Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1978_A1 D39.pdf [ 5.85MB ]
Metadata
JSON: 831-1.0094622.json
JSON-LD: 831-1.0094622-ld.json
RDF/XML (Pretty): 831-1.0094622-rdf.xml
RDF/JSON: 831-1.0094622-rdf.json
Turtle: 831-1.0094622-turtle.txt
N-Triples: 831-1.0094622-rdf-ntriples.txt
Original Record: 831-1.0094622-source.json
Full Text
831-1.0094622-fulltext.txt
Citation
831-1.0094622.ris

Full Text

CHANGES IN TESTIS CYCLIC NUCLEOTIDE METABOLISM • DURING TROUT SPERMATOGENESIS by JILLIAN FRANCES DAVIS B.Sc, University of Auckland, 1972 M.Sc, University 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 thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA • March, 1978 (c) J i l l i a n Frances Davis, 1978 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of B iochemis t ry The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 30.3.78 ABSTRACT The concentrations of c y c l i c GMP and c y c l i c AMP, i n rainbow trout (Salmo gairdnerii) t e s t i s , were determined during hormonally-induced spermatogenesis. In immature trout t e s t i s , c y c l i c GMP and c y c l i c AMP concentrations 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 occurred during mitotic a c t i v i t y and t e s t i s growth p r i o r to meiosis. C y c l i c AMP decreased 2 f o l d during th i s time. A further gradual 5 f o l d decrease i n c y c l i c GMP occurred during the remainder of spermatogen-e s i s . No s i g n i f i c a n t change i n c y c l i c AMP occurred during development afte r the i n i t i a l 2 f o l d decrease. 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 at both high (millimolar) and low (micromolar) sub-strate concentrations were determined i n trout t e s t i s during hormonally-induced spermatogenesis. When measured at m i l l i -molar substrate, c y c l i c GMP phosphodiesterase a c t i v i t i e s did not change s i g n i f i c a n t l y throughout spermatogenesis. C y c l i c AMP phosphodiesterase a c t i v i t i e s , measured at millimolar sub-strate, decreased about 50% p r i o r to meiosis and then incre-ased, during spermatid d i f f e r e n t i a t i o n , to atta i n a f i n a l a c t i v i t y s l i g h t l y greater than that observed i n immature t e s t i s . When measured at micromolar substrate, in the pre-sence of EGTA, c y c l i c GMP phosphodiesterase a c t i v i t i e s decreased about 50%, while c y c l i c AMP phosphodiesterase a c t i v i t i e s , measured under the same conditions, increased 20 f o l d during the course of spermatogenesis. A detailed study of c y c l i c GMP phosphodiesterase a c t i v i t i e s , measured 2 + at micromolar substrate, i n the presence and absence of 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 at the time of development at which the large decrease i n c y c l i c GMP i s observed. DEAE-cellulose p r o f i l e s of c y c l i c nucleotide phosphodi-esterase a c t i v i t i e s , from trout t e s t i s at d i f f e r e n t stages df development, showed two peaks of c y c l i c AMP a c t i v i t y and one peak of c y c l i c GMP a c t i v i t y . The l a t t e r cochromato-graphed with the f i r s t c y c l i c AMP a c t i v i t y peak. A large increase i n the f i r s t c y c l i c AMP phosphodiesterase peak occurred when spermatids were maturing, without a concurrent increase i n c y c l i c GMP phosphodiesterase a c t i v i t y . This indicates the induction of a s p e c i f i c , h i g h - a f f i n i t y c y c l i c AMP phosphodiesterase during the meiotic stage of t e s t i c u l a r development. Phosphodiesterase assays using micromolar substrate concentrations, on subcellular f r a c t i o n s , demonstrated that about 85% of c y c l i c AMP hydrolysis and 80% of c y c l i c GMP hydrolysis was soluble. Both peaks of c y c l i c AMP a c t i v i t y were observed i n the DEAE-cellulose p r o f i l e of a 100,000xg supernatant (soluble) f r a c t i o n from trout t e s t i s . The small amount of pa r t i c u l a t e c y c l i c AMP phosphodiesterase a c t i v i t y , i n the 100,000xg p e l l e t f r a c t i o n , was associated iii. mainly with the second peak on the DEAE-cellulose 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 showed only high a f f i n i t y c y c l i c AMP a c t i v i t y (apparent Kms for c y c l i c AMP of 1.1 uM and 0.3 itM) and both low and high a f f i n i t y c y c l i c GMP a c t i v i t y (apparent Kms for c y c l i c GMP of 22 0 yM and 8 pM). Ki n e t i c analyses of c y c l i c AMP hydrolysis by the two peaks of phosphodiesterase a c t i v i t i e s p u r i f i e d on DEAE-cellulose, confirmed the presence of high a f f i n i t y a c t i v i t i e s . Guanylate cyclase a c t i v i t e s were assayed i n the soluble and p a r t i c u l a t e fractions from .immature t e s t i s and from t e s t i s at d i f f e r e n t stages of hormonally-induced development. There was an approximate 1:2 r a t i o of soluble to p a r t i c u l a t e guanylate cyclase a c t i v i t i e s i n .immature and i n mature trout t e s t i s . A 3 f o l d decrease,in both soluble and part-i c u l a t e guanylate cyclase a c t i v i t i e s coincided with the 10 f o l d decrease i n c y c l i c GMP concentration observed i n maturing trout t e s t i s . Thus, i n trout t e s t i s during spermatogenesis, c y c l i c GMP concentrations r e f l e c t the developmental modulation of guanylate cyclase a c t i v i t y , rather than that of c y c l i c GMP phosphodiesterase. iv. TABLE OF CONTENTS Page Abstract . . . . . i Table of Contents iv L i s t of Tables v i i L i s t of Figures v i i i Acknowledgements xi Dedication x i i Abbreviations x i i i Introduction 1 A. Spermatogenesis 1 B. The role of c y c l i c AMP and c y c l i c GMP i n metabolism . . . 14 C. The ro l e of c y c l i c AMP and c y c l i c GMP i n spermatogenesis 17 D. Objectives of t h i s thesis 24 Materials and Methods 25 A. Materials. . . 25 B. . Methods 2 6 1. Chromatography materials preparation 2 6 2. Paper chromatography 27 3. Preparation of [ 1 ^ C] adenosine from [1!*C]AMP . 27 4. Fish husbandry 2 8 5. Laparotomy procedure for sex determination of f i s h 28 6. P i t u i t a r y extract preparation 29 7. C y c l i c nucleotide extraction 29 8. Determination of c y c l i c nucleotide concent-v rations by radioimmunoassay 31 9, Tissue 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 , . . , 10. DEAE-cellulose column chromatography 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 , , 11. C y c l i c n u c l e o t i d e phosphodiesterase assay 12. Tissue p r e p a r a t i o n f o r guanylate c y c l a s e assay 13. Guanylate c y c l a s e assay . . . . 14. P r o t e i n assay * • Results A. Testes growth r a t e during hormonally-induced spermatogenesis i n t r o u t B. C y c l i c GMP and c y c l i c AMP concentrations i n t r o u t t e s t i s during spermatogenesis . . . . C. 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 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 during t e s t i s development . . 4. DEAE-cellulose chromatographic f r a c t i o n -a t i o n of c y c l i c AMP and c y c l i c GMP phosphodiesterases 5. Soluble 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 ^ ionated on DEAE-cellulose 6. K i n e t i c analyses of DEAE-cellulose peak c y c l i c AMP phosphodiesterase a c t i v i t i e s vi. Page 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 . . . . . 89 1. Assay system 89 2. A c t i v i t i e s and properties during t e s t i s development 91 Discussion 98 References 117 Appendix 128 v i i . LIST OF TABLES Page TABLE I. C y c l i c GMP i n trout t e s t i s during hormonally-induced spermatogenesis (experiment 2) . ... . 46 TABLE I I . C y c l i c GMP i n trout t e s t i s during hormonally-induced spermatogenesis (experiment 3) . . . . 50 TABLE I I I . Total c y c l i c nucleotide phosphodiesterase a c t i v i t i e s i n trout t e s t i s during hormonally-induced spermatogenesis 65 TABLE IV. C y c l i c nucleotide phosphodiesterase a c t i v i t i e s measured at micromolar substrate i n trout t e s t i s during 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 at micromolar substrate i n trout t e s t i s during hormonally-induced spermatogenesis 71 TABLE VI. Guanylate cyclase s p e c i f i c a c t i v i t i e s i n trout t e s t i s during hormonally-induced spermatogenesis 92 TABLE VII. Total guanylate cyclase a c t i v i t i e s i n trout t e s t i s during hormonally-induced spermatogenesis . . , 93 v i i i . LIST OF FIGURES Page Figure 1. Light micrographs of trout t e s t i s during hormonally-induced spermatogenesis 8 Figure 2A. Sequential c e l l types and t h e i r character-i s t i c s during spermatogenesis 12 Figure 2B. T e s t i c u l a r enzyme a c t i v i e s related to s p e c i f i c c e l l types during spermatogenesis i n the rat 12 Figure 3. Testes growth rate during hormonally-induced spermatogenesis i n trout 42 Figure 4. Comparison of c y c l i c GMP concentration and testes wet weight during hormonally-induced spermatogenesis i n trout 47 Figure 5. C y c l i c AMP concentrations i n t e s t i s during hormonally-induced spermatogenesis i n trout. .52 Figure 6A. S t a b i l i t y of phosphodiesterase a c t i v i t i e s at 30° . . 56 Figure 6B. Phosphodiesterase a c t i v i t i e s as a function of Mg2+ concentration 56 Figure 7A. Phosphodiesterase a c t i v i t i e s i n the presence or absence of EGTA 58 Figure 7B. Phosphodiesterase a c t i v i t i e s as a function of pH 58 Figure 8. Hofstee p l o t of the rate of c y c l i c AMP hydrolysis by a t e s t i s homogenate from mature trout 61 Figure 9. Hofstee p l o t of the rate of c y c l i c GMP hydrolysis by the homogenate described i n the legend for Figure 8 63 Figure 10A. Total c y c l i c nucleotide phosphodiesterase a c t i v i t i e s i n trout t e s t i s during spermatogenesis 69 Figure 10B. C y c l i c nucleotide phosphodiesterase a c t i v i t i e s measured at micromolar substrate concentration i n trout t e s t i s during spermatogenesis . . . .69 ix. Page F i g u r e 11. 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 of 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 d u r i n g hormonally-induced spermatogenesis 73 F i g u r e 12A. D E A E - c e l l u l o s e p r o f i l e of c y c l i c AMP phospho-d 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 from 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 F i g u r e 12B. DEAE-cellul©se p r o f i l e of rechromatographed Peak I from the p r o f i l e of the t e s t i s homogenate d e s c r i b e d above . . .77 F i g u r e 13A. D E A E - c e l l u l o s e p r o f i l e o f t r o u t 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 o f 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 . . . . 7 9 F i g u r e 13B. D E A E - c e l l u l o s e p r o f i l e o f t r o u t 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 o f 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 F i g u r e 14. 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 h o r m o n a l l y - i n d u c e d f o r 8 weeks 82 F i g u r e 15A. Lineweaver-Burk 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 I from t e s t i s from t r o u t h o r m o n a l l y - i n d u c e d f o r 3 weeks 84 F i g u r e 15B. H o f s t e e p l o t o f t h e h y d r o l y s i s d e s c r i b e d above 84 F i g u r e 16A. Lineweaver-Burk 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 I from t e s t i s from t r o u t h o r m o n a l l y - i n d u c e d f o r 8 weeks 8 6 F i g u r e 16B. H o f s t e e p l o t o f t h e h y d r o l y s i s d e s c r i b e d above 8 6 F i g u r e 17A. T r i t o n X-100 e f f e c t on 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) and from t e s t i s o f t r o u t a f t e r 3 ( 3 P ) , 6 (6P), and 10 (10P) weeks o f t w i c e weekly hormone i n j e c t i o n s 95 X . Page Figure 17B. Comparison of the effects of salmon gonadotropin and Triton X-100 on the 100,000xg p e l l e t (OP) and the 100,000xg supernatant (OS) zero time trout t e s t i s guanylate cyclase a c t i v i t i e s 95 Figure 18. Comparison of guanylate cyclase act-i v i t i e s and c y c l i c GMP concentrations i n trout t e s t i s during hormonally-induced spermatogenesis 112 Figure 19A. Separation of c y c l i c AMP and c y c l i c GMP on a BioRad AG 1-X8 column 12 9 Figure 19B. Separation of c y c l i c AMP, c y c l i c GMP and 5'AMP on a DEAE-cellulose column 129 Figure 20A. Separation of c y c l i c nucleotides (A,G,C,U) on P a r t i s i l - 1 0 SAX 131 Figure 20B. Separation of c y c l i c nucleotides (A,G,C,U,I) on P a r t i s i l - 1 0 SAX 131 Figure 21A. Separation of 5'CMP, 5'AMP, and 5'UMP on P a r t i s i l - 1 0 SAX 134 Figure 21B. Separation of 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 XI, ACKNOWLEDGEMENTS The author wishes to express her appreciation to Professor M. Smith for his excellent supervision of t h i s work; to Bob, Shirley, Ev, Terry, Pat and Anne for t h e i r h e l p f u l discussions and encouragement; to Vivian for introducing me to f i s h culture; to Deena and Danny for help with radioimmunoassays; to Flora and to the Schonblom family for the happy times shared together; and to B i l l for his loving support. The Medical Research Council of Canada i s thanked for providing a Studentship to the author for the period 1974 - 75, and the Killam Foundation for a scholarship for the period 1975 - 78. DEDICATION This thesis i s dedicated to my Granny whose immense love and wisdom have so greatly enriched my l i f e . x i i i . c y c l i c AMP c y c l i c GMP AMP, GMP, CMP, UMP ATP, GTP DNA RNA pmol, ymol M uM, mM EGTA EDTA TCA DEAE-cellulose PEI-cellulose T r i s MIX POPOP PPO SCINTREX TTP A 2 5 i* xg ABBREVIATIONS adenosine 3',5'-monophosphate guanosine 3',5'-monophosphate the 5'-monophosphates of the ribonucleo-sides of adenine, guanine, cytosine and u r a c i l the 5'-triphosphates of the ribonucleo-sides of adenine and guanine deoxyribonucleic acid ribonucleic acid picomoles ( 1 0 ~ 1 2 ) ,.. . micromoles ( 1 0 ~ 6 ) molar (moles per l i t e r ) micromolar, millimolar ethylene g l y c o l bis (3-aminoethyl ether) N,N 1-tetraacetate ethylenediaminetetraacetate t r i c h l o r o a c e t i c acid diethylaminoethyl c e l l u l o s e polyethyleneithine • c e l l u l o s e tris(hydroxymethyl)aminomethane l-methyl-3-isobutyl xanthine 1.4- di[2-(5-phenyloxazolyl)]-benzene 2.5- diphenyl oxazole tradename for a universal 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 of 1:1 Triton X-100: Toluene, containing POPOP and PPO a s c i n t i l l a t i o n f l u i d consisting of Toluene:Triton X-100:PPO, 60:125:1.32 v/v/w amount of material with an o p t i c a l density of one i n a one cm pathlength times the force of gravity Curie revolutions per minute counts per minute degrees Centigrade v e l o c i t y substrate Michaelis Menton constant; the substrate concentration at which half maximal v e l o c i t y of an enzyme a c t i v i t y occurs apparent Km; the substrate concentration at which half maximal v e l o c i t y of an enzyme a c t i v i t y occurs, when there are multiple Kms for the same a c t i v i t y with d i f f e r i n g enzyme concentrations a n a l y t i c a l grade ion exchange res i n prepared by s i z i n g and pu r i f y i n g the standard DOWEX resi n of the same designation quaternary ammonium anion exchange re s i n ; medium pore size 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 into highly s p e c i a l i z e d haploid spermatozoa (1). In the vertebrate t e s t i s , there i s an ordered sequence of c y t o l o g i c a l and biochemical changes, which have been studied extensively (1). However, many d e t a i l s of the regulation of spermatogenesis remain unresolved. These include the control of DNA 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 t r a n s l a t i o n , the induction 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 the precise nature of hormonal control by the p i t u i t a r y gonadotropins and the t e s t i c u l a r androgens. Since the course of spermatogenesis, i n terms of s p e c i f i c timing of DNA, RNA and protein synthesis, i s known (1) possible roles for regulatory molecules, such as the c y c l i c nucleotides, i n the control of growth and d i f f e r e n t i a t i o n of t e s t i c u l a r c e l l s , can be investigated. There i s evidence for 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 l u t e i n i z i n g hormone (LH) (2, 3, 4). FSH and LH stimulate the production of c y c l i c AMP i n t h e i r respective target c e l l s , the S e r t o l i c e l l s (3) and the Leydig c e l l s (4)'. In FSH-stimulated S e r t o l i c e l l s , the c y c l i c AMP produced by adenylate cyclase has been shown to activate a c y c l i c AMP-dependent protein kinase and to r e s u l t i n de novo synthesis of a new protein, the androgen binding protein (5). In 2. LH-stimulated Leydig c e l l s , c y c l i c AMP has been shown to enhance the production of the major t e s t i c u l a r androgen, testosterone (6). A precise role for c y c l i c GMP has not yet been defined i n any system. In several c e l l l i n e s , c y c l i c GMP has been found to enhance mitosis ( 7 ) , and DNA (8) and RNA (9) synthesis. 