f f e r e n t i a t i o n o f secondary s e x c h a r a c t e r s o f t h e j u v e n i l e guppy has been a n a l y z e d by f i r s t removing t h e  104  p i t u i t a r y and s u b s e q u e n t l y t r e a t i n g such juveniles with testosterone.  hypophysectomized  Secondary sex  characters  i n c l u d i n g b o t h the l i p o p h o r e pigments and the gonopodium, a r e 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 m a i n t a i n e d i n the hypophysectomized end o f e x p e r i m e n t a l the sham-operated  period  j u v e n i l e s u n t i l the  ( e i g h t weeks).  During t h i s period,  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 t h e  hypophysectomized  j u v e n i l e s a r e 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 l i p o p h o r e s ) become d i f f e r e n t i a t e d  b u t the d i f f e r e n t i a t i o n i s n o t as complete as a d u l t c o n t r o l s . The i n c o m p l e t e d i f f e r e n t i a t i o n o f secondary sex  characters  may be due t o degree o f 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 o r t h a t the exogenous androgen  cannot l e a d t o  complete d i f f e r e n t i a t i o n o f secondary sex c h a r a c t e r s i n the absence o f p i t u i t a r y g o n a d o t r o p i n s . I t might a l s o be s u g g e s t e d t h a t t h e dose used inadequate.  However, the c o n c e n t r a t i o n o f  was  testosterone  (I ::2xic£ par t s ) used i n the experiment appears q u i t e s u f f i c i e n t when compared t o 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 p r o b a b l y n o t  the main r e a s o n f o r the i n c o m p l e t e d i f f e r e n t i a t i o n o f secondary sex c h a r a c t e r s . Data o b t a i n e d from hypophysectomy and  testosterone  t r e a t m e n t o f b o t h a d u l t and j u v e n i l e guppies suggest t h a t  105  secondary s e x c h a r a c t e r s a r e d i r e c t l y c o n t r o l l e d by t h e androgens and i n d i r e c t l y by t h e p i t u i t a r y g o n a d o t r o p i n s . d.  The b l o c k i n g a c t i o n o f ' m e t h a l l i b u r e ' on t h e e f f e c t s o f the g o n a d o t r o p i n s and t h e s i t e o f a c t i o n o f ' m e t h a l l i b u r e '  Blocking action of 'methallibure'. The  e f f e c t of 'methallibure  been p r e v i o u s l y r e p o r t e d .  1  on j u v e n i l e f i s h has n o t  No d e v e l o p m e n t a l changes o c c u r  i n t h e s t r u c t u r e o f t e s t i s o f j u v e n i l e guppy f o l l o w i n g • m e t h a l l i b u r e ' t r e a t m e n t (Table XX).  The number o f  s p e r m a t o g o n i a l c y s t s remains t h e same and these a r e n o t 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  o f t h e e f f e r e n t d u c t s and t h e main sperm d u c t i s checked. Secondary s e x c h a r a c t e r s do n o t appear.  A l l developmental  changes o f t h e j u v e n i l e t e s t i s a r e stopped i n t h e same f a s h i o n as i s e v i d e n t a f t e r hypophysectomy. Since  ' m e t h a l l i b u r e ' t r e a t m e n t produces i d e n t i c a l  e f f e c t s t o hypophysectomy i t i s c o n c l u d e d t h a t g o n a d o t r o p i n s e c r e t i o n o f t h e j u v e n i l e guppy i s e n t i r e l y b l o c k e d 'methallibure'.  In the j u v e n i l e s the gonadotropin s e c r e t i o n  was c o m p l e t e l y b l o c k e d because t h e ' m e t h a l l i b u r e ' was begun b e f o r e according  with  treatment  t h e gonadotrophs were d i f f e r e n t i a t e d ,  to Sokol  since  (1961) i t i s n o t u n t i l t h e 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 g r a n u l a t e d b a s o p h i l s appear i n t h e v e n t r a l r e g i o n o f meso-adenohypophysis. hypophysectomy ( ' m e t h a l l i b u r e '  Thus  chemical  t r e a t m e n t ) o f 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 o f gonad and appearance o f secondary sex c h a r a c t e r s  are  concerned. The  e f f e c t of  ' m e t h a l l i b u r e ' on the gonads  and  g o n a d a l f u n c t i o n s has been s t u d i e d i n o n l y t h r e e s p e c i e s teleost fish  (Hoar e t a_l 1967;  Wiebe 1968) .  