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Changes in testis cyclic nucleotide metabolism during trout spermatogenesis Davis, Jillian Frances 1978

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CHANGES IN TESTIS CYCLIC NUCLEOTIDE METABOLISM • DURING TROUT SPERMATOGENESIS by JILLIAN FRANCES DAVIS B.Sc,  U n i v e r s i t y o f Auckland, 1972  M . S c , U n i v e r s i t y of Auckland, 1974  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF .DOCTOR OF PHILOSOPHY  in -THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY -FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA • March, 1978 (c)  J i l l i a n Frances Davis, 1978  In p r e s e n t i n g t h i s  thesis  an advanced degree at  further  agree  fulfilment  of  the  requirements  the U n i v e r s i t y of B r i t i s h Columbia, I agree  the L i b r a r y s h a l l make it I  in p a r t i a l  freely  available  for  t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f  of  this  representatives. thesis for  It  financial  this  thesis  of  gain s h a l l not  Biochemistry  The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  30.3.78  or  i s understood that copying or p u b l i c a t i o n  written permission.  Department  that  reference and study.  f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s  for  be allowed without my  ABSTRACT  The c o n c e n t r a t i o n s o f c y c l i c GMP and c y c l i c AMP, i n rainbow  trout  (Salmo g a i r d n e r i i ) t e s t i s , were determined  d u r i n g hormonally-induced spermatogenesis.  In immature  t r o u t t e s t i s , c y c l i c GMP and c y c l i c AMP c o n c e n t r a t i o n s were approximately equal  (^2|imol/kg wet weight) .  An abrupt 10  f o l d decrease i n c y c l i c GMP o c c u r r e d d u r i n g m i t o t i c and t e s t i s  growth p r i o r  2 f o l d during this  to meiosis.  time.  Cyclic  esis.  No s i g n i f i c a n t  decrease  o f spermatogen-  change i n c y c l i c AMP o c c u r r e d d u r i n g  development a f t e r the i n i t i a l 2 f o l d Cyclic  AMP decreased  A further gradual 5 f o l d  i n c y c l i c GMP o c c u r r e d d u r i n g the remainder  activity  decrease.  AMP and c y c l i c GMP phosphodiesterase  activities,  measured a t both h i g h ( m i l l i m o l a r ) and low (micromolar)  sub-  s t r a t e c o n c e n t r a t i o n s were determined i n t r o u t t e s t i s d u r i n g hormonally-induced spermatogenesis. molar  When measured a t m i l l i -  s u b s t r a t e , c y c l i c GMP phosphodiesterase a c t i v i t i e s d i d  not change s i g n i f i c a n t l y  throughout spermatogenesis.  Cyclic  AMP phosphodiesterase a c t i v i t i e s , measured a t m i l l i m o l a r s t r a t e , decreased about 50% p r i o r  t o m e i o s i s and then  ased, d u r i n g spermatid d i f f e r e n t i a t i o n , activity testis.  slightly  to attain  sub-  incre-  a final  g r e a t e r than t h a t observed i n immature  When measured a t micromolar  s u b s t r a t e , i n the p r e -  sence o f EGTA, c y c l i c GMP phosphodiesterase  activities  decreased about  50%, w h i l e c y c l i c AMP  phosphodiesterase  a c t i v i t i e s , measured under t h e same c o n d i t i o n s , i n c r e a s e d 20 f o l d d u r i n g the course o f spermatogenesis.  A detailed  study of c y c l i c GMP phosphodiesterase a c t i v i t i e s , measured at micromolar  2+ s u b s t r a t e , i n the presence and absence o f Ca , 2+  showed there was no change i n Ca  -dependent c y c l i c  GMP  phosphodiesterase a c t i v i t y a t the time o f development a t which the l a r g e decrease i n c y c l i c GMP i s observed. DEAE-cellulose p r o f i l e s  o f c y c l i c n u c l e o t i d e phosphodi-  e s t e r a s e a c t i v i t i e s , from t r o u t t e s t i s a t d i f f e r e n t  stages  df development, showed two peaks o f c y c l i c AMP a c t i v i t y and one peak o f c y c l i c GMP a c t i v i t y .  The l a t t e r cochromato-  graphed w i t h the f i r s t c y c l i c AMP a c t i v i t y peak.  A large  i n c r e a s e i n the f i r s t c y c l i c AMP phosphodiesterase peak o c c u r r e d when spermatids were maturing, without a c o n c u r r e n t i n c r e a s e i n c y c l i c GMP phosphodiesterase a c t i v i t y .  This  i n d i c a t e s the i n d u c t i o n o f a s p e c i f i c , h i g h - a f f i n i t y  cyclic  AMP phosphodiesterase d u r i n g the m e i o t i c stage o f t e s t i c u l a r development. Phosphodiesterase  assays u s i n g micromolar  substrate  c o n c e n t r a t i o n s , on s u b c e l l u l a r f r a c t i o n s , demonstrated about  85% of c y c l i c AMP h y d r o l y s i s and 80% of c y c l i c  h y d r o l y s i s was s o l u b l e .  Both peaks o f c y c l i c AMP  that  GMP  activity  were observed i n the D E A E - c e l l u l o s e p r o f i l e o f a 100,000xg supernatant  ( s o l u b l e ) f r a c t i o n from t r o u t t e s t i s .  s m a l l amount o f p a r t i c u l a t e c y c l i c AMP activity,  i n the 100,000xg p e l l e t  The  phosphodiesterase  fraction,  was a s s o c i a t e d  iii. mainly w i t h the second peak on the D E A E - c e l l u l o s e p r o f i l e . K i n e t i c analyses of homogenate phosphodiesterases from mature t e s t i s activity  showed o n l y high a f f i n i t y c y c l i c  (apparent Kms  f o r c y c l i c AMP  o f 1.1  AMP  uM and 0.3 itM)  and both low and h i g h a f f i n i t y c y c l i c GMP  activity  Kms  K i n e t i c analyses  of  f o r c y c l i c GMP c y c l i c AMP  of 22 0 yM and 8 pM).  h y d r o l y s i s by the two peaks of phosphodiesterase  activities purified of high a f f i n i t y  on D E A E - c e l l u l o s e , confirmed the presence  activities.  Guanylate c y c l a s e a c t i v i t e s  were assayed i n the s o l u b l e  and p a r t i c u l a t e f r a c t i o n s from .immature testis  (apparent  testis  and  from  a t d i f f e r e n t stages of hormonally-induced development.  There was  an approximate 1:2  guanylate c y c l a s e a c t i v i t i e s trout t e s t i s .  r a t i o o f s o l u b l e to p a r t i c u l a t e i n .immature and i n mature  A 3 f o l d d e c r e a s e , i n both s o l u b l e and p a r t -  i c u l a t e guanylate c y c l a s e a c t i v i t i e s c o i n c i d e d w i t h the 10 f o l d decrease i n c y c l i c GMP maturing t r o u t t e s t i s .  c o n c e n t r a t i o n observed i n  Thus, i n t r o u t t e s t i s d u r i n g  spermatogenesis, c y c l i c GMP  concentrations r e f l e c t  developmental modulation of guanylate c y c l a s e r a t h e r than t h a t of c y c l i c GMP  the  activity,  phosphodiesterase.  iv. TABLE OF CONTENTS Page Abstract  . . . . .  Table of Contents  i iv  L i s t o f Tables  vii  L i s t of F i g u r e s  viii  Acknowledgements  xi  Dedication  x i i  Abbreviations  xiii  Introduction  1  A. Spermatogenesis B. The r o l e o f c y c l i c AMP and c y c l i c GMP i n metabolism . . .  1 14  C. The r o l e o f c y c l i c AMP and c y c l i c GMP i n spermatogenesis  17  D. O b j e c t i v e s o f t h i s t h e s i s  24  M a t e r i a l s and Methods  25  A. M a t e r i a l s . . .  25  B. . Methods  26  1. Chromatography m a t e r i a l s p r e p a r a t i o n  26  2. Paper chromatography  27  3. P r e p a r a t i o n o f [ ^C] adenosine from 1  [ *C]AMP 1!  4. F i s h husbandry 5. Laparotomy  .  27 28  procedure f o r sex d e t e r m i n a t i o n of  fish  28  6. P i t u i t a r y e x t r a c t p r e p a r a t i o n  29  7. C y c l i c n u c l e o t i d e e x t r a c t i o n 8. Determination of c y c l i c n u c l e o t i d e concent-v r a t i o n s by radioimmunoassay  29 31  9, T i s s u e p r e p a r a t i o n f o r c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e assay , . . ,  *  10. D E A E - c e l l u l o s e column chromatography o f c y c l i c n u c l e o t i d e phosphodiesterase activities , , 11. C y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e  assay  12. T i s s u e p r e p a r a t i o n f o r g u a n y l a t e c y c l a s e assay 13. G u a n y l a t e c y c l a s e assay . . . . 14. P r o t e i n assay Results A. T e s t e s growth r a t e d u r i n g h o r m o n a l l y - i n d u c e d spermatogenesis i n t r o u t B. C y c l i c GMP and c y c l i c AMP c o n c e n t r a t i o n s i n t r o u t t e s t i s d u r i n g spermatogenesis .... C. C y c l i c AMP and c y c l i c GMP p h o s p h o d i e s t e r a s e a c t i v i t i e s i n trout t e s t i s during spermatogenesis 1. Assay system 2. P r o p e r t i e s 3. A c t i v i t i e s d u r i n g t e s t i s development . . 4. D E A E - c e l l u l o s e chromatographic a t i o n o f c y c l i c AMP and c y c l i c phosphodiesterases  fractionGMP  5. S o l u b l e and p a r t i c u l a t e a c t i v i t i e s i o n a t e d on D E A E - c e l l u l o s e  fract^  6. K i n e t i c a n a l y s e s o f D E A E - c e l l u l o s e peak c y c l i c AMP p h o s p h o d i e s t e r a s e activities  •  vi.  Page D.  Guanylate cyclase  during  activities  spermatogenesis  in trout  testis  . . . . .  1. Assay system 2. A c t i v i t i e s and p r o p e r t i e s d u r i n g development  89 89  testis 91  Discussion  98  References  117  Appendix  128  vii. LIST OF TABLES Page TABLE I.  C y c l i c GMP i n t r o u t t e s t i s d u r i n g hormonallyinduced spermatogenesis (experiment 2) . ... . 46  TABLE I I . C y c l i c GMP i n t r o u t t e s t i s d u r i n g hormonallyinduced spermatogenesis (experiment 3) . . . .  50  TABLE I I I . T o t a l c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t i e s i n t r o u t t e s t i s d u r i n g hormonallyinduced spermatogenesis  65  TABLE IV.  C y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t i e s measured a t micromolar s u b s t r a t e i n t r o u t t e s t i s d u r i n g hormonally-induced spermatogenesis . . . .67  TABLE V.  C y c l i c GMP phosphodiesterase a c t i v i t i e s measured a t micromolar s u b s t r a t e i n t r o u t t e s t i s d u r i n g hormonally-induced spermatogenesis  71  Guanylate c y c l a s e s p e c i f i c a c t i v i t i e s i n t r o u t t e s t i s d u r i n g hormonally-induced spermatogenesis  92  TABLE VI.  TABLE V I I . T o t a l guanylate c y c l a s e a c t i v i t i e s i n t r o u t t e s t i s d u r i n g hormonally-induced spermatogenesis . . ,  93  viii.  LIST OF FIGURES F i g u r e 1.  L i g h t micrographs o f t r o u t t e s t i s d u r i n g hormonally-induced spermatogenesis character-  Page 8  F i g u r e 2A.  S e q u e n t i a l c e l l types and t h e i r i s t i c s d u r i n g spermatogenesis  F i g u r e 2B.  T e s t i c u l a r enzyme a c t i v i e s r e l a t e d t o s p e c i f i c c e l l types d u r i n g spermatogenesis i n the r a t  12  F i g u r e 3.  T e s t e s growth r a t e d u r i n g hormonallyinduced spermatogenesis i n t r o u t  42  F i g u r e 4.  Comparison o f c y c l i c GMP c o n c e n t r a t i o n and t e s t e s wet weight d u r i n g hormonallyinduced spermatogenesis i n t r o u t  47  12  F i g u r e 5.  C y c l i c AMP c o n c e n t r a t i o n s i n t e s t i s d u r i n g hormonally-induced spermatogenesis i n t r o u t . .52  F i g u r e 6A.  S t a b i l i t y o f phosphodiesterase at 30° . .  F i g u r e 6B. F i g u r e 7A. F i g u r e 7B. F i g u r e 8.  activities 56  Phosphodiesterase a c t i v i t i e s as a f u n c t i o n of Mg2+ c o n c e n t r a t i o n  56  Phosphodiesterase activities or absence o f EGTA  i n the presence 58  Phosphodiesterase of pH  as a f u n c t i o n  activities  58  Hofstee p l o t o f the r a t e o f c y c l i c AMP h y d r o l y s i s by a t e s t i s homogenate from mature t r o u t  61  Hofstee p l o t o f the r a t e o f c y c l i c GMP h y d r o l y s i s by the homogenate d e s c r i b e d i n the legend f o r F i g u r e 8  63  F i g u r e 10A. T o t a l c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t i e s i n t r o u t t e s t i s during spermatogenesis  69  F i g u r e 9.  F i g u r e 10B. C y c l i c n u c l e o t i d e phosphodiesterase activities measured a t micromolar s u b s t r a t e c o n c e n t r a t i o n i n t r o u t t e s t i s d u r i n g spermatogenesis . . . .69  ix. Page Figure  11.  F i g u r e 12A.  F i g u r e 12B.  F i g u r e 13A.  F i g u r e 13B.  F i g u r e 14.  F i g u r e 15A.  F i g u r e 15B. F i g u r e 16A.  F i g u r e 16B. F i g u r e 17A.  D E A E - c e l l u l o s e f r a c t i o n a t i o n o f c y c l i c AMP and c y c l i c GMP p h o s p h o d i e s t e r a s e a c t i v i t i e s in trout t e s t i s during hormonally-induced spermatogenesis  73  D E A E - c e l l u l o s e p r o f i l e o f c y c l i c AMP phosphod i e s t e r a s e s i n a t r o u t t e s t i s homogenate f r o m a t r o u t i n j e c t e d w i t h hormone f o r 8 weeks  77  DEAE-cellul©se p r o f i l e of rechromatographed Peak I f r o m t h e p r o f i l e o f t h e t e s t i s homogenate d e s c r i b e d above . .  DEAE-cellulose p r o f i l e of trout t e s t i s c y c l i c AMP p h o s p h o d i e s t e r a s e a c t i v i t i e s of a 100,000xg s u p e r n a t a n t f r a c t i o n from t e s t i s h o r m o n a l l y - i n d u c e d f o r 4 weeks . . .  .77  .79  DEAE-cellulose p r o f i l e of trout t e s t i s c y c l i c AMP p h o s p h o d i e s t e r a s e a c t i v i t i e s of a 100,000xg p a r t i c u l a t e f r a c t i o n from t h e t e s t i s d e s c r i b e d above  79  H o f s t e e p l o t o f t h e r a t e o f c y c l i c AMP h y d r o l y s i s by D E A E - c e l l u l o s e Peak I from t e s t i s from t r o u t hormonally-induced f o r 8 weeks  82  Lineweaver-Burk p l o t of the r a t e of c y c l i c AMP h y d r o l y s i s by D E A E - c e l l u l o s e P e a k I I from t e s t i s from t r o u t hormonally-induced f o r 3 weeks  84  Hofstee p l o t of the hydrolysis described above  84  Lineweaver-Burk p l o t of the r a t e of c y c l i c AMP h y d r o l y s i s by D E A E - c e l l u l o s e P e a k I I from t e s t i s from t r o u t hormonally-induced f o r 8 weeks  86  Hofstee p l o t of the hydrolysis described above  86  T r i t o n X-100 e f f e c t o n g u a n y l a t e c y c l a s e a c t i v i t y i n t h e 100,000xg p e l l e t from t e s t i s o f z e r o t i m e t r o u t (OP) a n d f r o m t e s t i s o f t r o u t a f t e r 3 ( 3 P ) , 6 ( 6 P ) , and 10 (10P) weeks o f t w i c e w e e k l y hormone injections  95  X.  Page  F i g u r e 17B. Comparison o f the e f f e c t s o f salmon gonadotropin and T r i t o n X-100 on the 100,000xg p e l l e t (OP) and the 100,000xg supernatant (OS) zero time t r o u t t e s t i s guanylate c y c l a s e a c t i v i t i e s F i g u r e 18.  F i g u r e 19A.  Comparison o f guanylate c y c l a s e a c t i v i t i e s and c y c l i c GMP c o n c e n t r a t i o n s i n t r o u t t e s t i s d u r i n g hormonally-induced spermatogenesis  112  S e p a r a t i o n o f c y c l i c AMP and c y c l i c GMP on a BioRad AG 1-X8 column  12 9  F i g u r e 19B. S e p a r a t i o n o f c y c l i c AMP, c y c l i c GMP and 5'AMP on a D E A E - c e l l u l o s e column F i g u r e 20A.  95  129  Separation of c y c l i c nucleotides on P a r t i s i l - 1 0 SAX  (A,G,C,U)  F i g u r e 20B.  Separation of c y c l i c nucleotides on P a r t i s i l - 1 0 SAX  (A,G,C,U,I) 131  F i g u r e 21A.  S e p a r a t i o n o f 5'CMP, 5'AMP, and 5'UMP on P a r t i s i l - 1 0 SAX  131  F i g u r e 21B. S e p a r a t i o n o f c y c l i c AMP and c y c l i c GMP on P a r t i s i l - 1 0 SAX  134 134  XI,  ACKNOWLEDGEMENTS  The  author wishes t o express her a p p r e c i a t i o n t o  P r o f e s s o r M. Smith f o r h i s e x c e l l e n t s u p e r v i s i o n o f t h i s work; t o Bob, S h i r l e y , Ev, T e r r y , Pat and Anne f o r t h e i r h e l p f u l d i s c u s s i o n s and encouragement; t o V i v i a n f o r i n t r o d u c i n g me t o f i s h c u l t u r e ; t o Deena and Danny f o r h e l p w i t h radioimmunoassays; t o F l o r a and t o the Schonblom f a m i l y f o r the happy times shared together;  and t o B i l l  f o r h i s l o v i n g support.  The M e d i c a l  Research C o u n c i l o f Canada i s thanked  f o r p r o v i d i n g a Studentship t o the author f o r the p e r i o d 1974  - 75, and t h e K i l l a m Foundation f o r a s c h o l a r s h i p  f o r the p e r i o d 1975 - 78.  DEDICATION  This t h e s i s i s dedicated  t o my Granny whose  immense love and wisdom have so g r e a t l y my  life.  enriched  xiii. ABBREVIATIONS cyclic  AMP  adenosine  3',5'-monophosphate  cyclic  GMP  guanosine  3',5'-monophosphate  AMP, GMP, CMP, UMP  the 5'-monophosphates o f the r i b o n u c l e o s i d e s o f adenine, guanine, c y t o s i n e and uracil  ATP, GTP  the 5 ' - t r i p h o s p h a t e s of the r i b o n u c l e o s i d e s o f adenine and guanine  DNA  deoxyribonucleic  RNA  ribonucleic acid  pmol, ymol  picomoles  M  molar  uM, mM  micromolar, m i l l i m o l a r  EGTA  e t h y l e n e g l y c o l b i s (3-aminoethyl ether) N,N -tetraacetate  (10~  1 2  acid  )  ,.. . micromoles ( 1 0 ~ ) 6  (moles p e r l i t e r )  1  EDTA  ethylenediaminetetraacetate  TCA  trichloroacetic acid  DEAE-cellulose  diethylaminoethyl  PEI-cellulose  polyethyleneithine • c e l l u l o s e  Tris  tris(hydroxymethyl)aminomethane  MIX  l - m e t h y l - 3 - i s o b u t y l xanthine  POPOP  1.4- d i [ 2 - ( 5 - p h e n y l o x a z o l y l ) ] - b e n z e n e  PPO  2.5- diphenyl oxazole  SCINTREX  tradename f o r a u n i v e r s a l l i q u i d s c i n t i l l a t i o n c o c k t a i l o f 1:1 T r i t o n X-100: Toluene, c o n t a i n i n g POPOP and PPO  TTP  a s c i n t i l l a t i o n f l u i d consisting of T o l u e n e : T r i t o n X-100:PPO, 6 0 : 1 2 5 : 1 . 3 2 v/v/w  A 5 2  xg  i*  cellulose  amount o f m a t e r i a l w i t h an o p t i c a l of one i n a one cm p a t h l e n g t h times the f o r c e o f g r a v i t y  density  Curie r e v o l u t i o n s per minute counts per minute degrees  Centigrade  velocity substrate M i c h a e l i s Menton constant; the s u b s t r a t e c o n c e n t r a t i o n a t which h a l f maximal v e l o c i t y o f an enzyme a c t i v i t y occurs apparent Km; the s u b s t r a t e c o n c e n t r a t i o n at which h a l f maximal v e l o c i t y o f an enzyme a c t i v i t y o c c u r s , when there are m u l t i p l e Kms f o r the same a c t i v i t y w i t h d i f f e r i n g enzyme c o n c e n t r a t i o n s a n a l y t i c a l grade i o n exchange r e s i n prepared by s i z i n g and p u r i f y i n g the standard DOWEX r e s i n o f the same d e s i g n a t i o n quaternary ammonium anion r e s i n ; medium pore s i z e  exchange  1. INTRODUCTION A.  Spermatogenesis Spermatogenesis i s a complex process of  sequential  developmental steps during which immature, d i p l o i d germ c e l l s d i f f e r e n t i a t e i n t o h i g h l y s p e c i a l i z e d h a p l o i d spermatozoa In the v e r t e b r a t e c y t o l o g i c a l and extensively  t e s t i s , there  biochemical  (1).  i s an ordered sequence of  changes, which have been s t u d i e d  However, many d e t a i l s of the r e g u l a t i o n of  spermatogenesis remain unresolved. of DNA  These i n c l u d e the c o n t r o l  r e p l i c a t i o n , t r a n s c r i p t i o n and  of c e l l u l a r d i f f e r e n t i a t i o n , and  t r a n s l a t i o n , the  Since  the  testicular  the course of spermatogenesis, i n terms of  s p e c i f i c t i m i n g of DNA,  RNA  and  p r o t e i n s y n t h e s i s , i s known  p o s s i b l e r o l e s f o r r e g u l a t o r y molecules, such as the nucleotides,  induction  the p r e c i s e nature of hormonal  c o n t r o l by the p i t u i t a r y gonadotropins and androgens.  (1).  i n the c o n t r o l of growth and  t e s t i c u l a r c e l l s , can be  (1)  cyclic  differentiation  of  investigated.  There i s evidence f o r the  involvement of c y c l i c  AMP  during mammalian spermatogenesis, as an i n t r a c e l l u l a r mediator of the p i t u i t a r y gonadotropin hormones, f o l l i c l e - s t i m u l a t i n g (FSH), and stimulate  l u t e i n i z i n g hormone (LH) the p r o d u c t i o n  (2, 3, 4).  of c y c l i c AMP  t a r g e t c e l l s , the S e r t o l i c e l l s In FSH-stimulated S e r t o l i c e l l s ,  (3) and  in their  FSH  cells  of a new  and  to r e s u l t i n de novo  p r o t e i n , the androgen b i n d i n g p r o t e i n  (4)'.  produced by  adenylate c y c l a s e has been shown to a c t i v a t e a c y c l i c dependent p r o t e i n kinase  LH  respective  the Leydig  the c y c l i c AMP  and  AMP-  synthesis  (5).  In  2. LH-stimulated L e y d i g c e l l s , c y c l i c AMP  has been shown t o  enhance the p r o d u c t i o n o f the major t e s t i c u l a r  androgen,  t e s t o s t e r o n e (6). A p r e c i s e r o l e f o r c y c l i c GMP i n any system.  In s e v e r a l c e l l  found to enhance m i t o s i s  has not y e t been d e f i n e d  l i n e s , c y c l i c GMP  ( 7 ) , and DNA  (8) and RNA  has been (9) s y n t h e s i s .  Since these a c t i v i t i e s are c h a r a c t e r i s t i c of c e l l s i n the e a r l y stages o f spermatogenesis, a r o l e f o r c y c l i c GMP i s p o s s i b l e and r e q u i r e s f u r t h e r  i n such c e l l s  investigation.  The c y t o l o g i c a l and b i o c h e m i c a l changes  observed i n germ  c e l l s d u r i n g spermatogenesis are b a s i c a l l y s i m i l a r f o r a l l vertebrates  (1).  The d i f f e r e n t c e l l types are c h a r a c t e r i z e d  by t h e i r s i z e , time o f appearance d u r i n g t e s t i s  development,  the appearance of t h e i r c y t o p l a s m i c and n u c l e a r c o n t e n t s , and t h e i r r e l a t i v e r a t e s of DNA,  RNA  and p r o t e i n s y n t h e s i s (1).  The i n f a n t i l e t e s t i s c o n t a i n s gonocytes which have a r i s e n from p r i m o r d i a l germ c e l l s .  A t a s p e c i f i c time i n development  the gonocytes m u l t i p l y and are transformed i n t o Spermatogonia  spermatogonia.  continue to m u l t i p l y by a s e r i e s of m i t o t i c  cell  d i v i s i o n s , the number of which v a r i e s from s p e c i e s to s p e c i e s . In mammals t h e r e are 4 t o 6 d i v i s i o n s , w h i l e i n f i s h t h e r e are about 10 to 12 d i v i s i o n s  (1, 10).  C e l l s d e r i v i n g from  one  p a r e n t a l spermatogonial c e l l remain p a r t i a l l y c o j o i n e d throughout d i f f e r e n t i a t i o n by c y t o p l a s m i c b r i d g e s (11).  The  cells  d e r i v i n g from one p a r e n t a l c e l l synchronously d i f f e r e n t i a t e i n t o primary spermatocytes  (1).  The primary spermatocytes  double t h e i r n u c l e a r DNA t o g i v e a t e t r a p l o i d and  (4n) content,  then have a long prophase p e r i o d d u r i n g which there i s  p a i r i n g of chromosomes and p o s s i b l e exchange o f g e n e t i c material.  