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 early stages of spermatogenesis, a r o l e for c y c l i c GMP i n such c e l l s i s possible and requires further investigation. The c y t o l o g i c a l and biochemical changes observed i n germ c e l l s during spermatogenesis are b a s i c a l l y similar for a l l vertebrates (1). The d i f f e r e n t c e l l types are characterized by th e i r s i z e , time of appearance during t e s t i s development, the appearance of t h e i r cytoplasmic and nuclear contents, and t h e i r r e l a t i v e rates of DNA, RNA and protein synthesis (1). The i n f a n t i l e t e s t i s contains gonocytes which have arisen from primordial germ c e l l s . At a s p e c i f i c time i n development the gonocytes multiply and are transformed into spermatogonia. Spermatogonia continue to multiply by a series of mitotic c e l l d i v i s i o n s , the number of which varies from species to species. In mammals there are 4 to 6 d i v i s i o n s , while i n f i s h there are about 10 to 12 d i v i s i o n s (1, 10). C e l l s deriving from one parental spermatogonial c e l l remain p a r t i a l l y cojoined through-out d i f f e r e n t i a t i o n by cytoplasmic bridges (11). The c e l l s deriving from one parental c e l l synchronously d i f f e r e n t i a t e into primary spermatocytes (1). The primary spermatocytes double th e i r nuclear DNA to give a t e t r a p l o i d (4n) content, and then have a long prophase period during which there i s pairing of chromosomes and possible exchange of genetic material. The primary spermatocyte f i n a l l y undergoes c e l l d i v i s i o n to give two secondary spermatocytes, each with a d i p l o i d (2n) complement of DNA. The secondary spermatocytes are shortlived and each divides without r e p l i c a t i o n of DNA to give r i s e to two spermatids with a haploid (n) content of DNA (1). This reductive d i v i s i o n i s termed meiosis. The spermatids so formed are non-dividing and only low level s of DNA synthesis can be detected (12). During the following stages of spermatid d i f f e r e n t i a t i o n , the volume of the spermatid i s greatly reduced by cytoplasmic loss and the nuclear DNA i s condensed (10). RNA synthesis ceases i n spermatids (13) while c e r t a i n proteins are synthesized during spermatid maturation (1, 14, 15). In the f i n a l d i f f e r e n t -i a t i o n step of transformation of spermatid into spermatozoa, new s t r u c t u r a l and enzymatic proteins are synthesized to form the propulsive t a i l and the complex headpiece c h a r a c t e r i s t i c of f u l l y mature sperm (1). (Figure 2A; pl3, sequence summary.) In vertebrates with d i f f e r e n t modes of sperm production, there are considerable differences i n t e s t i c u l a r structures (1) In f i s h , where there i s a r e l a t i v e l y short and intense breed-ing period, during which enormous quantities of sperm are released, a c y s t i c mode of sperm production i s used (16). The testes of salmonids, to which family the rainbow trout, 4. Salmo g a i r d n e r i i , belongs, are made up of elongated, branching tubular structures, known as lobules, within fibrous walls (17). The lobules contain within them 2 or more cysts of germ c e l l s surrounded by connective tissues(17). When the testes are immature, the cysts are e s s e n t i a l l y empty of germ c e l l s except for a small number of gonocytes or resting spermatogonia (18). As t e s t i c u l a r maturation begins, the gonocytes d i f f e r e n t i a t e into spermatogonia. In f i s h with several breeding seasons, the germ c e l l s which begin the second and following reproduct-ive cycles are resting spermatogonia (18) . These are germ c e l l s which remain undeveloped during the preceeding cycle. The spermatogonia divide rapidly f i l l i n g each cyst with a large number of germ c e l l s . These spermatogonia then d i f f e r -entiate v i a the sequence previously outlined to produce mature sperm. Within each cyst, a l l the germ c e l l s develop i n synchrony, but within a t e s t i s the various cysts may be at di f f e r e n t stages of development (19). In the mammal, male germ c e l l s are contained within the seminiferous tubules i n a concentric progression of maturity from periphery to the central lumen, into which mature sperm are continuously released a f t e r puberty (1). Except for i n pre-pubertal development, the entire length of the seminifer-ous tubule i s continually populated with germ c e l l s at various stages of development (1). In both mammals and fishe s , the male germ c e l l s are surrounded by a framework of fibrous connective tissue that 5. supports the blood vessels, lymphatics and nerves of the t e s t i s (1). The d i s t i n c t i v e component of th i s connective tissue i s the epitheloid c e l l which secretes t e s t i c u l a r androgen. These are termed Leydig c e l l s i n the mammalian t e s t i s (1) and lobule-boundary c e l l s or Leydig c e l l .homologues i n the f i s h t e s t i s (19). One other important somatic c e l l , the S e r t o l i c e l l , has been i d e n t i f i e d i n most vertebrate testes (19), but i s not ea s i l y i d e n t i f i e d 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 periphery of the seminiferous tubule (1). The S e r t o l i c e l l precursors are very sensitive to the endo-crine state of the animal and i n the rat continue to divide up to 20 days af t e r b i r t h (20). The S e r t o l i c e l l i s necessary for the early stages of germ c e l l maturation (1). I t produces an androgen binding protein (21) a f t e r FSH-stimulation and may provide nutrient support to developing germ c e l l s (22). S e r t o l i c e l l s also have considerable phagocytic 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 place a f t e r the release of sperm (22). When S e r t o l i c e l l s are observed i n fis h e s , they are found associated with s t r u c t u r a l elements of the cyst walls (19). In a l l vertebrates, both the development of the germ c e l l s and the a c t i v i t y of the Leydig and S e r t o l i c e l l s are under the control of the anterior part of the p i t u i t a r y gland, the adenohypophysis (1). In mammals, removal of the p i t u i t a r y (hypophysectomy) leads to the atrophy of maturing 6. germ c e l l s , degeneration of the seminiferous epithelium, and loss 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 species show a variety of degrees of degeneration afte r hypophysectomy (16). In salmonids, the transformation of spermatogonia into primary spermatocytes i s blocked aft e r hypophysectomy (24). In mammals, the p i t u i t a r y i s stimulated by a hypothalamic peptide releasing hormone to produce the glycoprotein gonado-tropins, FSH and LH (25), and the presence of both FSH and LH i s required for the maintenance of spermatogenesis (1). The target c e l l and action of each of these hormones has been mentioned e a r l i e r i n t h i s Introduction. In f i s h , the process of spermatogenesis i s also controlled by the p i t u i t a r y gland (17). However, p u r i f i c a t i o n studies of p i t u i t a r y extracts from both carp (26) and salmon (27) have not revealed the presence of more than one gonadotropin responsible for t e s t i s development i n f i s h . The major external factor regulating sexual maturation i n f i s h i s the photoperiod (28). In salmonids, the annual period of spermatogenic a c t i v i t y begins i n late spring and takes place over about 6 months, alternating with a period of involution and reconstitution of t e s t i c u l a r tissue during the winter months i n preparation for the next d i f f e r e n t i a t i o n period (18). In trout, appropriate manipulation of the number of hours of daylight per day can be used to accelerate spermatogenesis (28). 7. Another method of acceleration of the natural rate of spermatogenesis, i n the trout t e s t i s , i s by administering a series of inj e c t i o n s of salmon p i t u i t a r y gonadotropin extracts (29, 30). It has been shown that such a series of injections into sexually immature male rainbow trout, kept in water of the appropriate temperature, causes the growth and d i f f e r e n t i a t i o n of the immature testes (29, 30). This res u l t s i n a 500-1000 f o l d increase i n testes wet weight. The induced spermatogenesis biochemically and morphologically p a r a l l e l s that observed i n naturally maturing t e s t i s , but t e s t i s growth occurs at twice the rate (31). This i s probably due to the larger proportion of cysts 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 trout 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 at any time of the year, providing a convenient system for the study of biochemical changes during spermatogenesis. In a r t i f i c i a l hormonally-induced spermatogenesis i n trout, the testes weight doubling time i s about 1 week. This r e s u l t s i n a logarithmic increase i n testes weight for about 8 to 10 weeks u n t i l the f i n a l stages of t e s t i c u l a r development (4 to 5 weeks). In these f i n a l stages of spermat-ogenesis, a logarithmic decrease i n wet weight occurs, due to germ c e l l cytoplasmic loss and tissue degeneration (18). After 2 weeks of salmon p i t u i t a r y extract i n j e c t i o n s , the predominant germ c e l l type i s the spermatogonium- A t y p i c a l picture of the c e l l types present i n trout t e s t i s at t h i s stage i s shown i n Figure IA. Connective tissue separates 8. FIGURE 1 Upper l e f t . Light micrograph of a lobule from a trout t e s t i s after 2 weeks of salmon p i t u i t a r y extract i n j e c t i o n s . Several cysts of spermatogonia, with spherical nuclei containing multiple n u c l e o l i , are shown. Cysts are separated by connective tissues. xl,000 Upper r i g h t . A view of trout t e s t i s a f t e r 6 weeks of hormone injections shows primary spermatocytes (center f i e l d ) and spermatogonia (far r i g h t ) . xl,250 Lower l e f t . After 8 weeks of hormone i n j e c t i o n s , trout t e s t i s contains primary spermatocytes (lower l e f t ) and smaller secondary spermatocytes (lower r i g h t ) . A group of synchronous c e l l s undergoing the meiotic reduction d i v i s i o n can be seen i n the upper portion of the l i g h t micrograph. xl,250 Lower r i g h t . Trout t e s t i s a f t e r 12 weeks of hormone injections shows small, compact spermatids and some large resting spermatogonia. The breakdown of connective tissues between cysts i s apparent. XI,250 9. 10. the cysts of spermatogonia. It has been estimated that spermatogonia make up about 10-15% of the t o t a l wet weight of the immature t e s t i s (10). The other 85-90% i s presumed to be due to connective tissue and lymphatic and blood c e l l s . Spermatogonia are active i n DNA r e p l i c a t i o n (32) and c e l l d i v i s i o n r e s u l t i n g i n rapid t e s t i s growth. D i f f e r e n t i a t i o n into primary spermatocytes takes place during weeks 4 to 6 of hormonal induction (32). C e l l s i n the trout t e s t i s , at thi s stage of development, can be seen i n Figure IB. The primary spermatocytes d i f f e r e n t i a t e into secondary spermato-cytes, which i n turn produce early spermatids, from weeks 6 to 8 of hormonal induction (Figure IC). During weeks 8 to 10 spermatids d i f f e r e n t i a t e . At t h i s stage, the replacement of histones by protamine has been studied i n d e t a i l (33, 34, 35). After 10 to 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 trout t e s t i s and degeneration of t e s t i s tissue occurs, as seen i n Figure ID. The presence of spermatogonia at t h i s late stage of development r e f l e c t c e l l s which could i n i t i a t e the next spermatogenic cycle (18). In salmon, i t has been shown that spermatozoa produced from a similar 10 to 12 week series of i n j e c t i o n s of p i t u i t a r y gonadotropins into 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 naturally maturing f i s h (36). Induced spermatogenesis i n trout 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 i n germ c e l l s , since at any one given time there i s a predominance 11. of one germ c e l l type. Previous developmental studies 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 histones 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 trout t e s t i s (37). They are phosphorylated soon aft e r t h e i r synthesis and then bind to chromatin to cause i t s i n i t i a l condensation (33) . A protamine kinase, which i s stimulated 1.5 -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 trout t e s t i s at t h i s stage of development (38). Condensation of chromatin proceeds by a sequential release of histones, which may involve histone acetylation (34). The f i n a l transformation into sperm head nucleoprotamine i s clo s e l y linked to protamine dephosphorylation (35). Developmental studies during spermatogenesis have also been made i n rat t e s t i s (14, 15). Analysis of s p e c i f i c proteins during development has shown the formation of s p e c i f i c gene products i s associated with the appearance of s p e c i f i c c e l l types (15). This i s i l l u s t r a t e d i n the developmental patterns of enzymes shown i n Figure 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 sequential changes i n the rates of synthesis and phosphorylation of s p e c i f i c nuclear a c i d i c proteins have also been observed (15). I t has been suggested that i t i s these proteins which control the s e l e c t i v e a c t i v -ation or repression of genes during spermatogenesis (15). The c y c l i c nucleotides, c y c l i c AMP and c y c l i c GMP, modulate c e l l u l a r metabolism by stimulating the phosphorylation FIGURE 2 Sequential c e l l types and the i r c h a r a c t e r i s t i c s during spermatogenesis T e s t i c u l a r enzyme a c t i v i t i e s related to s p e c i f i c c e l l types during spermatogenesis i n the r a t (from reference 15) 13, A. S e q u e n t i a l c e l l types and t h e i r c h a r a c t e r i s t i c s duri-ng spermatogenesis -C e l l Type Gonocytes 'I Spermatogonia Spermatocytes a) Primary b) Secondary Spermatids Spermatozoa C h a r a c t e r i s t i c s 2n content of DNA < L i t t l e c e l l p r o l i f e r a t i o n 2n content of 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 s y n t h e s i s 4n content of DNA 2n content of DNA; ho DNA r e p l i c a t i o n , decreases RNA s y n t h e s i s n content of DNA DNA and RNA s y n t h e s i s ceases Histones r e p l a c e d by protamines F l a g e l l u m forms n content of DNA Sperm s p e c i f i c enzymes appear B. 