The  of  gonosomatic  i n d e x o f ' m e t h a l l i b u r e ' t r e a t e d a d u l t guppy i s markedly r e d u c e d . T h i s f i n d i n g i s i n a c c o r d w i t h Hoar e_t a_l (1967) and Wiebe (1968). In the a d u l t guppy, ' m e t h a l l i b u r e ' t r e a t m e n t b r i n g s about s i g n i f i c a n t changes i n the p e r c e n t d i f f e r e n t stages of spermatogenesis. treatment',  composition  After  of  e i g h t weeks o f  t h e r e are few c y s t s c o n t a i n i n g spermatogonia,  s p e r m a t o c y t e s , s p e r m a t i d s and sperm b u t spermatophores a r e present  i n abundance (Table X V I I ) .  completely  S i n c e hypophysectomy  b l o c k s the t r a n s f o r m a t i o n o f spermatogonia i n t o  s p e r m a t o c y t e s , the p r e s e n c e o f s p e r m a t o c y t e - c y s t s  in  • m e t h a l l i b u r e ' t r e a t e d t e s t i s s u g g e s t s t h a t a complete p i t u i t a r y b l o c k a g e o f g o n a d o t r o p i n 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^ p a r t s ) i n e i g h t weeks (Table  XIX).  Hoar e t a l (1967) and Wiebe (1968) c o n c l u d e d t h a t • m e t h a l l i b u r e ' e f f e c t i v e l y b l o c k s the p i t u i t a r y action.  gonadotropic  T h e i r 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  o f r e d u c t i o n d i v i s i o n and subsequent s p e r m i o g e n e s i s a r e blocked.  Wiebe (1968) found t h a t i n ' m e t h a l l i b u r e '  s e a p e r c h , Cymatogaster a g g r e g a t a , the spermatogonia s p e r m a t o c y t e s comprise 96% o f the l o b u l e a r e a  treated and  (10% o f  the  107  a r e a i n c o n t r o l ) and the spermatophores d i s a p p e a r .  Wiebe  (1968) f u r t h e r n o t e d t h a t i n t e r s t i t i a l c e l l s o f L e y d i g columnar e p i t h e l i a l •methallibure  1  and  ( S e r t o l i ) border c e l l s atrophy f o l l o w i n g  treatment.  The r e s u l t s o f 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 . the  ' m e t h a l l i b u r e ' t r e a t e d a d u l t guppy, spermatogonia  s p e r m a t o c y t e s comprise 3.4%  o f the t o t a l t e s t i s  Table X V I I ) .  The  and  area  (16% of the a r e a i n c o n t r o l ) and spermatophores a r e and cover 88% o f the t e s t i s a r e a  In  intact  (68% o f the a r e a i n c o n t r o l  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 t o be r e g r e s s e d ,  perhaps, even a s m a l l r e l e a s e o f  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). guppy the  I t seems l i k e l y t h a t i n the  ' m e t h a l l i b u r e ' t r e a t m e n t s u p p r e s s e s the  t r a n s f o r m a t i o n o f spermatogonia i n t o s p e r m a t o c y t e s (Table  XIX).  Wiebe (1968) demonstrated t h a t the b l o c k f o l l o w i n g ' m e t h a l l i b u r e ' t r e a t m e n t i s between p r i m a r y and spermatocytes.  The  secondary  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 t o two s p e c i e s o f f i s h - P o e c i l i a  reticulata  ( o v o v i v i p a r o u s , monthly b r e e d e r ) and Cymatogaster a g g r e g a t a (viviparous, seasonal  breeder).  In ' m e t h a l l i b u r e ' t r e a t e d male guppy, t h e r e i s a marked d e c r e a s e i n l i p o p h o r e p i g m e n t a t i o n o f the body and on the t a i l . t h a t the h e i g h t o f k i d n e y sex c h a r a c t e r )  b o t h on the  sides  Hoar e t a l (1967) demonstrated  t u b u l e o f s t i c k l e b a c k (a secondary  i s reduced f o l l o w i n g 'methallibure'  treatment.  