The primary  spermatocyte f i n a l l y  undergoes c e l l  d i v i s i o n t o g i v e two secondary spermatocytes, diploid  (2n) complement o f DNA.  The secondary spermatocytes  are s h o r t l i v e d and each d i v i d e s without to g i v e r i s e t o two spermatids DNA  (1).  spermatids  with a h a p l o i d  (n) content o f The  so formed a r e n o n - d i v i d i n g and o n l y low l e v e l s of  stages o f spermatid  (12).  During  the f o l l o w i n g  d i f f e r e n t i a t i o n , the volume o f the  i s g r e a t l y reduced  n u c l e a r DNA i s condensed spermatids  r e p l i c a t i o n of DNA  T h i s r e d u c t i v e d i v i s i o n i s termed m e i o s i s .  DNA s y n t h e s i s can be d e t e c t e d  spermatid  each with a  by c y t o p l a s m i c  (10).  l o s s and the  RNA s y n t h e s i s ceases i n  (13) w h i l e c e r t a i n p r o t e i n s a r e s y n t h e s i z e d d u r i n g  spermatid maturation  (1, 14, 15).  In the f i n a l  i a t i o n step o f t r a n s f o r m a t i o n o f spermatid  different-  i n t o spermatozoa,  new s t r u c t u r a l and enzymatic p r o t e i n s are s y n t h e s i z e d t o form the p r o p u l s i v e t a i l and the complex headpiece of  f u l l y mature sperm  characteristic  (1). (Figure 2A; p l 3 , sequence summary.)  In v e r t e b r a t e s w i t h d i f f e r e n t modes o f sperm p r o d u c t i o n , there a r e c o n s i d e r a b l e d i f f e r e n c e s i n t e s t i c u l a r s t r u c t u r e s (1) In f i s h , where there i s a r e l a t i v e l y s h o r t and i n t e n s e ing  breed-  p e r i o d , d u r i n g which enormous q u a n t i t i e s o f sperm are  r e l e a s e d , a c y s t i c mode o f sperm p r o d u c t i o n i s used (16). The  t e s t e s o f salmonids,  t o which f a m i l y the rainbow t r o u t ,  4. Salmo g a i r d n e r i i , belongs, a r e made up o f elongated,  branching  t u b u l a r s t r u c t u r e s , known as l o b u l e s , w i t h i n f i b r o u s w a l l s (17). The  l o b u l e s c o n t a i n w i t h i n them 2 o r more c y s t s o f germ c e l l s  surrounded  by c o n n e c t i v e t i s s u e s ( 1 7 ) .  When the t e s t e s a r e  immature, the c y s t s are e s s e n t i a l l y empty o f germ c e l l s f o r a s m a l l number o f gonocytes  or r e s t i n g spermatogonia (18).  As t e s t i c u l a r maturation b e g i n s , the gonocytes i n t o spermatogonia.  c y c l e s a r e r e s t i n g spermatogonia  c e l l s which remain undeveloped The  spermatogonia  differentiate  In f i s h w i t h s e v e r a l breeding  the germ c e l l s which begin the second ive  seasons,  and f o l l o w i n g r e p r o d u c t (18) .  These a r e germ  d u r i n g the preceeding  divide rapidly f i l l i n g  l a r g e number o f germ c e l l s .  except  cycle.  each c y s t w i t h a  These spermatogonia  then  differ-  e n t i a t e v i a the sequence p r e v i o u s l y o u t l i n e d t o produce mature sperm.  W i t h i n each c y s t , a l l the germ c e l l s develop i n  synchrony,  but w i t h i n a t e s t i s the v a r i o u s c y s t s may be a t  d i f f e r e n t stages o f development (19). In  the mammal, male germ c e l l s are c o n t a i n e d w i t h i n the  seminiferous tubules i n a concentric progression of maturity from p e r i p h e r y t o the c e n t r a l lumen, i n t o which mature sperm are c o n t i n u o u s l y r e l e a s e d a f t e r puberty  (1).  Except f o r  i n p r e - p u b e r t a l development, the e n t i r e l e n g t h o f the s e m i n i f e r ous t u b u l e i s c o n t i n u a l l y populated w i t h germ c e l l s a t v a r i o u s stages o f development ( 1 ) . In both mammals and f i s h e s , the male germ c e l l s a r e surrounded  by a framework o f f i b r o u s c o n n e c t i v e t i s s u e t h a t  5. supports the blood v e s s e l s , lymphatics testis  (1).  The d i s t i n c t i v e  t i s s u e i s the e p i t h e l o i d androgen. testis  and nerves o f the  component o f t h i s  c e l l which s e c r e t e s  connective  testicular  These are termed L e y d i g c e l l s i n the mammalian  (1) and lobule-boundary  c e l l s o r L e y d i g c e l l .homologues  i n t h e f i s h t e s t i s (19). One  other important  been i d e n t i f i e d easily  somatic c e l l , the S e r t o l i c e l l , has  i n most v e r t e b r a t e t e s t e s (19), but i s not  identified  i n a l l fishes  (10).  In the mammal, S e r t o l i  c e l l s l i n e the p e r i p h e r y of the s e m i n i f e r o u s t u b u l e ( 1 ) . The  S e r t o l i c e l l p r e c u r s o r s a r e very s e n s i t i v e t o the endo-  c r i n e s t a t e o f the animal and i n the r a t c o n t i n u e t o d i v i d e up t o 20 days a f t e r for  birth  (20).  The S e r t o l i  the e a r l y stages of germ c e l l maturation  cell (1).  produces an androgen b i n d i n g p r o t e i n (21) a f t e r  i s necessary It  FSH-stimulation  and may p r o v i d e n u t r i e n t support t o d e v e l o p i n g germ c e l l s (22). S e r t o l i c e l l s a l s o have c o n s i d e r a b l e p h a g o c y t i c a c t i v i t y and reabsorb r e s i d u a l bodies l e f t i n p l a c e a f t e r sperm  (22).  When S e r t o l i  c e l l s are observed  they are found a s s o c i a t e d w i t h s t r u c t u r a l  the r e l e a s e o f i n fishes,  elements o f the  c y s t w a l l s (19). In a l l v e r t e b r a t e s , both the development of the germ c e l l s and the a c t i v i t y o f the L e y d i g and S e r t o l i c e l l s are under the c o n t r o l o f the a n t e r i o r p a r t o f the p i t u i t a r y gland, the adenohypophysis pituitary  (1).  In mammals, removal o f the  (hypophysectomy) leads t o the atrophy o f maturing  6. germ c e l l s , degeneration of the s e m i n i f e r o u s e p i t h e l i u m , and l o s s of secondary sex c h a r a c t e r i s t i c s  (23).  In f i s h ,  d i f f e r e n t s p e c i e s show a v a r i e t y of degrees of degeneration a f t e r hypophysectomy of  spermatogonia  hypophysectomy  (16).  In salmonids, the t r a n s f o r m a t i o n  i n t o primary spermatocytes  i s blocked a f t e r  (24).  In mammals, the p i t u i t a r y i s s t i m u l a t e d by a hypothalamic p e p t i d e r e l e a s i n g hormone t o produce t r o p i n s , FSH and LH  the g l y c o p r o t e i n gonado-  (25), and the presence of both FSH  LH i s r e q u i r e d f o r the maintenance of spermatogenesis  and (1).  The t a r g e t c e l l and a c t i o n of each of these hormones has been mentioned of  e a r l i e r i n this Introduction.  spermatogenesis  (17).  In f i s h ,  i s a l s o c o n t r o l l e d by the p i t u i t a r y gland  However, p u r i f i c a t i o n s t u d i e s of p i t u i t a r y  from both carp  the process  (26) and salmon  extracts  (27) have not r e v e a l e d the  presence of more than one gonadotropin r e s p o n s i b l e f o r t e s t i s development i n f i s h . The major e x t e r n a l f a c t o r r e g u l a t i n g s e x u a l m a t u r a t i o n i n f i s h i s the p h o t o p e r i o d p e r i o d of spermatogenic  (28).  In salmonids, the annual  a c t i v i t y begins i n l a t e s p r i n g  and  takes p l a c e over about 6 months, a l t e r n a t i n g w i t h a p e r i o d of  i n v o l u t i o n and r e c o n s t i t u t i o n of t e s t i c u l a r t i s s u e d u r i n g  the w i n t e r months i n p r e p a r a t i o n f o r the next period  (18).  differentiation  In t r o u t , a p p r o p r i a t e m a n i p u l a t i o n of the  number of hours of d a y l i g h t per day can be used to a c c e l e r a t e spermatogenesis  (28).  7.  Another method of a c c e l e r a t i o n of the n a t u r a l r a t e of spermatogenesis,  i n the t r o u t t e s t i s , i s by a d m i n i s t e r i n g  a s e r i e s of i n j e c t i o n s of salmon p i t u i t a r y extracts  (29, 30).  gonadotropin  I t has been shown t h a t such a s e r i e s of  i n j e c t i o n s i n t o s e x u a l l y immature male rainbow t r o u t , kept i n water of the a p p r o p r i a t e temperature,  causes the growth  and d i f f e r e n t i a t i o n of the immature t e s t e s  (29, 30).  r e s u l t s i n a 500-1000 f o l d i n c r e a s e i n t e s t e s wet The  induced spermatogenesis  This  weight.  b i o c h e m i c a l l y and m o r p h o l o g i c a l l y  p a r a l l e l s t h a t observed i n n a t u r a l l y maturing t e s t i s growth occurs a t twice the r a t e  testis,  (31).  but  This i s  probably due t o the l a r g e r p r o p o r t i o n of c y s t s induced to d i f f e r e n t i a t e by the a r t i f i c i a l l y high dosage of gonadotropin. The induced t r o u t t e s t i s d i f f e r e n t i a t i o n can be i n i t i a t e d a t any time of the year, p r o v i d i n g a convenient system study of b i o c h e m i c a l changes d u r i n g In  f o r the  spermatogenesis.  a r t i f i c i a l hormonally-induced  spermatogenesis  t r o u t , the t e s t e s weight d o u b l i n g time i s about  in  1 week.  T h i s r e s u l t s i n a l o g a r i t h m i c i n c r e a s e i n t e s t e s weight f o r about  8 to 10 weeks u n t i l the f i n a l  development  (4 to 5 weeks).  stages of t e s t i c u l a r  In these f i n a l  stages of spermat-  ogenesis, a l o g a r i t h m i c decrease i n wet weight o c c u r s , due to  germ c e l l  c y t o p l a s m i c l o s s and t i s s u e degeneration  A f t e r 2 weeks of salmon p i t u i t a r y e x t r a c t the predominant germ c e l l  injections,  type i s the spermatogonium-  t y p i c a l p i c t u r e of the c e l l  (18).  A  types p r e s e n t i n t r o u t t e s t i s a t  t h i s stage i s shown i n F i g u r e IA.  Connective t i s s u e separates  8. FIGURE 1  Upper l e f t .  L i g h t micrograph o f a l o b u l e from a t r o u t  t e s t i s a f t e r 2 weeks o f salmon p i t u i t a r y e x t r a c t S e v e r a l c y s t s o f spermatogonia, w i t h s p h e r i c a l c o n t a i n i n g m u l t i p l e n u c l e o l i , are shown. separated by c o n n e c t i v e t i s s u e s . Upper r i g h t .  xl,000  A view o f t r o u t t e s t i s a f t e r 6 weeks o f  f i e l d ) and spermatogonia  (far r i g h t ) .  (center  xl,250  A f t e r 8 weeks o f hormone i n j e c t i o n s ,  t e s t i s c o n t a i n s primary spermatocytes s m a l l e r secondary spermatocytes of  nuclei  Cysts are  hormone i n j e c t i o n s shows primary spermatocytes  Lower l e f t .  injections.  trout  (lower l e f t ) and  (lower r i g h t ) .  synchronous c e l l s undergoing the m e i o t i c  A group  reduction  d i v i s i o n can be seen i n the upper p o r t i o n o f the l i g h t micrograph. Lower r i g h t .  xl,250 Trout t e s t i s a f t e r 12 weeks of hormone  i n j e c t i o n s shows s m a l l , compact spermatids and some l a r g e r e s t i n g spermatogonia.  The breakdown o f c o n n e c t i v e  t i s s u e s between c y s t s i s apparent.  XI,250  9.  10. the c y s t s of spermatogonia.  I t has been e s t i m a t e d t h a t  spermatogonia make up about 10-15% of the t o t a l wet of the immature t e s t i s  (10).  weight  The o t h e r 85-90% i s presumed  to be due to c o n n e c t i v e t i s s u e and lymphatic and b l o o d c e l l s . Spermatogonia  are a c t i v e i n DNA  replication  (32) and  d i v i s i o n r e s u l t i n g i n r a p i d t e s t i s growth.  cell  Differentiation  i n t o primary spermatocytes takes p l a c e d u r i n g weeks 4 to 6 of hormonal  induction  (32).  t h i s stage of development,  C e l l s i n the t r o u t t e s t i s , a t  can be seen i n F i g u r e IB.  primary spermatocytes d i f f e r e n t i a t e i n t o secondary  The  spermato-  c y t e s , which i n t u r n produce e a r l y spermatids, from weeks 6 to 8 of hormonal  induction  (Figure I C ) .  spermatids d i f f e r e n t i a t e .  During weeks 8 to 10  At t h i s stage, the replacement  of h i s t o n e s by protamine has been s t u d i e d i n d e t a i l 35).  (33, 34,  A f t e r 10 t o 12 weeks of hormone i n j e c t i o n s , mature  spermatids and sperm are the predominant  germ c e l l types i n  t r o u t t e s t i s and degeneration of t e s t i s t i s s u e o c c u r s , as seen i n F i g u r e ID.  The presence of spermatogonia a t t h i s  l a t e stage of development  r e f l e c t c e l l s which c o u l d  the next spermatogenic c y c l e  (18).  In salmon,  initiate  i t has been  shown t h a t spermatozoa produced from a s i m i l a r 10 to 12 week s e r i e s of i n j e c t i o n s of p i t u i t a r y gonadotropins i n t o  immature  salmon, were as e f f e c t i v e i n f e r t i l i z i n g eggs as those from n a t u r a l l y maturing f i s h  (36).  Induced spermatogenesis i n t r o u t t e s t i s i s a p a r t i c u l a r l y good system i n which to study developmental changes cells,  s i n c e a t any one g i v e n time t h e r e i s a  i n germ  predominance  11. of  one germ c e l l type.  P r e v i o u s developmental s t u d i e s have  concentrated on spermatid d i f f e r e n t i a t i o n and the replacement on chromatin of h i s t o n e s by protamines  (33, 34, 35).  After  about 7 to 8 weeks of hormone i n j e c t i o n s , protamines are f i r s t observed i n t r o u t t e s t i s  (37).  They are p h o s p h o r y l a t e d  soon a f t e r t h e i r s y n t h e s i s and then b i n d to chromatin to cause i t s i n i t i a l condensation s t i m u l a t e d 1.5  (33) .  A protamine k i n a s e , which i s  -2 f o l d by c y c l i c AMP,  has been p a r t i a l l y  p u r i f i e d from t r o u t t e s t i s a t t h i s stage of development  (38).  Condensation of chromatin proceeds by a s e q u e n t i a l r e l e a s e of h i s t o n e s , which may  involve histone acetylation  (34).  The  f i n a l t r a n s f o r m a t i o n i n t o sperm head nucleoprotamine i s c l o s e l y l i n k e d to protamine d e p h o s p h o r y l a t i o n (35). Developmental  s t u d i e s d u r i n g spermatogenesis have a l s o  been made i n r a t t e s t i s  (14, 15).  p r o t e i n s d u r i n g development  A n a l y s i s of s p e c i f i c  has shown the f o r m a t i o n o f s p e c i f i c  gene products i s a s s o c i a t e d with the appearance c e l l types  (15).  of s p e c i f i c  T h i s i s i l l u s t r a t e d i n the developmental  p a t t e r n s of enzymes shown i n F i g u r e 2B.  During  spermatogenesis  i n r a t t e s t i s , c h a r a c t e r i s t i c s e q u e n t i a l changes i n the r a t e s of  s y n t h e s i s and p h o s p h o r y l a t i o n of s p e c i f i c n u c l e a r a c i d i c  p r o t e i n s have a l s o been observed  (15).  I t has been suggested  t h a t i t i s these p r o t e i n s which c o n t r o l the s e l e c t i v e a t i o n or r e p r e s s i o n of genes d u r i n g spermatogenesis The c y c l i c n u c l e o t i d e s , c y c l i c AMP  and c y c l i c  activ-  (15). GMP,  modulate c e l l u l a r metabolism by s t i m u l a t i n g the p h o s p h o r y l a t i o n  FIGURE 2  Sequential c e l l during  types and t h e i r  characteristics  spermatogenesis  T e s t i c u l a r enzyme a c t i v i t i e s r e l a t e d t o s p e c i f i c c e l l types d u r i n g spermatogenesis r e f e r e n c e 15)  i n the r a t (from  13,  A.  Sequential  Cell  c e l l t y p e s and t h e i r characteristics duri-ng s p e r m a t o g e n e s i s -  Type  Gonocytes  'I Spermatogonia  Spermatocytes a) P r i m a r y b) S e c o n d a r y  Spermatids  Spermatozoa  B.  Characteristics 2n c o n t e n t o f DNA < Little cell proliferation 2n c o n t e n t o f DNA Rapid c e l l p r o l i f e r a t i o n A c t i v e DNA, RNA a n d ; p r o t e i n synthesis  4n c o n t e n t o f DNA 2n c o n t e n t o f DNA; ho DNA replication, RNA synthesis decreases c o n t e n t o f DNA DNA and RNA s y n t h e s i s c e a s e s H i s t o n e s r e p l a c e d by p r o t a m i n e s F l a g e l l u m forms n  n c o n t e n t o f DNA Sperm s p e c i f i c enzymes  appear  T e s t i c u l a r enzyme a c t i v i t i e s r e l a t e d t o s p e c i f i c • t y p e s d u r i n g spermatogenesis i n the r a t  cell  Spermatogonia Spermatids G o n o c y t e s \Spermatocytes Spermatozoa 400  800  r 300h  Heoo  200  H40O  c n  Q  1001-  H2oo c  100  Age,  days  120  200  14. of p r o t e i n s  (39).  Due  to the importance of  phosphorylation  of n u c l e a r p r o t e i n s i n the c o n t r o l of g e n e t i c  expression  d u r i n g spermatogenesis, the r o l e of c y c l i c AMP may  be c r u c i a l  B. The  f o r male germ c e l l  r o l e of c y c l i c AMP  and  cyclic  c y c l i c GMP  i n metabolism i n 1956,  and h i s a s s o c i a t e s , as the low molecular f a c t o r which mediated the i n t r a c e l l u l a r  by  Sutherland  weight, h e a t - s t a b l e effect  (40), an e x t e n s i v e  of  epinephrine  literature  been amassed i n d i c a t i n g the importance of c y c l i c AMP c e l l u l a r metabolic  r e g u l a t o r i n many t i s s u e s and  throughout the animal kingdom. Sutherland's  group, c y c l i c AMP  was  1  c y c l a s e , was  defined i n a l a t e r  same group, i n 1962 r e q u i r e d ATP NaF,  and,  different  and Mg  (42, 43). 2+ , was  organisms  which formed  of h y d r o l y s i s  to c y c l i c AMP,  by  manner, was  stimulated stimulated  by by  hormones.  of c y c l i c AMP  could a l t e r  the m e t a b o l i c  by Krebs and h i s a s s o c i a t e s , i n 1968,  s t a t e was  was  concentration determined  d u r i n g an i n v e s t i g a t i o n  on the r e g u l a t i o n of g l y c o g e n o l y s i s i n s k e l e t a l AMP  adenylate  They showed t h a t the enzyme  A mechanism by which a change i n the c e l l u l a r  Cyclic  (41).  s e r i e s of papers from the  p a r t i c u l a t e , was  in a tissue-specific  as a  shown to be degraded by  adenosine 5 -monophosphate as the product enzymatic c o n v e r s i o n of ATP  has  In the i n i t i a l papers from  an enzyme, c y c l i c n u c l e o t i d e phosphodiesterase,  The  GMP  differentiation.  Since the d i s c o v e r y of c y c l i c AMP,  and glucagon on r a t l i v e r  and  muscle  (44).  found to a c t i v a t e a c y c l i c AMP-dependent p r o t e i n  15. kinase by b i n d i n g to the r e g u l a t o r y subunit of the r e g u l a t o r y c a t a l y t i c holoenzyme causing d i s s o c i a t i o n of the r e g u l a t o r y subunit from the c a t a l y t i c subunit which was active  (44).  fully  C y c l i c AMP-dependent p r o t e i n k i n a s e s i n other  t i s s u e s have the same method of a c t i v a t i o n reviews  then  on c y c l i c AMP  metabolism e x i s t  (45, 46).  (47, 48,  49)  Many and  the  t o p i c w i l l not be f u r t h e r d i s c u s s e d except w i t h r e g a r d to spermatogenesis. F o l l o w i n g the d i s c o v e r y of c y c l i c AMP r o l e i n c e l l u l a r metabolic  and  i t s important  r e g u l a t i o n , evidence was  sought  f o r the e x i s t e n c e of other n a t u r a l l y o c c u r r i n g c y c l i c tides 1963  (50, 51).  C y c l i c GMP  (52) and has  was  i s o l a t e d from r a t u r i n e , i n  s i n c e been found  and p l a n t kingdom  (53, 54)  i n a l l p h y l a of the  In eucaryotes,  c o n c e n t r a t i o n s of c y c l i c GMP  have been found  (57), and  to be e l e v a t e d  several lectins  However, d i r e c t s t i m u l a t i o n of guanylate  diesterases kinases  (58, 59), c y c l i c GMP  (60, 61), and  (62, 63)  (56),  (7).  c y c l a s e s by any  these agents has not been c o n v i n c i n g l y demonstrated Guanylate c y c l a s e s  of  (54).  s p e c i f i c phospho-  c y c l i c GMP-dependent p r o t e i n  have a l s o been i d e n t i f i e d and  Guanylate c y c l a s e s d i f f e r from adenylate t h e i r s u b s t r a t e requirement  f o r GTP,  studied. cyclases i n  their divalent cation  2+ p r e f e r e n c e f o r Mn  animal  intracellular  i n response to a c e t y l c h o l i n e (55), p r o s t a g l a n d i n - F 2 a f i b r o b l a s t growth f a c t o r  nucleo-  , t h e i r frequent equal  distribution  between s o l u b l e and p a r t i c u l a t e f r a c t i o n s , and, i n s e n s i t i v i t y to p o l y p e p t i d e hormones (54).  their  Membrane-  bound guanylate c y c l a s e s have been shown to be by T r i t o n X-100  (64), by s p e c i f i c  lipids  activated  (65,  66)  and  by  2+ Ca  (67) .  S o l u b l e guanylate c y c l a s e s can be  by f r e e r a d i c a l and  forming agents, such as nitrosamines  nucleophiles,  (69).  Several  such as a z i d e , n i t r i t e and  c y c l a s e s by p r o s t a g l a n d i n s  c y c l i c GMP  activated  A c t i v a t i o n of guanylate  (and t h e i r m e t a b o l i c endoperoxide  been suggested to be r e l a t e d to t h e i r  a b i l i t y , as w e l l as t h e i r Cyclic  (70).  GMP  l i p i d character  oxidative  (54).  phosphodiesterase a c t i v i t i e s s p e c i f i c  have been r e p o r t e d ,  w h i l e there are other  d i e s t e r a s e s which can h y d r o l y z e  both c y c l i c GMP  and  w i t h v a r y i n g a f f i n i t i e s f o r each c y c l i c n u c l e o t i d e In a number of mammalian t i s s u e s , micromolar of c y c l i c GMP  (68),  hydroxylamine  s o l u b l e guanylate c y c l a s e s can be  spontaneously by a i r o x i d a t i o n  products) has  activated  were shown to s t i m u l a t e  phosphodiesterase-catalyzed  2-3  for phospho-  cyclic (60,  AMP  71).  concentrations  f o l d the r a t e of  h y d r o l y s i s of micromolar conc2+  e n t r a t i o n s of c y c l i c AMP  (72).  i n the presence of a s p e c i f i c  I t has  been shown t h a t Ca  ,  calcium-dependent phosphodi-  e s t e r a s e p r o t e i n a c t i v a t o r (73)  increases  more than c y c l i c AMP  (74).  hydrolysis  c y c l i c GMP  Since  this  hydrolysis calcium-  dependent r e g u l a t o r p r o t e i n i s i n excess of phosphodiesterases i n most t i s s u e s  (75), i t has  a t i o n of c y c l i c GMP u l a r calcium Cyclic  been suggested t h a t the r e g u l -  h y d r o l y s i s i s dependent on the  concentration  intracell-  (54).  GMP-dependent p r o t e i n kinases  have been shown to  be a c t i v a t e d by a somewhat s i m i l a r mechanism to c y c l i c  AMP-  dependent p r o t e i n k i n a s e s , although i n the holoenzyme c o n s i s t s of two u n i t s , w h i l e i n the of each s u b u n i t has  r e g u l a t o r y and  former case, the  (76).  l a t t e r case, each two  catalytic  holoenzyme has  c y c l i c GMP-dependent p r o t e i n k i n a s e s and  activation  inhibits  S e v e r a l e x c e l l e n t reviews on 54)  and  Cyclic  CMP  t o p i c w i l l not  to  spermatogenesis.  and  The  role  avail-  further  i n some mammalian t i s s u e s  (78),  but role  systems.  