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 to 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 Spe rmatogon i a Spe rmat i d s Gonocytes \Spermatocytes Spermatozoa Q 400 300h 200 1001-800 Heoo r c n H40O H2oo c 100 120 200 Age, days 14. of proteins (39). Due to the importance of phosphorylation of nuclear proteins i n the control of genetic expression during spermatogenesis, the role of c y c l i c AMP and c y c l i c GMP may be c r u c i a l for male germ c e l l d i f f e r e n t i a t i o n . B. The role of c y c l i c AMP and c y c l i c GMP i n metabolism Since the discovery of c y c l i c AMP, i n 1956, by Sutherland and his associates, as the low molecular weight, heat-stable factor which mediated the i n t r a c e l l u l a r e f f e c t of epinephrine and glucagon on rat l i v e r (40), an extensive l i t e r a t u r e has been amassed in d i c a t i n g the importance of c y c l i c AMP as a c e l l u l a r metabolic regulator i n many tissues and organisms throughout the animal kingdom. In the i n i t i a l papers from Sutherland's group, c y c l i c AMP was shown to be degraded by an enzyme, c y c l i c nucleotide phosphodiesterase, which formed adenosine 51-monophosphate as the product of hydrolysis (41). The enzymatic conversion of ATP to c y c l i c AMP, by adenylate cyclase, was defined i n a l a t e r series of papers from the same group, i n 1962 (42, 43). They showed that the enzyme 2 + required ATP and Mg , was p a r t i c u l a t e , was stimulated by NaF, and, i n a t i s s u e - s p e c i f i c manner, was stimulated by d i f f e r e n t hormones. A mechanism by which a change i n the c e l l u l a r concentration of c y c l i c AMP could a l t e r the metabolic state was determined by Krebs and his associates, i n 1968, during an investigation on the regulation of glycogenolysis i n s k e l e t a l muscle (44). C y c l i c AMP was found to activate a c y c l i c AMP-dependent protein 15. kinase by binding to the regulatory subunit of the regulatory-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 regulatory subunit from the c a t a l y t i c subunit which was then f u l l y active (44). C y c l i c AMP-dependent protein kinases i n other tissues have the same method of a c t i v a t i o n (45, 46). Many reviews on c y c l i c AMP metabolism exi s t (47, 48, 49) and the topic w i l l not be further discussed except with regard to spermatogenesis. Following the discovery of c y c l i c AMP and i t s important role i n c e l l u l a r metabolic regulation, evidence was sought for the existence of other naturally occurring c y c l i c nucleo-tides (50, 51). C y c l i c GMP was i s o l a t e d from rat urine, i n 1963 (52) and has since been found i n a l l phyla of the animal and plant kingdom (53, 54) In eucaryotes, i n t r a c e l l u l a r concentrations of c y c l i c GMP have been found to be elevated i n response to acetylcholine (55), prostaglandin-F2a (56), f i b r o b l a s t growth factor (57), and several l e c t i n s (7). However, d i r e c t stimulation of guanylate cyclases by any of these agents has not been convincingly demonstrated (54). Guanylate cyclases (58, 59), c y c l i c GMP s p e c i f i c phospho-diesterases (60, 61), and c y c l i c GMP-dependent protein kinases (62, 63) have also been i d e n t i f i e d and studied. Guanylate cyclases d i f f e r from adenylate cyclases i n th e i r substrate requirement for GTP, t h e i r divalent cation 2+ preference for Mn , t h e i r frequent equal d i s t r i b u t i o n between soluble and p a r t i c u l a t e f r a c t i o n s , and, t h e i r i n s e n s i t i v i t y to polypeptide hormones (54). Membrane-bound guanylate cyclases have been shown to be activated by Triton X-100 (64), by s p e c i f i c l i p i d s (65, 66) and by 2+ Ca (67) . Soluble guanylate cyclases can be activated by free r a d i c a l forming agents, such as nitrosamines (68), and nucleophiles, such as azide, n i t r i t e and hydroxylamine (69). Several soluble guanylate cyclases can be activated spontaneously by a i r oxidation (70). A c t i v a t i o n of guanylate cyclases by prostaglandins (and t h e i r metabolic endoperoxide products) has been suggested to be related to t h e i r oxidative a b i l i t y , as well as t h e i r l i p i d character (54). C y c l i c GMP phosphodiesterase a c t i v i t i e s s p e c i f i c for c y c l i c GMP have been reported, while there are other phospho-diesterases which can hydrolyze both c y c l i c GMP and c y c l i c AMP with varying a f f i n i t i e s for each c y c l i c nucleotide (60, 71). In a number of mammalian tissues, micromolar concentrations of c y c l i c GMP were shown to stimulate 2-3 f o l d the rate of phosphodiesterase-catalyzed hydrolysis of micromolar conc-2+ entrations of c y c l i c AMP (72). I t has been shown that Ca , i n the presence of a s p e c i f i c calcium-dependent phosphodi-esterase protein activator (73) increases c y c l i c GMP hydrolysis more than c y c l i c AMP hydrolysis (74). Since t h i s calcium-dependent regulator protein i s i n excess of phosphodiesterases i n most tissues (75), i t has been suggested that the regul-ation of c y c l i c GMP hydrolysis i s dependent on the i n t r a c e l l -ular calcium concentration (54). C y c l i c GMP-dependent protein kinases have been shown to be activated by a somewhat sim i l a r mechanism to c y c l i c AMP-dependent protein kinases, although i n the l a t t e r case, each holoenzyme consists of two regulatory and two c a t a l y t i c sub-units, while i n the former case, the holoenzyme has only one of each subunit (76). In many tissues, a protein modulator has been observed, which enhances c y c l i c GMP a c t i v a t i o n of c y c l i c GMP-dependent protein kinases and i n h i b i t s c y c l i c TAMP act i v a t i o n of c y c l i c TAMP-dependent protein kinases (77). Several excellent reviews on c y c l i c GMP metabolism are a v a i l -able (53, 54) and t h i s topic w i l l not be covered further except i n r e l a t i o n to spermatogenesis. C y c l i c CMP and c y t i d y l a t e cyclases have also been recently i d e n t i f i e d i n some mammalian tissues (78), but very l i t t l e i s known about th i s c y c l i c nucleotide's r o l e i n b i o l o g i c a l systems. C. The role of c y c l i c AMP and c y c l i c GMP i n spermatogenesis A study of i n t r a c e l l u l a r concentrations of c y c l i c AMP and c y c l i c GMP during spermatogenesis i n the r a t t e s t i s (79) showed that both c y c l i c nucleotide concentrations were elev-ated i n i n f a n t i l e t e s t i s ( c y c l i c AMP 20 pmol/mg protein; c y c l i c GMP 0.6 pmol/mg protein) and both decreased at about the time of the onset of the meiotic reduction d i v i s i o n i n germ c e l l s (20-25 days afte r b i r t h ) . C y c l i c AMP continued to decrease u n t i l the time of the appearance of spermatids (about 35 days afte r birth) and then increased steadily i n maturing t e s t i s to a f i n a l adult concentration of about half the i n i t i a l concentration. C y c l i c GMP continued to decrease 18. throughout maturation, to a concentration about one-sixth of the i n i t i a l concentration. This study also u t i l i z e d immuno-fluorescent probes for c y c l i c AMP and c y c l i c GMP. This technique i s assumed to l o c a l i z e c y c l i c nucleotides associated with c y c l i c nucleotide receptor proteins i n the tissue because any free c y c l i c nucleotide should be l o s t during the treatment process of the unfixed frozen tissues (80) . During the infan-t i l e phase of r a t t e s t i c u l a r development, fluorescence within seminiferous tubules was intense, for both c y c l i c nucleotides, and nuclear staining patterns of c e l l s close to the tubular wall i . e . S e r t o l i c e l l s and spermatogonia, were observed for both c y c l i c nucleotides. This tubular wall staining was observed throughout maturation, (79). In another study, of c y c l i c AMP immuriofluorescent l o c a l i z a t i o n i n developing r a t t e s t i s (81), the c y c l i c AMP tubular staining pattern was shown to p e r s i s t one month following hypophysectomy i n adult rats, despite undetectable plasma concentrations of FSH and LH. In the investigation i n which both c y c l i c nucleotides were l o c a l i z e d , c y c l i c AMP fluorescence was not detected i n germ c e l l s i n l a t e r stages of t e s t i c u l a r development, i . e . meiosis and spermatid d i f f e r e n t i a t i o n , while c y c l i c GMP was observed associated with germ c e l l types during most stages of development (79) . In p a r t i c u l a r , c y c l i c GMP was found associated with nuclear elements, s p e c i f i c a l l y with the chromosomes of prophase spermatocytes during meiosis. C y c l i c AMP was not observed on chromosomes at any stage of develop-ment- These res u l t s suggested nuclear roles for both 19. c y c l i c AMP and c y c l i c GMP i n i n f a n t i l e development, and also, a s p e c i f i c nuclear role for c y c l i c GMP at meiosis. Developmental studies of the adenylate and guanylate cyclases, i n rat t e s t i s , have indicated some c o r r e l a t i o n of th e i r s p e c i f i c a c t i v i t i e s with the observed changes i n c y c l i c nucleotide concentrations (82, 83). Steiner and his assoc-iates showed a c o r r e l a t i o n between t e s t i s tissue c y c l i c GMP concentration and soluble.guanylate cyclase a c t i v i t y during rat t e s t i c u l a r d i f f e r e n t i a t i o n (82) . These workers con-cluded that elevated c y c l i c GMP concentrations i n the i n f a n t i l e r a t t e s t i s r e f l e c t , i n part, the a c t i v i t y of an inducible soluble guanylate cyclase (82). Braun and his associates have also i d e n t i f i e d a soluble guanylate cyclase a c t i v i t y i n r a t t e s t i s , which was most active i n premeiotic stages of development (83). This guanylate cyclase was l o c a l i z e d to a non-germ c e l l tubular component of the immature r a t t e s t i s (83). In rat t e s t i s depleted of germ c e l l s by X - i r r a d i a t i o n before b i r t h , two s p e c i f i c membrane-bound adenylate cyclases have been i d e n t i f i e d (84). One i s sen s i t i v e to FSH and i s present i n the tubular S e r t o l i c e l l s and perhaps i n spermatogonial c e l l s and thecQther:lis^sensitive to LH and i s present i n the Leydig c e l l s (85) . A d i s t i n c t i v e germ ce l l - a s s o c i a t e d soluble adenylate cyclase has been detected i n the seminiferous tubules of rat s , at the time of the appearance of 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 increased about 2 f o l d during the period of spermatid development into mature sperm and reached maximal values i n the t e s t i s of adult r a t s . 20. The timing of the appearance of t h i s soluble adenylate cyclase i n r at t e s t i s correlates with the observed increases i n c y c l i c AMP concentrations (79). This 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 stimulatory e f f e c t 2+ 2+ of Mn could be potentiated by Ca (85). In epididymal 2+ sperm, the Mn - s e n s i t i v e adenylate cyclase was found to be associated with "mitochondrial" and "microsomal" p a r t i c u l a t e 2+ fractions (83) . I t was suggested that t h i s Mn - s e n s i t i v e adenylate cyclase may play a r o l e i n the transformation of spermatid c e l l s into spermatozoa, perhaps i n the formation of the sperm t a i l (83). In support of t h i s statement i s the previous observation that " c y c l i c AMP i s absolutely necessary for f l a g e l l a formation and hence m o t i l i t y , i n c y c l i c AMP-d e f i c i e n t mutants of Esoherichia c o l i and Salmonella typhimurium" (.87) . Sperm of both invertebrates and vertebrates have been 2+ 2+ shown to contain equal amounts of Mg - or Mn -dependent 2+ p a r t i c u l a t e adenylate cyclase, but p a r t i c u l a t e Mn -dependent guanylate cyclase was several hundred f o l d more active i n invertebrate sperm than i n vertebrate sperm (88). The extremely active guanylate cyclase i n sea urchin sperm was found to be l o c a l i z e d primarily i n the f l a g e l l a r plasma membrane (89). Sperm from several species of f i s h (salmon and herring) contained appreciable amounts of adenylate cyclase but no detectable guanylate cyclase (88). Developmental studies on c y c l i c nucleotide phosphodiester-ase a c t i v i t i e s during spermatogenesis are scarce. This i s probably due to the complexity of multiple, interconvertible phosphodiesterase a c t i v i t i e s which are observed i n most tissues (90). One investigation of t o t a l c y c l i c AMP phospho-diesterase a c t i v i t i e s during r a t spermatogenesis showed a 5 f o l d increase i n a c t i v i t i e s from day 2 0 to day 50 after b i r t h (91). C y c l i c AMP phosphodiesterase a c t i v i t i e s then remained constant through to f u l l maturity (80 days afte r b i r t h ) . In t h i s study, day 20 aft e r b i r t h was the f i r s t time 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 determined. Studies using the phosphodiesterase i n h i b i t o r s caffeine, theophylline and papaverine showed an increase i n r e s p i r a t i o n and mobility of bovine spermatozoa, an e f f e c t also produced by c y c l i c GMP and di b u t y r y l c y c l i c AMP (92). These studies suggested the presence of an active c y c l i c nucleotide phospho-diesterase i n mammalian sperm. A highly s p e c i f i c t e s t i c u l a r c y c l i c AMP phosphodiesterase was found associated with sexual maturation i n the r a t and rabbit (93), and i n the ram (94). In the r a t , one form of c y c l i c AMP phosphodiesterase, with a Km of 6.5 x 10" 5 M was i d e n t i f i e d i n immature t e s t i s and a second, high a f f i n i t y enzyme, with a Km of 2.5 x 10~'6 M, appeared i n coincidence with the appearance of mature sperm (91) An active c y c l i c AMP phosphodiesterase was found i n f i s h sperm (salmon and herring) but very low c y c l i c GMP hydrolysis was detected (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 nucleotide concentrations may be regulated by two factors other than th e i r s y n t h e s i s b y t h e c y c l a s e s a n d t h e i r d e g r a d a t i o n by t h e p h o s p h o d i e s t e r a s e s . T h e s e f a c t o r s a r e e x t r a c e l l u l a r e x c r e t -i o n ( 9 5 , 96) and i n t r a c e l l u l a r b i n d i n g t o i n a c t i v a t a b l e c y c l i c n u c l e o t i d e b i n d i n g p r o t e i n s ( 9 7 ) . The p o s s i b l e i m p o r t a n c e o f t h e r o l e o f e x c r e t i o n o f c y c l i c n u c l e o t i d e s i n t h e r e g u l a t i o n o f 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 , i n t e s t i s c e l l s , i s unknown. The c o n c e n t r a t i o n o f c y c l i c n u c l e o t i d e b i n d i n g p r o t e i n s i n t e s t i s h a s b e e n i n v e s t i g a t e d f r o m t h e p o i n t o f v i e w o f s t u d y i n g c y c l i c TAMP-dependent p r o t e i n k i n a s e a c t i v i t y d u r i n g s p e r m a t o -g e n e s i s ( 9 8 , 9 9 ) . I t h a s b e e n s u g g e s t e d , o n t h e b a s i s o f s t u d i e s o n t h e r e g u l a t i o n o f b o v i n e h e a r t p r o t e i n k i n a s e s (97) t h a t , a d e p h o s p h o r y l a t e d f o r m o f h o l o e n z y m e c o u l d be r e s i s t a n t t o c y c l i c A M P - i n d u c e d d i s s o c i a t i o n a n d c o u l d s e r v e as a c y c l i c n u c l e o t i d e s i n k , r e n d e r i n g c y c l i c AMP u n a v a i l a b l e f o r p r o t e i n k i n a s e a c t i v a t i o n , a s w e l l a s r e s i s t a n t t o h y d r o l y s i s by 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 . R e c e n t l y , i t h a s b e e n d e m o n s t r a t e d , u s i n g c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e s s e p a r a t e d by DEAE-c e l l u l o s e c h r o m a t o g r a p h y (Type I k i n a s e e l u t e d b y 0.1 M N a C l a n d T ype I I k i n a s e e l u t e d b y a b o u t 0.2 M N a C l ) f r o m 5 r a t and 2 b o v i n e t i s s u e s , t h a t a u t o p h o s p h o r y l a t i o n o f t h e r e g u l a t o r y s u b u n i t b y t h e c a t a l y t i c s u b u n i t o c c u r r e d w i t h a l l Type I I enzymes b u t n o t w i t h T ype I enzymes ( 1 0 0 ) . A n e g a t i v e i n f l u -e n c e o f Type I I c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e s , on l y m p h o c y t e m i t o g e n e s i s , h a s b e e n o b s e r v e d ( 1 0 1 ) . I n d e v e l -o p m e n t a l s t u d i e s