108  Wiebe (1968) found t h a t ' m e t h a l l i b u r e ' causes the a t r o p h y o f secondary sex m o d i f i c a t i o n s on Cymatogaster male a n a l fin.  These r e s u l t s i n d i c a t e t h a t ' m e t h a l l i b u r e '  treatment  causes r e d u c t i o n i n g o n a d a l s t e r o i d o g e n e s i s . Since  ' m e t h a l l i b u r e ' t r e a t m e n t o f the a d u l t guppy  does not l e a d t o complete r e g r e s s i o n o f gametogenetic  and  s t e r o i d o g e n e t i c t i s s u e s o f the t e s t i s , as i s e v i d e n t i n the absence o f the p i t u i t a r y , i t i s c o n c l u d e d t h a t does n o t c o m p l e t e l y b l o c k the r e l e a s e o f the  'methallibure'  pituitary  gonadotropins. The  s i t e of a c t i o n of  'methallibure'  Although i t i s recognised  that 'methallibure'  blocks  the a c t i o n o f p i t u i t a r y g o n a d o t r o p i n s on b o t h the gametog e n e t i c and e n d o c r i n e t i s s u e s o f t e s t i s Wiebe 1968), the s i t e o f a c t i o n o f known.  (Hoar e t a l  1967;  ' m e t h a l l i b u r e ' i s not  ' M e t h a l l i b u r e ' t r e a t m e n t o f b o t h 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 o f the aldehyde f u c h s i n p o s i t i v e granules gonado tropic'ce l i s .  from the c y t o p l a s m o f the  pituitary  As i n a l l s i m i l a r s t u d i e s , t h e r e i s a  problem o f i n t e r p r e t a t i o n ; i s a c e l l w h i c h i s d e p l e t e d granules  i n a c t i v e , or i s the r a t e o f s y n t h e s i s o f the  s i m p l y e q u a l t o , or l e s s than the r a t e o f r e l e a s e ?  of granules  Examination  o f the mean c e l l diameter o f the gonadotrophs and o f the number o f c e l l s i n d i c a t e s t h a t t h e r e i s a v e r y  total  significant  (p< 0.001) r e d u c t i o n i n the s i z e o f the gonadotrophs o f  the  t r e a t e d f i s h compared w i t h t h o s e o f the c o n t r o l s i n b o t h the  109  a d u l t and j u v e n i l e s t a g e s which would t e n d t o 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 g o n a d o t r o p i c b l o c k i n g a c t i o n o f • m e t h a l l i b u r e ' may be e f f e c t i v e i n one o f two ways:  (a)  i t may b l o c k the h y p o t h a l a m i c c o n t r o l over g o n a d o t r o p i c hormone s y n t h e s i s , o r , (b) i t may  d i r e c t l y b l o c k the s y n t h e s i s  o f 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 e n d o c r i n e c e l l s . The h y p o t h a l a m i c n u c l e u s l a t e r a l i s t u b e r i s ( F l o r e n t i n 1934; H i l d 1950; Stahl 1957; B i l l e n s t e i n 1962; Oztan 1963)  Brehm  (NLT)  1958;  and p r e - o p t i c n u c l e u s  (PON)  (see Dodd, P e r k s and Dodd 1966 f o r r e v i e w ) 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 o f the s e x u a l c y c l e . and B a l l  Olivereau  (1966) demonstrated by means o f a u t o t r a n s p l a n t s o f  the p i t u i t a r y t h a t an i n t i m a t e c o n t a c t w i t h the hypothalamus i s n e c e s s a r y f o r the maintenance  o f gonadotrophs.  It is  p o s s i b l e t h a t ' m e t h a l l i b u r e ' b l o c k s the s y n t h e s i s o r r e l e a s e o f f a c t o r s produced by these n u c l e i .  However, most  o f the above a u t h o r s d e s c r i b e changes i n the appearance  of  the neurons o f the n u c l e i or changes i n the amount o f 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 s e x u a l c y c l e .  I n the p r e s e n t i n v e s t i g a t i o n no  such  changes a r e 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 o f g o n a d o t r o p h i c a c t i v i t y , o f f e r i n g no s u p p o r t f o r the f i r s t hypothesis.  