of c y c l i c AMP  A study of and  covered  i s known about t h i s c y c l i c n u c l e o t i d e ' s  in biological  C.  (77).  metabolism are be  of  c y t i d y l a t e c y c l a s e s have a l s o been  recently identified very l i t t l e  c y c l i c GMP  this  except i n r e l a t i o n  one  c y c l i c TAMP  a c t i v a t i o n of c y c l i c TAMP-dependent p r o t e i n kinases  (53,  only  In many t i s s u e s , a p r o t e i n modulator  been observed, which enhances c y c l i c GMP  able  sub-  c y c l i c GMP  and  intracellular  c y c l i c GMP  i n spermatogenesis  c o n c e n t r a t i o n s of c y c l i c  d u r i n g spermatogenesis i n the  AMP  rat testis  (79)  showed t h a t both c y c l i c n u c l e o t i d e c o n c e n t r a t i o n s were e l e v ated i n i n f a n t i l e t e s t i s c y c l i c GMP the  0.6  time of  germ c e l l s  the  pmol/mg p r o t e i n ) and onset of  the  (20-25 days a f t e r  to decrease u n t i l the (about 35  ( c y c l i c AMP  days a f t e r  time of  20  pmol/mg p r o t e i n ;  both decreased a t  about  meiotic reduction d i v i s i o n birth). the  b i r t h ) and  Cyclic  AMP  appearance of  in  continued spermatids  then i n c r e a s e d s t e a d i l y  in  maturing t e s t i s to a f i n a l a d u l t c o n c e n t r a t i o n of about h a l f the  i n i t i a l concentration.  Cyclic  GMP  continued to  decrease  18. throughout maturation,  to a c o n c e n t r a t i o n about o n e - s i x t h of  the i n i t i a l c o n c e n t r a t i o n .  T h i s study  f l u o r e s c e n t probes f o r c y c l i c AMP technique  i s assumed t o l o c a l i z e  a l s o u t i l i z e d immuno-  and c y c l i c GMP.  This  c y c l i c nucleotides associated  with c y c l i c n u c l e o t i d e r e c e p t o r p r o t e i n s i n the t i s s u e because any  f r e e c y c l i c n u c l e o t i d e should be l o s t d u r i n g the treatment  process  of the u n f i x e d f r o z e n t i s s u e s (80) .  t i l e phase of r a t t e s t i c u l a r seminiferous and  During  the i n f a n -  development, f l u o r e s c e n c e w i t h i n  t u b u l e s was i n t e n s e , f o r both c y c l i c n u c l e o t i d e s ,  n u c l e a r s t a i n i n g p a t t e r n s of c e l l s c l o s e t o the t u b u l a r  w a l l i . e . S e r t o l i c e l l s and spermatogonia, were observed f o r both c y c l i c n u c l e o t i d e s .  T h i s t u b u l a r w a l l s t a i n i n g was  observed throughout maturation, (79). c y c l i c AMP testis  immuriofluorescent  (81), the c y c l i c AMP  shown t o p e r s i s t  In another study, of  localization  i n developing r a t  t u b u l a r s t a i n i n g p a t t e r n was  one month f o l l o w i n g hypophysectomy i n a d u l t  rats,  d e s p i t e undetectable  LH.  In the i n v e s t i g a t i o n i n which both c y c l i c  were l o c a l i z e d ,  c y c l i c AMP  germ c e l l s i n l a t e r meiosis  plasma c o n c e n t r a t i o n s  nucleotides  f l u o r e s c e n c e was not d e t e c t e d i n  stages of t e s t i c u l a r  and spermatid  of FSH and  differentiation,  development, i . e .  w h i l e c y c l i c GMP  observed a s s o c i a t e d with germ c e l l types d u r i n g most of development  (79) .  In p a r t i c u l a r ,  was  stages  c y c l i c GMP was found  a s s o c i a t e d w i t h n u c l e a r elements, s p e c i f i c a l l y with the chromosomes of prophase spermatocytes d u r i n g m e i o s i s .  Cyclic  AMP was not observed on chromosomes a t any stage of development-  These r e s u l t s  suggested n u c l e a r r o l e s f o r both  19.  c y c l i c AMP  and c y c l i c GMP  also, a specific  i n i n f a n t i l e development, and  n u c l e a r r o l e f o r c y c l i c GMP  at meiosis.  Developmental s t u d i e s o f the adenylate and  guanylate  c y c l a s e s , i n r a t t e s t i s , have i n d i c a t e d some c o r r e l a t i o n of their  specific  a c t i v i t i e s w i t h the observed  nucleotide concentrations  (82, 83).  changes i n c y c l i c  S t e i n e r and h i s assoc-  i a t e s showed a c o r r e l a t i o n between t e s t i s  tissue cyclic  c o n c e n t r a t i o n and s o l u b l e . g u a n y l a t e c y c l a s e a c t i v i t y rat testicular  differentiation  (82) .  c l u d e d t h a t e l e v a t e d c y c l i c GMP infantile rat testis reflect, i n d u c i b l e s o l u b l e guanylate  These workers con-  c o n c e n t r a t i o n s i n the  c y c l a s e (82).  Braun and h i s  a s o l u b l e guanylate c y c l a s e  i n r a t t e s t i s , which was most a c t i v e i n p r e m e i o t i c  stages o f development localized  during  i n p a r t , the a c t i v i t y of an  a s s o c i a t e s have a l s o i d e n t i f i e d activity  GMP  (83).  T h i s guanylate c y c l a s e was  to a non-germ c e l l t u b u l a r component  immature r a t t e s t i s  (83).  of the  In r a t t e s t i s d e p l e t e d of germ  c e l l s by X - i r r a d i a t i o n b e f o r e b i r t h ,  two s p e c i f i c  bound adenylate c y c l a s e s have been i d e n t i f i e d  membrane-  (84).  One i s  s e n s i t i v e to FSH and i s p r e s e n t i n the t u b u l a r S e r t o l i and perhaps i n spermatogonial  c e l l s and t h e c Q t h e r : l i s ^ s e n s i t i v e  to LH and i s present i n the L e y d i g c e l l s A distinctive  cells  (85) .  germ c e l l - a s s o c i a t e d s o l u b l e adenylate  c y c l a s e has been d e t e c t e d i n the s e m i n i f e r o u s t u b u l e s of rats,  a t the time o f the appearance o f spermatid c e l l s (83).  The s p e c i f i c  a c t i v i t y of the enzyme i n c r e a s e d about 2 f o l d  d u r i n g the p e r i o d of spermatid development i n t o mature and reached maximal values i n the t e s t i s of a d u l t  rats.  sperm  20. The  timing of the appearance of t h i s s o l u b l e adenylate  i n r a t t e s t i s c o r r e l a t e s with AMP  concentrations  (79).  cyclase  the observed i n c r e a s e s i n c y c l i c  T h i s enzyme was  found to be  insens-  2+ l t i v e to Mg , f l u o r i d e , FSH and LH, and the s t i m u l a t o r y e f f e c t 2+ 2+ of Mn c o u l d be p o t e n t i a t e d by Ca (85). In epididymal 2+ sperm, the Mn  - s e n s i t i v e adenylate  a s s o c i a t e d with  "mitochondrial"  c y c l a s e was  and  found to  be  "microsomal" p a r t i c u l a t e 2+  fractions  (83) .  adenylate  c y c l a s e may  spermatid  c e l l s i n t o spermatozoa, perhaps i n the  of the sperm t a i l previous  I t was  suggested t h a t t h i s Mn  p l a y a r o l e i n the t r a n s f o r m a t i o n  (83).  observation  -sensitive  In support  and  formation  of t h i s statement i s the  t h a t " c y c l i c AMP  f o r f l a g e l l a formation  of  i s a b s o l u t e l y necessary  hence m o t i l i t y , i n c y c l i c  d e f i c i e n t mutants of E s o h e r i c h i a c o l i and  Salmonella  AMPtyphimurium"  (.87) . Sperm of both i n v e r t e b r a t e s and  v e r t e b r a t e s have been 2+ 2+ shown to c o n t a i n equal amounts of Mg - or Mn -dependent 2+ p a r t i c u l a t e adenylate  c y c l a s e , but p a r t i c u l a t e Mn  guanylate c y c l a s e was  s e v e r a l hundred f o l d more a c t i v e i n  i n v e r t e b r a t e sperm than i n v e r t e b r a t e  sperm  (88).  -dependent  The  extremely a c t i v e guanylate c y c l a s e i n sea u r c h i n sperm found to be membrane and  l o c a l i z e d p r i m a r i l y i n the f l a g e l l a r plasma  (89).  herring)  was  Sperm from s e v e r a l s p e c i e s of f i s h  contained  appreciable  but no d e t e c t a b l e guanylate c y c l a s e  (salmon  amounts of adenylate  cyclase  (88).  Developmental s t u d i e s on c y c l i c n u c l e o t i d e  phosphodiester-  ase a c t i v i t i e s d u r i n g spermatogenesis are s c a r c e .  This i s  probably due to the complexity of m u l t i p l e ,  interconvertible  phosphodiesterase a c t i v i t i e s which are observed i n most tissues  (90).  One  i n v e s t i g a t i o n of t o t a l c y c l i c AMP  phospho-  d i e s t e r a s e a c t i v i t i e s d u r i n g r a t spermatogenesis  showed a  5 f o l d i n c r e a s e i n a c t i v i t i e s from day 2 0 to day  50  birth  (91).  remained  C y c l i c AMP  after  phosphodiesterase a c t i v i t i e s  then  constant through to f u l l m a t u r i t y (80 days a f t e r  birth).  In t h i s study, day 20 a f t e r b i r t h was  the f i r s t  sampled, so i n f a n t i l e t e s t i s a c t i v i t i e s were not  time  determined.  S t u d i e s u s i n g the phosphodiesterase i n h i b i t o r s  caffeine,  t h e o p h y l l i n e and papaverine showed an i n c r e a s e i n r e s p i r a t i o n and m o b i l i t y of bovine spermatozoa, by c y c l i c GMP  an e f f e c t a l s o  and d i b u t y r y l c y c l i c AMP  (92).  produced  These s t u d i e s  suggested the presence of an a c t i v e c y c l i c n u c l e o t i d e phosphod i e s t e r a s e i n mammalian sperm.  A highly s p e c i f i c  c y c l i c AMP  found a s s o c i a t e d w i t h s e x u a l  phosphodiesterase was  maturation i n the r a t and r a b b i t In  the r a t , one form of c y c l i c AMP  Km of 6.5  x 10"  5  M was  (93), and i n the ram  i d e n t i f i e d i n immature t e s t i s and a x 10~'  6  M,  i n c o i n c i d e n c e w i t h the appearance of mature sperm  An a c t i v e c y c l i c AMP  phosphodiesterase was  (91)  found i n f i s h sperm  (salmon and h e r r i n g ) but very low c y c l i c GMP detected  (94).  phosphodiesterase, w i t h a  second, h i g h a f f i n i t y enzyme, w i t h a Km of 2.5 appeared  testicular  hydrolysis  was  (88).  The o v e r a l l balance of i n t r a c e l l u l a r c y c l i c n u c l e o t i d e c o n c e n t r a t i o n s may  be r e g u l a t e d by two  f a c t o r s other than  their  synthesis  by t h e c y c l a s e s  phosphodiesterases. ion  and t h e i r  binding  nucleotides  nucleotide  has  (98, 9 9 ) .  studies that,  kinase  the point  2 bovine subunit  activity  cyclic  studying  during  spermato-  Recently,  chromatography  serve  unavailable  i t has been  (Type I k i n a s e  e l u t e d by about  as a  subunit  occurred  Type I enzymes  ence o f Type I I c y c l i c lymphocyte mitogenesis,  by  0.1 M  from  with  A negative  (101).  AMP-dependent p r o t e i n  activities,  Type  throughout d i f f e r e n t i a t i o n ,  NaCl  a l l Type I I  opmental studies of c y c l i c  I kinase  DEAE-  5 r a t and  AMP-dependent p r o t e i n k i n a s e s ,  i n rat testis,  cyclic  of the regulatory  (100).  has been observed  cyclic  for protein  separated  M NaCl)  (97)  demonstrated,  e l u t e d by  autophosphorylation  by t h e c a t a l y t i c  enzymes b u t n o t w i t h  0.2  of  be r e s i s t a n t  as w e l l as r e s i s t a n t t o h y d r o l y s i s by  tissues, that  from b i r t h  i n testis  on t h e b a s i s  and c o u l d AMP  The  protein kinases  AMP-dependent p r o t e i n k i n a s e s  Type I I k i n a s e  proteins  of view of  heart  cyclic  i s unknown.  form o f holoenzyme could  phosphodiesterases. cyclic  cells,  I t has been suggested,  sink, rendering  activation,  of excretion of  binding  AMP-induced d i s s o c i a t i o n  cellulose and  nucleotide  a dephosphorylated  cyclic  using  i n testis  on t h e r e g u l a t i o n o f b o v i n e  nucleotide  AMP  of the role  TAMP-dependent p r o t e i n k i n a s e  genesis  to  of cyclic  been i n v e s t i g a t e d from  cyclic  to inactivatable cyclic  i n the regulation of i n t r a c e l l u l a r  concentrations,  concentration  binding  excret-  proteins (97).  The p o s s i b l e i m p o r t a n c e cyclic  by t h e  These f a c t o r s a r e e x t r a c e l l u l a r  ( 9 5 , 96) a n d i n t r a c e l l u l a r  nucleotide  degradation  remained  while  influ-  on  In develkinase constant  Type I I k i n a s e  increased  from b i r t h  onwards to o b t a i n an a d u l t p r o f i l e  the time of the f i r s t r e d u c t i v e  d i v i s i o n s (98).  at  It is  p o s s i b l e t h a t Type II c y c l i c AMP-dependent p r o t e i n k i n a s e s may  be  important i n the c o n t r o l of e f f e c t i v e  c y c l i c AMP  i n male germ c e l l s d u r i n g  and  intracellular  after  meiosis.  In another i n v e s t i g a t i o n on the changes i n p r o t e i n k i n a s e activities  i n r a t t e s t i s during  spermatogenesis, t o t a l  activities  ( i n c l u d i n g both c y c l i c AMP-dependent and  cyclic  AMP-independent p r o t e i n kinases) decreased at about days a f t e r b i r t h  and  35-45 days of age increase  then a sharp i n c r e a s e o c c u r r e d  (99).  This increase  and  of b i o c h e m i c a l events such as, the c y c l i c AMP  concentrations  10-20 around  c o r r e l a t e d with  i n the c y c l i c AMP-dependent versus c y c l i c  independent p r o t e i n kinase r a t i o ,  kinase  AMP-  a l s o w i t h the  increase  (79) , i n c r e a s e s  in  an  timing  intracellular  i n plasma and  test-  2+ lcular  testosterone  (102), the i n c r e a s e  s o l u b l e adenylate c y c l a s e high a f f i n i t y c y c l i c AMP was  (83), and  i n Mn  -sensitive  a l s o the i n c r e a s e  phosphodiesterase a c t i v i t y  suggested t h a t a l l the t e s t i c u l a r  i n the  (91) .  It  enzyme a c t i v i t y . i n c r e a s e s  were r e l a t e d to the appearance of spermatids  (99).  While very a c t i v e c y c l i c AMP-dependent p r o t e i n k i n a s e s have been observed i n sperm of s e v e r a l mammalian  species,  c y c l i c GMP-dependent p r o t e i n k i n a s e s have not been found No  developmental s t u d i e s of c y c l i c GMP-dependent p r o t e i n  a c t i v i t i e s or of c y c l i c GMP-binding p r o t e i n s , i n t e s t i s any  vertebrate  or i n v e r t e b r a t e ,  have been  reported.  (88)  ?  kinase from  D. O b j e c t i v e s of t h i s t h e s i s The GMP  o b j e c t i v e s of t h i s t h e s i s were to determine c y c l i c  and c y c l i c 7AMP c o n c e n t r a t i o n s i n the t e s t i s of the rainbow  t r o u t Salmo g a i r d n e r i i d u r i n g spermatogenesis and to i n v e s t i gate the developmental modulation of the enzymes r e g u l a t i n g these c o n c e n t r a t i o n s . c y c l i c GMP  P a r t i c u l a r emphasis was  metabolism, due  placed  to the l a c k of i n f o r m a t i o n on  c y c l i c n u c l e o t i d e ' s r o l e i n growth and d i f f e r e n t i a t i o n . p r e v i o u s l y d i s c u s s e d , a r t i f i c i a l hormonally-induced  germ c e l l maturation  events  and  throughout the maturing t e s t i s .  stages  Consequently, changes chronolog-  and c o r r e l a t e d with d i s t i n c t stages of germ c e l l  differentiation.  As  take p l a c e as an ordered sequence of  i n the b i o c h e m i s t r y of the t i s s u e can be f o l l o w e d ically  this  spermato-  genesis i n t r o u t i s a w e l l - d e f i n e d system, i n which the of  on  growth  MATERIALS AND METHODS  A. MATERIALS A l l r a d i o c h e m i c a l s were from New England N u c l e a r . [ H]adenosine 3  activity  1  (specific  39.8 C i p e r mmol) and '[ H] guanosine 3',5'-mono3  phosphate, per  3 5 - m o n o p h o s p h a t e , ammonium s a l t  ammonium s a l t  ( s p e c i f i c a c t i v i t y 8.8 o r 10 C i  mmol) were p u r i f i e d by chromatography  on AG 1-X8 r e s i n ,  as d e s c r i b e d i n Methods. [ H]guanosine 3  purified  by paper chromatography,  [ ^C]adenosine 1  ( s p e c i f i c a c t i v i t y 19 C i per mmol) was as d e s c r i b e d i n Methods.  5 *-monophosphate, diammonium s a l t  (specific  a c t i v i t y 0.422 C i per mmol) was converted t o [ C ] a d e n o s i n e 14  as d e s c r i b e d i n Methods. C y c l i c AMP, d y c l i c  GMP, ATP, GTP, adenosine, guanosine,  phosphocreatine, c r e a t i n e phosphokinase  ( r a b b i t muscle,  Type I) and MIX were a l l purchased from Sigma Chemical Co. Cyclic  n u c l e o t i d e phosphodiesterase (beef heart) was bought  from Calbiochem and made up t o 5 mg/ml i n H 0 and s t o r e d 2  f r o z e n a t -20° i n s m a l l a l i q u o t s . E. c o l i a l k a l i n e phosphatase was purchased from PL Biochemi c a l s and was made up as a 0.37 mg/ml s o l u t i o n i n H 2 O and s t o r e d f r o z e n a t -20° i n s m a l l a l i q u o t s . Bovine serum albumin, F r a c t i o n V, L o t 400427, was bought from Calbiochem. A l l o t h e r chemicals were reagent grade.  26. T r i t o n X-100 (tradename o f Rohm and Haas f o r i s o - o c t y l phenoxy-polyethoxyethanol) was s u p p l i e d by J.T. Baker Co. SCINTREX (tradename  for a universal liquid  c o c k t a i l o f 1:1 T r i t o n X-100:Toluene,  scintillation  c o n t a i n i n g PPO and  POPOP) was purchased from J.T. Baker Co. C y c l i c GMP radioimmunoassay C o l l a b o r a t i v e Research.  k i t s were purchased from  The c y c l i c AMP k i t was bought  from New England N u c l e a r .  B. METHODS 1. Chromatography m a t e r i a l s p r e p a r a t i o n DEAE-cellulose  (Whatman DE 32, m i c r o g r a n u l a r ) was  washed w i t h 0.5 N NaOH (10 volumes), d i s t i l l e d H 0 (20 2  volumes), 1 M sodium a c e t a t e , pH 6.5 (10 volumes), and then e q u i l i b r a t e d w i t h column b u f f e r , i . e . 20 mM sodium pH 6.5 c o n t a i n i n g 4 mM  acetate,  2-mercaptoethanol.  BioRad AG 1-X8, 200-400 mesh, c h l o r i d e form, r e s i n was prepared as d e s c r i b e d p r e v i o u s l y  (103).  F o r c y c l i c GMP  sample p u r i f i c a t i o n , the AG 1-X8 r e s i n was then poured  into  columns (0.7 x 3.5 cm) and prewashed w i t h 5 N f o r m i c a c i d (15 ml), f o l l o w e d by d i s t i l l e d H 0 2  (15 m l ) .  F o r the  c y c l i c AMP sample p u r i f i c a t i o n s , the AG 1-X8 r e s i n was prewashed w i t h 5 N f o r m i c a c i d  (10 volumes) f o l l o w e d by  d i s t i l l e d H 0, i n a l a r g e g l a s s - s i n t e r e d 2  For  funnel.  c y c l i c GMP p h o s p h o d i e s t e r a s e assays, the AG 1-X8  r e s i n was prepared as d e s c r i b e d p r e v i o u s l y resuspended i n d i s t i l l e d H 0 2  (103) and'was  (1:2 v/v r e s i n t o H 0) a f t e r 2  a d d i t i o n o f 1 N formic a c i d t o a pH o f 2.5. AMP phosphodiesterase  For c y c l i c  assays, the AG 1-X8 r e s i n was r e s u s p -  ended i n d i s t i l l e d H 0  (1:2 v/v r e s i n t o H 0) a f t e r a d d i t i o n  2  2  o f g l a c i a l a c e t i c a c i d t o a pH o f 3.7.  2. Paper chromatography A l l paper chromatography was on Whatman 40 paper u s i n g the descending  system a t 23°.  The s o l v e n t used was i s o -  propanol:NH :H 0 (7:1:2 v / v / v ) . 3  2  N u c l e o t i d e s and n u c l e o s i d e s  were l o c a t e d by shortwave u l t r a v i o l e t absorbance u s i n g a hand-held M i n e r a l i g h t from U l t r a v i o l e t Products, U l t r a v i o l e t absorbing  California.  r e g i o n s which contained r a d i o a c t i v e  compounds were c u t out and e l u t e d by the method o f Heppel (104).  The e l u a t e  (0.5 ml) was added t o 5 ml o f TTP  s c i n t i l l a t i o n f l u i d and the r a d i o a c t i v i t y determined i n a liquid scintillation  spectrometer.  3. P r e p a r a t i o n o f [ **C] adenosine from 1  [ C]AMP ltf  100  [^CjAMP  ( s p e c i f i c a c t i v i t y 0. 422 C i p e r mmol),  y l (0.0018 mg) was incubated w i t h E. c o l i  phosphatase  alkaline  (0.05 mg/ml) and 8 mM T r i s . H C l , pH 7.8, i n a  t o t a l volume o f 130 y l , a t 30° f o r 135 min.  This  was cochromatographed on paper w i t h adenosine  (1 A 5 ^ u n i t )  and AMP  (1 A 5 ^ u n i t ) and developed 2  previous s e c t i o n .  2  as d e s c r i b e d i n the  Using these c o n d i t i o n s there was 100%  c o n v e r s i o n o f [ *C]AMP t o [ ^C] adenosine. ll  mixture  1  28. 4. F i s h husbandry Immature rainbow t r o u t Salmo g a i r d n e r i i , 17-20 cm i n length,  from the Sun V a l l e y T r o u t Farm i n M i s s i o n ,  Columbia, were kept i n 200 l i t e r , aquaria.  British  self-cleaning, fibreglass  The r e c i r c u l a t i n g water was aerated  and kept a t  9-12° and a 13 hour l i g h t and 11 hour dark c y c l e was used. The ets,  f i s h were f e d three  times a week, on Oregon M o i s t P e l l -  from the Moore-Clark CO., La Conor, Washington. Immature male t r o u t were s e l e c t e d by laparotomy and  were i n j e c t e d i n t r a p e r i t o n e a l l y , twice a week, w i t h 0.1 ml of a crude salmon p i t u i t a r y e x t r a c t .  I n j e c t i o n s were  continued f o r 12 weeks, d u r i n g which time, a t r e g u l a r i n t e r v a l s , f i s h were k i l l e d , by a blow on the head, b e f o r e  testes  were d i s s e c t e d .  5. Laparotomy procedure f o r sex d e t e r m i n a t i o n of f i s h The 62 mg/1  f i s h were a n a e s t h e t i z e d  i n an aerated  t r i c a i n e methane s u l f o n a t e  minutes and then p l a c e d  solution of  (MS 222) f o r s e v e r a l  i n a nylon suspension rack.  m a i n t a i n a n a e s t h e s i a and oxygen supply, a tube was in  the mouth and a s o l u t i o n of MS 222 (50 mg/1)  u a l l y f l u s h e d over the g i l l s .  was  A ventral mid-line  To placed continincision  was then made, s t a r t i n g 0.5 cm p o s t e r i o r t o the p e c t o r a l f i n s and running c a u d a l l y  f o r 5 cm.  The f l a p s o f t i s s u e  on e i t h e r s i d e o f the i n c i s i o n were extended u s i n g and  a b l u n t probe used to examine the gonads.  were i d e n t i f i e d by t h e i r t r a n s l u c e n t  retractors  The males  t h r e a d l i k e t e s t e s , each  about 8 cm long and 1 mm  thick.  The o v a r i e s i n the female  t r o u t were y e l l o w i s h and were c h a r a c t e r i z e d by a marked t a p e r i n g from the a n t e r i o r to the p o s t e r i o r end. wound was  c l o s e d w i t h 5-0  The  s i l k s u t u r e s (Ethicon) and the  f i s h r e t u r n e d to the a q u a r i a .  The whole procedure took  no more than 5 minutes and r o u t i n e l y t h e r e was  9 0-100%  survival.  6. P i t u i t a r y e x t r a c t p r e p a r a t i o n P i t u i t a r i e s were c o l l e c t e d from f r e s h l y - k i l l e d , spawning  chinook salmon, Oncorhynchus tschawytscha, i n l a t e Sept-  ember, a t the Green R i v e r Hatchery, Auburn,  Washington.  A core, c o n t a i n i n g the b r a i n and the u n d e r l y i n g p i t u i t a r y gland, was  removed from the salmon head.  The  pituitary  gland, a s m a l l s p h e r i c a l g l a n d , average weight 85 mg,  was  scooped out o f the core, f r o z e n immediately w i t h s o l i d and s t o r e d a t -80°.  