o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e a c t i v i t i e s , i n r a t t e s t i s , T ype I k i n a s e r e m a i n e d c o n s t a n t f r o m b i r t h t h r o u g h o u t d i f f e r e n t i a t i o n , w h i l e T ype I I k i n a s e increased from b i r t h onwards to obtain an adult p r o f i l e at the time of the f i r s t reductive d i v i s i o n s (98). I t i s possible that Type II c y c l i c AMP-dependent protein kinases may be important i n the control of e f f e c t i v e i n t r a c e l l u l a r c y c l i c AMP i n male germ c e l l s during and a f t e r meiosis. In another investigation on the changes i n protein kinase a c t i v i t i e s i n r a t t e s t i s during spermatogenesis, t o t a l kinase a c t i v i t i e s (including both c y c l i c AMP-dependent and c y c l i c AMP-independent protein kinases) decreased at about 10-20 days after b i r t h and then a sharp increase occurred around 35-45 days of age (99). This increase correlated with an increase i n the c y c l i c AMP-dependent versus c y c l i c AMP-independent protein kinase r a t i o , and also with the timing of biochemical events such as, the increase i n i n t r a c e l l u l a r c y c l i c AMP concentrations (79) , increases i n plasma and t e s t -2+ l c u l a r testosterone (102), the increase i n Mn - s e n s i t i v e soluble adenylate cyclase (83), and also the increase i n the high a f f i n i t y c y c l i c AMP phosphodiesterase a c t i v i t y (91) . I t was suggested that a l l the t e s t i c u l a r enzyme ac t i v i t y . i n c r e a s e s were related to the appearance of spermatids (99). While very active c y c l i c AMP-dependent protein kinases have been observed i n sperm of several mammalian species, c y c l i c GMP-dependent protein kinases have not been found (88) ? No developmental studies of c y c l i c GMP-dependent protein kinase a c t i v i t i e s or of c y c l i c GMP-binding proteins, i n t e s t i s from any vertebrate or invertebrate, have been reported. D. Objectives of t h i s thesis The objectives of t h i s thesis were to determine c y c l i c GMP and c y c l i c 7AMP concentrations i n the t e s t i s of the rainbow trout Salmo g a i r d n e r i i during spermatogenesis and to i n v e s t i -gate the developmental modulation of the enzymes regulating these concentrations. P a r t i c u l a r emphasis was placed on c y c l i c GMP metabolism, due to the lack of information on t h i s c y c l i c nucleotide's role i n growth and d i f f e r e n t i a t i o n . As previously discussed, a r t i f i c i a l hormonally-induced spermato-genesis i n trout i s a well-defined system, i n which the stages of germ c e l l maturation take place as an ordered sequence of events throughout the maturing t e s t i s . Consequently, changes i n the biochemistry of the tissue can be followed chronolog-i c a l l y and correlated with d i s t i n c t stages of germ c e l l growth and d i f f e r e n t i a t i o n . MATERIALS AND METHODS A. MATERIALS A l l radiochemicals were from New England Nuclear. [ 3H]adenosine 35 1-monophosphate, ammonium s a l t ( s p e c i f i c a c t i v i t y 39.8 C i per mmol) and '[ 3H] guanosine 3',5'-mono-phosphate, ammonium s a l t ( s p e c i f i c a c t i v i t y 8.8 or 10 C i per mmol) were p u r i f i e d by chromatography on AG 1-X8 re s i n , as described i n Methods. [ 3H]guanosine ( s p e c i f i c a c t i v i t y 19 Ci per mmol) was p u r i f i e d by paper chromatography, as described i n Methods. [ 1^C]adenosine 5 *-monophosphate, diammonium s a l t ( s p e c i f i c a c t i v i t y 0.422 C i per mmol) was converted to [ 1 4C]adenosine as described i n Methods. C y c l i c AMP, d y c l i c GMP, ATP, GTP, adenosine, guanosine, phosphocreatine, creatine phosphokinase (rabbit muscle, Type I) and MIX were a l l purchased from Sigma Chemical Co. C y c l i c nucleotide phosphodiesterase (beef heart) was bought from Calbiochem and made up to 5 mg/ml i n H 20 and stored frozen at -20° i n small aliquots. E. c o l i a l k a l i n e phosphatase was purchased from PL Biochem-i c a l s and was made up as a 0.37 mg/ml solution i n H 2 O and stored frozen at -20° i n small aliquots. Bovine serum albumin, Fraction V, Lot 400427, was bought from Calbiochem. A l l other chemicals were reagent grade. 26. Tr i t o n X-100 (tradename of Rohm and Haas for i s o - o c t y l -phenoxy-polyethoxyethanol) was supplied by J.T. Baker Co. SCINTREX (tradename for a universal 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 of 1:1 Triton X-100:Toluene, containing PPO and POPOP) was purchased from J.T. Baker Co. C y c l i c GMP radioimmunoassay k i t s were purchased from Collaborative Research. The c y c l i c AMP k i t was bought from New England Nuclear. B. METHODS 1. Chromatography materials preparation DEAE-cellulose (Whatman DE 32, microgranular) was washed with 0.5 N NaOH (10 volumes), d i s t i l l e d H 20 (20 volumes), 1 M sodium acetate, pH 6.5 (10 volumes), and then equilibr a t e d with column buffer, i . e . 20 mM sodium acetate, pH 6.5 containing 4 mM 2-mercaptoethanol. BioRad AG 1-X8, 200-400 mesh, chloride form, r e s i n was prepared as described previously (103). For c y c l i c GMP sample p u r i f i c a t i o n , the AG 1-X8 resi n was then poured into columns (0.7 x 3.5 cm) and prewashed with 5 N formic acid (15 ml), followed by d i s t i l l e d H 20 (15 ml). For 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 resi n was prewashed with 5 N formic acid (10 volumes) followed by d i s t i l l e d H20, i n a large glass-sintered funnel. For c y c l i c GMP phosphodiesterase assays, the AG 1-X8 res i n was prepared as described previously (103) and'was resuspended i n d i s t i l l e d H 20 (1:2 v/v re s i n to H20) after addition of 1 N formic acid to a pH of 2.5. For c y c l i c AMP phosphodiesterase assays, the AG 1-X8 re s i n was resusp-ended i n d i s t i l l e d H 20 (1:2 v/v re s i n to H20) af t e r addition of g l a c i a l acetic acid to a pH of 3.7. 2. Paper chromatography A l l paper chromatography was on Whatman 40 paper using the descending system at 23°. The solvent used was i s o -propanol:NH 3:H 20 (7:1:2 v/v/v). Nucleotides and nucleosides were located by shortwave u l t r a v i o l e t absorbance using a hand-held Mineralight from U l t r a v i o l e t Products, C a l i f o r n i a . U l t r a v i o l e t absorbing regions which contained radioactive compounds were cut out and eluted by the method of Heppel (104). The eluate (0.5 ml) was added to 5 ml of 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 l i q u i d s c i n t i l l a t i o n spectrometer. 3. Preparation of [ 1 **C] adenosine from [^CjAMP [ltfC]AMP ( s p e c i f i c a c t i v i t y 0. 422 C i per mmol), 100 y l (0.0018 mg) was incubated with E. c o l i a l k a l i n e phosphatase (0.05 mg/ml) and 8 mM Tris.HCl, pH 7.8, i n a t o t a l volume of 130 y l , at 30° for 135 min. This mixture was cochromatographed on paper with adenosine (1 A 2 5^ unit) and AMP (1 A 2 5^ unit) and developed as described i n the previous section. Using these conditions there was 100% conversion of [ll*C]AMP to [ 1 ^ C] adenosine. 28. 4. Fish husbandry Immature rainbow trout Salmo g a i r d n e r i i , 17-20 cm i n length, from the Sun Valley Trout Farm i n Mission, B r i t i s h Columbia, were kept i n 200 l i t e r , s e l f - c l e a n i n g , fibreglass aquaria. The r e c i r c u l a t i n g water was aerated and kept at 9-12° and a 13 hour l i g h t and 11 hour dark cycle was used. The f i s h were fed three times a week, on Oregon Moist P e l l -ets, from the Moore-Clark CO., La Conor, Washington. Immature male trout were selected by laparotomy and were injected i n t r a p e r i t o n e a l l y , twice a week, with 0.1 ml of a crude salmon p i t u i t a r y extract. Injections were continued for 12 weeks, during which time, at regular i n t e r -vals, f i s h were k i l l e d , by a blow on the head, before testes were dissected. 5. Laparotomy procedure for sex determination of f i s h The f i s h were anaesthetized i n an aerated solution of 62 mg/1 t r i c a i n e methane sulfonate (MS 222) for several minutes and then placed i n a nylon suspension rack. To maintain anaesthesia and oxygen supply, a tube was placed in the mouth and a solution of MS 222 (50 mg/1) was contin-u a l l y flushed over the g i l l s . A ventral mid-line i n c i s i o n was then made, s t a r t i n g 0.5 cm posterior to the pectoral f i n s and running caudally for 5 cm. The flaps of tissue on either side of the i n c i s i o n were extended using retractors and a blunt probe used to examine the gonads. The males were i d e n t i f i e d by t h e i r translucent threadlike testes, each about 8 cm long and 1 mm thick. The ovaries i n the female trout were yellowish and were characterized by a marked tapering from the anterior to the posterior end. The wound was closed with 5-0 s i l k sutures (Ethicon) and the f i s h returned to the aquaria. The whole procedure took no more than 5 minutes and routinely there was 9 0-100% su r v i v a l . 6. P i t u i t a r y extract preparation 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 , spawn-ing chinook salmon, Oncorhynchus tschawytscha, i n late Sept-ember, at the Green River Hatchery, Auburn, Washington. A core, containing the brain and the underlying p i t u i t a r y gland, was removed from the salmon head. The p i t u i t a r y gland, a small spherical gland, average weight 85 mg, was scooped out of the core, frozen immediately with s o l i d C0 2, and stored at -80°. Three volumes of saline (1.25%) were added to thawed p i t u i t a r y glands and the mixture homogenized i n a Waring blender, at high speed, for 2 min. The solution was then centrifuged at 11,250 rpm for 15 min i n a S o r v a l l SS 34 rotor. The supernatant was stored at -20° i n 5 ml aliquots. Before i n j e c t i o n , the thawed extract was c l a r i -f i e d by centrifugation at maximum speed on an International bench top centrifuge for 1-2 min. 7. C y c l i c nucleotide extraction Testes were dissected from hormonally-induced trout 30. at weekly i n t e r v a l s , over a 10-12 week t e s t i s maturation time, and immediately placed i n l i q u i d nitrogen. Samples were stored frozen at -80°. Two d i f f e r e n t procedures were used for extraction of c y c l i c nucleotides. For the c y c l i c GMP determinations, each sample was quickly weighed and placed i n 1% perchloric acid (1 ml; about 5 volumes) which contained 0.5 pmol [ 3 H ] c y c l i c GMP (10 C i per mmol). The tissue was homogenized i n a Potter-Elvjhem homogenizer, for 30 sec, and the homogenate centrifuged at 10,000xg for 10 min. The supernatant solution was adjusted to pH 7.0 with 6 N KOH and 1 M Tris.HCl, pH 7.5 added to a f i n a l buffer concentration of 0.1 M and recentrifuged at 10,000xg for 20 min. The supernatant solution was then applied to an AG 1-X8 column (0.7 x 3.5 cm), prewashed as described e a r l i e r i n Methods. After sample adsorption, the column was washed with 0.1 N formic a c i d (10 ml) followed by 2 N formic acid (12 ml) and then 5 N formic acid (14 ml). The 5 N formic acid eluate, containing c y c l i c GMP (Appendix ; Figure 19A) was rotary evaporated at 36°, redissolved i n d i s t i l l e d H 20 and re-rotary evaporated at 36°. The c y c l i c GMP f r a c t i o n was f i n a l l y redissolved i n 50 mM sodium acetate, pH 6.2 (1 ml). Total recovery of [ 3 H ] c y c l i c GMP through the extraction and p u r i f i -cation procedure was 60-70%. Under the rotary evaporation conditions described above, c y c l i c GMP was not degraded, as shown by 100% recovery of [ 3 H ] c y c l i c GMP cochromatographing with c y c l i c GMP on paper. For the c y c l i c AMP determinations,the c y c l i c nucleotide were extracted by homogenization i n 5% TCA (1 ml; 5 volumes) containing 0.25 pmol [ 3 H ] c y c l i c AMP (39.8 C i per mmol) and centrifuged at 10,000xg for 10 min. The supernatant was extracted 4 times with d i e t h y l ether (about 6 ml; 4 volumes) (This was a more rapid method of acid removal than the KClOi* p r e c i p i t a t i o n method.) The r e s u l t i n g aqueous layer was applied to AG 1-X8 columns. The r e s i n had been prewashed with 5 N formic acid by a batch method, as described e a r l i e r i n Methods. The column was then washed with 0.1 N formic acid (10 ml) followed by 2 N formic acid (12 ml). The 2 N formic acid eluate, containing c y c l i c AMP (Appendix ; Figure l ^ A ) , was rotary evaporated as described above. Total recovery of [ 3 H ] c y c l i c AMP through the extraction and p u r i f i c a t i o n procedure was 60-70%. 8. Determination of c y c l i c nucleotide concentrations by  r adio immuno ass ay C y c l i c nucleotide concentrations were determined by the radioimmunoassay method developed by Steiner (112 ) using radioimmunoassay k i t s purchased from Collaborative Research for c y c l i c GMP assays and by New England Nuclear for c y c l i c AMP assays. C y c l i c GMP was routinely determined i n the range of 0.5-10 pmol. C y c l i c AMP was determined i n the range of 1-20 pmol. A l l k i t s (except 1 from Collab-orative Research) gave 40-50% maximum binding of 1 2 5 I - c y c l i c GMP or 1 2 5 I - c y c l i c AMP to t h e i r respective antibodies and 32. l i n e a r standard curves over the ranges detailed above. A l l samples were assayed i n duplicate, at 2-3 d i l u t i o n s and a portion of each sample was subjected to phosphodiesterase treatment. The conditions for the phosphodiesterase hydro-l y s i s were 0.025 unit of beef heart c y c l i c nucleotide phos-phodiesterase, 30 mM Tris.HCl, 6 mM MgCl2 and sample incubated i n a f i n a l volume of 50 y l for 20 min at 37°. The reaction was terminated by heating i n a temperature heat block at 100° for 1 min. Samples with c y c l i c nucleotide "blank" values, a f t e r phosphodiesterase treatment, above 10% of the untreated sample, were not used. About 5% of samples had unacceptable blanks. 9. Tissue preparation for c y c l i c nucleotide phosphodiesterase  assay Testes were excised from hormonally-induced trout and were placed on i c e , weighed and minced with a sc a l p e l . They were then homogenized i n 4 volumes of buffer, by 10 passes of a motordriven pestle i n a l o o s e - f i t t i n g glass-Teflon homogenizer. Homogenizing buffer was 10 mM Tris.HCl, pH 7.5, 0.25 M sucrose, 4 mM 2-mercaptoethanol, 2 mM MgCl2 (buffer A) for a l l t o t a l phosphodiesterase a c t i v i t i e s and DEAE-cellulose separations. The homogenate was f i l t e r e d through 4 layers of cheesecloth to remove connective tissue. 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 apply-ing manual pressure from the anterior abdomen toward the p o s t e r i o r l y located genital-anal opening. Sperm were homogenized i n 9 volumes of buffer A and f i l t e r e d through 4 layers of cheesecloth. Total sperm a c t i v i t i e s were expressed as pmol c y c l i c nucleotide hydrolyzed / min / mg protein. Protein i n sperm suspensions was 1.3% of the wet weight, compared with 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 t e s t i s . Since i t was necessary to assay a large volume of sperm suspension, i n order to make a protein determination, the t u r b i d i t y i n such assays may have increased the apparent protein concentration. 