On the o t h e r hand, a decrease i n the number and  s i z e o f gonadotroph  c e l l s as w e l l as a d e p l e t i o n o f 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 t o the second h y p o t h e s i s .  110  L a r g e b l o o d s i n u s e s a r e found i n the v e n t r a l r e g i o n o f meso-adenohypophysis o f ' m e t h a l l i b u r e ' t r e a t e d  fish  (group I I ) and i n t h o s e c o n t r o l groups i n w h i c h the gonadotrophs a r e d e v e l o p e d and presumably f u n c t i o n a l (groups I and I V ) .  The r o l e o f s i n u s e s o r c a p i l l a r i e s i s n o t c l e a r  a l t h o u g h 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 . gonadotrophs may  An i n c r e a s e d b l o o d s u p p l y t o the  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 o f  g o n a d o t r o p i c hormones, and the c o n t r o l o f hypothalamus over the i n t r i n s i c e n d o c r i n e c e l l s o f the p i t u i t a r y may c o n t r o l l e d i n t h i s way  ( L e a t h e r l a n d 1967).  be  Thus a n e g a t i v e  feed-back mechanism f o l l o w i n g a d e c l i n e i n t e s t o s t e r o n e o r e s t r o g e n p r o d u c t i o n a f t e r t r e a t m e n t w i t h ' m e t h a l l i b u r e ' would r e s u l t i n an enlargement o f the b l o o d s i n u s e s c o n t r o l l e d by the hypothalamus.  An enlargement o f 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 t h o s e f i s h a c t i v e l y s e c r e t i n g g o n a d o t r o p i c 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 o r appearance o f the somatotrophs i n the a d u l t g r o u p s ' ( I and I I ) , w h i c h may be e x p e c t e d i n more or l e s s f u l l y grown f i s h .  There were,  however, fewer o f t h e s e 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 o f the experiment (group I I I ) compared w i t 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 I V ) .  Somatotrophs were n o t i d e n t i f i e d i n any o f the ' m e t h a l l i b u r e ' t r e a t e d j u v e n i l e f i s h , w h i c h 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 o f the compound on the p r o d u c t i o n o f growth  Ill  hormone i n the j u v e n i l e f i s h . •methallibure  1  The  v e r y l i m i t e d growth o f  t r e a t e d j u v e n i l e s compared w i t h the  the  controls  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 s y n t h e s i s and/or r e l e a s e . The  thyrotrophs  o f the a d u l t ' m e t h a l l i b u r e '  treated  fish  a r e b o t h s i g n i f i c a n t l y l a r g e r and more numerous than those o f the c o n t r o l s .  The  thyrotrophs  o f the  'methallibure'  t r e a t e d j u v e n i l e f i s h a r e 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 b e g i n n i n g  of  e x p e r i m e n t , b u t 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 . the  'methallibure'  I t i s p r o b a b l y more c o n s i s t e n t to compare 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 w h i c h are o f a s i m i l a r w e i g h t r a t h e r than group IV c o n t r o l s o f a s i m i l a r age. 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  a c t i o n s , one production  ' M e t h a l l i b u r e ' has been shown  a t the l e v e l o f the t h y r o i d b l o c k i n g  distinct  the  o f the t h y r o i d hormone, and s i m u l t a n e o u s l y  the l e v e l o f the p i t u i t a r y and/or hypothalamus t e n d i n g cause t h y r o i d i n v o l u t i o n (Walpole 1965;  i n 'methallibure'  s i z e of  t r e a t e d a d u l t and  i s i n d i c a t i v e o f an i n c r e a s e d TSH  to  T u l l o c h e t a l 1963).  However, the c l e a r i n c r e a s e i n number and thyrotrophs  at  production  the  juvenile fish  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)  p i t u i t a r y thyrotrophs the  'methallibure'  and  the number and  s i z e of  the  w i t h i n the f i v e groups, the TEH  t r e a t e d a d u l t group i s g r e a t e r  of  than t h a t  of  the c o n t r o l s .  