Three volumes o f s a l i n e  C0 , 2  (1.25%) were  added t o thawed p i t u i t a r y glands and the mixture homogenized i n a Waring b l e n d e r , a t h i g h speed, f o r 2 min. was  then c e n t r i f u g e d a t 11,250 rpm  SS 34 r o t o r . aliquots.  The supernatant was  The  solution  f o r 15 min i n a S o r v a l l s t o r e d a t -20° i n 5 ml  B e f o r e i n j e c t i o n , the thawed e x t r a c t was  clari-  f i e d by c e n t r i f u g a t i o n a t maximum speed on an I n t e r n a t i o n a l bench top c e n t r i f u g e f o r 1-2  min.  7. C y c l i c n u c l e o t i d e e x t r a c t i o n Testes were d i s s e c t e d from hormonally-induced t r o u t  30. a t weekly i n t e r v a l s , over a 10-12 time, and  immediately p l a c e d  were s t o r e d f r o z e n a t -80°.  week t e s t i s  i n l i q u i d nitrogen. Two  determinations,  placed  each sample was  i n 1% p e r c h l o r i c a c i d  contained  0.5  t i s s u e was  and  1 M T r i s . H C l , pH M and  7.5  about 5 volumes) which  cm),  adjusted  to pH  7.0  then a p p l i e d to an AG  formic a c i d (10 ml)  earlier  the column was  c y c l i c GMP  The  (Appendix  ; Figure  19A)  evaporated a t 36°, r e d i s s o l v e d i n d i s t i l l e d H 0 2  evaporated a t 36°.  The  r e d i s s o l v e d i n 50 mM  sodium a c e t a t e ,  of  [ H ] c y c l i c GMP 3  c y c l i c GMP  f r a c t i o n was  60-70%.  conditions  above, c y c l i c GMP  shown by  100%  w i t h c y c l i c GMP  column  i n Methods.  recovery  of  on paper.  pH  6.2  N and  acid eluate, was  rotary  and  re-rotary  finally  (1 ml).  Total  through the e x t r a c t i o n and p u r i f i -  c a t i o n procedure was described  1-X8  The  washed w i t h 0.1  5 N formic  KOH  concentration  f o l l o w e d by 2 N formic a c i d (12 ml)  then 5 N formic a c i d (14 ml).  min.  with 6 N  added to a f i n a l b u f f e r  prewashed as d e s c r i b e d  A f t e r sample a d s o r p t i o n ,  recovery  The  homogenizer, f o r  r e c e n t r i f u g e d a t 10,000xg f o r 20 min.  supernatant s o l u t i o n was  containing  and  the homogenate c e n t r i f u g e d a t 10,000xg f o r 10  supernatant s o l u t i o n was  (0.7 x 3.5  cyclic  (10 C i per mmol).  3  The  of 0.1  q u i c k l y weighed  (1 ml;  pmol [ H ] c y c l i c GMP  F o r the  homogenized i n a P o t t e r - E l v j h e m  30 sec, and  Samples  d i f f e r e n t procedures were  used f o r e x t r a c t i o n of c y c l i c n u c l e o t i d e s . GMP  maturation  Under the r o t a r y was  [ H ] c y c l i c GMP 3  evaporation  not degraded,  as  cochromatographing  For the c y c l i c AMP  determinations,the  were e x t r a c t e d by homogenization i n 5% TCA c o n t a i n i n g 0.25  pmol [ H ] c y c l i c AMP  (39.8  c e n t r i f u g e d a t 10,000xg f o r 10 min.  The  3  e x t r a c t e d 4 times with d i e t h y l ether (This was  cyclic  nucleotide  (1 ml;  5 volumes)  C i per mmol) and supernatant  (about 6 ml;  4 volumes)  a more r a p i d method of a c i d removal than the KClOi*  p r e c i p i t a t i o n method.) a p p l i e d to AG  1-X8  The  columns.  r e s u l t i n g aqueous l a y e r The  i n Methods. (10 ml)  The  column was  then washed with  0.1  N  formic a c i d e l u a t e , c o n t a i n i n g c y c l i c AMP  T o t a l recovery  of  [ H ] c y c l i c AMP 3  p u r i f i c a t i o n procedure was  The  (Appendix  r o t a r y evaporated as d e s c r i b e d  earlier formic  f o l l o w e d by 2 N formic a c i d (12 ml).  F i g u r e l ^ A ) , was  was  r e s i n had been prewashed  w i t h 5 N formic a c i d by a batch method, as d e s c r i b e d  acid  was  2 N  ;  above.  through the e x t r a c t i o n and  60-70%.  8. Determination of c y c l i c n u c l e o t i d e c o n c e n t r a t i o n s  by  r adio immuno ass ay C y c l i c nucleotide concentrations  were determined by  the radioimmunoassay method developed by S t e i n e r  (112 )  using radioimmunoassay k i t s purchased from C o l l a b o r a t i v e Research f o r c y c l i c GMP f o r c y c l i c AMP  assays and by New  assays.  C y c l i c GMP  i n the range of 0.5-10 pmol. the range of 1-20  pmol.  was  r o u t i n e l y determined  C y c l i c AMP  A l l kits  England Nuclear  (except  was  determined i n  1 from C o l l a b -  o r a t i v e Research) gave 40-50% maximum b i n d i n g o f GMP  or  1 2 5  I - c y c l i c AMP  1 2 5  I-cyclic  to t h e i r r e s p e c t i v e a n t i b o d i e s  and  32. l i n e a r standard curves over the ranges d e t a i l e d above. samples were assayed i n d u p l i c a t e , a t 2-3 p o r t i o n o f each sample was treatment.  All  d i l u t i o n s and a  s u b j e c t e d to phosphodiesterase  The c o n d i t i o n s f o r the phosphodiesterase hydro-  l y s i s were 0.025 u n i t of beef h e a r t c y c l i c n u c l e o t i d e phosp h o d i e s t e r a s e , 30 mM  T r i s . H C l , 6 mM  MgCl2 and sample incubated  i n a f i n a l volume o f 50 y l f o r 20 min a t 37°. was  terminated by h e a t i n g i n a temperature  f o r 1 min.  The  reaction  heat b l o c k a t 100°  Samples w i t h c y c l i c n u c l e o t i d e "blank" v a l u e s ,  a f t e r phosphodiesterase treatment, above 10% of the u n t r e a t e d sample, were not used.  About 5% of samples had  unacceptable  blanks.  9. T i s s u e p r e p a r a t i o n f o r c y c l i c n u c l e o t i d e phosphodiesterase assay Testes were e x c i s e d from hormonally-induced t r o u t  and  were p l a c e d on i c e , weighed and minced w i t h a s c a l p e l .  They  were then homogenized i n 4 volumes of b u f f e r , by 10 passes of a motordriven p e s t l e i n a l o o s e - f i t t i n g homogenizer. 0.25  Homogenizing b u f f e r was  M sucrose, 4 mM  2-mercaptoethanol,  glass-Teflon  10 mM 2 mM  T r i s . H C l , pH MgCl2  7.5,  ( b u f f e r A)  f o r a l l t o t a l phosphodiesterase a c t i v i t i e s and D E A E - c e l l u l o s e separations.  The homogenate was  f i l t e r e d through 4 l a y e r s  of c h e e s e c l o t h to remove c o n n e c t i v e t i s s u e . Sperm were c o l l e c t e d from f r e s h l y - k i l l e d f i s h by a p p l y i n g manual p r e s s u r e from the a n t e r i o r abdomen toward p o s t e r i o r l y l o c a t e d g e n i t a l - a n a l opening.  the  Sperm were  homogenized i n 9 volumes of b u f f e r A and f i l t e r e d 4 layers of cheesecloth. expressed  through  T o t a l sperm a c t i v i t i e s were  as pmol c y c l i c n u c l e o t i d e hydrolyzed / min / mg  protein.  P r o t e i n i n sperm suspensions  was 1.3% o f the  wet weight, compared w i t h 8-12% of the wet weight i n immature t e s t i s samples, and 5-8% of the wet weight i n maturing  testis.  Since i t was necessary to  assay a l a r g e volume o f sperm suspension, a p r o t e i n d e t e r m i n a t i o n , the t u r b i d i t y have i n c r e a s e d the apparent  i n order to make  i n such assays  may  protein concentration.  10. D E A E - c e l l u l o s e column chromatography of c y c l i c n u c l e o t i d e phosphodiesterase  activities  C h e e s e c l o t h - f i l t e r e d homogenates o f t r o u t t e s t e s i n b u f f e r A were d i r e c t l y columns 20 mM  a p p l i e d to D E A E - c e l l u l o s e  (DE 32)  (0.5 x 10.5 cm or 0.8 x 22 cm) p r e - e q u i l i b r a t e d i n  sodium a c e t a t e , pH 6.5, b u f f e r c o n t a i n i n g 4 mM  2-mercaptoethanol.  A f t e r sample a d s o r p t i o n , columns were  washed w i t h s e v e r a l column volumes o f t h i s b u f f e r . i n i t i a l wash contained no phosphodiesterase linear  g r a d i e n t from 20 mM  The  activity.  t o 1 M sodium a c e t a t e , pH  A 6.5,  was then a p p l i e d , w i t h a flow r a t e of 2 6 ml per hour and a t o t a l g r a d i e n t volume o f 20 0 ml. was 4.5 ml.  Column f r a c t i o n  volume  (The e x c e p t i o n to t h i s was i n F i g u r e 11A, i n  which case there was a 100 ml t o t a l g r a d i e n t volume and a 2.2 ml column f r a c t i o n  volume.)  Each c o l l e c t i o n  tube  c o n t a i n e d 50 u l , (or 25 y l i n the case o f the 2.2 ml column  f r a c t i o n volume) o f a 0.2 M MgCl  2  and 100 mg/ml bovine serum  albumin s o l u t i o n to give a f i n a l c o n c e n t r a t i o n o f 1.8 mM M g C l  2  i n the 4.5 ml  and 0.9 mg bovine serum albumin/ml.  (In  the absence o f t h i s s t a b i l i z i n g s o l u t i o n , phosphodiesterase a c t i v i t i e s i n DEAE-cellulose  column f r a c t i o n s were very low.)  F r a c t i o n s c o n t a i n i n g phosphodiesterase a c t i v i t y were pooled and  dialyzed against  10 mM T r i s . H C l , pH 7.5, 1 mM M g C l , 2  0.4 mM d i t h i o t h r e i t o l i n 50% g l y c e r o l (v/v).  The d i a l y s a t e s  were a l i q u o t e d i n t o small volumes and s t o r e d a t -20° f o r f u t u r e k i n e t i c analyses.  F r a c t i o n s were s t a b l e w i t h  respect  to c y c l i c AMP h y d r o l y s i s , but n o t f o r c y c l i c GMP h y d r o l y s i s , f o r a t l e a s t 6 months. For D E A E - c e l l u l o s e natant and s o n i c a t e d  chromatography o f 100,000xg super-  10 0,000xg p e l l e t t r o u t t e s t i s f r a c t i o n s  (Figure 13A and B), the t e s t e s from 2 f i s h  (630 mg/fish)  were e x c i s e d and homogenized i n 7 ml o f b u f f e r A and then passed through 4 l a y e r s o f c h e e s e c l o t h .  From t h i s homo-  genate, 3 ml was d i r e c t l y a p p l i e d t o a DEAE-cellulose and  the phosphodiesterase  put a s i d e was  activities  column  f r a c t i o n a t e d , 1 ml was  f o r t o t a l a c t i v i t y assays and the remaining 3 ml  c e n t r i f u g e d a t 100,000xg f o r 1 hr.  The supernatant was  p i p e t t e d o f f and s t o r e d a t -20° f o r f u t u r e chromatography.  DEAE-cellulose  The 100,000xg p e l l e t was washed w i t h  b u f f e r A, rehomogenized and r e c e n t r i f u g e d a t 100,000xg f o r 1 hr.  The r e s u l t i n g supernatant was d i s c a r d e d  and the  p e l l e t resuspended i n the o r i g i n a l volume o f b u f f e r A. A f t e r s o n i c a t i o n a t 30 sec/ml by a Branson s o n i f i e r  fitted  with a m i c r o t i p  (Model W 185),  P l a i n v i e w , N.Y.,  of 5, the s o l u b i l i z e d p e l l e t was f o r 20 min -20°  and  the supernatant  of D E A E - c e l l u l o s e  r e c e n t r i f u g e d at 30,000xg  p i p e t t e d o f f and  f o r f u t u r e DEAE-cellulose  stored at  chromatography.  columns was  Preparation  as d e s c r i b e d f o r t o t a l  homogenates, except t h a t column s i z e was the t o t a l g r a d i e n t volume was 2 ml were c o l l e c t e d .  at a s e t t i n g  100  ml.  0.8  testis  x 16 cm,  and  Column f r a c t i o n s o f  F r a c t i o n tubes c o n t a i n e d  25 y l of  the  s t a b i l i z i n g s o l u t i o n d e s c r i b e d above. A l l DEAE-cellulose  columns and r e l a t e d o p e r a t i o n s were  c a r r i e d out at 4°.  11. C y c l i c n u c l e o t i d e phosphodiesterase The  two-step assay f o r c y c l i c n u c l e o t i d e phosphodiest-  erase a c t i v i t y was 103,  assay  105)  s i m i l a r to t h a t d e s c r i b e d p r e v i o u s l y  w i t h the m o d i f i c a t i o n s of the use o f EV  (60,  coli  a l k a l i n e phosphatase, i n s t e a d of snake venom n u c l e o t i d a s e , f o r the second i n c u b a t i o n , and  a s m a l l e r assay volume of  100 y l . An  a p p r o p r i a t e a l i q u o t o f enzyme, to o b t a i n 10-40%  c o n v e r s i o n of s u b s t r a t e to product, to 30 min  at 30° i n 10 mM  MgCl2/ 4 mM 5 x 10"  8  M  was  T r i s . H C l , pH  incubated 7.5  or 8.0,  for 5 2  mM  2-mercaptoethanol b u f f e r c o n t a i n i n g about [ H ] c y c l i c AMP 3  volume of 100  yl.  or  [ H ] c y c l i c GMP, 3  in a total  Assays were i n s i l i c o n i z e d  tubes 2+  (10 x 1.2 (100  yM)  cm). was  When i n d i c a t e d , EGTA (250 yM)  i n c l u d e d i n t h i s volume.  or  For h i g h e r  Ca substrate  c o n c e n t r a t i o n s , the i n d i c a t e d amount o f u n l a b e l e d c y c l i c n u c l e o t i d e was i n c l u d e d .  Chromatographic  p r o f i l e s were  assayed a t pH 8.0 f o r maximum assay a c t i v i t y . assays were a t pH 7.5 t o approximate  A l l other  physiological  conditions.  The r e a c t i o n was terminated by h e a t i n g i n a temperature heat b l o c k a t 100° f o r 90 sec.  (For the f i n a l Table V.  c y c l i c GMP phosphodiesterase assays, t e r m i n a t i o n was f o r 60 sec a t 100° s i n c e a lower blank was thus obtained.) Tubes were c o o l e d on i c e , r e - e q u i l i b r a t e d t o 30° and 10 y l of E_. c o l i a l k a l i n e phosphatase f u r t h e r 2 0 min i n c u b a t i o n .  (0.37 mg/ml) added f o r a  T h i s r e a c t i o n was terminated  by the a d d i t i o n o f 1 ml o f AG 1-X8 r e s i n s l u r r y a t the a p p r o p r i a t e pH i . e . pH 2.5 f o r c y c l i c GMP assays and pH 3.7 f o r c y c l i c AMP assays. s t i r r e d w i t h a magnetic made.  These r e s i n s l u r r i e s were  s t i r r e r w h i l e a d d i t i o n s were being  Assay tubes were l e f t t o stand f o r a t l e a s t 10 min  a f t e r r e s i n s l u r r y a d d i t i o n s and then c e n t r i f u g e d a t 3,000xg f o r 5 min.  A 0.4 ml a l i q u o t o f the r e s u l t i n g supernatant  s o l u t i o n was added to 5 ml o f TTP o r a 0.6 ml a l i q u o t t o 5 ml o f SCINTREX s c i n t i l l a t i o n  f l u i d s and the r a d i o a c t i v i t y  determined. Recoveries o f n u c l e o s i d e s i n the supernatant were determined by [ H] guanosine 3  fraction  o r [ "*C] adenosine recovery 1  and found t o be 80% and 90% r e s p e c t i v e l y .  Resin s l u r r i e s  a t the above pHs gave blanks o f the r e s p e c t i v e unbound [ H ] c y c l i c n u c l e o t i d e o f 2-14% f o r c y c l i c GMP and 2-8% f o r 3  c y c l i c AMP.  Blanks were c o n s i s t e n t w i t h i n assays to - 0.5%,  but v a r i e d between assays w i t h i n c r e a s i n g blanks  correlating  t o i n c r e a s i n g l e n g t h o f time a f t e r p u r i f i c a t i o n o f the c y c l i c n u c l e o t i d e s on AG 1-X8 c y c l i c GMP  columns.  [ H] 3  Due to the low  phosphodiesterase a c t i v i t i e s i n column f r a c t i o n s  a f t e r D E A E - c e l l u l o s e s e p a r a t i o n s , a combination o f 0.2 ml o f 95% e t h a n o l p l u s 0.8 ml o f AG 1-X8 r e s i n s l u r r y was to terminate c y c l i c GMP  used  assays, s i n c e the recovery o f guano-  s i n e under these c o n d i t i o n s was 90-95%, w h i l e the c y c l i c blank was unchanged.  GMP  D u p l i c a t e s were l e s s c o n s i s t e n t w i t h  the a d d i t i o n o f a l c o h o l , so t o t a l c y c l i c GMP measured u s i n g the 1 ml of AG 1-X8  a c t i v i t i e s were  r e s i n s l u r r y a t pH 2.5,  to terminate the second r e a c t i o n , and a c t i v i t i e s were c o r r e c t e d f o r the 80% guanosine r e c o v e r y .  C y c l i c AMP  activities  were a l s o c o r r e c t e d f o r the 90% adenosine r e c o v e r y .  12. T i s s u e p r e p a r a t i o n f o r guanylate c y c l a s e assay T r o u t t e s t e s were homogenized i n 10 volumes o f 10 mM T r i s . H C l , pH 7.5, 0.25 M sucrose, 4 mM e t h a n o l and 1 mg/ml bovine serum albumin  (2-3 ml) 2-mercapto-  (buffer B).  Homogenization c o n d i t i o n s were the same as those d e s c r i b e d f o r c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t i e s .  The  homogenate was c e n t r i f u g e d a t 100,000xg f o r 1 h r a t 4°, and the supernatant p i p e t t e d o f f and s t o r e d f r o z e n a t -20°. The p e l l e t was resuspended  i n 1 ml o f b u f f e r B and t h i s  then c e n t r i f u g e d a t 100,000xg  f o r 1 hr a t 4°.  supernatant was d i s c a r d e d and the f i n a l p e l l e t  The  was  second  resuspended  i n 1 ml o f b u f f e r B and s t o r e d f r o z e n a t -20°. Before  38. guanylate c y c l a s e assay, the thawed p e l l e t suspension was homogenized as d e s c r i b e d  13.  Guanylate c y c l a s e The  above.  assay  i n c u b a t i o n mixture f o r the assay o f guanylate  l a s e a c t i v i t i e s contained  50 mM T r i s . H C l b u f f e r , pH 7.5,  2 mM M n C l , 1.6 mM MIX, 1 mM c y c l i c GMP, 0.1-0.25 mM 2  GTP  (specific activity  cpm p e r assay t u b e ) ,  cyc-  [a P] 3 2  10-30 cpm p e r pmol; 300,000-400,000  15 mM phosphocreatine, 12.25 u n i t s o f  c r e a t i n e phosphokinase,  [ H ] c y c l i c GMP 3  (25 n C i ; 13,000 cpm  per assay tube) and 40-250 yg o f t e s t i s p r o t e i n , i n a f i n a l volume o f 100 y l .  When i n d i c a t e d , 1 y l of T r i t o n X-100  was added t o make a f i n a l c o n c e n t r a t i o n  o f 1% T r i t o n X-100,  o r , 15 y l o f salmon p i t u i t a r y e x t r a c t was added. assay blank value was o b t a i n e d using without t e s t i s p r o t e i n . 37°  f o r various  lengths  incubation  The mixtures  Incubations were c a r r i e d o u t a t o f time  (10-30 min).  The r e a c t i o n  was i n i t i a t e d by the a d d i t i o n o f t e s t i s p r o t e i n and was terminated by the a d d i t i o n o f 20 y l o f a s o l u t i o n o f EDTA (30 mM) t o a t t a i n a f i n a l c o n c e n t r a t i o n  o f 5 mM.  The  r e a c t i o n mixture was d i l u t e d t o 0.5 ml w i t h d i s t i l l e d H 0, 2  v o r t e x mixed and t r a n s f e r r e d t o a BioRad AG 50W-X2, 200-400 mesh, hydrogen form, r e s i n column  (0.7 x 4 cm) prepared by  p i p e t t i n g 2 ml o f a 50% v/v suspension o f the r e s i n i n H 0 2  i n t o the column.  A f t e r adsorption  o f the 0.5 ml, the  column was e l u t e d w i t h 0.4 ml o f H 0 and the 0.9 ml t o t a l 2  i n i t i a l e l u a t e added t o 5 ml o f SCINTREX f o r r a d i o a c t i v i t y  determination.  About 40-60% o f the [ a P ] G T P was recovered 32  in this fraction.  The column was then e l u t e d with an a d d i t -  i o n a l 1 ml o f H 2 O and t h i s e l u a t e chromatographed d i r e c t l y onto a n e u t r a l alumina  (Woelm, a c t i v i t y grade I) column  (0.7 x 2.5 cm) made up by p i p e t t i n g 2 ml o f a 50% v/v suspe n s i o n o f n e u t r a l alumina i n 0.1 M T r i s . H C l , pH 7.4, i n t o the column.  The alumina column was then e l u t e d with 4 ml  of 0.1 M T r i s . H C l , pH 7.4. SCINTREX and r a d i o a c t i v i t y mined.  E l u a t e s were added t o 10 ml of (both  [ P ] and [ H]) was d e t e r 3 2  3  A l l r e s u l t s were c o r r e c t e d f o r the recovery o f [ H] 3  c y c l i c GMP and were c a l c u l a t e d as pmol c y c l i c GMP formed from the s p e c i f i c a c t i v i t y o f the GTP used i n each experiment. A f t e r d i l u t i o n o f [ a P ] G T P w i t h u n l a b e l e d GTP, the f i n a l GTP 32  c o n c e n t r a t i o n was determined by absorbance a t 252 nm, u s i n g an e x t i n c t i o n c o e f f i c i e n t o f 13, 800 M~ .  The o v e r a l l  1  recovery o f c y c l i c GMP, a f t e r l o s s e s d u r i n g i n c u b a t i o n and the two stage column procedure, was 45-65%.  The blank o f  the assay, under these c o n d i t i o n s , ranged from 0.006-0.05% of the added The  [a P]GTP. 32  above assay i s a s l i g h t m o d i f i c a t i o n o f a p r e v i o u s l y  r e p o r t e d assay  (106).  In the l a t t e r case, the f i r s t column  e l u a t e was 1 ml (compared with 0.9 ml above) and the f i n a l 0.1 M T r i s . H C l e l u a t e was 2 ml (compared w i t h 4 ml above). The  s l i g h t m o d i f i c a t i o n s were made i n the present  research  because the o v e r l a p o f GTP and c y c l i c GMP was g r e a t e r on the AG  50W-X2 columns prepared  by the present  author,  and a l s o  the m a j o r i t y o f [ P ] c y c l i c GMP was found i n the 3rd and 4th 3 2  milliliter the  o f f the n e u t r a l alumina column ( t h i s  first milliliter  includes  adsorbed from the AG 5 0W-X2 column  onto the n e u t r a l alumina  column).  14. P r o t e i n assay P r o t e i n c o n c e n t r a t i o n s were assayed by the method of Lowry e t a l (107) w i t h bovine serum albumin as the s t a n d a r d .  41. RESULTS  A. Testes growth r a t e d u r i n g hormonally-induced in  spermatogenesis  trout A t e s t e s weight d o u b l i n g time o f about 1 week i s observed  in  rainbow t r o u t , Salmo g a i r d n e r i i ,  w i t h a salmon p i t u i t a r y e x t r a c t  i n j e c t e d twice weekly  (10).  This r e s u l t s i n a  l o g a r i t h m i c t e s t e s weight i n c r e a s e d u r i n g the f i r s t weeks of t e s t i c u l a r m a t u r a t i o n . of  8 to 10  However, the growth r a t e  hormonally-induced t e s t e s i s not completely  from i n d i v i d u a l to i n d i v i d u a l d u r i n g development  synchronous (Table I ) .  A l s o , there are l a r g e v a r i a t i o n s i n i n i t i a l t e s t e s weights between d i f f e r e n t batches o f t r o u t . weights  Thus, zero time t e s t e s  (sum o f the weight o f both t e s t i s ) v a r i e d between  about 30 and 130 mg.  T h i s appeared to be r e l a t e d to the  age o f the f i s h , o r the season i n which they were o b t a i n e d , or  to the l e n g t h o f time they were kept i n the l a b o r a t o r y  a q u a r i a b e f o r e the s t a r t o f experiments.  Batches o f t r o u t  w i t h l a r g e zero time t e s t e s produced p r o p o r t i o n a l l y t e s t e s throughout  large  development.  F i g u r e 3 shows the response to hormone i n j e c t i o n o f a batch o f t r o u t whose i n i t i a l  t e s t e s weights were i n the range  of  30 to 60 mg.  The p l a t e a u and l o s s o f weight a t the end  of  the growth p e r i o d i s a consequence  of germ c e l l c y t o p l a s m i c  l o s s and t e s t i c u l a r t i s s u e degeneration i n the f i n a l stages of  spermatogenesis.  The r e l a t i o n s h i p between d u r a t i o n of  hormone treatment and t e s t e s weight permits the r e p o r t i n g o f  FIGURE 3  Testes growth r a t e during hormonally-induced spermatogenesis i n t r o u t Weights o f t e s t e s  ( i . e . weight o f a p a i r o f t e s t e s )  from  rainbow t r o u t , Salmo g a i r d n e r i i , maintained under the conditions  described  i n Materials  and Methods, are p l o t t e d  as a f u n c t i o n o f days f o l l o w i n g the i n i t i a t i o n o f twice weekly i n j e c t i o n s o f salmon p i t u i t a r y e x t r a c t . An average f i s h weighed  about 250 gm and was  about 2 6 cm  long. Each p o i n t represents  an average weight o f the p a i r of  t e s t e s from a sampling o f 2 or 3 f i s h .  changes i n c y c l i c n u c l e o t i d e b i o c h e m i s t r y d u r i n g  development  with r e f e r e n c e to e i t h e r time or t e s t e s weight.  