10. DEAE-cellulose column chromatography of c y c l i c nucleotide  phosphodiesterase a c t i v i t i e s Cheesecloth-filtered homogenates of trout testes i n buffer A were d i r e c t l y applied to DEAE-cellulose (DE 32) columns (0.5 x 10.5 cm or 0.8 x 22 cm) pre-equilibrated i n 20 mM sodium acetate, pH 6.5, buffer containing 4 mM 2-mercaptoethanol. After sample adsorption, columns were washed with several column volumes of th i s buffer. The i n i t i a l wash contained no phosphodiesterase a c t i v i t y . A li n e a r gradient from 20 mM to 1 M sodium acetate, pH 6.5, was then applied, with a flow rate of 2 6 ml per hour and a t o t a l gradient volume of 20 0 ml. Column f r a c t i o n volume was 4.5 ml. (The exception to t h i s was i n Figure 11A, i n which case there was a 100 ml t o t a l gradient 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 contained 50 ul, (or 25 y l i n the case of the 2.2 ml column f r a c t i o n volume) of 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 concentration i n the 4.5 ml of 1.8 mM MgCl 2 and 0.9 mg bovine serum albumin/ml. (In the absence of t h i s s t a b i l i z i n g solution, phosphodiesterase a c t i v i t i e s i n DEAE-cellulose column fractions were very low.) Fractions containing phosphodiesterase a c t i v i t y were pooled and dialyzed against 10 mM Tris.HCl, pH 7.5, 1 mM MgCl 2, 0.4 mM d i t h i o t h r e i t o l i n 50% glycerol (v/v). The dialysates were aliquoted into small volumes and stored at -20° for future k i n e t i c analyses. Fractions were stable with respect to c y c l i c AMP hydrolysis, but not for c y c l i c GMP hydrolysis, for at l e a s t 6 months. For DEAE-cellulose chromatography of 100,000xg super-natant and sonicated 10 0,000xg p e l l e t trout t e s t i s fractions (Figure 13A and B), the testes from 2 f i s h (630 mg/fish) were excised and homogenized i n 7 ml of buffer A and then passed through 4 layers of cheesecloth. From th i s homo-genate, 3 ml was d i r e c t l y applied to a DEAE-cellulose column and the phosphodiesterase a c t i v i t i e s fractionated, 1 ml was put aside for t o t a l a c t i v i t y assays and the remaining 3 ml was centrifuged at 100,000xg for 1 hr. The supernatant was pipetted o f f and stored at -20° for future DEAE-cellulose chromatography. The 100,000xg p e l l e t was washed with buffer A, rehomogenized and recentrifuged at 100,000xg for 1 hr. The r e s u l t i n g supernatant was discarded and the p e l l e t resuspended i n the o r i g i n a l volume of buffer A. After sonication at 30 sec/ml by a Branson s o n i f i e r f i t t e d with a microtip (Model W 185), Plainview, N.Y., at a setting of 5, the s o l u b i l i z e d p e l l e t was recentrifuged at 30,000xg for 20 min and the supernatant pipetted o f f and stored at -20° for future DEAE-cellulose chromatography. Preparation of DEAE-cellulose columns was as described for t o t a l t e s t i s homogenates, except that column size was 0.8 x 16 cm, and the t o t a l gradient volume was 100 ml. Column fractions of 2 ml were coll e c t e d . Fraction tubes contained 25 y l of the s t a b i l i z i n g solution described above. A l l DEAE-cellulose columns and related operations were ca r r i e d out at 4°. 11. C y c l i c nucleotide phosphodiesterase assay The two-step assay for c y c l i c nucleotide phosphodiest-erase a c t i v i t y was s i m i l a r to that described previously (60, 103, 105) with the modifications of the use of EV c o l i a l k a l i n e phosphatase, instead of snake venom nucleotidase, for the second incubation, and a smaller assay volume of 100 y l . An appropriate aliquot of enzyme, to obtain 10-40% conversion of substrate to product, was incubated for 5 to 30 min at 30° i n 10 mM Tris.HCl, pH 7.5 or 8.0, 2 mM MgCl2/ 4 mM 2-mercaptoethanol buffer containing about 5 x 10" 8 M [ 3 H ] c y c l i c AMP or [ 3 H ] c y c l i c GMP, i n a t o t a l volume of 100 y l . Assays were i n s i l i c o n i z e d tubes 2+ (10 x 1.2 cm). When indicated, EGTA (250 yM) or Ca (100 yM) was included i n t h i s volume. For higher substrate concentrations, the indicated amount of unlabeled c y c l i c nucleotide was included. Chromatographic p r o f i l e s were assayed at pH 8.0 for maximum assay a c t i v i t y . A l l other assays were at pH 7.5 to approximate physiological conditions. The reaction was terminated by heating i n a temperature heat block at 100° for 90 sec. (For the f i n a l Table V. c y c l i c GMP phosphodiesterase assays, termination was for 60 sec at 100° since a lower blank was thus obtained.) Tubes were cooled on i c e , re-equilibrated to 30° and 10 y l of E_. c o l i a l k a line phosphatase (0.37 mg/ml) added for a further 2 0 min incubation. This reaction was terminated by the addition of 1 ml of AG 1-X8 re s i n s l u r r y at the appropriate pH i . e . pH 2.5 for c y c l i c GMP assays and pH 3.7 for c y c l i c AMP assays. These r e s i n s l u r r i e s were s t i r r e d with a magnetic s t i r r e r while additions were being made. Assay tubes were l e f t to stand for at le a s t 10 min after r e s i n s l u r r y additions and then centrifuged at 3,000xg for 5 min. A 0.4 ml aliquot of the r e s u l t i n g supernatant solution was added to 5 ml of TTP or a 0.6 ml aliquot to 5 ml of 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 of nucleosides i n the supernatant f r a c t i o n were determined by [ 3H] guanosine or [ 1 "*C] adenosine recovery and found to be 80% and 90% respectively. Resin s l u r r i e s at the above pHs gave blanks of the respective unbound [ 3 H ] c y c l i c nucleotide of 2-14% for c y c l i c GMP and 2-8% for c y c l i c AMP. Blanks were consistent within assays to - 0.5%, but varied between assays with increasing blanks cor r e l a t i n g to increasing length of time a f t e r p u r i f i c a t i o n of the [3H] c y c l i c nucleotides on AG 1-X8 columns. Due to the low c y c l i c GMP phosphodiesterase a c t i v i t i e s i n column fractions after DEAE-cellulose separations, a combination of 0.2 ml of 95% ethanol plus 0.8 ml of AG 1-X8 r e s i n s l u r r y was used to terminate c y c l i c GMP assays, since the recovery of guano-sine under these conditions was 90-95%, while the c y c l i c GMP blank was unchanged. Duplicates were less consistent with the addition of alcohol, so t o t a l c y c l i c GMP a c t i v i t i e s were measured using the 1 ml of AG 1-X8 r e s i n s l u r r y at pH 2.5, to terminate the second reaction, and a c t i v i t i e s were corr-ected for the 80% guanosine recovery. C y c l i c AMP a c t i v i t i e s were also corrected for the 90% adenosine recovery. 12. Tissue preparation for guanylate cyclase assay Trout testes were homogenized i n 10 volumes (2-3 ml) of 10 mM Tris.HCl, pH 7.5, 0.25 M sucrose, 4 mM 2-mercapto-ethanol and 1 mg/ml bovine serum albumin (buffer B). Homogenization conditions were the same as those described for c y c l i c nucleotide phosphodiesterase a c t i v i t i e s . The homogenate was centrifuged at 100,000xg for 1 hr at 4°, and the supernatant pipetted o f f and stored frozen at -20°. The p e l l e t was resuspended i n 1 ml of buffer B and t h i s was then centrifuged at 100,000xg for 1 hr at 4°. The second supernatant was discarded and the f i n a l p e l l e t resuspended i n 1 ml of buffer B and stored frozen at -20°. Before 38. guanylate cyclase assay, the thawed p e l l e t suspension was homogenized as described above. 13. Guanylate cyclase assay The incubation mixture for the assay of guanylate cyc-lase a c t i v i t i e s contained 50 mM Tris.HCl buffer, pH 7.5, 2 mM MnCl 2, 1.6 mM MIX, 1 mM c y c l i c GMP, 0.1-0.25 mM [a 3 2P] GTP ( s p e c i f i c a c t i v i t y 10-30 cpm per pmol; 300,000-400,000 cpm per assay tube), 15 mM phosphocreatine, 12.25 units of creatine phosphokinase, [ 3 H ] c y c l i c GMP (25 nCi; 13,000 cpm per assay tube) and 40-250 yg of t e s t i s protein, i n a f i n a l volume of 100 y l . When indicated, 1 y l of Trit o n X-100 was added to make a f i n a l concentration of 1% Trit o n X-100, or, 15 y l of salmon p i t u i t a r y extract was added. The assay blank value was obtained using incubation mixtures without t e s t i s protein. Incubations were c a r r i e d out at 37° for various lengths of time (10-30 min). The reaction was i n i t i a t e d by the addition of t e s t i s protein and was terminated by the addition of 20 y l of a solution of EDTA (30 mM) to atta i n a f i n a l concentration of 5 mM. The reaction mixture was di l u t e d to 0.5 ml with d i s t i l l e d H20, vortex mixed and transferred to a BioRad AG 50W-X2, 200-400 mesh, hydrogen form, re s i n column (0.7 x 4 cm) prepared by pipetting 2 ml of a 50% v/v suspension of the resi n i n H 20 into the column. After adsorption of the 0.5 ml, the column was eluted with 0.4 ml of H 20 and the 0.9 ml t o t a l i n i t i a l eluate added to 5 ml of SCINTREX for r a d i o a c t i v i t y determination. About 40-60% of the [a 3 2P]GTP was recovered i n t h i s f r a c t i o n . The column was then eluted with an addit-ional 1 ml of H 2 O and thi s eluate chromatographed d i r e c t l y onto a neutral alumina (Woelm, a c t i v i t y grade I) column (0.7 x 2.5 cm) made up by pipetting 2 ml of a 50% v/v susp-ension of neutral alumina i n 0.1 M Tris.HCl, pH 7.4, into the column. The alumina column was then eluted with 4 ml of 0.1 M Tris.HCl, pH 7.4. Eluates were added to 10 ml of SCINTREX and r a d i o a c t i v i t y (both [ 3 2P] and [ 3H]) was deter-mined. A l l r e s u l t s were corrected for the recovery of [3H] c y c l i c GMP and were calculated 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 of the GTP used i n each experiment. Af t e r d i l u t i o n of [a 3 2P]GTP with unlabeled GTP, the f i n a l GTP concentration was determined by absorbance at 252 nm, using an extinction c o e f f i c i e n t of 13, 800 M~1. The o v e r a l l recovery of c y c l i c GMP, after losses during incubation and the two stage column procedure, was 45-65%. The blank of the assay, under these conditions, ranged from 0.006-0.05% of the added [a 3 2P]GTP. The above assay i s a s l i g h t modification of a previously reported assay (106). In the l a t t e r case, the f i r s t column eluate was 1 ml (compared with 0.9 ml above) and the f i n a l 0.1 M Tris.HCl eluate was 2 ml (compared with 4 ml above). The s l i g h t modifications were made i n the present research because the overlap of GTP and c y c l i c GMP was greater on the AG 50W-X2 columns prepared by the present author, and also the majority of [ 3 2 P ] c y c l i c GMP was found i n the 3rd and 4th m i l l i l i t e r o f f the neutral alumina column (this includes the f i r s t m i l l i l i t e r adsorbed from the AG 5 0W-X2 column onto the neutral alumina column). 14. Protein assay Protein concentrations were assayed by the method of Lowry et a l (107) with bovine serum albumin as the standard. 41. RESULTS A. Testes growth rate during hormonally-induced spermatogenesis  i n trout A testes weight doubling time of about 1 week i s observed i n rainbow trout, Salmo g a i r d n e r i i , injected twice weekly with a salmon p i t u i t a r y extract (10). This r e s u l t s i n a logarithmic testes weight increase during the f i r s t 8 to 10 weeks of t e s t i c u l a r maturation. However, the growth rate of hormonally-induced testes i s not completely synchronous from i n d i v i d u a l to i n d i v i d u a l during development (Table I ) . Also, there are large variations i n i n i t i a l testes weights between d i f f e r e n t batches of trout. Thus, zero time testes weights (sum of the weight of both t e s t i s ) varied between about 30 and 130 mg. This appeared to be related to the age of the f i s h , or the season i n which they were obtained, or to the length of time they were kept i n the laboratory aquaria before the s t a r t of experiments. Batches of trout with large zero time testes produced proportionally large testes throughout development. Figure 3 shows the response to hormone i n j e c t i o n of a batch of trout whose i n i t i a l testes weights were i n the range of 30 to 60 mg. The plateau and loss of weight at the end of the growth period i s a consequence of germ c e l l cytoplasmic loss and t e s t i c u l a r tissue degeneration i n the f i n a l stages of spermatogenesis. The re l a t i o n s h i p between duration of hormone treatment and testes weight permits the reporting of FIGURE 3 Testes growth rate during hormonally-induced  spermatogenesis i n trout Weights of testes ( i . e . weight of a pair of testes) from rainbow trout, Salmo g a i r d n e r i i , maintained under the conditions described i n Materials and Methods, are plotted as a function of days following the i n i t i a t i o n of twice weekly inje c t i o n s of salmon p i t u i t a r y extract. An average f i s h weighed about 250 gm and was about 2 6 cm long. Each point represents an average weight of the p a i r of testes from a sampling of 2 or 3 f i s h . changes i n c y c l i c nucleotide biochemistry during development with reference to either time or testes weight. B. C y c l i c GMP and c y c l i c TAMP concentrations i n trout t e s t i s  during spermatogenesis C y c l i c nucleotides have been determined by receptor protein binding displacement (108), enzymatic cycli n g (109), protein kinase a c t i v a t i o n (110), high pressure ion exchange chromatography (111), and radioimmunoassay (112). . In i n i t i a l experiments (Appendix ) , high pressure ion exchange chromatography was investigated, but was rejected due to i t s i n a b i l i t y to detect the amount of c y c l i c GMP available from trout t e s t i s . Subsequently, radioimmunoassay was found to be a p a r t i c u l a r l y sensitive and convenient technique, and was used i n a l l measurements of c y c l i c GMP and c y c l i c AMP reported i n th i s thesis. The c y c l i c GMP i n trout t e s t i s was determined i n 3 experimental batches of hormonally-induced rainbow trout. Testes were rapidl y excised and frozen i n l i q u i d nitrogen to minimize post-mortem a l t e r a t i o n of the c y c l i c nucleotide concentration. In the f i r s t experiment, nucleotide extracts were not p u r i f i e d before c y c l i c GMP was measured. However, after treatment with, beef heart c y c l i c nucleotide phosphodi-esterase, some c y c l i c GMP samples had high radioimmunoassay blank values (greater than 10% of the untreated sample). Therefore, i n subsequent experiments, c y c l i c GMP extracted from t e s t i s was p u r i f i e d before radioimmunoassay. From the available p u r i f i c a t i o n techniques for c y c l i c nucleotides from b i o l o g i c a l sources,i.e. adsorption chrom-atography on neutral alumina (113), thin-layer chromatography on PEI-cellulose (114), inorganic s a l t c o p r e c i p i t a t i o n using ZnCC>3 and BaSCH (115), or ion exchange chromatography on cation exchange resins, such as DOWEX 50 (116), and anion exchange resins, such as DOWEX 1 and 2 (117), anion exchange was used iinthis research because of i t s r e l a t i v e ease and speed. In a l l experiments subsequent to the f i r s t , trout t e s t i s nucleotide samples were p u r i f i e d by anion exchange chromatography on AG 1-X8 columns, as described i n Materials and Methods. Only a li m i t e d number of samples were assayed i n the f i r s t experiment (data not shown), but since high c y c l i c GMP concentrations (1 ymol per kg te s t i s ) were observed early i n development and not i n mature t e s t i s , a more thorough sampling of testes at early stages of development was made i n the second experiment (Table I and Figure 4). In experiment 2, c y c l i c GMP was found to be elevated i n zero time t e s t i s and in t e s t i s during premeiotic p r o l i f e r a t i o n of spermatogonia (Figure 4). P r i o r to the time of the meiotic reduction d i v i s i o n i n trout 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 of the meiotic reduction d i v i s i o n i n hormonally-induced trout t e s t i s , at between 5 to 6 weeks after the i n i t i a t i o n of hormone i n j e c t i o n , was determined i n a study of predominant c e l l types during development, th e i r DNA content and t h e i r [ 3H]thymidine . TABLE I 46. CYCLIC GMP IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS (EXPERIMENT 2) A. Samples from testes with an average weight for the time of hormonal induction Day of Total Testes C y c l i c GMPa C y c l i c GMPa Hormonal Wet Weight (umol/kg wet (pmol/mg Induction (mg) weight) protein) 0 37 1. 93 19 56 1.45 13 10 92 1.00 10 93 1.93 20 15 100 1.51 19 24 300 1.12 13 30 500 0. 39 4.0 39 880 0.12 1.3 45 1400 b 0.08 0.8 1400C 0.08 0.9 70 7000 b 0.06 1.1 7000 c 0. 04 0.7 87 9200 b 0. 04 0.8 9200 c 0. 03 0.6 98 d 0. 