I n t h e j u v e n i l e groups, the TEH i s l a r g e s t  i n t h e f i s h o f group TV w h i c h a r e r a p i d l y d e v e l o p i n g and which, d u r i n g t h e c o u r s e o f t h e experiment, a c h i e v e d s e x u a l m a t u r i t y , and s m 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 t h e b e g i n n i n g o f the experiment.  The t h y r o i d f o l l i c l e s o f t h e  ' m e t h a l l i b u r e ' t r e a t e d groups have a much r e d u c e d  colloid  c o n t e n t compared w i t h t h e c o n t r o l s , c o n s i s t e n t w i t h a d e c r e a s e d t h y r o i d hormone p r o d u c t i o n . The apparent h y p e r a c t i v i t y o f the p i t u i t a r y thyrotrophs i n the ' m e t h a l l i b u r e ' t r e a t e d f i s h thus appears t o r e s u l t from a f a l l i n t h y r o i d hormone p r o d u c t i o n , and t h a t o f the c o n t r o l group (IV) from an i n c r e a s e d t h y r o i d a c t i v i t y a t t h i s s t a g e of  the sexual c y c l e  ( S t o l k 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 b o t h t h e number and mean c e l l diameter o f gonadotrophs, an-, i n c r e a s e i n t h e number and mean c e l l diameter o f t h e t h y r o t r o p h s and a decrease i n t h e number o f somatotrophs o f t r e a t e d j u v e n i l e s .  113 SUMMARY 1.  Hypophysectomy o f 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 c o n t a i n s o n l y  spermatogonia and spermatophores  because the s p e r m a t o c y t e s ,  s p e r m a t i d s and sperm p r e s e n t a t the time o f o p e r a t i o n t r a n s f o r m i n t o spermatophores. spermatogonia and t h e i r cease.  The m i t o t i c d i v i s i o n s o f  transformation into  I t i s c o n c l u d e d t h a t the d i v i s i o n  and t h e i r  of  spermatocytes spermatogonia  t r a n s f o r m a t i o n i n t o spermatocytes a r e  pituitary-  dependent b u t the t r a n s f o r m a t i o n o f s p e r m a t o c y t e s , s p e r m a t i d s and sperm i n t o spermatophores 2.  are p i t u i t a r y - i n d e p e n d e n t .  I n the absence o f the p i t u i t a r y ,  the  spermatophores  r u p t u r e 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 o f b e i n g d i s c h a r g e d , a r e phagocytosed w i t h i n the r e g r e s s e d e f f e r e n t d u c t s and the main sperm d u c t . r e l e a s e o f spermatophores  I t seems t h a t the  i s under the c o n t r o l o f the  pituitary. 3.  A f t e r hypophysectomy, S e r t o l i  c e l l s and e p i t h e l i a l  interstitial  c e l l s l i n i n g the e f f e r e n t d u c t s and the  main sperm d u c t r e g r e s s . hypophysectomized  cells,  Methyl t e s t o s t e r o n e treatment of  a n i m a l s causes the h y p e r t r o p h y o f 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 d u c t s and the main sperm duct; r e g r e s s e d 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  are  i t i s concluded that  a l s o r e s t o r e d to normal.  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  Sertoli  c e l l s l i n i n g the  d u c t a r e m a i n t a i n e d by androgens and n o t by gonadotropins.  cells  pituitary  114  4.  Hypophysectomy and m e t h y l t e s t o s t e r o n e t r e a t m e n t  of the a d u l t guppy do n o t b r i n g about any change i n the s t r u c t u r e o f the gonopodium. morphogenesis  I t suggests t h a t once  o f the gonopodium i s complete, i t becomes  independent o f the p i t u i t a r y and the androgens.  The  l i p o p h o r e s , on the o t h e r hand, become obscure or  entirely  d i s a p p e a r i n the absence o f the p i t u i t a r y b u t t h e r e i s moderate r e c o v e r y i n the c o n t e n t o f l i p o p h o r e s a f t e r testosterone treatment. are r e g u l a t e d by 5.  