B. C y c l i c GMP during  and c y c l i c TAMP c o n c e n t r a t i o n s i n t r o u t  testis  spermatogenesis  C y c l i c n u c l e o t i d e s have been determined by r e c e p t o r p r o t e i n b i n d i n g displacement (108), enzymatic c y c l i n g protein kinase a c t i v a t i o n chromatography initial  chromatography inability  ( 1 1 0 ) , h i g h p r e s s u r e i o n exchange  (111), and radioimmunoassay  experiments was  (Appendix )  (112). .  In  , h i g h p r e s s u r e i o n exchange  i n v e s t i g a t e d , but was  r e j e c t e d due to i t s  to d e t e c t the amount o f c y c l i c GMP  trout t e s t i s .  (109),  available  Subsequently, radioimmunoassay  was  from  found to  be a p a r t i c u l a r l y s e n s i t i v e and c o n v e n i e n t technique, and was  used i n a l l measurements o f c y c l i c GMP  reported i n t h i s  and c y c l i c  AMP  thesis.  The c y c l i c GMP  i n t r o u t t e s t i s was  determined i n 3  e x p e r i m e n t a l batches o f hormonally-induced rainbow  trout.  Testes were r a p i d l y e x c i s e d and f r o z e n i n l i q u i d  nitrogen  to minimize post-mortem a l t e r a t i o n o f the c y c l i c  nucleotide  concentration.  In the f i r s t experiment, n u c l e o t i d e  were not p u r i f i e d b e f o r e c y c l i c GMP  measured.  However,  a f t e r treatment with, beef h e a r t c y c l i c n u c l e o t i d e  phosphodi-  e s t e r a s e , some c y c l i c GMP  samples  was  extracts  had h i g h  radioimmunoassay  blank v a l u e s (greater than 10% of the u n t r e a t e d sample). T h e r e f o r e , i n subsequent experiments, c y c l i c GMP from t e s t i s was  p u r i f i e d before  radioimmunoassay.  extracted  From the a v a i l a b l e p u r i f i c a t i o n techniques f o r c y c l i c n u c l e o t i d e s from b i o l o g i c a l s o u r c e s , i . e . a d s o r p t i o n chromatography on n e u t r a l alumina on P E I - c e l l u l o s e ZnCC>3 and BaSCH  (113), t h i n - l a y e r  chromatography  (114), i n o r g a n i c s a l t c o p r e c i p i t a t i o n (115), o r  using  i o n exchange chromatography on  c a t i o n exchange r e s i n s , such as DOWEX 50 (116), and anion exchange r e s i n s , such as DOWEX 1 and 2 (117), anion exchange was used i i n t h i s r e s e a r c h because o f i t s r e l a t i v e ease and speed.  In a l l experiments subsequent  t e s t i s n u c l e o t i d e samples chromatography  t o the f i r s t ,  trout  were p u r i f i e d by anion exchange  on AG 1-X8 columns,  as d e s c r i b e d i n M a t e r i a l s  and Methods. Only a l i m i t e d number o f samples were assayed i n the first  experiment  concentrations development of  (data not shown), but s i n c e h i g h c y c l i c GMP  (1 ymol per kg t e s t i s ) were observed e a r l y i n  and not i n mature t e s t i s ,  a more thorough  t e s t e s a t e a r l y stages o f development  second experiment  sampling  was made i n the  (Table I and F i g u r e 4).  In experiment 2,  c y c l i c GMP was found t o be e l e v a t e d i n zero time t e s t i s and in  t e s t i s d u r i n g p r e m e i o t i c p r o l i f e r a t i o n o f spermatogonia  (Figure 4).  P r i o r t o the time o f the m e i o t i c r e d u c t i o n  d i v i s i o n i n t r o u t t e s t i s , an abrupt 10 f o l d decrease i n t e s t i s c y c l i c GMP was observed  (Figure 4 ) .  The onset o f the m e i o t i c  r e d u c t i o n d i v i s i o n i n hormonally-induced t r o u t t e s t i s , a t between 5 t o 6 weeks a f t e r the i n i t i a t i o n o f hormone i n j e c t i o n , was  determined i n a study o f predominant  development,  t h e i r DNA content and t h e i r  cell  types d u r i n g  [ H]thymidine . 3  46. TABLE I  CYCLIC GMP IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS  (EXPERIMENT 2)  A. Samples from t e s t e s w i t h an average weight f o r the time o f hormonal i n d u c t i o n Day o f T o t a l Testes Hormonal Wet Weight Induction (mg) 0  C y c l i c GMP (umol/kg wet weight)  37 56 92 93 100 300 500 880 1400 1400C 7000 7000 9200 9200  10 15 24 30 39 45  b  70  b  c  87  b  c  98  d  a  1. 93 1.45 1.00 1.93 1.51 1.12 0. 39 0.12 0.08 0.08 0.06 0. 04 0. 04 0. 03 0. 15  C y c l i c GMP (pmol/mg protein)  a  19 13 10 20 19 13 4.0 1.3 0.8 0.9 1.1 0.7 0.8 0.6 12e  B. Samples from t e s t e s w i t h an abnormal weight f o r the time o f hormonal i n d u c t i o n 15 24 30 39 a  400 780 150 160  0.30 0.14 0.81 0.85  3.1 1.4 12 10  Each sample assayed f o r c y c l i c GMP i n d u p l i c a t e , a t 2-3 d i l u t i o n s , i n 2 radioimmunoassays and the mean value o f these r e s u l t s recorded. D u p l i c a t e range ±10%. O v e r a l l range f o r the same sample a t d i f f e r e n t d i l u t i o n s was ±30% b, c Two samples from the same t e s t e s , assayed as above d Sperm e Assuming a 1.3% p r o t e i n content, as i n o t h e r sperm samples  FIGURE 4  Comparison o f c y c l i c GMP c o n c e n t r a t i o n and t e s t e s weight d u r i n g hormonally-induced spermatogenesis i n t r o u t C y c l i c GMP' e x t r a c t e d and assayed as d e s c r i b e d i n M a t e r i a l s and Methods, was measured i n t e s t i s from rainbow  trout,  Salmo g a i r d n e r i i , grown as d e s c r i b e d i n the legend f o r F i g u r e 3.  The average wet weight growth curve i s as i n  F i g u r e 3. Values f o r c y c l i c GMP were o b t a i n e d from t e s t e s w i t h an average wet weight f o r the time o f hormonal  induction.  Each b i o l o g i c a l sample was assayed i n d u p l i c a t e , a t 2-3 dilutions, the  i n 2 radioimmunoassays  sum o f these r e s u l t s  and the mean v a l u e o f  recorded.  The range between  d u p l i c a t e s i n the same radioimmunoassay  was i 10%.  The o v e r a l l range between v a l u e s o b t a i n e d f o r the same b i o l o g i c a l sample a t d i f f e r e n t d i l u t i o n s was - 30%.  48.  20 Days  of  40 60 80 hormonal induction  incorporation was  (10).  Although the exact t i m i n g o f m e i o s i s  not determined i n the p r e s e n t s t u d i e s , i t i s obvious  the abrupt decrease i n c y c l i c GMP e r a t i o n o f germ c e l l s had ceased  o c c u r r e d b e f o r e the p r o l i f (Figure 4 ) .  f o l l o w i n g the l a r g e decrease i n c y c l i c GMP, e l a t i o n between t e s t e s weight and c y c l i c GMP observed:.: (Table IA and F i g u r e 4) . c y c l i c GMP  that  During the stages an i n v e r s e c o r r c o n c e n t r a t i o n was  Only those v a l u e s f o r  o b t a i n e d from t e s t e s w i t h the average weight f o r  the time o f hormonal i n d u c t i o n were used i n F i g u r e 4 .  In  t e s t e s which were not d e v e l o p i n g a t the average growth r a t e , as shown i n F i g u r e 3, c y c l i c GMP  c o n c e n t r a t i o n s a g a i n showed an  i n v e r s e r e l a t i o n s h i p to t o t a l t e s t e s wet weight The c y c l i c GMP 0.15  (Table I B ) .  c o n c e n t r a t i o n observed i n sperm o f  ymol/kg wet weight i s s i m i l a r to a p r e v i o u s r e p o r t of  c y c l i c GMP  i n t r o u t sperm of 0.2-0.3 ymol/kg wet weight  (129).  The d i f f i c u l t i e s i n o b t a i n i n g an a c c u r a t e p r o t e i n c o n c e n t r a t ion  i n sperm suspensions  ( M a t e r i a l s and Methods) puts  on the e x p r e s s i o n of c y c l i c GMP In experiment i n c y c l i c GMP  i n pmol/mg p r o t e i n .  2, r a t h e r l a r g e v a r i a t i o n s were observed  i n e a r l y developmental  samples from t e s t e s of  approximately the same t o t a l wet weight  (Table I ) .  Such  v a r i a t i o n s were a l s o observed i n e a r l y developmental i n the t h i r d experiment GMP  was  found t o be  (Table I I ) .  In experiment  samples 3, c y c l i c  e l e v a t e d i n a l l samples from zero time  to 4 weeks of hormonal i n d u c t i o n h i g h e r than c y c l i c GMP (Table I ) .  doubt  (Table I I ) , and  slightly  i n e a r l y samples i n experiment  In experiment  3, c y c l i c GMP  2.  again decreased  TABLE I I  CYCLIC GMP IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS Day o f Hormonal Induction  (EXPERIMENT 3)  T o t a l Testes Wet Weight (mg) '  C y c l i c GMP (umol/kg wet weight) a  0  30  21  130  3.20  28  110  1. 80  28  34 0  2.20  35  910  0.26  49  630  0.38  63  16000  0.03  66  10000  0. 08  2. 36  Each sample assayed i n d u p l i c a t e , a t 2 d i l u t i o n s , i n 2 radioimmunoassays and the mean value o f these r e s u l t s recorded. D u p l i c a t e range ± 10%. O v e r a l l range f o r the same sample a t d i f f e r e n t d i l u t i o n s was ± 30%.  d r a m a t i c a l l y a f t e r about 4 t o 5 weeks o f hormone i n j e c t i o n s (Table I I ) .  The c o n c e n t r a t i o n o f c y c l i c GMP  to about one-tenth of i t s o r i g i n a l the  again decreased  concentration.  f o l l o w i n g stages o f t e s t i s development,  During  t h e r e was  i n v e r s e c o r r e l a t i o n between t e s t e s weight and c y c l i c concentration  an GMP  (Table II) .  In experiment  3, t e s t i s c y c l i c GMP  was  compared w i t h c y c l i c  GMP  i n another t r o u t t i s s u e , namely the l i v e r .  GMP  was  measured i n both immature  Liver  cyclic  (3 weeks o f hormonal i n d u c t i o n )  and mature (12 weeks o f hormonal i n d u c t i o n ) f i s h , and found to be 0.22  t  0.07  and 0.11  respectively.  ± 0.01  umol/kg wet weight  I t i s not c l e a r i f t h i s  l i v e r c y c l i c GMP  i s significant.  100%  liver,  reduction i n trout  The t r o u t l i v e r c y c l i c  GMP  c o n c e n t r a t i o n s are somewhat h i g h e r than those observed i n r a t l i v e r of 0.04-0.07 umol/kg wet weight l i v e r In experiment 4, c y c l i c AMP  was  determined i n t r o u t  d u r i n g hormonally-induced spermatogenesis AMP  ( 128).  (Figure 5 ) .  testis Cyclic  decreased 2 f o l d a t a p r e m e i o t i c stage o f t e s t i s develop-  ment, a t about, o r p r i o r t o the time of the 10 f o l d decrease i n c y c l i c GMP  (Figure 4 ) .  C y c l i c AMP  and c y c l i c GMP  conc-  e n t r a t i o n s i n zero time t e s t i s were approximately equal a t about 2 umol/kg wet weight t e s t i s  (Figures 4 and 5 ) .  After  the 2: foTd p r e m e i o t i c decrease i n t r o u t t e s t i s c y c l i c AMP, significant  changes i n c y c l i c AMP  no  concentrations i n developing  t e s t i s o c c u r r e d d u r i n g the remainder o f spermatogenesis  (Figure 5)  FIGURE 5  C y c l i c AMP c o n c e n t r a t i o n s i n t e s t i s d u r i n g induced spermatogenesis  hormonally-  i n trout  C y c l i c AMP, e x t r a c t e d and assayed as d e s c r i b e d i n M a t e r i a l and Methods, was measured i n t e s t i s from rainbow t r o u t , Salmo g a i r d n e r i i , grown as d e s c r i b e d i n the legend f o r F i g u r e 3. Values  f o r c y c l i c AMP were o b t a i n e d from t e s t e s w i t h an  average wet weight f o r the time o f hormonal i n d u c t i o n . Each p o i n t r e p r e s e n t s a b i o l o g i c a l sample assayed i n d u p l i c a t e , a t 2-3 d i l u t i o n s ,  i n 2 radioimmunoassays and  the mean v a l u e o f the sum o f these r e s u l t s  recorded.  The range between d u p l i c a t e s i n the same radioimmunoassay was ^ 10%.  The o v e r a l l range between values o b t a i n e d f o r  the same b i o l o g i c a l sample a t d i f f e r e n t d i l u t i o n s was * 30  [  Days of H o r m o n a l  Induction  54. C. C y c l i c AMP and c y c l i c GMP phosphodiesterase in trout t e s t i s during  1. Assay  activities  spermatogenesis  system  A r a d i o i s o t o p e assay system p r o v i d e s the maximum sensitivity  f o r c y c l i c n u c l e o t i d e phosphodiesterase assays  (118) and was t h e r e f o r e used i n t h i s r e s e a r c h .  In t h i s  method,  [ H ] c y c l i c n u c l e o t i d e i s h y d r o l y z e d t o the c o r r e s -  ponding  5 - n u c l e o t i d e , which i s then converted t o a n u c l e o -  3  1  s i d e by the a d d i t i o n o f excess phosphatase.  In many cases  (60, 103, 105) the excess 5 - n u c l e o t i d a s e p r e s e n t i n snake 1  venom has been u t i l i z e d i n t h i s second i n c u b a t i o n . present research,  c o l i a l k a l i n e phosphatase  In the  was used.  S e p a r a t i o n o f n u c l e o s i d e from c y c l i c n u c l e o t i d e can be accomplished by passage over an alumina column (113), a DOWEX 1 o r 2 column graphy  (119, 120), by t h i n - l a y e r chromato-  (121), by paper chromatography  of DOWEX 1 r e s i n acidification  (123).  (122) o r by batch use  The l a t t e r method, m o d i f i e d by  o f the r e s i n p r i o r t o i t s use, r e s u l t e d i n  q u a n t i t a t i v e r e c o v e r i e s o f adenosine, guanosine major metabolites (103, 105).  and t h e i r  T h i s assay system p r o v i d e s  a simple, r a p i d and r e l i a b l e method by which c y c l i c AMP o r c y c l i c GMP phosphodiesterases can be determined,  even i n  crude t i s s u e p r e p a r a t i o n s , and, was thus adopted  f o r use.  The temperature f o r comparison  o f 30° f o r a c t i v i t y assays was chosen  of r e s u l t s with previous c y c l i c nucleotide  phosphodiesterase s t u d i e s i n f i s h sperm (88).  DEAE-cellulose p r o f i l e s maximum assay a c t i v i t y .  were assayed a t pH 8.0 f o r  A l l o t h e r assays were a t pH 7.5.  F o l l o w i n g assay c o n d i t i o n s p r e v i o u s l y d e s c r i b e d (119), a 2+ Mg  c o n c e n t r a t i o n o f 2 mM was used.  When i n c l u d e d , EGTA  was p r e s e n t to ..eliminate the p o s s i b l e e f f e c t s o f v a r y i n g amounts o f Ca  2+ -dependent phosphodiesterase p r o t e i n 2+  in tissue preparations.  When i n c l u d e d , Ca  was p r e s e n t t o  i n v e s t i g a t e the e f f e c t s of such an a c t i v a t o r . b u f f e r c o n t a i n e d 2-mercaptoethanol to s t a b i l i z e  because  activator  The assay  i t had been shown  the enzyme a c t i v i t y from other t i s s u e s  (12 4).  2. P r o p e r t i e s C y c l i c AMP  and c y c l i c GMP phosphodiesterases i n t r o u t  t e s t i s were s t a b l e , a t the assay temperature  o f 30°, f o r at  l e a s t 30 min, a t 0.1M s u b s t r a t e , as shown .in F i g u r e 6A.  In 2+  one t e s t i s homogenate,a study o f enzyme requirement f o r Mg 2+ showed an approximate f o r c y c l i c AMP respectively to  Mg  optimum o f 8-10 mM and 2-10  ,  mM  and c y c l i c GMP phosphodiesterase a c t i v i t i e s ,  (Figure 6B).  8.0, f o r both c y c l i c AMP  A broad pH optimum from pH 7.5 and c y c l i c GMP  phosphodiesterase  a c t i v i t i e s , was determined i n one t r o u t t e s t i s homogenate (Figure 7B) .  In the same t e s t i s homogenate, (Figure :7A) ...  .' EGTA was shown to reduce both c y c l i c AMP  and c y c l i c  GMP  phosphodiesterase a c t i v i t i e s , measured a t micromolar s u b t r a t e by about 20%.  Due to the c o m p l e x i t i e s o f m u l t i p l e 2+  forms, d e t a i l e d developmental pH o p t i m a  were not made.  s t u d i e s o f Mg  optima  enzyme and  56. FIGURE 6  A. S t a b i l i t y  o f phosphodiesterase  Phosphodiesterase testis, pituitary  activities  a c t i v i t i e s , measured i n a homogenate o f  from t r o u t which had been i n j e c t e d w i t h a salmon e x t r a c t f o r 8 weeks and maintained  d e s c r i b e d i n M a t e r i a l s and Methods. was  4g.  Phosphodiesterase  i n standard  O—O  T o t a l t e s t e s weight  a c t i v i t i e s were assayed, a t 30°,  C y c l i c nucleotide concentration  EGTA was not i n c l u d e d i n assays.  cyclic  under c o n d i t i o n s  i n c u b a t i o n mixtures a t pH 7.5, as d e s c r i b e d i n  M a t e r i a l s and Methods. 0.1 mM.  a t 30°  •—•  cyclic  was AMP  GMP  2+  B. Phosphodiesterase  activities  as -a f u n c t i o n o f Mg  concentration Phosphodiesterase  activities,  i n the homogenate  above, were assayed, a t 30°, i n standard  described  i n c u b a t i o n mixes 2+  at pH 7.5, with v a r y i n g c o n c e n t r a t i o n s i n M a t e r i a l s and Methods. was 0.1 mM.  o f Mg  C y c l i c nucleotide  EGTA was not i n c l u d e d .  , as d e s c r i b e d concentration  Symbols as above.  58. FIGURE 7  A. Phosphodiesterase  1  activities  i n the presence or absence  of EGTA Phosphodiesterase  a c t i v i t i e s , measured i n a t e s t i s  homogenate,  from t r o u t which had been i n j e c t e d w i t h a salmon p i t u i t a r y e x t r a c t f o r 4 weeks and maintained i n M a t e r i a l s and Methods. Phosphodiesterase standard  T o t a l t e s t e s weight 629  mg.  a c t i v i t i e s were assayed, a t 30°, i n  i n c u b a t i o n mixtures a t pH 7.5, as d e s c r i b e d i n  M a t e r i a l s and Methods. 1 yM.  under c o n d i t i o n s d e s c r i b e d  C y c l i c nucleotide concentration  When p r e s e n t , EGTA c o n c e n t r a t i o n was 250 A—A  cyclic  yM.  AMP  c y c l i c AMP A—A  cyclic  o—o  c y c l i c GMP  p l u s EGTA  GMP  B. Phosphodiesterase Phosphodiesterase  was  p l u s EGTA  a c t i v i t i e s as a f u n c t i o n o f pH  a c t i v i t i e s , i n the homogenate  above, were assayed, a t 30°, i n standard  described  incubation  mixtures,  a t v a r y i n g pHs, as d e s c r i b e d i n M a t e r i a l s and Methods. C y c l i c n u c l e o t i d e c o n c e n t r a t i o n was 1 yM. was i n c l u d e d i n a l l assays.  EGTA (250 yM)  Symbols as above.  60. The a f f i n i t i e s of phosphodiesterase a c t i v i t i e s f o r c y c l i c AMP  and c y c l i c GMP  were determined  t e s t i s homogenate ( F i g u r e s 8 and 9). only h i g h a f f i n i t y c y c l i c AMP  3.  Activities  during t e s t i s  Hofstee p l o t s showed  activities  a h i g h and low a f f i n i t y c y c l i c GMP  i n a mature t r o u t  (Figure 8) and  activity  both  (Figure 9 ) .  development  A c t i v i t i e s were measured throughout development a t both saturating  ( m i l l i m o l a r ) and s u b s a t u r a t i n g (micromolar) s u b s t r a t e  c o n c e n t r a t i o n s , to d e t e c t t o t a l and h i g h a f f i n i t y components, respectively.  T o t a l c y c l i c AMP  phosphodiesterase  activities  measured i n the presence or absence of EGTA (Table I I I ) decreased 50% p r i o r to m e i o s i s .  T h i s was  f o l l o w e d by an i n c r e a s e  d u r i n g spermatid d i f f e r e n t i a t i o n r a i s i n g t o t a l c y c l i c  AMP  phosphodiesterase a c t i v i t y to s l i g h t l y above t h a t observed i n immature t e s t i s . about 2 0-40%  EGTA decreased t o t a l c y c l i c AMP  i n zero time and mature t e s t i s but had an  f i c a n t e f f e c t on a c t i v i t i e s i n t e s t i s T o t a l c y c l i c GMP relatively  activities  j u s t p r i o r to meiosis.  phosphodiesterase  activities  remained  c o n s t a n t throughout development, i n the absence  of EGTA (Table I I I ) . enhance c y c l i c GMP t e s t i s about  The presence o f EGTA appeared  to  phosphodiesterase a c t i v i t i e s i n immature  30%, but throughout  the f o l l o w i n g stages o f  development r e s u l t e d i n about a 2 0% decrease i n c y c l i c activities. AMP  insigni-  The r a t h e r minor e f f e c t of EGTA on both  and c y c l i c GMP  phosphodiesterase  testis  GMP cyclic  a c t i v i t i e s d i d not show  any important changes d u r i n g development i n the c o n c e n t r a t i o n 2+ or  the a c t i v i t y of a Ca  -dependent phosphodiesterase  protein  FIGURE 8  Hofstee p l o t o f the r a t e o f c y c l i c AMP h y d r o l y s i s by a t e s t i s homogenate  (minus the l,000xg p e l l e t ) from t r o u t  hormonally-induced f o r 10 weeks, as d e s c r i b e d and Methods.  T o t a l t e s t e s weight 9.8 g.  were s t o r e d f r o z e n a t -20° f o r a week before  i n Materials  Homogenates kinetic analysis  Homogenates were c e n t r i f u g e d a t l , 0 00xg f o r 10 min a f t e r thawing.  The i n s o l u b l e l i p i d p e l l e t was d i s c a r d e d  supernatant assayed f o r a c t i v i t y .  The u n i t o f v e l o c i t y i s  nmol c y c l i c AMP hydrolyzed/min/mg p r o t e i n . 30°, i n standard  Assays were a t  i n c u b a t i o n mixtures a t pH 7.5, as d e s c r i b e d  i n M a t e r i a l s and Methods. a l l assays.  and the  Substrate  EGTA (250 yM) was i n c l u d e d i n  concentrations  v a r i e d from 0.1-500 yM  1  t  63.  FIGURE 9  Hofstee p l o t o f the r a t e o f c y c l i c GMP h y d r o l y s i s by the homogenate d e s c r i b e d  i n the legend f o r F i g u r e  8.  The  u n i t of v e l o c i t y i s nmol c y c l i c GMP hydrolyzed/min/mg p r o t e i n . Assays were a t 30°, i n standard as d e s c r i b e d  i n c u b a t i o n mixtures a t pH 7.5,  i n M a t e r i a l s and Methods.  i n c l u d e d i n a l l assays. from 5 - 1,000  uM.  Substrate  EGTA (25 0 uM)  concentrations  was  varied  64.  V  TABLE I I I TOTAL CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITIES IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS  Week o f Hormonal Induction  T o t a l Testes Wet Weight (mg)  C y c l i c AMP Activity Minus, P l u s _ EGTA EGTA  C y c l i c GMP Activity Minus Plus EGTA EGTA c b  0  70  1210  1010'  570  740  2  80  1390  1260  600  910  3  480  650  630  450  490  4  890  930  850  520  500  5  1500  770  560  430  350  6  3000  1000  510  510  300  8  4100  1560  1060  630  590  10  9800  1720  1210  560  480  12  450  a  90  Trout t e s t e s were e x c i s e d , p l a c e d i n l i q u i d n i t r o g e n and s t o r e d a t -80 u n t i l one complete developmental s e r i e s of t e s t e s had been c o l l e c t e d . T r o u t were hormonally-induced by a twice weekly i n j e c t i o n o f a salmon p i t u i t a r y e x t r a c t , as d e s c r i b e d i n M a t e r i a l s and Methods. Crude homogenates of the thawed and weighed t e s t e s were prepared i n ^buffer A and assayed f o r c v c l i c AMP and c y c l i c GMP phosphodiesterase a c t i v i t i e s , a t 30 , i n standard i n c u b a t i o n mixtures at pH 7.5, as d e s c r i b e d i n M a t e r i a l s and Methods. Millimolar s u b s t r a t e c o n c e n t r a t i o n s were used. A c t i v i t y u n i t s are pmol c y c l i c n u c l e o t i d e hydrolyzed/min/mg p r o t e i n . Values are the means o f d u p l i c a t e s (± 10%) from the same homogenate. T h i s sample c o n s i s t e d o f sperm, obtained M a t e r i a l s and Methods k No EGTA i n assay mixture C  -EGTA(250  uM) i n assay mixture  as d e s c r i b e d i n  66. activator i n trout The s p e c i f i c GMP  testis. a c t i v i t i e s o f sperm c y c l i c AMP  p h o s p h o d i e s t e r a s e s , measured a t m i l l i m o l a r  and  cyclic  substrate  c o n c e n t r a t i o n s , are r e s p e c t i v e l y 3 and 6 f o l d lower than the specific  a c t i v i t i e s i n mature t e s t i s  (Table I I I ) .  