15 12e B. Samples from testes with an abnormal weight for the time of hormonal induction 15 400 0.30 3.1 24 780 0.14 1.4 30 150 0.81 12 39 160 0.85 10 a Each sample assayed for c y c l i c GMP i n duplicate, at 2-3 d i l u t i o n s , i n 2 radioimmunoassays and the mean value of these r e s u l t s recorded. Duplicate range ±10%. Overall range for the same sample at 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 testes, assayed as above d Sperm e Assuming a 1.3% protein content, as in other sperm samples FIGURE 4 Comparison of c y c l i c GMP concentration and testes weight  during hormonally-induced spermatogenesis i n trout C y c l i c GMP' extracted and assayed as described i n Materials 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 described i n the legend for Figure 3. The average wet weight growth curve i s as i n Figure 3. Values for c y c l i c GMP were obtained from testes with an average wet weight for the time of hormonal induction. Each b i o l o g i c a l sample was assayed i n duplicate, at 2-3 d i l u t i o n s , i n 2 radioimmunoassays and the mean value of the sum of these re s u l t s recorded. The range between duplicates i n the same radioimmunoassay was i 10%. The o v e r a l l range between values obtained for the same b i o l o g i c a l sample at d i f f e r e n t d i l u t i o n s was - 30%. 4 8 . 2 0 4 0 6 0 8 0 D a y s of h o r m o n a l i n d u c t i o n incorporation (10). Although the exact timing of meiosis was not determined i n the present studies, i t i s obvious that the abrupt decrease i n c y c l i c GMP occurred before the p r o l i f -eration of germ c e l l s had ceased (Figure 4). During the stages following the large decrease i n c y c l i c GMP, an inverse corr-e l a t i o n between testes weight and c y c l i c GMP concentration was observed:.: (Table IA and Figure 4) . Only those values for c y c l i c GMP obtained from testes with the average weight for the time of hormonal induction were used i n Figure 4 . In testes which were not developing at the average growth rate, as shown i n Figure 3, c y c l i c GMP concentrations again showed an inverse re l a t i o n s h i p to t o t a l testes wet weight (Table IB). The c y c l i c GMP concentration observed i n sperm of 0.15 ymol/kg wet weight i s s i m i l a r to a previous report of c y c l i c GMP i n trout 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 obtaining an accurate protein concentrat-ion i n sperm suspensions (Materials and Methods) puts doubt on the expression of c y c l i c GMP i n pmol/mg protein. In experiment 2, rather large variations were observed i n c y c l i c GMP i n early developmental samples from testes of approximately the same t o t a l wet weight (Table I ) . Such variations were also observed i n early developmental samples i n the t h i r d experiment (Table I I ) . In experiment 3, c y c l i c GMP was found to be elevated i n a l l samples from zero time to 4 weeks of hormonal induction (Table I I ) , and s l i g h t l y higher than c y c l i c GMP i n early samples i n experiment 2. (Table I ) . In experiment 3, c y c l i c GMP again decreased TABLE II CYCLIC GMP IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS (EXPERIMENT 3) Day of Total Testes C y c l i c GMPa Hormonal Wet Weight (umol/kg wet Induction (mg) ' weight) 0 30 2. 36 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 Each sample assayed i n duplicate, at 2 d i l u t i o n s , i n 2 radioimmunoassays and the mean value of these results recorded. Duplicate range ± 10%. Overall range for the same sample at d i f f e r e n t d i l u t i o n s was ± 30%. dramatically af t e r about 4 to 5 weeks of hormone injec t i o n s (Table I I ) . The concentration of c y c l i c GMP again decreased to about one-tenth of i t s o r i g i n a l concentration. During the following stages of t e s t i s development, there was an inverse c o r r e l a t i o n between testes weight and c y c l i c GMP concentration (Table II) . In experiment 3, t e s t i s c y c l i c GMP was compared with c y c l i c GMP i n another trout t i s s u e , namely the l i v e r . Liver c y c l i c GMP was measured i n both immature (3 weeks of hormonal induction) and mature (12 weeks of hormonal induction) f i s h , and found to be 0.22 t 0.07 and 0.11 ± 0.01 umol/kg wet weight l i v e r , respectively. I t i s not clear i f t h i s 100% reduction i n trout l i v e r c y c l i c GMP i s s i g n i f i c a n t . The trout l i v e r c y c l i c GMP concentrations are somewhat higher than those observed i n rat l i v e r of 0.04-0.07 umol/kg wet weight l i v e r ( 128). In experiment 4, c y c l i c AMP was determined i n trout t e s t i s during hormonally-induced spermatogenesis (Figure 5). C y c l i c AMP decreased 2 f o l d at a premeiotic stage of t e s t i s develop-ment, at about, or p r i o r to 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-entrations i n zero time t e s t i s were approximately equal at about 2 umol/kg wet weight t e s t i s (Figures 4 and 5). After the 2: foTd premeiotic decrease i n trout t e s t i s c y c l i c AMP, no s i g n i f i c a n t changes i n c y c l i c AMP concentrations i n developing t e s t i s occurred during the remainder of spermatogenesis (Figure 5) FIGURE 5 Cy c l i c AMP concentrations i n t e s t i s during hormonally- induced spermatogenesis i n trout C y c l i c AMP, extracted and assayed as described i n Material 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 described i n the legend for Figure 3. Values for c y c l i c AMP were obtained from testes with an average wet weight for the time of hormonal induction. Each point represents a b i o l o g i c a l sample assayed i n duplicate, at 2-3 d i l u t i o n s , i n 2 radioimmunoassays and the mean value of the sum of these re s u l t s recorded. The range between duplicates i n the same radioimmunoassay was ^ 10%. The o v e r a l l range between values obtained for the same b i o l o g i c a l sample at d i f f e r e n t d i l u t i o n s was * 30 [ Days of Hormonal Induction 54. C. 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 trout t e s t i s during spermatogenesis 1. Assay system A radioisotope assay system provides the maximum s e n s i t i v i t y for c y c l i c nucleotide phosphodiesterase assays (118) and was therefore used i n thi s research. In thi s method, [ 3 H ] c y c l i c nucleotide i s hydrolyzed to the corres-ponding 5 1-nucleotide, which i s then converted to a nucleo-side by the addition of excess phosphatase. In many cases (60, 103, 105) the excess 5 1-nucleotidase present i n snake venom has been u t i l i z e d i n thi s second incubation. In the present research, c o l i a l k a l i n e phosphatase was used. Separation of nucleoside from c y c l i c nucleotide can be accomplished by passage over an alumina column (113), a DOWEX 1 or 2 column (119, 120), by thin-layer chromato-graphy (121), by paper chromatography (122) or by batch use of DOWEX 1 re s i n (123). The l a t t e r method, modified by a c i d i f i c a t i o n of the re s i n p r i o r to i t s use, resulted i n quantitative recoveries of adenosine, guanosine and t h e i r major metabolites (103, 105). This assay system provides a simple, rapid and r e l i a b l e method by which c y c l i c AMP or c y c l i c GMP phosphodiesterases can be determined, even i n crude tissue preparations, and, was thus adopted for use. The temperature of 30° for a c t i v i t y assays was chosen for comparison of results with previous c y c l i c nucleotide phosphodiesterase studies i n f i s h sperm (88). DEAE-cellulose p r o f i l e s were assayed at pH 8.0 for maximum assay a c t i v i t y . A l l other assays were at pH 7.5. Following assay conditions previously described (119), a 2+ Mg concentration of 2 mM was used. When included, EGTA was present to ..eliminate the possible e f f e c t s of varying 2 + amounts of Ca -dependent phosphodiesterase protein activator 2+ i n tissue preparations. When included, Ca was present to investigate the e f f e c t s of such an activator. The assay buffer contained 2-mercaptoethanol because i t had been shown to s t a b i l i z e the enzyme a c t i v i t y from other tissues (12 4). 2. Properties C y c l i c AMP and c y c l i c GMP phosphodiesterases i n trout t e s t i s were stable, at the assay temperature of 30°, for at le a s t 30 min, at 0.1M substrate, as shown .in Figure 6A. In 2+ one t e s t i s homogenate,a study of enzyme requirement for Mg , 2+ showed an approximate Mg optimum of 8-10 mM and 2-10 mM for 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 , respectively (Figure 6B). A broad pH optimum from pH 7.5 to 8.0, for 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 , was determined i n one trout 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 at micromolar subtrate by about 20%. Due to the complexities of multiple enzyme 2+ forms, detailed developmental studies of Mg optima and pH optima were not made. 56. FIGURE 6 A. S t a b i l i t y of phosphodiesterase a c t i v i t i e s at 30° Phosphodiesterase a c t i v i t i e s , measured i n a homogenate of t e s t i s , from trout which had been injected with a salmon p i t u i t a r y extract for 8 weeks and maintained under conditions described i n Materials and Methods. Total testes weight was 4g. Phosphodiesterase a c t i v i t i e s were assayed, at 30°, i n standard incubation mixtures at pH 7.5, as described i n Materials and Methods. C y c l i c nucleotide concentration was 0.1 mM. EGTA was not included i n assays. • — • c y c l i c AMP O—O c y c l i c GMP 2 + B. Phosphodiesterase a c t i v i t i e s as -a function of Mg concentration Phosphodiesterase a c t i v i t i e s , i n the homogenate described above, were assayed, at 30°, i n standard incubation mixes 2+ at pH 7.5, with varying concentrations of Mg , as described i n Materials and Methods. C y c l i c nucleotide concentration was 0.1 mM. EGTA was not included. Symbols as above. 58. FIGURE 7 A. Phosphodiesterase 1 a c t i v i t i e s 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 trout which had been injected with a salmon p i t u i t a r y extract for 4 weeks and maintained under conditions described i n Materials and Methods. Total testes weight 629 mg. Phosphodiesterase a c t i v i t i e s were assayed, at 30°, i n standard incubation mixtures at pH 7.5, as described i n Materials and Methods. C y c l i c nucleotide concentration was 1 yM. When present, EGTA concentration was 250 yM. A — A c y c l i c AMP c y c l i c AMP plus EGTA A — A c y c l i c GMP o—o c y c l i c GMP plus EGTA B. Phosphodiesterase a c t i v i t i e s as a function of pH Phosphodiesterase a c t i v i t i e s , i n the homogenate described above, were assayed, at 30°, i n standard incubation mixtures, at varying pHs, as described i n Materials and Methods. C y c l i c nucleotide concentration was 1 yM. EGTA (250 yM) was included i n a l l assays. 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 for c y c l i c AMP and c y c l i c GMP were determined i n a mature trout t e s t i s homogenate (Figures 8 and 9). Hofstee plots showed only high a f f i n i t y c y c l i c AMP a c t i v i t i e s (Figure 8) and both a high and low a f f i n i t y c y c l i c GMP a c t i v i t y (Figure 9). 3. A c t i v i t i e s during t e s t i s development A c t i v i t i e s were measured throughout development at both saturating (millimolar) and subsaturating (micromolar) substrate concentrations, to detect t o t a l and high a f f i n i t y components, respectively. Total c y c l i c AMP phosphodiesterase a c t i v i t i e s measured i n the presence or absence of EGTA (Table III) decr-eased 50% p r i o r to meiosis. This was followed by an increase during 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 that observed i n immature t e s t i s . EGTA decreased t o t a l c y c l i c AMP a c t i v i t i e s about 2 0-40% i n zero time and mature t e s t i s but had an i n s i g n i -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 just p r i o r to meiosis. Total c y c l i c GMP phosphodiesterase a c t i v i t i e s remained r e l a t i v e l y constant throughout development, i n the absence of EGTA (Table I I I ) . The presence of EGTA appeared to enhance c y c l i c GMP phosphodiesterase a c t i v i t i e s i n immature t e s t i s about 30%, but throughout the following stages of t e s t i s development resulted i n about a 2 0% decrease i n c y c l i c GMP a c t i v i t i e s . The rather minor e f f e c t of EGTA on 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 did not show any important changes during development i n the concentration 2+ or the a c t i v i t y of a Ca -dependent phosphodiesterase protein FIGURE 8 Hofstee p l o t of the rate of c y c l i c AMP hydrolysis by a t e s t i s homogenate (minus the l,000xg p e l l e t ) from trout hormonally-induced for 10 weeks, as described i n Materials and Methods. Total testes weight 9.8 g. Homogenates were stored frozen at -20° for a week before k i n e t i c analysis Homogenates were centrifuged at l,0 00xg for 10 min a f t e r thawing. The insoluble l i p i d p e l l e t was discarded and the supernatant assayed for a c t i v i t y . The unit of v e l o c i t y i s nmol c y c l i c AMP hydrolyzed/min/mg protein. Assays were at 30°, i n standard incubation mixtures at pH 7.5, as described i n Materials and Methods. EGTA (250 yM) was included i n a l l assays. Substrate concentrations varied from 0.1-500 yM 1 t 6 3 . FIGURE 9 Hofstee p l o t of the rate of c y c l i c GMP hydrolysis by the homogenate described i n the legend for Figure 8. The unit of v e l o c i t y i s nmol c y c l i c GMP hydrolyzed/min/mg protein. Assays were at 30°, i n standard incubation mixtures at pH 7.5, as described i n Materials and Methods. EGTA (25 0 uM) was included i n a l l assays. Substrate concentrations varied from 5 - 1,000 uM. 64 . V TABLE III TOTAL CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITIES IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS Week of Total Testes C y c l i c AMP C y c l i c GMP Hormonal Wet Weight A c t i v i t y A c t i v i t y Induction (mg) Minus, Plus_ Minus Plus EGTA EGTA EGTA b EGTA c 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 a 450 90 Trout testes were excised, placed i n l i q u i d nitrogen and stored at -80 u n t i l one complete developmental series of testes had been c o l l e c t e d . Trout were hormonally-induced by a twice weekly i n j e c t i o n of a salmon p i t u i t a r y extract, as described i n Materials and Methods. Crude homogenates of the thawed and weighed testes were prepared i n ^buffer A and assayed for 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 , at 30 , i n standard incubation mixtures at pH 7.5, as described i n Materials and Methods. Millimolar substrate concentrations were used. A c t i v i t y units are pmol c y c l i c nucleotide hydrolyzed/min/mg protein. Values are the means of duplicates (± 10%) from the same homogenate. This sample consisted of sperm, obtained as described i n Materials and Methods k No EGTA i n assay mixture C-EGTA(250 uM) i n assay mixture 66. activator i n trout t e s t i s . The s p e c i f i c a c t i v i t i e s of sperm c y c l i c AMP and c y c l i c GMP phosphodiesterases, measured at millimolar substrate concentrations, are respectively 3 and 6 f o l d lower than the s p e c i f i c 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 nucleotide phosphodiesterase s p e c i f i c a c t i v i t i e s i n sperm and i n t e s t i s tissue 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 i n measuring protein i n sperm suspensions (Materials and Methods). C y c l i c AMP and c y c l i c GMP phosphodiesterases, i n t e s t i s homogenates from trout at d i f f e r e n t stages of spermatogenesis, were measured at micromolar substrate, i n the presence of EGTA (Table IV). As seen i n the t o t a l c y c l i c AMP a c t i v i t i e s (Table I I I ) , a 50% decrease of the small amount of i n i t i a l c y c l i c AMP phosphodiesterase, measured at micromolar substrate, occurs i n a premeiotic stage of spermatogenesis. This i s followed by a 20 to 40 f o l d increase i n c y c l i c AMP a c t i v i t y , measured at micromolar substrate, during the l a t e r stages of development.