I t i s c o n c l u d e d t h a t the l i p o p h o r e s  androgens.  Exogenous m e t h y l t e s t o s t e r o n e appears t o have a  d i r e c t s p e r m a t o k i n e t i c e f f e c t on the t e s t i s o f hypophysectomized rapidly  animal.  Spermatogonial cysts d i v i d e  and t r a n s f o r m i n t o spermatocytes b u t l a t e r s t a g e s  o f spermatogenesis do n o t appear. 6. mitotic  Hypophysectomy o f the j u v e n i l e guppy p r e v e n t s the  d i v i s i o n o f spermatogonia i n the t e s t i s ; no o t h e r  s t a g e s o f spermatogenesis appear and the i n t e r s t i t i a l and S e r t o l i c e l l s a r e n o t d i f f e r e n t i a t e d . t r e a t m e n t o f the hypophysectomized spermatogenesis  Testosterone  j u v e n i l e s do n o t  ( i n c o n t r a s t t o the a d u l t ) and the  c e l l s and S e r t o l i c e l l s a r e n o t e v i d e n t .  cells  initiate interstitial  I t i s concluded  t h a t the spermatogenesis and the d i f f e r e n t i a t i o n o f the interstitial  c e l l s and S e r t o l i c e l l s a r e under the c o n t r o l  of p i t u i t a r y g o n a d o t r o p i n s o n l y .  115  7.  In the absence o f the p i t u i t a r y , the sperm d u c t s  do not d i f f e r e n t i a t e and the secondary sex c h a r a c t e r s do appear i n the j u v e n i l e guppy.  Following  testosterone  t r e a t m e n t the sperm ducts a r e w e l l d i f f e r e n t i a t e d and secondary sex c h a r a c t e r s become e v i d e n t .  not  the  I t i s concluded  t h a t the androgens cause the d i f f e r e n t i a t i o n and maintenance o f the sperm d u c t s and the secondary sex  characters  ( s i m i l a r t o the a d u l t ) . 8.  The  'methallibure  1  t e s t i s o f the a d u l t guppy t r e a t e d w i t h c o n t a i n s few c y s t s o f e a r l i e r s t a g e s  spermatogenesis.  The  of  spermatophores r e m a i n 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 r e d u c e d i n s i z e b u t t h e r e a r e no changes i n S e r t o l i c e l l s interstitial cells.  and  There i s a marked d e c r e a s e i n the amount  of lipophore pigmentation.  Since  'methallibure'  treatment  o f the a d u l t guppy does n o t l e a d t o complete r e g r e s s i o n s o f 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 o f  testis,  as i s e v i d e n t i n the absence o f the p i t u i t a r y , i t seems t h a t ' m e t h a l l i b u r e ' does not c o m p l e t e l y  b l o c k the r e l e a s e o f  p i t u i t a r y gonadotropins. 9.  No d e v e l o p m e n t a l changes o c c u r i n the t e s t i s o f  the j u v e n i l e guppy f o l l o w i n g ' m e t h a l l i b u r e ' t r e a t m e n t . secondary sex c h a r a c t e r s a r e not d i f f e r e n t i a t e d .  The  Since  ' m e t h a l l i b u r e ' produces i d e n t i c a l e f f e c t s t o hypophysectomy, i t i s concluded that gonadotropin s e c r e t i o n of j u v e n i l e guppies i s e n t i r e l y b l o c k e d w i t h  the  'methallibure'.  116  10.  ' M e t h a l l i b u r e ' t r e a t m e n t does n o t cause any  change i n t h e s i z e o r appearance o f t h e c e l l s o f p r o - and meta-adenohypophysis  o f t h e a d u l t o r j u v e n i l e guppies b u t  the c e l l t y p e s o f meso-adenohypophysis t h y r o t r o p s and somatotrophs)  (gonadotrophs,  show changes.  A c l e a r decrease  i n b o t h t h e number and mean c e l l diameter o f gonadotrophs, and an i n c r e a s e i n t h e number and mean c e l l diameter o f t h e thyrotrophs  i s evident i n 'methallibure'  juvenile fish,  t r e a t e d a d u l t and  whereas a decrease i s n o t i c e d i n t h e number  o f somatotrophs o f t h e j u v e n i l e s .  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