Direct  comparison between the c y c l i c n u c l e o t i d e phosphodiesterase specific  a c t i v i t i e s i n sperm and i n t e s t i s t i s s u e i s poss-  i b l y i n v a l i d due to the d i f f i c u l t y sperm suspensions ( M a t e r i a l s and C y c l i c AMP  and c y c l i c GMP  i n measuring p r o t e i n i n  Methods).  phosphodiesterases, i n t e s t i s  homogenates from t r o u t a t d i f f e r e n t stages o f spermatogenesis, were measured a t micromolar s u b s t r a t e , i n the presence o f EGTA (Table I V ) .  As seen i n the t o t a l c y c l i c AMP  (Table I I I ) , a 50% decrease o f the s m a l l amount o f c y c l i c AMP  activities initial  p h o s p h o d i e s t e r a s e , measured a t micromolar  substrate,  occurs i n a p r e m e i o t i c stage o f spermatogenesis.  This i s  f o l l o w e d by a 20 to 40 f o l d i n c r e a s e i n c y c l i c AMP  activity,  measured a t micromolar s u b s t r a t e , d u r i n g the l a t e r  stages of  development.(Table I V ) .  C y c l i c AMP  phosphodiesterase a c t -  i v i t i e s were not measured a t micromolar s u b s t r a t e throughout development  i n the absence o f EGTA.  As seen i n F i g u r e  EGTA caused a 2 0% r e d u c t i o n i n c y c l i c AMP  7A,  phosphodiesterase  a c t i v i t y , measured a t micromolar s u b s t r a t e , i n a t e s t i s homogenate:..from one stage: o f development.  I t i s possible  t h a t , i n the absence o f EGTA, a s l i g h t l y g r e a t e r i n c r e a s e i n h i g h a f f i n i t y c y c l i c AMP  phosphodiesterase a c t i v i t y would  observed d u r i n g spermatogenesis.  be  TABLE I V  CYCLIC NUCLEOTIDE PHOSPHODIESTERASE A C T I V I T I E S MEASURED AT MICROMOLAR SUBSTRATE IN TROUT T E S T I S DURING HORMONALLYINDUCED  SPERMATOGENESIS  Week o f Hormonal Induction  T o t a l Testes Wet W e i g h t (mg)  2  220  48  20  2.4  3  250  43  17  2.5  4  630  26  14  1.9  6  7100  430  16  27  10  9800  1000  11  91  310  3  12  b  C y c l i c AMP C y c l i c GMP Activity Activity  A/G Ratio  124  L e g e n d a s i n T a b l e I I I , e x c e p t a s s a y s were m e a s u r e d a t micromolar substrate concentrations. Assay mixtures a l l contain EGTA (250 uM). A c t i v i t y u n i t s a r e pmol c y c l i c n u c l e o t i d e hydrolyzed/min/mg p r o t e i n , The r a t i o activity  o f c y c l i c AMP  to cyclic  GMP  phosphodiesterase  T h i s sample c o n s i s t e d o f sperm, o b t a i n e d M a t e r i a l s and Methods  as d e s c r i b e d i n  a  68. C y c l i c GMP phosphodiesterase a c t i v i t y , measured a t micromolar  s u b s t r a t e , i n the presence o f EGTA, decreases l e s s  50% p r o g r e s s i v e l y throughout spermatogenesis  than  (Table I V ) .  The r a t i o o f c y c l i c AMP t o c y c l i c GMP h y d r o l y s i s , measured a t micromolar  s u b s t r a t e , changes from about 2 t o 124 d u r i n g  testis differentiation.  T h i s i n d i c a t e s the s p e c i f i c  induction  of a h i g h a f f i n i t y c y c l i c AMP phosphodiesterase i n a l a t e stage o f spermatogenesis.  In a study by Drummond and h i s  a s s o c i a t e s , i n which c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t i e s i n salmon sperm were measured, a t micromolar s t r a t e c o n c e n t r a t i o n s , a c y c l i c AMP to c y c l i c GMP  sub-  activity  r a t i o o f 124 was a l s o observed (88). The s e q u e n t i a l changes i n c y c l i c n u c l e o t i d e phosphodie s t e r a s e a c t i v i t i e s , d u r i n g spermatogenesis graphed  i n F i g u r e 10.  Total  i n t r o u t , are  (Figure 10A) and h i g h a f f i n i t y  (Figure 10B) phosphodiesterase a c t i v i t i e s , i n the presence of EGTA, are shown both as a f u n c t i o n o f time o f hormonal i n d u c t i o n and i n r e l a t i o n t o the stage o f germ c e l l entiation.  differ-  The i n d u c t i o n o f a h i g h a f f i n i t y c y c l i c  AMP  phosphodiesterase, a t about m e i o s i s , i s e v i d e n t i n F i g u r e 10B. 2+ Since i t had been shown t h a t Ca , i n the presence o f a 2+ Ca -dependent phosphodiesterase p r o t e i n a c t i v a t o r , i n c r e a s e s the a c t i v i t y o f c y c l i c GMP h y d r o l y s i s more so than c y c l i c  AMP 2+  hydrolysis  (74), a more d e t a i l e d study o f the e f f e c t s o f Ca  and EGTA on h i g h a f f i n i t y c y c l i c GMP phosphodiesterase a c t ivities  i n d e v e l o p i n g t r o u t t e s t i s , was undertaken.  r e s u l t s o f t h i s study are summarized i n Table V.  The Only i n  FIGURE 10  A. Total, c y c l i c n u c l e o t i d e phosphodiesterase trout testis  during  1  activities in  spermatogenesis  T e s t i s homogenates, from t r o u t i n j e c t e d twice weekly w i t h a salmon p i t u i t a r y e x t r a c t and maintained  as d e s c r i b e d i n  M a t e r i a l s and Methods, were assayed f o r c y c l i c phosphodiesterase mixtures a t pH  a c t i v i t i e s , a t 30°, i n standard  7.5,  i n c l u d e d i n a l l assays.  The  1 mM.  activity  EGTA (250  O —  data from Table I I I .  O cyclic  • — •  yM)  u n i t s are pmol  c y c l i c n u c l e o t i d e hydrolyzed/min/mg p r o t e i n . illustrates  incubation  as d e s c r i b e d i n M a t e r i a l s and Methods.  C y c l i c n u c l e o t i d e c o n c e n t r a t i o n was was  nucleotide  T h i s graph  cyclic  AMP  GMP  B. C y c l i c n u c l e o t i d e phosphodiesterase  a c t i v i t i e s ^ measured  a t micromolar s u b s t r a t e c o n c e n t r a t i o n s , i n t r o u t  testis  d u r i n g s pe rma to gene s i s Legend as above, except t h a t c y c l i c n u c l e o t i d e was  1 yM.  concentration  T h i s graph i l l u s t r a t e s data from Tables  Symbols as above.  IV and  V.  70.  Spermatogonia  Spermatids  Spermatocytes  2 Week  4 Hormonal  of  Hormonal  2 vVeek  6  of  4  8  10  Induction  6  8 Induction  10  TABLE V CYCLIC GMP PHOSPHODIESTERASE.ACTIVITIES MEASURED AT MICROMOLAR SUBSTRATE IN TROUT TESTIS DURING HORMONALLYIN DU CE D"SPE RMATOGENESIS  Week o f Hormonal Induction  T o t a l Testes Wet Weight (mg)  C y c l i c GMP Phosphodiesterase Activities No A d d i t i o n Plus Plus , EGTA i-Ca a  2+  0  30  43  27  45  2  80  27  24  29  3  410  29  23  27  3  1000  19  18  21  6  2500  14  12  13  7  9000  20  15  22  8  28000  18  17  19  10  18000  40  18  41  !  Legend as i n Table I I I , except assays were measured a t micromolar s u b s t r a t e c o n c e n t r a t i o n s f  a  EGTA  (250 uM) i n assay mixture  Ca  (100 pM) i n assay mixture  72. two cases, zero time and completely mature t e s t i s , was marked i n h i b i t i o n respectively). was  by EGTA noted  (30 and 55%  a  inhibition,  In a l l other stages of development, there  a 12-25% i n h i b i t i o n  by EGTA.  Activity  i n the  presence  2+ of  Ca  was  approximately the same as t h a t observed with no  additions.  There was  no s p e c i f i c  phosphodiesterase a c t i v i t y ,  high a f f i n i t y c y c l i c  GMP  i n the presence or absence of  2+ Ca  , a s s o c i a t e d w i t h the p r e m e i o t i c stage i n which the l a r g e  decrease i n c y c l i c GMP  c o n c e n t r a t i o n was  4. D E A E - c e l l u l o s e chromatographic and c y c l i c GMP  observed  ( F i g u r e 4).  •fractionation' of c y c l i c  phosphodiesterases  When chromatographed on D E A E - c e l l u l o s e , t r o u t  testis  homogenates, from a l l stages of development, y i e l d two c y c l i c n u c l e o t i d e phosphodiesterase e l u t e d by about 0.35 a c e t a t e , pH at  6.5  AMP  M  fractions.  (Peak I) and 0.65  (Figure 11).  active  These are  M'(Peak II) sodium  Column f r a c t i o n s were assayed  low s u b s t r a t e c o n c e n t r a t i o n s (- 0.1  uM) .  One  profile  2+ (Figure 11B)  was  assayed i n the presence o f Ca  i n the presence of EGTA (250 uM)  or c y c l i c GMP  uM),  or w i t h standard assay  i n c u b a t i o n mixtures w i t h no a d d i t i o n s . c y c l i c AMP  (100  No d i f f e r e n c e i n  phosphodiesterase a c t i v i t i e s were  observed under these d i f f e r e n t assay conditions,, ( data not 2+ shown).  T h i s i n d i c a t e s the l o s s of Ca  -dependent phospho-  d i e s t e r a s e p r o t e i n a c t i v a t o r d u r i n g D E A E - c e l l u l o s e column purification, E a r l y attempts  as has been p r e v i o u s l y been r e p o r t e d (75). a t f r a c t i o n a t i o n of bovine h e a r t a c t i v i t i e s ,  73. FIGURE 11 DEAE-cellulose  f r a c t i o n a t i o n o f c y c l i c AMP and c y c l i c GMP  phosphodiesterase a c t i v i t i e s i n t r o u t t e s t i s  during  hormonally-induced spermatogenesis DEAE-cellulose diesterases described A.  p r o f i l e s of c y c l i c nucleotide  phospho-  i n t r o u t t e s t i s homogenates prepared as  i n M a t e r i a l s and Methods.  Two weeks o f hormonal i n d u c t i o n .  Average t e s t e s weight  14 6 mg; 2 80 mg i n homogenate form a p p l i e d t o a column o f 0.5 x 10.5 cm s i z e . B.  Four weeks o f hormonal i n d u c t i o n . 629  Average t e s t e s weight  mg; 540 mg i n homogenate form a p p l i e d to a column o f  1 x 15 cm s i z e . C.  Column f r a c t i o n s i z e 2.2 ml.  Column f r a c t i o n s i z e 4.5 ml.  S i x weeks o f hormonal i n d u c t i o n .  Average t e s t e s weight  3550 mg; 666 mg i n homogenate form a p p l i e d t o a column o f 0.8 x 22 cm s i z e . D.  Column f r a c t i o n s i z e 4.5 ml.  E i g h t weeks o f hormonal i n d u c t i o n .  Average t e s t e s weight  40 30 mg; 6 66 mg i n homogenate form a p p l i e d t o a column o f 0.8 x 22 cm s i z e .  Column f r a c t i o n s i z e 4.5 ml.  For a l l p r o f i l e s , a f t e r sample a p p l i c a t i o n columns were washed w i t h 20 mM sodium a c e t a t e , pH 6.5 b u f f e r 4 mM 2-mercaptoethanol.  The i n i t i a l wash contained  phosphodiesterase a c t i v i t y . pH  containing  2 0 mM - 1 M sodium  no  acetate,  6.5 l i n e a r g r a d i e n t s were s t a r t e d a t tube 10 and c o n t -  inued  through t o tube 55, a t a flow r a t e o f 2 6 ml/hr.  T o t a l g r a d i e n t volume was 2 00 ml f o r p r o f i l e s B, C and D. T o t a l g r a d i e n t volume f o r A was 100 ml.  F r a c t i o n 25 and  f r a c t i o n 38 correspond t o 0.35 and 0.65 M sodium  acetate,  respectively. Phosphodiesterase a c t i v i t i e s were assayed i n 50 y l column fraction aliquots standard  (or 25 y l f o r p r o f i l e A ) , a t 30°, i n  i n c u b a t i o n m i x t u r e s , a t pH 8.0, as d e s c r i b e d i n  M a t e r i a l s and Methods. EGTA (250 yM) was i n a l l assays. • — • c y c l i c AMP (3 x 10 M; A and B. 1.5 x 10~ M; C and D) 7  O — O c y c l i c GMP  7  (3 x 10" M; A and B. 1.6 x 10" M; C and D) 7  7  74.  Fraction  Numbe  75.  on D E A E - c e l l u l o s e ,  were u n s u c c e s s f u l due  to t h i s l o s s (125) .  A t e a r l y stages of t r o u t t e s t i s development 11A  and  at 0.35  11B)  an a c t i v e c y c l i c GMP  M sodium a c e t a t e .  prolonged  phosphodiesterase  T h i s a c t i v i t y was  eluted  unstable  storage a t -20°, i n c o n t r a s t to the c y c l i c  a c t i v i t y which e l u t e d a t t h i s p o s i t i o n . esterase a c t i v i t y eluting found to h y d r o l y z e In l a t e 11D)  (Figures  a t 0.65  c y c l i c AMP  was  almost e x c l u s i v e l y .  stages of spermatogenesis (Figures 11C  Peak I I c y c l i c AMP w h i l e c y c l i c GMP  AMP  phosphodi- •.  M sodium a c e t a t e  a l a r g e i n c r e a s e i n the r e l a t i v e  decreased.  The  on  phosphodiesterase  and  s i z e o f Peak I to activity  h y d r o l y s i s i s unchanged or  slightly  Q u a n t i t a t i v e comparisons between  stages of development cannot be made, due  occurs,  different  to the  differing  amounts of homogenates a p p l i e d to columns and the l a c k of enzyme r e c o v e r y  data.  Each f r a c t i o n . t u b e contained  bovine serum albumin p r e v e n t i n g enzyme - p r o t e i n . ~ The  2 peaks  a c e t a t e , i n F i g u r e 11B,  may  a t about 0.3-0.4 M sodium  be a p a r t i a l s e p a r a t i o n of  ( p r o f i l e not shown).  3 weeks of hormone  However, the 100,000xg  supernatant  profile,  F i g u r e 11B,  d i d not show t h i s double peak (Figure  i n F i g u r e 11D,  was  two  a l s o seen on a p r o f i l e  of t e s t i s homogenate from a t r o u t a f t e r  A p o r t i o n (33%)  of  the accurate measurement of  enzyme forms, s i n c e t h i s e f f e c t was  injections  4mg  from the same t e s t i s homogenate as i n  of Peak I, from the p r o f i l e  13A). described  rechromatographed on D E A E - c e l l u l o s e  o n l y one peak, cochromatographing at the o r i g i n a l  and  Peak I  76. p o s i t i o n , was  observed  (Figures 12A and 12B).  l i v e r , i t has been demonstrated AMP  column, when  on D E A E - c e l l u l o s e , gave a p r o f i l e which  c o n t a i n e d both the o r i g i n a l  low a f f i n i t y a c t i v i t y , i n a  M sodium a c e t a t e c u t , p l u s a h i g h a f f i n i t y a c t i v i t y , .in  a 0.6 was  t h a t the low a f f i n i t y c y c l i c  phosphodiesterase peak from a B i o - G e l A-5m  rechromatographed  0.3  In the r a t  M sodium a c e t a t e c u t (60).  observed i n the p r e s e n t case  a 50% l o s s i n c y c l i c AMP c y c l i c GMP ography  No such Peak I I a c t i v i t y (Figure 12B).  of Peak I (Figure 12B).  t h e r e f o r e was  was  a c t i v i t y and about a 90% l o s s of  a c t i v i t y , observed i n F i g u r e 11D,  the rechromatographed  There  Cyclic  Peak I p r o f i l e , was  not i n c l u d e d i n F i g u r e 12.  on  GMP  rechromat-  activity, in  v e r y low,  and,  The cause of the  a c t i v i t y l o s s e s i s not known.  5. S o l u b l e and p a r t i c u l a t e a c t i v i t i e s f r a c t i o n a t e d on DEAE-cellulose When the 100,000xg supernatant from a t r o u t t e s t i s homogenate, was D E A E - c e l l u l o s e , both c y c l i c AMP identified  (soluble)  fraction,  chromatographed on  phosphodiesterase peaks  i n the t o t a l homogenate p r o f i l e , were observed  (Figure 13A).  Cyclic  GMP  phosphodiesterase a c t i v i t y  observed i n the Peak I p o s i t i o n , but was t h e r e f o r e was  not i n c l u d e d i n F i g u r e 13A.  extremely low,  and,  There was  2.5  f o l d i n c r e a s e i n Peak I I to Peak I c y c l i c AMP  (Figure 11B)  s o n i c a t e d 100,000xg p e l l e t  a  activity ratio in  the s o l u b l e p r o f i l e , .as compared w i t h the same- r a t i o homogenate p r o f i l e  was  i n the  from the same t e s t i s .  (particulate) fraction,  from  A  FIGURE 12  A. D E A E - c e l l u l o s e p r o f i l e o f c y c l i c AMP  phosphodiesterases  i n a t e s t i s homogenate from a t r o u t i n j e c t e d w i t h a salmon pituitary  e x t r a c t f o r 8 weeks ( d e t a i l e d d e s c r i p t i o n i n the  legend f o r F i g u r e 11D). assayed  Phosphodiesterase  i n 10 y l column f r a c t i o n  standard i n c u b a t i o n mixtures, M a t e r i a l s and Methods. Cyclic  a c t i v i t i e s were  a l i q u o t s , a t 30°, i n  a t pH 8.0, as d e s c r i b e d i n  EGTA (250 yM) was i n a l l assays.  AMP c o n c e n t r a t i o n was 1.5 x 10" M. 7  B. D E A E - c e l l u l o s e p r o f i l e o f rechromatographed,-peak I , from the p r o f i l e o f the t e s t i s homogenate d e s c r i b e d above. Peak I a c t i v i t y  from the above p r o f i l e was pooled, and-;  concentrated as d e s c r i b e d i n M a t e r i a l s and Methods, and a p o r t i o n o f the concentrate a p p l i e d t o a D E A E - c e l l u l o s e column.  Conditions f o r DEAE-cellulose  d e s c r i b e d i n M a t e r i a l s and Methods.  f r a c t i o n a t i o n as Phosphodiesterase  a c t i v i t i e s assayed as d e s c r i b e d above, except AMP c o n c e n t r a t i o n was 0.5 x 10" M. 7  that c y c l i c  79. FIGURE 13  A. D E A E - c e l l u l o s e p r o f i l e of t r o u t t e s t i s c y c l i c  AMP  phosphodiesterase a c t i v i t i e s i n a 100,000xg supernatant fraction Average  from t e s t i s hormonally-induced f o r 4 weeks. (from 2 f i s h ) t o t a l t e s t e s weight of 62 9  B. D E A E - c e l l u l o s e p r o f i l e of t r o u t t e s t i s c y c l i c  mg.  AMP  phosphodiesterase a c t i v i t i e s i n a 100,000xg p a r t i c u l a t e fraction  from the t e s t i s d e s c r i b e d above.  The c o n d i t i o n s f o r both s e p a r a t i o n p r o f i l e s described i n d e t a i l g r a d i e n t was  i n M a t e r i a l s and Methods.  from f r a c t i o n  column f r a c t i o n  10 to f r a c t i o n  50.  were as The 50 u l  a l i q u o t s were assayed f o r phosphodiesterase  a c t i v i t i e s , a t 30°, i n standard i n c u b a t i o n mixtures a t pH  8.0,  as d e s c r i b e d i n M a t e r i a l s and Methods. _7  assay c o n c e n t r a t i o n was  1.5  i n c l u d e d i n assay mixtures.  x 10  M.  Cyclic  EGTA (250 uM)  was  AMP  81. the same t r o u t t e s t i s homogenate, contained mainly Peak I I c y c l i c AMP  activity  (Figure 13B).  The p a r t i c u l a t e f r a c t i o n ,  from t r o u t t e s t i s homogenate,  c o n t a i n e d about 15% o f the c y c l i c AMP a c t i v i t i e s and 20% of the c y c l i c GMP a c t i v i t i e s , measured a t micromolar The s p e c i f i c c y c l i c AMP  phosphodiesterase phosphodiesterase  substrate concentrations.  a c t i v i t y o f the 100,000xg p e l l e t was 21 pmol  hydrolyzed/min/mg p r o t e i n , about  50% of t h a t o f  the 100,000xg supernatant a t 43 pmol c y c l i c AMP min/mg p r o t e i n .  For c y c l i c GMP  hydrolyzed/  h y d r o l y s i s , the s p e c i f i c  a c t i v i t y o f the 100,000xg p e l l e t was 3.7 pmol c y c l i c  GMP  hydrolyzed/min/mg p r o t e i n , about 75% of t h a t o f the 100,000xg supernatant a t 4.8 pmol c y c l i c GMP hydrolyzed/min/mg p r o t e i n .  6. K i n e t i c analyses o f c y c l i c AMP  phosphodiesterase  activities  f r a c t i o n a t e d on D E A E - c e l l u l o s e K i n e t i c analyses on the c y c l i c AMP  phosphodiesterase  a c t i v i t i e s , i n the peaks o f a c t i v i t y o b t a i n e d from DEAEcellulose,  were made i n order t o i n v e s t i g a t e t h e i r s u b s t r a t e  affinities.  Two types o f data e x p r e s s i o n were used, i . e .  Lineweaver-Burk p l o t s and Hofstee  plots.  A Hofstee p l o t of Peak I c y c l i c AMP activities,  phosphodiesterase  from a mature t e s t i s homogenate, r e v e a l e d a low  apparent Km^ o b t a i n e d from the l i n e a r  slope i n F i g u r e 14.  Hofstee p l o t s o f Peak I I a c t i v i t i e s , from both p r e m e i o t i c and mature t e s t i s homogenates;, i n d i c a t e d s i m i l a r .low; apparent Kms  (Figures 15B and 16B).  Peak I a c t i v i t y  from  FIGURE 14  Hofstee p l o t o f the irate of c y c l i c AMP h y d r o l y s i s by D E A E - c e l l u l o s e Peak I from t e s t i s from t r o u t hormonallyinduced f o r 8 weeks f o r F i g u r e 11D).  ( d e t a i l e d d e s c r i p t i o n i n the legend The u n i t of v e l o c i t y i s pmol/min/10 y l .  Assays were a t 30°, i n standard i n c u b a t i o n mixtures a t pH 7.5, as d e s c r i b e d i n M a t e r i a l s and Methods. (250 yM) was i n c l u d e d i n a l l assays. e n t r a t i o n ranged from 0.05 - 5 yM.  EGTA  S u b s t r a t e conc-  FIGURE 15  A. Lineweaver-Burk  p l o t o f the r a t e o f c y c l i c AMP  hydr-  o l y s i s o f D E A E - c e l l u l o s e Peak I I from t e s t i s from t r o u t hormonally-induced f o r 3 weeks ( t o t a l t e s t e s weight 2 50 The u n i t o f v e l o c i t y i s pmol/min/10 y l .  Assays were a t 30°,  i n standard i n c u b a t i o n m i x t u r e s , a t pH 7.5, M a t e r i a l s and Methods. assays.  EGTA (250 yM) was  mg).  as d e s c r i b e d i n included i n a l l  S u b s t r a t e c o n c e n t r a t i o n ranged from 0.05  - 5 yM.  B. Hofstee p l o t o f the h y d r o l y s i s d e s c r i b e d above. The Lineweaver-Burk 0.8  yM and 2.3 yM.  of 0.5  yM and 4.4  p l o t g i v e s two apparent Kms  of  The Hofstee p l o t g i v e s two apparent yM.  Kms  FIGURE 16  A. Lineweaver-Burk  p l o t o f the r a t e of c y c l i c AMP  by D E A E - c e l l u l o s e Peak I I from t e s t i s  hydrolysis  from t r o u t hormonally-  induced f o r 8 weeks ( d e t a i l e d d e s c r i p t i o n i n the legend f o r F i g u r e 11D).  The u n i t o f v e l o c i t y i s pmol/min/10 u l .  Assays were a t 30°, i n standard i n c u b a t i o n mixtures a t pH as d e s c r i b e d i n M a t e r i a l s and Methods. included i n a l l assays. from 0.05  - 5  EGTA (250 yM)  S u b s t r a t e c o n c e n t r a t i o n ranged  yM.  B. Hofstee p l o t o f the h y d r o l y s i s d e s c r i b e d above. The Lineweaver-Burk  was  p l o t g i v e s a Km  - 1.0  The Hofstee p l o t g i v e s an apparent Km of 0.7  yM..  yM. .  7.5  87.  p r e m e i o t i c t e s t i s homogenate was too low t o o b t a i n r e l i a b l e k i n e t i c data  from.  The n o n - l i n e a r i t y o f both Lineweaver-Burk and Hofstee  (Figure 15A)  (Figures 14, 15B and 16B) p l o t s of c y c l i c  phosphodiesterase a c t i v i t i e s  i s commonly  AMP  observed i n  k i n e t i c s t u d i e s on phosphodiesterase a c t i v i t i e s i n v a r i o u s tissues  (60, 90, 133).  Two apparent Km values c o u l d be  o b t a i n e d from the two s l o p e s on both the p l o t and the Hofstee p l o t o f c y c l i c AMP  Lineweaver-Burk  h y d r o l y s i s by  Peak I I phosphodiesterase from the p r e m e i o t i c t e s t i s homogenate  (Figures 15A and 15B).  The apparent Kms  from a l l  p l o t s corresponded t o phosphodiesterase a c t i v i t i e s w i t h high a f f i n i t y f o r c y c l i c  AMP.  89.  D. Guanylate: c y c l a s e a c t i v i t i e s i n t r o u t t e s t i s  during  spermatogenesis  1. Assay system In crude p r e p a r a t i o n s , guanylate  cyclase a c t i v i t i e s  u s u a l l y s m a l l compared to the a c t i v i t i e s of  interfering  enzymes, such as n u c l e o s i d e t r i p h o s p h a t a s e s ,  cyclic  t i d e phosphodiesterases,  enzymes c a t a l y z e , may effective all  i t s p o s s i b l e degradation  products,  Assays u t i l i z i n g r a d i o a c t i v e GTP  GMP  (126).  contaminating  cochromatograph w i t h c y c l i c GMP,  method o f s e p a r a t i n g .::.! c y c l i c GMP  sensitivity.  nucleo-  n u c l e o t i d a s e s and deaminases  Since the products-, of the r e a c t i o n s which these  are  from GTP  i s imperative  an and (126).  p r o v i d e the maximum  I f [ a P ] G T P i s used, s e p a r a t i o n of 32  cyclic  from a l l other p u r i n e n u c l e o t i d e s and i n o r g a n i c phosphate,  i s required.  