(Table IV). C y c l i c AMP phosphodiesterase act-i v i t i e s were not measured at micromolar substrate throughout development i n the absence of EGTA. As seen i n Figure 7A, EGTA caused a 2 0% reduction i n c y c l i c AMP phosphodiesterase a c t i v i t y , measured at micromolar substrate, i n a t e s t i s homogenate:..from one stage: of development. I t i s possible that, i n the absence of EGTA, a s l i g h t l y greater increase i n high 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 be observed during spermatogenesis. TABLE IV CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITIES MEASURED  AT MICROMOLAR SUBSTRATE IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS Week of T o t a l Testes C y c l i c AMP C y c l i c GMP A/G Hormonal Wet Weight A c t i v i t y A c t i v i t y R a t i o a I n d u c t i o n (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 12 b 310 3 124 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 . Assay mixtures a l l c o n t a i n EGTA (250 uM). 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 , The r a t i o o f c y c l i c AMP to c y c l i c GMP phosphodiesterase a c t i v i t y 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 as d e s c r i b e d i n M a t e r i a l s and Methods 68. C y c l i c GMP phosphodiesterase a c t i v i t y , measured at micro-molar substrate, i n the presence of EGTA, decreases less than 50% progressively throughout spermatogenesis (Table IV). The r a t i o of c y c l i c AMP to c y c l i c GMP hydrolysis, measured at micromolar substrate, changes from about 2 to 124 during t e s t i s d i f f e r e n t i a t i o n . This indicates the s p e c i f i c induction of a high a f f i n i t y c y c l i c AMP phosphodiesterase i n a late stage of spermatogenesis. In a study by Drummond and his associates, i n which c y c l i c nucleotide phosphodiesterase a c t i v i t i e s i n salmon sperm were measured, at micromolar sub-strate concentrations, a c y c l i c AMP to c y c l i c GMP a c t i v i t y r a t i o of 124 was also observed (88). The sequential changes i n c y c l i c nucleotide phosphodi-esterase a c t i v i t i e s , during spermatogenesis i n trout, are graphed i n Figure 10. Total (Figure 10A) and high 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 function of time of hormonal induction and i n r e l a t i o n to the stage of germ c e l l d i f f e r -e n t i a t i o n . The induction of a high a f f i n i t y c y c l i c AMP phosphodiesterase, at about meiosis, i s evident i n Figure 10B. 2+ Since i t had been shown that Ca , i n the presence of a 2 + Ca -dependent phosphodiesterase protein activator, increases the a c t i v i t y of c y c l i c GMP hydrolysis more so than c y c l i c AMP 2+ hydrolysis (74), a more detailed study of the e f f e c t s of Ca and EGTA on high a f f i n i t y c y c l i c GMP phosphodiesterase act-i v i t i e s i n developing trout t e s t i s , was undertaken. The results of t h i s study are summarized i n Table V. Only i n FIGURE 10 A. Total, c y c l i c nucleotide phosphodiesterase a c t i v i t i e s i n  trout t e s t i s during 1 spermatogenesis Testis homogenates, from trout injected twice weekly with a salmon p i t u i t a r y extract and maintained as described i n Materials and Methods, were assayed for c y c l i c nucleotide phosphodiesterase a c t i v i t i e s , at 30°, in standard incubation mixtures at pH 7.5, as described i n Materials and Methods. C y c l i c nucleotide concentration was 1 mM. EGTA (250 yM) was included i n a l l assays. The a c t i v i t y units are pmol c y c l i c nucleotide hydrolyzed/min/mg protein. This graph i l l u s t r a t e s data from Table I I I . • — • c y c l i c AMP O — O c y c l i c GMP B. C y c l i c nucleotide phosphodiesterase a c t i v i t i e s ^ measured  at micromolar substrate concentrations, i n trout t e s t i s during s pe rma to gene s i s Legend as above, except that c y c l i c nucleotide concentration was 1 yM. This graph i l l u s t r a t e s data from Tables IV and V. Symbols as above. 70. S p e r m a t o g o n i a S p e r m a t i d s Spermatocytes 2 4 6 8 10 W e e k of H o r m o n a l I n d u c t i o n 2 4 6 8 10 vVeek of H o r m o n a l I n d u c t i o n TABLE V CYCLIC GMP PHOSPHODIESTERASE.ACTIVITIES MEASURED AT  MICROMOLAR SUBSTRATE IN TROUT TESTIS DURING HORMONALLY-IN DU CE D"SPE RMATOGENESIS Week of Total Testes C y c l i c GMP Phosphodiesterase Hormonal Wet Weight A c t i v i t i e s Induction (mg) No Addition Plus Plus , EGTA a i-Ca 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 at micromolar substrate concentrations 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 a marked i n h i b i t i o n by EGTA noted (30 and 55% i n h i b i t i o n , r e s p e ctively). In a l l other stages of development, there was a 12-25% i n h i b i t i o n by EGTA. A c t i v i t y i n the presence 2+ of Ca was approximately the same as that observed with no additions. There was no s p e c i f i c high a f f i n i t y c y c l i c GMP phosphodiesterase a c t i v i t y , i n the presence or absence of 2+ Ca , associated with the premeiotic stage i n which the large decrease i n c y c l i c GMP concentration was observed (Figure 4). 4. DEAE-cellulose chromatographic •fractionation' of c y c l i c AMP and c y c l i c GMP phosphodiesterases When chromatographed on DEAE-cellulose, trout t e s t i s homogenates, from a l l stages of development, y i e l d two active c y c l i c nucleotide phosphodiesterase f r a c t i o n s . These are eluted by about 0.35 M (Peak I) and 0.65 M'(Peak II) sodium acetate, pH 6.5 (Figure 11). Column fractions were assayed at low substrate concentrations (- 0.1 uM) . One p r o f i l e 2+ (Figure 11B) was assayed i n the presence of Ca (100 uM), i n the presence of EGTA (250 uM) or with standard assay incubation mixtures with no additions. No difference i n c y c l i c AMP or c y c l i c GMP 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). This indicates the loss of Ca -dependent phospho-diesterase protein activator during DEAE-cellulose column p u r i f i c a t i o n , as has been previously been reported (75). Early attempts at fr a c t i o n a t i o n of bovine heart 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 of 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 trout t e s t i s during hormonally-induced spermatogenesis DEAE-cellulose p r o f i l e s of c y c l i c nucleotide phospho-diesterases i n trout t e s t i s homogenates prepared as described i n Materials and Methods. A. Two weeks of hormonal induction. Average testes weight 14 6 mg; 2 80 mg i n homogenate form applied to a column of 0.5 x 10.5 cm si z e . Column f r a c t i o n size 2.2 ml. B. Four weeks of hormonal induction. Average testes weight 629 mg; 540 mg i n homogenate form applied to a column of 1 x 15 cm si z e . Column f r a c t i o n size 4.5 ml. C. Six weeks of hormonal induction. Average testes weight 3550 mg; 666 mg i n homogenate form applied to a column of 0.8 x 22 cm si z e . Column f r a c t i o n size 4.5 ml. D. Eight weeks of hormonal induction. Average testes weight 40 30 mg; 6 66 mg i n homogenate form applied to a column of 0.8 x 22 cm si z e . Column f r a c t i o n size 4.5 ml. For a l l p r o f i l e s , after sample application columns were washed with 20 mM sodium acetate, pH 6.5 buffer containing 4 mM 2-mercaptoethanol. The i n i t i a l wash contained no phosphodiesterase a c t i v i t y . 2 0 mM - 1 M sodium acetate, pH 6.5 l i n e a r gradients were started at tube 10 and cont-inued through to tube 55, at a flow rate of 2 6 ml/hr. Total gradient volume was 2 00 ml for p r o f i l e s B, C and D. Total gradient volume for A was 100 ml. Fraction 25 and fr a c t i o n 38 correspond to 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 f r a c t i o n aliquots (or 25 y l for p r o f i l e A), at 30°, i n standard incubation mixtures, at pH 8.0, as described i n Materials and Methods. EGTA (250 yM) was i n a l l assays. • — • c y c l i c AMP (3 x 10 7M; A and B. 1.5 x 10~7M; C and D) O — O c y c l i c GMP (3 x 10"7M; A and B. 1.6 x 10"7M; C and D) 74. Fraction N u m b e 75. on DEAE-cellulose, were unsuccessful due to t h i s loss (125) . At early stages of trout t e s t i s development (Figures 11A and 11B) an active c y c l i c GMP phosphodiesterase eluted at 0.35 M sodium acetate. This a c t i v i t y was unstable on prolonged storage at -20°, i n contrast to the c y c l i c AMP a c t i v i t y which eluted at t h i s p o s i t i o n . The phosphodi- •. esterase a c t i v i t y e l u t i n g at 0.65 M sodium acetate was found to hydrolyze c y c l i c AMP almost exclusively. In late stages of spermatogenesis (Figures 11C and 11D) a large increase i n the r e l a t i v e size of Peak I to Peak II c y c l i c AMP phosphodiesterase a c t i v i t y occurs, while c y c l i c GMP hydrolysis i s unchanged or s l i g h t l y decreased. Quantitative comparisons between d i f f e r e n t stages of development cannot be made, due to the d i f f e r i n g amounts of homogenates applied to columns and the lack of enzyme recovery data. Each fraction.tube contained 4mg of bovine serum albumin preventing the accurate measurement of enzyme - protein.~ The 2 peaks at about 0.3-0.4 M sodium acetate, i n Figure 11B, may be a p a r t i a l separation of two enzyme forms, since t h i s e f f e c t was also seen on a p r o f i l e of t e s t i s homogenate from a trout after 3 weeks of hormone inject i o n s ( p r o f i l e not shown). However, the 100,000xg supernatant p r o f i l e , from the same t e s t i s homogenate as i n Figure 11B, did not show thi s double peak (Figure 13A). A portion (33%) of Peak I, from the p r o f i l e described i n Figure 11D, was rechromatographed on DEAE-cellulose and only one peak, cochromatographing at the o r i g i n a l Peak I 76. po s i t i o n , was observed (Figures 12A and 12B). In the r a t l i v e r , i t has been demonstrated that the low a f f i n i t y c y c l i c AMP phosphodiesterase peak from a Bio-Gel A-5m column, when rechromatographed on DEAE-cellulose, gave a p r o f i l e which contained 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 0.3 M sodium acetate cut, plus a high a f f i n i t y a c t i v i t y , .in a 0.6 M sodium acetate cut (60). No such Peak II a c t i v i t y was observed i n the present case (Figure 12B). There was a 50% loss i n c y c l i c AMP a c t i v i t y and about a 90% loss of c y c l i c GMP a c t i v i t y , observed i n Figure 11D, on rechromat-ography of Peak I (Figure 12B). C y c l i c GMP a c t i v i t y , i n the rechromatographed Peak I p r o f i l e , was very low, and, therefore was not included i n Figure 12. The cause of the a c t i v i t y losses i s not known. 5. Soluble and p a r t i c u l a t e a c t i v i t i e s fractionated on  DEAE-cellulose When the 100,000xg supernatant (soluble) f r a c t i o n , from a trout t e s t i s homogenate, was chromatographed on DEAE-cellulose, both c y c l i c AMP phosphodiesterase peaks i d e n t i f i e d i n the t o t a l homogenate p r o f i l e , were observed (Figure 13A). C y c l i c GMP phosphodiesterase a c t i v i t y was observed i n the Peak I p o s i t i o n , but was extremely low, and, therefore was not included i n Figure 13A. There was a 2.5 f o l d increase i n Peak II to Peak I c y c l i c AMP a c t i v i t y r a t i o i n the soluble p r o f i l e , .as compared with the same- r a t i o i n the homogenate p r o f i l e (Figure 11B) from the same t e s t i s . A sonicated 100,000xg p e l l e t (particulate) f r a c t i o n , from FIGURE 12 A. DEAE-cellulose p r o f i l e of c y c l i c AMP phosphodiesterases i n a t e s t i s homogenate from a trout injected with a salmon p i t u i t a r y extract for 8 weeks (detailed description i n the legend for Figure 11D). Phosphodiesterase a c t i v i t i e s were assayed i n 10 y l column f r a c t i o n aliquots, at 30°, i n standard incubation mixtures, at pH 8.0, as described i n Materials and Methods. EGTA (250 yM) was i n a l l assays. C y c l i c AMP concentration was 1.5 x 10"7M. B. DEAE-cellulose p r o f i l e of rechromatographed,-peak I, from the p r o f i l e of the t e s t i s homogenate described 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 described i n Materials and Methods, and a portion of the concentrate applied to a DEAE-cellulose column. Conditions for DEAE-cellulose fr a c t i o n a t i o n as described i n Materials and Methods. Phosphodiesterase a c t i v i t i e s assayed as described above, except that c y c l i c AMP concentration was 0.5 x 10"7M. 79. FIGURE 13 A. DEAE-cellulose p r o f i l e of trout 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 f r a c t i o n from t e s t i s hormonally-induced for 4 weeks. Average (from 2 fish) t o t a l testes weight of 62 9 mg. B. DEAE-cellulose p r o f i l e of trout 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 p a r t i c u l a t e f r a c t i o n from the t e s t i s described above. The conditions for both separation p r o f i l e s were as described i n d e t a i l i n Materials and Methods. The gradient was from f r a c t i o n 10 to f r a c t i o n 50. 50 u l column f r a c t i o n aliquots were assayed for phosphodiesterase a c t i v i t i e s , at 30°, i n standard incubation mixtures at pH 8.0, as described i n Materials and Methods. C y c l i c AMP _7 assay concentration was 1.5 x 10 M. EGTA (250 uM) was included i n assay mixtures. 81. the same trout t e s t i s homogenate, contained mainly Peak II c y c l i c AMP a c t i v i t y (Figure 13B). The p a r t i c u l a t e f r a c t i o n , from trout t e s t i s homogenate, contained about 15% of the c y c l i c AMP phosphodiesterase a c t i v i t i e s and 20% of the c y c l i c GMP phosphodiesterase a c t i v i t i e s , measured at micromolar substrate concentrations. The s p e c i f i c a c t i v i t y of the 100,000xg p e l l e t was 21 pmol c y c l i c AMP hydrolyzed/min/mg protein, about 50% of that of the 100,000xg supernatant at 43 pmol c y c l i c AMP hydrolyzed/ min/mg protein. For c y c l i c GMP hydrolysis, the s p e c i f i c a c t i v i t y of the 100,000xg p e l l e t was 3.7 pmol c y c l i c GMP hydrolyzed/min/mg protein, about 75% of that of the 100,000xg supernatant at 4.8 pmol c y c l i c GMP hydrolyzed/min/mg protein. 6. Kinetic analyses of c y c l i c AMP phosphodiesterase a c t i v i t i e s  fractionated on DEAE-cellulose Kinetic 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 of a c t i v i t y obtained from DEAE-c e l l u l o s e , were made i n order to investigate t h e i r substrate a f f i n i t i e s . Two types of data expression were used, i . e . Lineweaver-Burk plots and Hofstee p l o t s . A Hofstee p l o t of Peak I c y c l i c AMP phosphodiesterase a c t i v i t i e s , from a mature t e s t i s homogenate, revealed a low apparent Km^ obtained from the l i n e a r slope i n Figure 14. Hofstee p l o t s of Peak II a c t i v i t i e s , from both premeiotic and mature t e s t i s homogenates;, indicated similar .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 of the irate of c y c l i c AMP hydrolysis by DEAE-cellulose Peak I from t e s t i s from trout hormonally-induced for 8 weeks (detailed description i n the legend for Figure 11D). The unit of v e l o c i t y i s pmol/min/10 y l . Assays were at 30°, i n standard incubation mixtures at pH 7.5, as described i n Materials and Methods. EGTA (250 yM) was included i n a l l assays. Substrate conc-entration ranged from 0.05 - 5 yM. FIGURE 15 A. Lineweaver-Burk p l o t of the rate of c y c l i c AMP hydr-o l y s i s of DEAE-cellulose Peak II from t e s t i s from trout hormonally-induced for 3 weeks (t o t a l testes weight 2 50 mg). The unit of v e l o c i t y i s pmol/min/10 y l . Assays were at 30°, i n standard incubation mixtures, at pH 7.5, as described i n Materials and Methods. EGTA (250 yM) was included in a l l assays. Substrate concentration ranged from 0.05 - 5 yM. B. Hofstee p l o t of the hydrolysis described above. The Lineweaver-Burk p l o t gives two apparent Kms of 0.8 yM and 2.3 yM. The Hofstee p l o t gives two apparent Kms of 0.5 yM and 4.4 yM. FIGURE 16 A. Lineweaver-Burk p l o t of the rate of c y c l i c AMP hydrolysis by DEAE-cellulose Peak II from t e s t i s from trout hormonally-induced for 8 weeks (detailed description i n the legend for Figure 11D). The unit of v e l o c i t y i s pmol/min/10 u l . Assays were at 30°, i n standard incubation mixtures at pH 7.5 as described i n Materials and Methods. EGTA (250 yM) was included i n a l l assays. Substrate concentration ranged from 0.05 - 5 yM. B. Hofstee p l o t of the hydrolysis described above. The Lineweaver-Burk p l o t gives a Km - 1.0 yM. The Hofstee p l o t gives an apparent Km of 0.7 yM.. . 