With  [ H] 3  or  [ C]GTP as s u b s t r a t e , a d d i t i o n a l llf  s e p a r a t i o n from a l l p u r i n e n u c l e o s i d e s , bases and u r i c i s necessary.  acid  Methods f o r such s e p a r a t i o n s are the same  as those mentioned f o r c y c l i c n u c l e o t i d e p u r i f i c a t i o n . particularly activities  popular method uses  f o r assaying guanylate  [ a P ] G T P as s u b s t r a t e and  of r e a c t i o n products A modification  32  on n e u t r a l alumina  cyclase  purification  (127).  of the l a t t e r method, has been developed  to p r o v i d e g r e a t e r s e n s i t i v i t y cyclase a c t i v i t i e s  A  (106).  f o r the assay of  guanylate  The m o d i f i e d method u t i l i z e s a  combination of DOWEX 50 i o n exchange chromatography and n e u t r a l alumina a d s o r p t i o n chromatography, i n the s e p a r a t i o n of  90.  reaction products.  T h i s method was  chosen f o r the p r e s e n t  r e s e a r c h , due t o i t s low b l a n k o f [ a P ] G T P i n the  purified  32  [ P ] c y c l i c GMP 3 2  fraction,  i t s ease o f performance, i t s  r e l i a b i l i t y and f o r the p r e v i o u s r i g o r o u s i d e n t i f i c a t i o n - o f the  [ P] 32  p r o d u c t as c y c l i c GMP  (106).  Assay c o n d i t i o n s and i n c u b a t i o n m i x t u r e s c l o s e l y owed t h o s e d e s c r i b e d i n the paper w h i c h d e t a i l e d the s t a g e column p r o c e d u r e  (106).  folltwo-  I n t h i s paper a c c u r a t e and  r e l i a b l e g u a n y l a t e c y c l a s e a s s a y s were c a r r i e d o u t i n a v a r i e t y of t i s s u e s .  <:The assay temperature o f 37°  was  used, because g u a n y l a t e c y c l a s e a c t i v i t i e s i n f i s h sperm (88)  had been c a r r i e d o u t a t 37°. I n the p r e s e n t r e s e a r c h , the i n c u b a t i o n c o n t e n t s o u t -  lined previously  (106) were m o d i f i e d t o i n c l u d e a c y c l i c  nucleotide phosphodiesterase i n h i b i t o r , l - m e t h y l - 3 - i s o b u t y l x a n t h i n e , and a GTP  r e g e n e r a t i n g system, c o n s i s t i n g o f 15  mM  p h o s p h o c r e a t i n e and 12.25 u n i t s o f c r e a t i n e phosphokinase  per  assay tube.  an  The GTP  r e g e n e r a t i n g system was  found t o be  a b s o l u t e r e q u i r e m e n t f o r the d e t e c t i o n o f the t o t a l g u a n y l a t e c y c l a s e a c t i v i t i e s i n t r o u t t e s t i s homogenates. the GTP  Without  r e g e n e r a t i n g system o n l y 4% o f the g u a n y l a t e c y c l a s e  a c t i v i t y p r e s e n t was  detected.  The m o d i f i e d i n c u b a t i o n  m i x t u r e a l s o c o n t a i n e d 2-mercaptoethanol  to prevent  spont-  aneous a i r o x i d a t i o n o f the s o l u b l e g u a n y l a t e c y c l a s e , as has been r e p o r t e d i n o t h e r . t i s s u e s ( 7 0 ) .  91. 2. A c t i v i t i e s  and  p r o p e r t i e s d u r i n g t e s t i s development  Guanylate c y c l a s e a c t i v i t i e s were measured i n 100,000xg p e l l e t  (particulate) fraction  supernatant ( s o l u b l e ) f r a c t i o n , from zero time t r o u t , and of and  VII).  The  p a r t i c u l a t e and  weeks of t e s t i c u l a r iculate period  and  and  soluble s p e c i f i c  maturation  (Table V I ) .  The  m a j o r i t y of  the  10 weeks (Tables  a 2  an  and  soluble a c t i v i t i e s  activity  approximate 3 f o l d decrease i n both t o t a l  i n the  (Table V I I ) .  Not  third time, activity (Table  correlate  spermatogenesis.  A guanylate c y c l a s e p a r t i c u l a t e to s o l u b l e r a t i o specific  and  (Tables VI  and  VII).  t o t a l p a r t i c u l a t e guanylate c y c l a s e a c t i v i t y appeared  Table V I I ) , 2.9  to m e i o s i s  resulting  at t h i s stage of  of  1.9  t o t a l a c t i v i t i e s i n zero time  i n f u l l y mature t e s t i s  increase p r i o r  VI),  particulate  p r e s e n t guanylate c y c l a s e study to  observed f o r  same  enough t e s t e s were  enzyme decreases w i t h a p r e c i s e stage of  t e s t i s and  part-  decreases i n both  f o l d decrease i n p a r t i c u l a t e s p e c i f i c  and  was  10  Both t o t a l  During t h i s  a 3 f o l d decrease i n s o l u b l e s p e c i f i c  VI  activities  t o t a l a c t i v i t i e s , o c c u r r e d between the  and  the  6,  extract injections  s i x t h week of hormone i n j e c t i o n s .  sampled  3,  s o l u b l e a c t i v i t i e s decreased 4 f o l d over the  and  there was  100,000xg  4 f o l d , r e s p e c t i v e l y , during the  (Table V I I ) .  specific  i n the  t r o u t t e s t i s homogenates  from t r o u t a f t e r  twice weekly salmon p i t u i t a r y  decreased 6 f o l d and  and  of  and  the  The to  (week 3 of hormonal i n d u c t i o n ;  i n a p a r t i c u l a t e to s o l u b l e r a t i o development.  T r i t o n X-100, a n o n - i o n i c d e t e r g e n t , has  been  of  TABLE VI  GUANYLATE CYCLASE SPECIFIC' ACTIVITIES IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS  Week o f Hormonal Induction  T o t a l Testes Wet Weight (mg)  Guanylate C y c l a s e A c t i v i t i e s 100,000xg 100,000xg Supernatant Pellet  0  130  14. 8 + 0.9  27.9 + 2.0  3  350  11.7 + 0.8  21.8 + 2.4  6  3300  4.3 + 0.4  9.7 + 0.7  10  22000  3.9 + 0.7  4.7 + 0.2  Crude homogenates o f t r o u t t e s t i s were prepared i n b u f f e r and 100,000xg supernatant and p e l l e t f r a c t i o n s o b t a i n e d , a d e s c r i b e d i n M a t e r i a l s and Methods.  Activity  assays were  at 37°, i n standard i n c u b a t i o n mixtures a t pH 7.5, as d e s c r i b e d i n M a t e r i a l s and Methods. pmol c y c l i c GMP formed/min/mg p r o t e i n .  Activity  u n i t s are  Values a r e the  average o f d u p l i c a t e assays on the same p r e p a r a t i o n . The - v a l u e i n d i c a t e s the range between assay d u p l i c a t e s .  TABLE V I I  TOTAL GUANYLATE CYCLASE ACTIVITIES IN TROUT TESTIS 1  DURING HORMONALLY-INDUCED SPERMATOGENESIS  Week o f Hormonal Induction  T o t a l Guanylate C y c l a s e A c t i v i t i e s 100,000xg 100,000xg Totaia Supernatant Pellet  0  0.38  0.70  1.08  3  0.37  1.04  1.41  6  0.12  0.40  0.52  10  0.09  0.19  0.28  Legend as f o r Table V I , except t h a t a c t i v i t y u n i t s are pmol c y c l i c GMP formed/min/mg  t e s t i s wet weight.  T o t a l o b t a i n e d by the summation o f the 100,000xg and supernatant a c t i v i t i e s . was n o t assayed.  T o t a l homogenate  pellet  activity  shown to a c t i v a t e guanylate c y c l a s e a c t i v i t i e s i n other tissues was  (64), s p e c i f i c a l l y  i n the p a r t i c u l a t e f r a c t i o n s .  i n v e s t i g a t e d w i t h regard  to i t s e f f e c t on t r o u t  guanylate c y c l a s e a c t i v i t i e s d u r i n g (Figure 17A).  T r i t o n X-100  It  testis  spermatogenesis  (1% concentration)  stimulated  p a r t i c u l a t e guanylate c y c l a s e a c t i v i t i e s , a t a l l stages of t r o u t t e s t i s development, approximately 2 f o l d The  s o l u b l e guanylate c y c l a s e a c t i v i t y ,  was  also stimulated  by T r i t o n X-100  l a t t e r s t i m u l a t i o n was  1.8  (Figure  17A).  i n zero time t e s t i s ,  (Figure 17B).  The  f o l d , compared w i t h 2.2  fold  s t i m u l a t i o n , by T r i t o n X-100, of p a r t i c u l a t e guanylate cyclase a c t i v i t y The  i n zero time t e s t i s .  e f f e c t of a gonadotropin e x t r a c t on guanylate  activities,  i n zero time t e s t i s , was  (Figure 17B).  also  investigated  (The gonadotropin e x t r a c t used was  salmon p i t u i t a r y p r e p a r a t i o n  Methods.)  the  used i n the i n i t i a t i o n  enhancement of t r o u t spermatogenesis, as d e s c r i b e d M a t e r i a l s and  The  cyclase  standard  and in  gonadotropin e x t r a c t caused  an apparent 10 f o l d s t i m u l a t i o n of the s o l u b l e zero time a c t i v i t y and  an apparent 2 f o l d s t i m u l a t i o n of the p a r t i c u l a t e  zero time a c t i v i t y .  A d d i t i o n of both T r i t o n X-100  and  gonadotropin e x t r a c t , to the p a r t i c u l a t e f r a c t i o n r e s u l t e d i n a s l i g h t l y l e s s than a d d i t i v e i n c r e a s e stimulation occurred extract  (Figure 17B).  No  i . e . 3.2  s t i m u l a t i o n of  fold  activity  w i t h the a d d i t i o n of heat-denatured gonadotropin (data not  e x t r a c t was  shown).  However, when the  assayed without any  gonadotropin  t e s t i s p r o t e i n present, a  95. FIGURE 17  A. T r i t o n X-100  e f f e c t on guanylate c y c l a s e a c t i v i t y i n  the 100,000xg p e l l e t from t e s t i s o f zero time t r o u t  (OP),  and from t e s t i s of t r o u t a f t e r 3 (3P), 6 (6P), and 10  (10P)  weeks of twice weekly hormone i n j e c t i o n s , as d e s c r i b e d i n M a t e r i a l s and Methods.  T o t a l t e s t e s weights  and  tissue  p r e p a r a t i o n as d e s c r i b e d i n the legend f o r Table V I . A c t i v i t i e s were assayed a t 37°, i n standard i n c u b a t i o n m i x t u r e s , as d e s c r i b e d i n M a t e r i a l s and Methods. bars i n d i c a t e the range between assay  Error  duplicates.  •  No a d d i t i o n s  W$  P l u s 1% T r i t o n X-100  (x)  B. Comparison o f the e f f e c t s of salmon gonadotropin  and  T r i t o n X-100 supernatant activities.  on the 100,000xg p e l l e t (OS)  (OP)  and the 100,000xg  zero time t r o u t t e s t i s guanylate c y c l a s e  A c t i v i t i e s were assayed as d e s c r i b e d above.  Symbols as above, p l u s : fZ3  P l u s salmon gonadotropin (h) (15 y l crude p i t u i t a r y e x t r a c t ) T r i t o n X-100 gonadotropin  (1%) and (hx)  salmon  c o n s i d e r a b l e endogenous g u a n y l a t e c y c l a s e a c t i v i t y was observed;  4.23 pmol/min/15 y l o f g o n a d o t r o p i n e x t r a c t ,  11.3 pmol/min/mg g o n a d o t r o p i n e x t r a c t p r o t e i n .  or  Since the  u n s t i m u l a t e d s o l u b l e g u a n y l a t e c y c l a s e a c t i v i t y was lower t h a n t h e u n s t i m u l a t e d p a r t i c u l a t e a c t i v i t y , t h e same a d d i t i o n o f g o n a d o t r o p i n e x t r a c t t o e a c h , produced a g r e a t e r apparent s t i m u l a t i o n o f the s o l u b l e a c t i v i t y . a c t i v i t y are t o t a l l y a t t r i b u t a b l e  Both increases  t o the endogenous  c y c l a s e a c t i v i t y i n the g o n a d o t r o p i n  extract.  in  guanylate  98. DISCUSSION C y c l i c GMP and c y c l i c AMP c o n c e n t r a t i o n s i n t r o u t t e s t i s d u r i n g spermatogenesis C y c l i c GMP c o n c e n t r a t i o n s i n immature t r o u t t e s t e s were r e v e a l e d t o be h i g h  (about 2 ymol/kg t e s t i s wet weight) and  equal t o c y c l i c 7AMP c o n c e n t r a t i o n s i n immature t r o u t t e s t e s . In most v e r t e b r a t e t i s s u e s c y c l i c GMP c o n c e n t r a t i o n s a r e i n the range o f 0.05-0.1 ymol/kg wet weight  (53) and c y c l i c AMP  c o n c e n t r a t i o n s are about 10-50 f o l d h i g h e r t i s s u e s , i n c l u d i n g lung, cerebellum  (64).  and lymph, c y c l i c  and c y c l i c AMP c o n c e n t r a t i o n s are approximately the normal c y c l i c AMP c o n c e n t r a t i o n range  GMP  equal and i n  (53, 64).  v e r t e b r a t e t i s s u e , namely the r e t i n a , extremely GMP c o n c e n t r a t i o n s are observed  In s e v e r a l  In one  high  cyclic  (about 100 ymol/kg wet weight)  and t h e r e i s a c y c l i c GMP t o c y c l i c AMP r a t i o o f 100 (141). In one i n v e r t e b r a t e t i s s u e , namely the male c r i c k e t u c t i v e accessory gland, very h i g h c y c l i c GMP  reprod-  concentrations  are a l s o found, 40-100 ymol/kg wet weight, w h i l e c y c l i c AMP c o n c e n t r a t i o n s are only 0.02-0.3 ymol/kg wet weight  (130).  The h i g h c y c l i c GMP c o n c e n t r a t i o n s i n the l a t t e r case were shown to be a s s o c i a t e d w i t h the gland i t s e l f and n o t the sperm i t c o n t a i n s , s i n c e high c y c l i c GMP was a l s o i n the r e p r o d u c t i v e glands  observed  from c a s t r a t e d c r i c k e t s  In immature t r o u t t e s t i s i t has been estimated spermatogonial  (130). that  germ c e l l s make up about 10-15% o f the t o t a l  wet weight and the remainder i s due to connective t i s s u e s and the c e l l s w i t h i n t h i s component such as L e y d i g c e l l homologues and b l o o d and nerve c e l l s  (10).  One cannot  which o f these c e l l types may have an e s p e c i a l l y  conclude  high  c o n c e n t r a t i o n o f one AMP,  or both.  However, over a p e r i o d of a 5-10  rease i n t e s t i c u l a r cell  99. or c y c l i c  c y c l i c n u c l e o t i d e . , c y c l i c GMP  wet  fold  inc-  weight, d u r i n g which time the germ  component would be i n c r e a s i n g r a p i d l y , c y c l i c  c o n c e n t r a t i o n s remained high  (Figure 4 ) .  q u i t e p o s s i b l e t h a t e l e v a t e d c y c l i c GMP  GMP  Therefore, i t i s concentrations  are  c h a r a c t e r i s t i c of spermatogonia, as w e l l as perhaps other cell  types p r e s e n t i n the immature t r o u t t e s t i s . S e v e r a l l i n e s o f r e s e a r c h have suggested  GMP  may  a c t as a p o s i t i v e s i g n a l  act  as a negative s i g n a l  (131,  (7, 8) w h i l e c y c l i c AMP  132)  i n the c e l l  l i n g the growth of c u l t u r e d f i b r o b l a s t s . e n t r a t i o n s of c y c l i c GMP  and  that c y c l i c  c y c l i c AMP  for control-  R e c i p r o c a l conchave been  d u r i n g the c e l l c y c l e of N o v i k o f f hepatoma c e l l s E l e v a t e d c y c l i c GMP  may  observed (95).  d u r i n g m i t o s i s and S phase were c o n s i d e r e d  to be c o n s i s t e n t w i t h p o t e n t i a l modulatory r o l e s f o r c y c l i c GMP  in proliferation  r e c i p r o c a l c y c l i c GMP  (95).  There was  and c y c l i c AMP  no evidence  for  concentrations i n trout  t e s t i s d u r i n g development.  In f a c t i n immature t e s t i s  the  c o n c e n t r a t i o n s were e q u a l .  However, the h e t e r o g e n e i t y  of  cell  types p r e s e n t i n immature t r o u t t e s t i s prevents  the  o b s e r v a t i o n of r e l a t i v e c y c l i c n u c l e o t i d e c o n c e n t r a t i o n s i n specific  cell  hypothesis  types.  Goldberg  s t a t e s i n the Y i n Yang  of c y c l i c n u c l e o t i d e r e g u l a t i o n , t h a t the  p r o p o r t i o n s of c e l l u l a r c y c l i c GMP important  and c y c l i c AMP  than t h e i r a c t u a l c o n c e n t r a t i o n s  (5 3).  relative  may  be more  The  Yang h y p o t h e s i s , based on the a n c i e n t o r i e n t a l concept dualism between opposing  n a t u r a l f o r c e s which may  enter  Yin of a into  100. a mutual i n t e r a c t i o n t h a t r e s u l t s i n s y n t h e s i s , was to e x p l a i n the s t r i k i n g l y of c y c l i c GMP (53, 133,  antagonistic regulatory  and c y c l i c AMP  134).  both c y c l i c GMP  used  influences  i n several biological  systems-.  In more .recent r e s e a r c h (54) the r o l e s of and c y c l i c AMP  i n regulation i n various c e l l  types has become a s u b j e c t of c o n s i d e r a b l e c o n t r o v e r s y . There i s growing evidence t h a t the c y c l i c n u c l e o t i d e s do not act  alone as modulatory 2+  modulators  such as Ca  c e l l u l a r event  e f f e c t o r s but may  act i n concert with  . . and l i p i d s to promote or i n h i b i t a  (54).  The most s t r i k i n g  aspect of the p r e s e n t study o f c y c l i c  nucleotides i n developing trout t e s t i s  i s the sharp . (during  1 week of the 12 week process) 10 f o l d decrease i n c y c l i c a f t e r about 4 weeks.(Figure  4).  GMP  T h i s decrease occurs a t the  middle of the l o g a r i t h m i c growth of the t r o u t t e s t i s  (Figure 3)  s h o r t l y b e f o r e the onset of m e i o s i s a t weeks 5 t o 6 (10). Although t h e r e i s u n c e r t a i n t y about the e x a c t t i m i n g of m e i o s i s i n the p r e s e n t study, i t i s obvious from F i g u r e 4 t h a t the decrease i n c y c l i c GMP  o c c u r r e d b e f o r e the p r o l i f e r -  a t i o n o f germ c e l l s had ceased. of  the decrease i n c y c l i c GMP  f o r the onset of m e i o s i s .  The magnitude and t i m i n g  suggest t h a t i t may  specific  to determine whether spermatogonia  c e l l types r a p i d l y  have s i g n i f i c a n t l y g r e a t e r  than primary  spermatocytes.  In r a t t e s t i s , the b i n d i n g of immunofluorescent c y c l i c GMP  to prophase  critical  To s u b s t a n t i a t e t h i s h y p o t h e s i s  i t would be important t o i s o l a t e  c o n c e n t r a t i o n s of c y c l i c GMP  be  chromosomes i n primary  a n t i b o d i e s to  spermatocytes(79)  i s c o n s i s t e n t w i t h an important r o l e f o r c y c l i c GMP  at meiosis.  101. A f t e r the sharp 10 f o l d decrease i n c y c l i c GMP testis  a t the onset of m e i o s i s , c y c l i c GMP  decreased  a l l y another 5 f o l d d u r i n g the remainder of (Figure 4 ) .  i n trout  spermatogenesis  A f t e r the 2 f o l d decrease i n c y c l i c AMP  trout t e s t i s prior  to m e i o s i s  change s i g n i f i c a n t l y  gradu-  (Figure 5 ) , c y c l i c AMP  in d i d not  d u r i n g the f o l l o w i n g stages of m e i o t i c  r e d u c t i o n and spermatid  differentiation.  The c y c l i c n u c l e o t i d e c o n c e n t r a t i o n s i n d e v e l o p i n g t r o u t t e s t i s can be compared w i t h those i n r a t t e s t i s d u r i n g maturation GMP  (79).  Elevated i n i t i a l  and c y c l i c AMP  c o n c e n t r a t i o n s of both  were a l s o observed i n the r a t t e s t i s , but  the i n i t i a l  c y c l i c AMP  c o n c e n t r a t i o n was  the i n i t i a l  c y c l i c GMP  concentration.  40 f o l d h i g h e r than Both c y c l i c n u c l e o -  t i d e s decreased about 2 f o l d a t the time of the f i r s t tive divisions i n rat testis. c y c l i c GMP  cyclic  The p a r t i c u l a r l y  reduc-  elevated  c o n c e n t r a t i o n s observed i n t r o u t t e s t i s d u r i n g  e a r l y development and the s t r i k i n g  decrease i n these concent-  r a t i o n s a t the onset o f m e i o s i s appear to be s p e c i a l to t h i s system.  The l a r g e m u l t i p l i c a t i o n of spermatogonial germ  c e l l s and the synchrony of germ c e l l t e s t i s may  c o n t i b u t e to these o b s e r v a t i o n s .  remainder o f r a t t e s t i c u l a r GMP  were seen, but t h e r e was  ration. c y c l i c GMP which may  development i n the t r o u t  There was  development,'fluctuations i n c y c l i c an o v e r a l l  decrease i n concent-  a l a r g e r and: c l e a r t r e n d of decrease i n  i n t r o u t t e s t i s d u r i n g spermatid result  During the  differentiation,  from the homogeneity of germ c e l l  present i n trout t e s t i s .  T h e r e - i s much  type  g r e a t e r germ  102. c e l l heterogeneity decreased  i n r a t testis„(1, 10).  Cyclic  AMP  s t e a d i l y i n r a t t e s t i s u n t i l the time of  the  appearance o f spermatids  (79).  During  spermatid  e n t i a t i o n the c y c l i c AMP  c o n c e n t r a t i o n i n c r e a s e d to about  h a l f of the immature r a t t e s t i s c o n c e n t r a t i o n . AMP  d i d not i n c r e a s e i n t r o u t t e s t i s d u r i n g  d i f f e r e n t i a t i o n , suggesting may than  p l a y a more important o  f  differ-  Cyclic  spermatid  that this c y c l i c  nucleotide  r o l e i n spermatids of mammals  fish.  I t would be d e s i r a b l e to combine c y c l i c n u c l e o t i d e terminations  in trout testis  , with  n u c l e o t i d e b i n d i n g s t u d i e s and c e l l  immunofluorescent  cyclic  s e p a r a t i o n methods, to  determine the predominant s i t e s of the c y c l i c and  de-  nucleotides  t h e i r receptor proteins. The  o b s e r v a t i o n of the l a r g e and  c y c l i c GMP  abrupt  decrease i n  e a r l y i n the development of t r o u t t e s t i s ,  init-  i a t e d an i n v e s t i g a t i o n i n t o some of the f a c t o r s which be important rations.  i n the c o n t r o l of c e l l u l a r c y c l i c GMP  In p a r t i c u l a r c y c l i c GMP  i t i e s and guanylate t e s t i s were s t u d i e d .  may  concent-  phosphodiesterase  c y c l a s e a c t i v i t i e s i n developing  activtrout  E x t r u s i o n of c y c l i c n u c l e o t i d e s i s  another p o t e n t i a l l y important nucleotide concentrations  method of c o n t r o l l i n g  cyclic  i n t r o u t t e s t i s , but t h i s was  i n v e s t i g a t e d i n the present  research.  not  103. C y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t i e s i n t r o u t t e s t i s during  spermatogenesis  The f a c t t h a t the t o t a l c y c l i c GMP  phosphodiesterase  activ-  i t y i n t r o u t t e s t i s d i d not change s i g n i f i c a n t l y  throughout  spermatogenesis  relation  (Table I I I ) i n d i c a t e d no obvious  between c y c l i c GMP concentration. on c y c l i c GMP  phosphodiesterase a c t i v i t y and c y c l i c  T h i s c o n c l u s i o n was  GMP  confirmed by the s t u d i e s  phosphodiesterases measured w i t h  micromolar  s u b s t r a t e c o n c e n t r a t i o n d u r i n g development ' (Tables IV and and the p r o f i l e s cellulose  V)  of phosphodiesterase a c t i v i t y on DEAE-  (Figure 11).  These s t u d i e s showed t h a t t h e r e  no i n d u c t i o n of a h i g h a f f i n i t y c y c l i c GMP  was  phosphodiesterase  a t the time of the l a r g e decrease i n c y c l i c GMP c o n c e n t r a t i o n . EGTA i n h i b i t e d t o t a l c y c l i c AMP  and c y c l i c GMP  phosphodi-  e s t e r a s e a c t i v i t i e s i n t r o u t t e s t i s from 10 to 50% a t d i f f e r ent  stages of development,  c y c l i c GMP .  except f o r a 30% s t i m u l a t i o n of  phosphodiesterases i n immature t e s t i s . .  The i n h i b i t i o n  (Table I I I ) .  .  ".  by EGTA i n d i c a t e s the presence of an a c t i v e Ca  b i n d i n g a c t i v a t o r of c y c l i c n u c l e o t i d e phosphodiesterase .  . .  a c t i v i t i e s in trout t e s t i s .  .  2+  A Ca  - b i n d i n g p r o t e i n capable  of a c t i v a t i n g c y c l i c n u c l e o t i d e phosphodiesterases has been i s o l a t e d from sea u r c h i n sperm  (133) but t h i s p r o t e i n  activ-  a t o r had no e f f e c t on endogenous sea u r c h i n sperm c y c l i c n u c l e o t i d e phosphodiesterases i s o l a t e d on D E A E - c e l l u l o s e . 2+  I t i s apparent i n s e v e r a l t i s s u e s t h a t the Ca e i n may  -binding prot-  be i n v o l v e d w i t h other m e t a b o l i c f u n c t i o n s , such as  2+  104. p r o t e i n p h o s p h o r y l a t i o n (134), b e s i d e s phosphodiesterase  2+ activation.  The p o s s i b i l i t y  of the i n d u c t i o n of a Ca  dependent high a f f i n i t y c y c l i c GMP  phosphodiesterase  activity  a s s o c i a t e d w i t h the l a r g e decrease i n c y c l i c GMP  i n devel-  oping t r o u t t e s t i s was  No change  investigated  2+ i n the Ca was  . -dependent c y c l i c GMP  . .  phosphodiesterase  activity  found a t the" onset o f meiosis.. Both c y c l i c AMP  and c y c l i c  measured a t micromolar immature t r o u t t e s t i s stage of development i n c y c l i c AMP rat  (Table V ) .  GMP  phosphodiesterase  activities  s u b s t r a t e c o n c e n t r a t i o n s were h i g h e r i n than i n t e s t i s (Figure 10B).  and c y c l i c GMP  a t the  spermatocyte  In a study of changes  phosphodiesterase a c t i v i t e s i n  l i v e r and lung t i s s u e s , h i g h e r a c t i v i t i e s were found i n  f e t a l than i n neonatal t i s s u e s , and the a c t i v i t i e s  i n neonatal  t i s s u e s were i n t u r n higher than those i n a d u l t t i s s u e s A r e v e r s e p a t t e r n was  observed  i n developing b r a i n  In a l l these t i s s u e s the magnitude of c y c l i c GMP  (135).  (135).  phosphodi-  e s t e r a s e a c t i v i t i e s a t d i f f e r e n t stages of development, c o r r e l a t e d w e l l w i t h the magnitude of cyclic.GMP kinase a c t i v i t i e s  (136).  On the b a s i s of h i g h c y c l i c  phosphodiesterase and p r o t e i n k i n a s e a c t i v i t i e s lung and a d u l t b r a i n , i t was u l a t e d by c y c l i c GMP  of  in fetal  were important i n these t i s s u e s  (13 5).  p r o t e i n kinase a c t i v i t i e s i n  c e l l s d u r i n g development might r e v e a l the  a c t i o n of c y c l i c GMP  GMP  hypothesized t h a t a c t i o n s mod-  An i n v e s t i g a t i o n of c y c l i c GMP trout testis  protein  a t the onset of m e i o s i s .  site  105. The most s t r i k i n g esterase a c t i v i t i e s  change i n c y c l i c n u c l e o t i d e phosphodii n developing trout t e s t i s  t i o n of a c y c l i c AMP  phosphodiesterase w i t h h i g h s u b s t r a t e  a f f i n i t y , which appears  a t about week 5 of  (Table IV and F i g u r e s 10B, c y c l i c AMP  i s the induc-  spermatogenesis  11C and 11D).  phosphodiesterase a c t i v i t y  A high a f f i n i t y  a s s o c i a t e d w i t h the  onset of m a t u r i t y has been found i n r a t and r a b b i t t e s t i s and i n ram  sperm  (94).  D E A E - c e l l u l o s e chromatography of t r o u t t e s t i s at d i f f e r e n t c y c l i c AMP  stages of development  illustrated  phosphodiesterase a c t i v i t y ,  a l s o h y d r o l y s e d c y c l i c GMP  ( F i g u r e 11).  development the induced h i g h a f f i n t y e s t e r a s e a c t i v i t y was of  the c y c l i c AMP  of  homogenates two peaks of  the f i r s t of which During  c y c l i c AMP  spermatid phosphodi-  observed t o c o e l u t e w i t h the  peaks  n o t a b l e t h a t there was c y c l i c GMP  (Figures 11C  and 11D).  first  It is  no c o - i n d u c t i o n of a h i g h a f f i n i t y  phosphodiesterase a c t i v i t y .  D E A E - c e l l u l o s e peak c y c l i c AMP  K i n e t i c analyses  phosphodiesterase  activ-  i t i e s from mature t r o u t t e s t i s homogenate (Figure  11D)  confirmed the presence of h i g h a f f i n i t y a c t i v i t i e s  i n both  peaks kinetic  (Figures 15 and 16). p l o t s may  (93)  result  The n o n - l i n e a r i t y of these  from m u l t i p l e enzyme s p e c i e s i n  one peak, from n e g a t i v e c o - o p e r a t i v i t y of a s i n g l e enzyme s p e c i e s , or from i n t e r c o n v e r t i b l e forms of with d i f f e r i n g  activities  phosphodiesterase  (60, 90).  Although ho l o w - a f f i n i t y c y c l i c AMP was observed i n mature t r o u t t e s t i s  phosphodiesterase  (Figure 8), such an  106. activity  i s o b v i o u s l y p r e s e n t i n immature t e s t i s , where  c y c l i c AMP phosphodiesterase a c t i v i t i e s were a t l e a s t 4 0 f o l d h i g h e r when measured w i t h m i l l i m o l a r c y c l i c AMP w i t h micromolar  s u b s t r a t e (Tables I I I and I V ) .  be l i t t l e or no low a f f i n i t y c y c l i c AMP activity  than  There may  phosphodiesterase  i n mature t e s t i s , where a c t i v i t i e s measured w i t h  m i l l i m o l a r c y c l i c AMP were o n l y about 2 0% h i g h e r than measured w i t h micromolar  s u b s t r a t e (Tables I I I and I V ) .  T h i s i n d i c a t e s t h a t the induced h i g h a f f i n i t y c y c l i c phosphodiesterase i s the predominant c y c l i c AMP esterase a c t i v i t y  AMP  phosphodi-  p r e s e n t i n mature t e s t i s .  Both a low and h i g h a f f i n i t y c y c l i c GMP a c t i v i t y were observed  phosphodiesterase  i n mature t r o u t t e s t i s  C y c l i c GMP phosphodiesterase a c t i v i t i e s ment were about  those  (Figure 9 ) .  throughout  develop-  4 0 f o l d h i g h e r when measured w i t h m i l l i m o l a r  c y c l i c GMP, than when measured w i t h micromolar (Tables I I I and I V ) .  cyclic  GMP  T h i s i n d i c a t e s t h a t the low a f f i n i t y  phosphodiesterase a c t i v i t y  i s predominant throughout  sperm-  atogenesis . S o l u b l e . c y c l i c AMP and c y c l i c GMP  phosphodiesterase,  a c t i v i t i e s , i . e . those found i n the 100,000xg supernatant f r a c t i o n , when measured a t micromolar ions were about  substrate concentrat-  85% and 80%, r e s p e c t i v e l y , o f s i m i l a r l y  measured t o t a l homogenate a c t i v i t i e s .  A soluble  fraction  from t r o u t t e s t i s homogenate c o n t a i n e d both peaks o f c y c l i c n u c l e o t i d e phosphodiesterase a c t i v i t y cellulose  (Figure 13A).  The 2.5 f o l d  e l u t e d from DEAEi n c r e a s e i n the r a t i o  107. of  Peak  I I t o Peak  profile  I cyclic  as compared w i t h  enate p r o f i l e  from  TAMP a c t i v i t y  t h e same r a t i o  t h e same t e s t i s  partial  conversion  o f Peak  aration  or storage  a t -20°.  of  I I t o Peak  I cyclic  Peak  on D E A E - c e l l u l o s e preparations mild  after  treatment  The  trout  testis  second peak  second  cellulose  AMP  profiles  gland  the present  study  were  i.e.a first  and c y c l i c  cyclic  AMP  (137).  observed  after  AMP  pellet  fraction  from  mainly  activity  i n the  phosphodi-  (Figure 13B).  In  phosphodiesterases  (60)  p e a k o n DEAE-  exclusively  similar  on t r o u t t e s t i s  AMP  GMP  from the  GMP  reproductive  t o those  cyclic  and a second peak  forcyclic  GMP,  c o n c e n t r a t i o n s were  i n the reproductive accessory  No a p p r e c i a b l e c h a n g e s i n t h e s p e c i f i c  observed  nucleotide  peak h y d r o l y z i n g  The o b s e r v a t i o n o f no  specificity  e v e n when c y c l i c  a t 4° o r  o f male c r i c k e t  homogenates  cyclic  distinct  isolated  profiles  phosphodiesterases,  with  i n rat liver  cyclic  phosphodiesterase  was  activity  fraction.  DEAE-cellulose accessory  contained  studies of r a t liver  cyclic  prep-  (60).  e l u t e d from DEAE-cellulose  100,000xg p e l l e t  in  phosphodiesterase  i n t h e 100,000xg  homogenate,  fractionation the  AMP  s m a l l amount o f p a r t i c u l a t e  esterase a c t i v i t y  reflect  Such an i n c r e a s e i n t h e r a t i o  o f homogenates  trypsin  homog-  I I on supernatant  has been observed  storage  with  i n the total  ( F i g u r e 11B), may  I t o Peak  profiles  i n the soluble  both  specific for phosphodiesterase  i n the trout high  gland  testis,  i s also o f male  activity  or  crickets. kinetic  108. p r o p e r t i e s of accessory gland c y c l i c GMP  phosphodiesterases  were seen d u r i n g a developmental p e r i o d over which c y c l i c c o n c e n t r a t i o n s rose more than 500  fold  GMP  (137).  The c y c l i c n u c l e o t i d e phosphodiesterase s t u d i e s i n t r o u t t e s t i s do not i n d i c a t e a s e l e c t i v e r o l e f o r phosphodiesterases i n modulating c y c l i c GMP  concentrations during  spermatogenesis.  Guanylate c y c l a s e a c t i v i t i e s i n t r o u t t e s t i s d u r i n g spe rmatogene s i s Guanylate c y c l a s e a c t i v i t i e s i n t r o u t t e s t i s were i n v e s t i g a t e d to determine  t h e i r r o l e i n r e g u l a t i n g c y c l i c GMP  e n t r a t i o n s d u r i n g spermatogenesis.  conc-  A direct correlation  found between guanylate c y c l a s e a c t i v i t i e s and c y c l i c GMP  was con-  c e n t r a t i o n s i n d e v e l o p i n g t r o u t t e s t i s , as i s shown i n F i g u r e 18.  Surveys of guanylate eye l a s e a c t i v i t i e s i n r a t t i s s u e s  have demonstrated  a c o r r e l a t i o n between guanylate c y c l a s e  a c t i v i t y and c y c l i c GMP  c o n c e n t r a t i o n (82, 138,  139).  In immature and mature t r o u t t e s t i s , a p a r t i c u l a t e to s o l u b l e guanylate c y c l a s e a c t i v i t y r a t i o of 1.9 (Tables VI and V I I ) . was  was  observed  A s i m i l a r p a r t i c u l a t e to s o l u b l e  ratio  observed i n a study of guanylate c y c l a s e a c t i v i t i e s i n  immature and a d u l t r a t t e s t i s  (82).  Both p a r t i c u l a t e  s o l u b l e guanylate c y c l a s e s p e c i f i c a c t i v i t i e s d u r i n g " t r o u t t e s t i s maturation  and  decreased  (Table V I ) , but the p a r t i c -  u l a t e enzyme showed a t o t a l a c t i v i t y i n c r e a s e j u s t p r i o r to meiosis  (Table V I I ) .  r o l e f o r c y c l i c GMP  T h i s c o u l d be r e l a t e d t o a s p e c i f i c  i n the nucleus a t m e i o s i s .  more thorough i n v e s t i g a t i o n o f guanylate c y c l a s e d u r i n g spermatogenesis  However, a activities  i s needed to s u b s t a n t i a t e t h i s i d e a .  FIGURE 18 Comparison GMP  of guanylate c y c l a s e a c t i v i t i e s and  c o n c e n t r a t i o n s i n t r o u t t e s t i s d u r i n g hormonally-  induced  spermatogenesis.  Guanylate c y c l a s e was ent  cyclic  assayed, i n t r o u t t e s t i s a t d i f f e r -  stages of development,  Methods.  as d e s c r i b e d i n M a t e r i a l s  Data from Table VI.  pmol c y c l i c GMP  Units of a c t i v i t y  formed/min/mg p r o t e i n .  Cyclic  of development,  are  GMP  c e n t r a t i o n s assayed, i n t r o u t t e s t i s a t d i f f e r e n t  and  constages  as d e s c r i b e d i n M a t e r i a l s and Methods.  Data from Table IA.  110.  Spermatogonia  2 Week  of  Spermatocytes  Spermatids  4 6 8 Hormonal Induction  10  111. T r i t o n X-100  (a n o n - i o n i c  detergent) a c t i v a t e d p a r t i c u l a t e  guanylate c y c l a s e a c t i v i t i e s i n t r o u t t e s t i s , a t a l l stages of development, about 2 f o l d .  This indicates that  the  p a r t i c u l a t e enzyme had  the same membrane environment through-  out spermatogenesis.  S o l u b l e guanylate c y c l a s e a c t i v i t y i n  immature t r o u t t e s t i s was same extent there may  a c t i v a t e d by T r i t o n X-100  as the p a r t i c u l a t e enzyme.  p a r t i c u l a t e enzyme.  and  others  I t has  soluble  been suggested by  Goldberg  t h a t the s o l u b l e form of guanylate c y c l a s e  o r i g i n a t e from c e l l membranes importance of the two a l i a n t i s s u e s , has  (54).  the  T h i s suggests t h a t  be a c l o s e r e l a t i o n i n form between the  and  to  Discussion  on  may  the  forms of guanylate c y c l a s e , i n mamm-  s t r e s s e d the p o s s i b l e p h y s i o l o g i c a l  r e g u l a t i o n of the two  forms  (140).  Increased  particulate  and decreased s o l u b l e guanylate c y c l a s e a c t i v i t i e s have been found i n r e g e n e r a t i n g tomas (140).  r a t l i v e r , f e t a l r a t l i v e r , and  These o b s e r v a t i o n s  l e d to the  speculation  t h a t p a r t i c u l a t e guanylate c y c l a s e a c t i v i t y may w i t h t i s s u e growth and  hepa-  be  t h a t s o l u b l e a c t i v i t y may  be  associated assoc-  iated with acquired  f u n c t i o n s of d i f f e r e n t i a t i o n .  r e l a t i v e l y constant  r a t i o of p a r t i c u l a t e to s o l u b l e guanyl-  ate c y c l a s e a c t i v i t y d u r i n g support t h i s  The  t r o u t t e s t i s development does not  hypothesis.  No d i r e c t e f f e c t of a hormonal or p h y s i o l o g i c a l agent on guanylate c y c l a s e a c t i v i t y has been c o n v i n c i n g l y  demonstrated.  The  f i n d i n g of an apparent s t i m u l a t i o n of both p a r t i c u l a t e  and  s o l u b l e guanylate c y c l a s e a c t i v i t i e s i n immature t r o u t  112. t e s t i s , by a salmon p i t u i t a r y was  gonadotropin e x t r a c t  shown to be due t o endogenous a c t i v i t y  preparation.  (Figure 16),  i n the hormone  The s o l u b l e t e s t i s guanylate c y c l a s e , presum-  ably not the p h y s i o l o g i c a l form which would respond t o gonadotropin s t i m u l a t i o n , was  a p p a r e n t l y s t i m u l a t e d to a  g r e a t e r extent than the p a r t i c u l a t e enzyme.  Hormone s t i m -  u l a t i o n of guanylate c y c l a s e s i n o t h e r t i s s u e s has been r e p o r t e d t o be suspect due t o the i m p u r i t i e s of the prepara t i o n s used  (54).  In these cases a l s o , the s o l u b l e guanylate  c y c l a s e was  a p p a r e n t l y s t i m u l a t e d to a g r e a t e r extent than  the p a r t i c u l a t e enzyme.  Conclusions T h i s study of t r o u t spermatogenesis  has r e v e a l e d t h a t  d u r i n g t e s t i s development t h e r e are t h r e e s t r i k i n g  and  p r e c i s e l y timed changes r e l a t e d to c y c l i c n u c l e o t i d e metabolism.  These are a decrease i n c y c l i c GMP c o n c e n t r a t i o n ,  a decrease i n guanylate c y c l a s e a c t i v i t i e s and the i n d u c t i o n of a h i g h a f f i n i t y c y c l i c AMP  phosphodiesterase.  I t i s reasonable to assume t h a t t h e r e i s a c o n n e c t i o n between the decrease i n c y c l i c GMP  c o n c e n t r a t i o n , the  decrease i n guanylate c y c l a s e a c t i v i t y and the onset of meiosis  (Figure 18).  C l e a r l y f u r t h e r s t u d i e s should be  d i r e c t e d towards the i d e n t i f i c a t i o n the decrease i n c y c l i c GMP  of the c e l l s i n which  c o n c e n t r a t i o n occurs and  an  i n v e s t i g a t i o n of the guanylate c y c l a s e a c t i v i t i e s i n those cells.  I f these experiments  c o n f i r m the presumed  113. r e l a t i o n s h i p , then i t w i l l be o f g r e a t  i n t e r e s t to define  the nature o f the decreased a c t i v i t y o f the guanylate c y c l a s e s , and the source and nature o f the s i g n a l which induces the change.  A l s o of importance w i l l be an  i n v e s t i g a t i o n o f the t a r g e t o f the c y c l i c GMP i n the c e l l s where the c o n c e n t r a t i o n  changes.  A detailed  study o f the c y c l i c GMP-binding p r o t e i n s and c y c l i c dependent p r o t e i n kinase during  a c t i v i t i e s i n trout  testis  spermatogenesis may r e v e a l e x c i t i n g r e s u l t s .  The  i n d u c t i o n o f a high a f f i n i t y c y c l i c AMP phospho-  diesterase i n trout t e s t i s during  spermatogenesis  (Figure 10B) i s not unique t o the developing 94).  GMP-  trout testis(93,  I d e n t i f i c a t i o n of^ and s t u d i e s on, the c e l l type i n  t r o u t t e s t i s i n which the i n d u c t i o n takes p l a c e provide  could  relevant insights into c y c l i c nucleotide  u l a t i o n during t h i s type,  spermatogenesis.  should  reg-  Investigations of  be p o s s i b l e i n t r o u t t e s t i s , but i t  may be t h a t s t u d i e s on the mammalian t e s t i s , w i t h b e t t e r d e f i n e d endocrinology  and c y t o l o g y may be more  fruitful  at t h i s time; i d e a l l y , d e t a i l e d s t u d i e s on both systems should  be c a r r i e d o u t .  I t seems c l e a r from t h i s i n t r o d u c t o r y  study t h a t a  d e t a i l e d study o f the involvement o f c y c l i c i n t e s t i s development w i l l p r o v i d e interesting results.  nucleotides  both s u r p r i s i n g and  114. REFERENCES 1. C o u r o t , M.,  Hocereau-de r e v i e r s ,  (1970) I n : The T e s t i s . W.R.  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APPENDIX  V a r i o u s methods f o r the s e p a r a t i o n o f c y c l i c were i n v e s t i g a t e d d u r i n g t h i s r e s e a r c h  nucleotides  (Figure 19).  s e p a r a t i o n o f c y c l i c AMP from c y c l i c GMP, i n t r o u t  The testis  samples, was r o u t i n e l y performed by e l u t i o n o f AG 1-X8 r e s i n columns w i t h formic a c i d (Figure 19A). d e t a i l e d i n the legend  Under the c o n d i t i o n s  f o r F i g u r e 19A, i t was found t h a t  c y c l i c GMP contamination i n the c y c l i c AMP f r a c t i o n was 2%, and  c y c l i c AMP contamination i n the c y c l i c . GMP f r a c t i o n was  4%. Separation  o f c y c l i c AMP and c y c l i c GMP on D E A E - c e l l u l o s e  columns was a l s o i n v e s t i g a t e d (Figure 19B).  While t h e  s e p a r a t i o n was e x c e l l e n t , t h i s method was t o o time-consuming f o r r o u t i n e sample p u r i f i c a t i o n s .  On  DEAE-cellulose  f r a c t i o n a t i o n , w i t h a l i n e a r g r a d i e n t o f 0 - 0.1 M c y c l i c AMP and c y c l i c GMP separated  NEUHCO3,  from 5'AMP (Figure 19B).  C y c l i c AMP, c y c l i c GMP and 5'AMP e l u t e d a t 0.02, 0.04, and 0.06  M  NH4HCO3,  pH 7.8, r e s p e c t i v e l y .  Both c y c l i c AMP and  c y c l i c GMP c o u l d be e l u t e d by batch e l u t i o n w i t h 0.05 M NHitHCO3, without e l u t i o n o f 5'AMP. Separation  o f c y c l i c n u c l e o t i d e s , by h i g h pressure  liquid  chromatography on P a r t i s i l - 1 0 SAX, was w e l l i n v e s t i g a t e d ,  (Fig-  ure 20)to determine i f t h i s method was s e n s i t i v e enough t o quantitate trout t e s t i s  c y c l i c AMP and c y c l i c GMP.  c y c l i c AMP and c y c l i c GMP separated the maximum s e n s i t i v i t y  obtained  Although  w e l l on P a r t i s i l - 1 0 SAX,  under the o p e r a t i n g  12 6.  FIGURE 1 9 A.  S e p a r a t i o n of c y c l i c AMP 1-X8  AG cm)  by  (formate) 2 0 0 - 4 0 0  and  c y c l i c GMP  on  a BioRad  mesh, r e s i n column;. ( 0 . 3 5  e l u t i o n w i t h formic a c i d .  under handpump p r e s s u r e .  E l u t i o n was  performed  two  the  r e s u l t s graphed as a composite  elution profiles. ( 1 2 ml;  formic a c i d  C y c l i c AMP  with 0 . 1 N formic a c i d I  I  !H  B.  (DE  3 2 ) column  and  units  1 4 ml  AMP  cyclic  GMP  Symbols as  by  AMP  on  with  7.8.  Column  2 . 7 ml.  100  and  F i v e absorbance  their  specific  measured i n mMHO and  comparison w i t h standards.  9 0 - 1 0 0 % ; c y c l i c GMP above, p l u s :  E3  a DEAE-  elution  determined by wavelength scans of  v e r t e d to N H it H C O 3 by  The  t o t a l g r a d i e n t volume was  C o n d u c t i v i t y was  r e c o v e r y was  and  0 . 1 M NHi+HC03, pH  of each compound were a p p l i e d  peak tubes.  90%.  hr,  was  ml).  c y c l i c GMP  column f r a c t i o n volume was  elution positions  AMP  per  (10  ( 0 . 5 x 1 0 . 2 cm)  a l i n e a r g r a d i e n t from 0 flow r a t e was  c y c l i c GMP  75% r e c o v e r y ) .  cyclic  S e p a r a t i o n of c y c l i c AMP,  cellulose  ml  ( 1 4 ml;  AMP  of  eluted with 2 N  was  85% recovery) and  eluted with 5 N formic a c i d prewash was  ml.  identical  3  the  2  Column f r a c t i o n s i z e was  [ H ] c y c l i c n u c l e o t i d e s were chromatographed on separate columns and  0.7  x  the con-  Cyclic  r e c o v e r y was  85 -  I  10  20 Fraction  30 Number  FIGURE 20 A. S e p a r a t i o n o f c y c l i c n u c l e o t i d e s SAX  (Reeve-Angel Co., U.S.A).  (A,G,C,U) on P a r t i s i l  Operating  conditions:-  Column: PXS - 1025 SAX; 4.6 mm x 25 cm Column Temperature: Ambient Mobile Phase: 0.0 07 M K H P 0 , pH 3.2 2  4  Flow Rate: 1.1 ml p e r min P r e s s u r e : Pump s e t t i n g 40; 200 - 840 p s i D e t e c t i o n : UV A^^nm; 100% s e n s i t i v i t y  0.01 A^^nm  Peaks: a. c y c l i c CMP (0.05 A^^nm t o t a l ) b. c y c l i c AMP  "  c. c y c l i c UMP  "  d. c y c l i c GMP  "  B. S e p a r a t i o n o f c y c l i c n u c l e o t i d e s P a r t i s i l - 1 0 SAX.  Operating  (A,G,C,U,I) on  conditions:-  Column: PXS - 1025 SAX; 4.6 x 25 cm Column Temperature: Ambient Mobile  Phase: 0.003 M K H P 0 , pH 3.2 2  4  Flow Rate: 1.1 ml p e r min Pressure:  200 - 840 p s i  D e t e c t i o n : UV A^^nm; 100% s e n s i t i v i t y  0.01 A  Peaks: a. c y c l i c CMP (0.05 A^^nm t o t a l ) b. c y c l i c AMP  "  c. c y c l i c IMP  "  d. c y c l i c UMP e. c y c l i c GMP  "  2 5 4  nm  c o n d i t i o n s used, was 50 pmol/10 y l sample, about 10-100 f o l d higher than the amount o f c y c l i c AMP o r c y c l i c GMP i n such a sample from t r o u t t e s t i s .  Furthermore,  cycli  AMP was shown t o o v e r l a p w i t h 5'CMP and c y c l i c GMP t o o v e r l a p w i t h 5'AMP, as shown i n F i g u r e s 21A and 21B). Therefore,  complete s e p a r a t i o n of these n u c l e o t i d e s from  c y c l i c AMP and c y c l i c GMP would be r e q u i r e d before  these  c y c l i c n u c l e o t i d e s c o u l d be q u a n t i t a t e d i n b i o l o g i c a l samples, even i f the system's s e n s i t i v i t y  was a c c e p t a b l e  FIGURE 21 A. S e p a r a t i o n SAX.  o f 5 CMP, 5'AMP and 5'UMP on P a r t i s i l - 1 0  Operating  1  conditions:-  Column: PXS - 1025 SAX; 4.6 mm x 25 cm Column Temperature: Mobile  Ambient  Phase: 0.005 M KH P0 , pH 3.2 2  4  Flow Rate: 1.1 ml p e r min Pressure:  200 - 840 p s i  D e t e c t i o n : UV A^^nm; 100% s e n s i t i v i t y Peaks: a. 5 CMP (0.13 A  nm  total)  (0.05  "  )  c. 5'UMP (0.13  "  )  1  b. 5'AMP  2 5 4  0.005 A^^nm  B. S e p a r a t i o n o f c y c l i c AMP and c y c l i c GMP on P a r t i s i l SAX.  Operating  c o n d i t i o n s as d e s c r i b e d  Peaks: a. c y c l i c AMP b. c y c l i c GMP  (0.025 A  2 5 4  nm  total)  above.  

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