87. premeiotic t e s t i s homogenate was too low to obtain r e l i a b l e k i n e t i c data from. The non-linearity of both Lineweaver-Burk (Figure 15A) and Hofstee (Figures 14, 15B and 16B) plots of c y c l i c AMP phosphodiesterase a c t i v i t i e s i s commonly observed i n k i n e t i c studies on phosphodiesterase a c t i v i t i e s i n various tissues (60, 90, 133). Two apparent Km values could be obtained from the two slopes on both the Lineweaver-Burk plot and the Hofstee p l o t of c y c l i c AMP hydrolysis by Peak II phosphodiesterase from the premeiotic t e s t i s homo-genate (Figures 15A and 15B). The apparent Kms from a l l plots corresponded to phosphodiesterase a c t i v i t i e s with high a f f i n i t y for c y c l i c AMP. 89. D. Guanylate: cyclase a c t i v i t i e s i n trout t e s t i s during  spermatogenesis 1. Assay system In crude preparations, guanylate cyclase a c t i v i t i e s are usually small compared to the a c t i v i t i e s of i n t e r f e r i n g enzymes, such as nucleoside triphosphatases, c y c l i c nucleo-tide phosphodiesterases, nucleotidases and deaminases (126). Since the products-, of the reactions which these contaminating enzymes catalyze, may cochromatograph with c y c l i c GMP, an e f f e c t i v e method of separating .::.! c y c l i c GMP from GTP and a l l i t s possible degradation products, i s imperative (126). Assays u t i l i z i n g radioactive GTP provide the maximum s e n s i t i v i t y . If [a 3 2P]GTP i s used, separation of c y c l i c GMP from a l l other purine nucleotides and inorganic phosphate, i s required. With [3H] or [ l l fC]GTP as substrate, additional separation from a l l purine nucleosides, bases and u r i c acid i s necessary. Methods for such separations are the same as those mentioned for c y c l i c nucleotide p u r i f i c a t i o n . A p a r t i c u l a r l y popular method for assaying guanylate cyclase a c t i v i t i e s uses [a 3 2P]GTP as substrate and p u r i f i c a t i o n of reaction products on neutral alumina (127). A modification of the l a t t e r method, has been developed to provide greater s e n s i t i v i t y for the assay of guanylate cyclase a c t i v i t i e s (106). The modified method u t i l i z e s a combination of DOWEX 50 ion exchange chromatography and neutral alumina adsorption chromatography, i n the separation of 90. r e a c t i o n products. This method was chosen f o r the present research, due to i t s low blank of [a 3 2P]GTP i n the p u r i f i e d [ 3 2 P ] c y c l i c GMP f r a c t i o n , i t s ease of performance, i t s r e l i a b i l i t y and f o r the previous r i g o r o u s i d e n t i f i c a t i o n - of the [ 3 2P] product 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 mixtures c l o s e l y f o l l -owed those described i n the paper which d e t a i l e d the two-stage column procedure (106). In t h i s paper accurate and r e l i a b l e guanylate c y c l a s e assays were c a r r i e d out i n a v a r i e t y of t i s s u e s . <:The assay temperature of 37° was used, because guanylate 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 out at 37°. In the present research, the i n c u b a t i o n contents out-l i n e d p r e v i o u s l y (106) were modified to i n c l u d e a c y c l i c n u c l e o t i d e 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 xanthine, and a GTP regenerating system, c o n s i s t i n g of 15 mM phosphocreatine and 12.25 u n i t s of c r e a t i n e phosphokinase per assay tube. The GTP regenerating system was found to be an absolute requirement f o r the d e t e c t i o n of the t o t a l guanylate cyc 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. Without the GTP regenerating system only 4% of the guanylate c y c l a s e a c t i v i t y present was detected. The modified i n c u b a t i o n mixture a l s o contained 2-mercaptoethanol to prevent spont-aneous a i r o x i d a t i o n of the s o l u b l e guanylate c y c l a s e , as has been reported 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 properties during t e s t i s development Guanylate cyclase a c t i v i t i e s were measured i n the 100,000xg p e l l e t (particulate) f r a c t i o n and in the 100,000xg supernatant (soluble) f r a c t i o n , of trout t e s t i s homogenates from zero time trout, and from trout a f t e r 3, 6, and 10 weeks of twice weekly salmon p i t u i t a r y extract injections (Tables VI and VII). The p a r t i c u l a t e and soluble s p e c i f i c a c t i v i t i e s decreased 6 f o l d and 4 f o l d , respectively, during the 10 weeks of t e s t i c u l a r maturation (Table VI). Both t o t a l part-i c u l a t e and soluble a c t i v i t i e s decreased 4 f o l d over the same period (Table VII). The majority of the decreases i n both s p e c i f i c and t o t a l a c t i v i t i e s , occurred between the t h i r d and s i x t h week of hormone i n j e c t i o n s . During t h i s time, there was a 2 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 a c t i v i t y and a 3 f o l d decrease i n soluble s p e c i f i c a c t i v i t y (Table VI), and an approximate 3 f o l d decrease i n both t o t a l p a r t i c u l a t e and soluble a c t i v i t i e s (Table VII). Not enough testes were sampled i n the present guanylate cyclase study to correlate the enzyme decreases with a precise stage of spermatogenesis. A guanylate cyclase p a r t i c u l a t e to soluble r a t i o of 1.9 was observed for s p e c i f i c and t o t a l a c t i v i t i e s in zero time t e s t i s and i n f u l l y mature t e s t i s (Tables VI and VII). The t o t a l p a r t i c u l a t e guanylate cyclase a c t i v i t y appeared to increase p r i o r to meiosis (week 3 of hormonal induction; Table VII), r e s u l t i n g i n a p a r t i c u l a t e to soluble r a t i o of 2.9 at t h i s stage of development. Triton X-100, a non-ionic detergent, has been TABLE VI GUANYLATE CYCLASE SPECIFIC' ACTIVITIES IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS Week of Total Testes Guanylate Cyclase A c t i v i t i e s Hormonal Wet Weight 100,000xg 100,000xg Induction (mg) Supernatant P e l l e t 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 of trout t e s t i s were prepared i n buffer and 100,000xg supernatant and p e l l e t fractions obtained, a described i n Materials and Methods. A c t i v i t y assays were at 37°, i n standard incubation mixtures at pH 7.5, as described i n Materials and Methods. A c t i v i t y units are pmol c y c l i c GMP formed/min/mg protein. Values are the average of duplicate assays on the same preparation. The - value indicates the range between assay duplicates. TABLE VII TOTAL GUANYLATE CYCLASE1 ACTIVITIES IN TROUT TESTIS DURING HORMONALLY-INDUCED SPERMATOGENESIS Week of Total Guanylate Cyclase A c t i v i t i e s Hormonal 100,000xg 100,000xg Totaia Induction Supernatant P e l l e t 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 for Table VI, except that a c t i v i t y units are pmol c y c l i c GMP formed/min/mg t e s t i s wet weight. Total obtained by the summation of the 100,000xg p e l l e t and supernatant a c t i v i t i e s . Total homogenate a c t i v i t y was not assayed. shown to activate guanylate cyclase a c t i v i t i e s i n other tissues (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 fractions. I t was investigated with regard to i t s e f f e c t on trout t e s t i s guanylate cyclase a c t i v i t i e s during spermatogenesis (Figure 17A). Triton X-100 (1% concentration) stimulated p a r t i c u l a t e guanylate cyclase a c t i v i t i e s , at a l l stages of trout t e s t i s development, approximately 2 f o l d (Figure 17A). The soluble guanylate cyclase a c t i v i t y , i n zero time t e s t i s , was also stimulated by Triton X-100 (Figure 17B). The l a t t e r stimulation was 1.8 f o l d , compared with 2.2 f o l d stimulation, 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 i n zero time t e s t i s . The e f f e c t of a gonadotropin extract on guanylate cyclase a c t i v i t i e s , i n zero time t e s t i s , was also investigated (Figure 17B). (The gonadotropin extract used was the standard salmon p i t u i t a r y preparation used i n the i n i t i a t i o n and enhancement of trout spermatogenesis, as described i n Materials and Methods.) The gonadotropin extract caused an apparent 10 f o l d stimulation of the soluble zero time a c t i v i t y and an apparent 2 f o l d stimulation of the p a r t i c u l a t e zero time a c t i v i t y . Addition of both Triton X-100 and gonadotropin extract, to the p a r t i c u l a t e f r a c t i o n resulted i n a s l i g h t l y less than additive increase i . e . 3.2 f o l d stimulation (Figure 17B). No stimulation of a c t i v i t y occurred with the addition of heat-denatured gonadotropin extract (data not shown). However, when the gonadotropin extract was assayed without any t e s t i s protein present, a 95. FIGURE 17 A. T r i t o n X-100 e f f e c t on guanylate cyclase 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 of zero time trout (OP), and from t e s t i s of trout 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 described i n Materials and Methods. Total testes weights and tissue preparation as described i n the legend for Table VI. A c t i v i t i e s were assayed at 37°, i n standard incubation mixtures, as described i n Materials and Methods. Error bars indicate the range between assay duplicates. • No additions W$ Plus 1% Tr i t o n X-100 (x) B. Comparison of the effects of 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 trout t e s t i s guanylate cyclase a c t i v i t i e s . A c t i v i t i e s were assayed as described above. Symbols as above, plus: fZ3 Plus salmon gonadotropin (h) (15 y l crude p i t u i t a r y extract) Tri t o n X-100 (1%) and salmon gonadotropin (hx) cons ide r ab l e endogenous guanyla te c y c l a s e a c t i v i t y was observed; 4.23 pmol/min/15 y l of gonadotropin e x t r a c t , or 11.3 pmol/min/mg gonadot ropin e x t r a c t p r o t e i n . S ince the uns t imula ted s o l u b l e guanyla te c y c l a s e a c t i v i t y was lower than the uns t imula t ed p a r t i c u l a t e a c t i v i t y , the same a d d i t i o n of gonadotropin e x t r a c t to each, produced a g rea te r apparent s t i m u l a t i o n of the s o l u b l e a c t i v i t y . Both inc reases i n 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 to the endogenous guanyla te c y c l a s e a c t i v i t y i n the gonadotropin e x t r a c t . 98. DISCUSSION Cy c l i c GMP and c y c l i c AMP concentrations i n trout t e s t i s  during spermatogenesis C y c l i c GMP concentrations i n immature trout testes were revealed to be high (about 2 ymol/kg t e s t i s wet weight) and equal to c y c l i c 7AMP concentrations in immature trout testes. In most vertebrate tissues c y c l i c GMP concentrations are i n the range of 0.05-0.1 ymol/kg wet weight (53) and c y c l i c AMP concentrations are about 10-50 f o l d higher (64). In several tissues, including lung, cerebellum and lymph, c y c l i c GMP and c y c l i c AMP concentrations are approximately equal and i n the normal c y c l i c AMP concentration range (53, 64). In one vertebrate tissue, namely the r e t i n a , extremely high c y c l i c GMP concentrations are observed (about 100 ymol/kg wet weight) and there i s a c y c l i c GMP to c y c l i c AMP r a t i o of 100 (141). In one invertebrate tissue, namely the male c r i c k e t reprod-uctive accessory gland, very high c y c l i c GMP concentrations are also found, 40-100 ymol/kg wet weight, while c y c l i c AMP concentrations are only 0.02-0.3 ymol/kg wet weight (130). The high c y c l i c GMP concentrations i n the l a t t e r case were shown to be associated with the gland i t s e l f and not the sperm i t contains, since high c y c l i c GMP was also observed i n the reproductive glands from castrated crickets (130). In immature trout t e s t i s i t has been estimated that spermatogonial germ c e l l s make up about 10-15% of the t o t a l wet weight and the remainder i s due to connective tissues and the c e l l s within t h i s component such as Leydig c e l l homo-logues and blood and nerve c e l l s (10). One cannot conclude which of these c e l l types may have an es p e c i a l l y high 99. concentration of one c y c l i c nucleotide. , c y c l i c GMP or c y c l i c AMP, or both. However, over a period of a 5-10 f o l d i nc-rease i n t e s t i c u l a r wet weight, during which time the germ c e l l component would be increasing r a p i d l y , c y c l i c GMP concentrations remained high (Figure 4). Therefore, i t i s quite possible that elevated c y c l i c GMP concentrations are c h a r a c t e r i s t i c of spermatogonia, as well as perhaps other c e l l types present i n the immature trout t e s t i s . Several l i n e s of research have suggested that c y c l i c GMP may act as a p o s i t i v e signal (7, 8) while c y c l i c AMP may act as a negative signal (131, 132) i n the c e l l for control-l i n g the growth of cultured f i b r o b l a s t s . Reciprocal conc-entrations of c y c l i c GMP and c y c l i c AMP have been observed during the c e l l cycle of Novikoff hepatoma c e l l s (95). Elevated c y c l i c GMP during mitosis and S phase were considered to be consistent with p o t e n t i a l modulatory roles for c y c l i c GMP i n p r o l i f e r a t i o n (95). There was no evidence for r e c i p r o c a l c y c l i c GMP and c y c l i c AMP concentrations i n trout t e s t i s during development. In f a c t i n immature t e s t i s the concentrations were equal. However, the heterogeneity of c e l l types present i n immature trout t e s t i s prevents the observation of r e l a t i v e c y c l i c nucleotide concentrations i n s p e c i f i c c e l l types. Goldberg states i n the Yin Yang hypothesis of c y c l i c nucleotide regulation, that the r e l a t i v e proportions of c e l l u l a r c y c l i c GMP and c y c l i c AMP may be more important than t h e i r actual concentrations (5 3). The Yin Yang hypothesis, based on the ancient o r i e n t a l concept of a dualism between opposing natural forces which may enter into 100. a mutual in t e r a c t i o n that r e s u l t s i n synthesis, was used to explain the s t r i k i n g l y antagonistic regulatory influences of c y c l i c GMP and c y c l i c AMP i n several b i o l o g i c a l systems-. (53, 133, 134). In more .recent research (54) the roles of both c y c l i c GMP and c y c l i c AMP i n regulation i n various c e l l types has become a subject of considerable controversy. There i s growing evidence that the c y c l i c nucleotides do not act alone as modulatory effectors but may act i n concert with 2+ . . modulators such as Ca and l i p i d s to promote or i n h i b i t a c e l l u l a r event (54). The most s t r i k i n g aspect of the present study of 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 GMP afte r about 4 weeks.(Figure 4). This decrease occurs at the middle of the logarithmic growth of the trout t e s t i s (Figure 3) shortly before the onset of meiosis at weeks 5 to 6 (10). Although there i s uncertainty about the exact timing of meiosis i n the present study, i t i s obvious from Figure 4 that the decrease i n c y c l i c GMP occurred before the p r o l i f e r -ation of germ c e l l s had ceased. The magnitude and timing of the decrease i n c y c l i c GMP suggest that i t may be c r i t i c a l for the onset of meiosis. To substantiate th i s hypothesis i t would be important to i s o l a t e s p e c i f i c c e l l types rapi d l y to determine whether spermatogonia have s i g n i f i c a n t l y greater concentrations of c y c l i c GMP than primary spermatocytes. In r a t t e s t i s , the binding of immunofluorescent antibodies to c y c l i c GMP to prophase chromosomes i n primary spermatocytes(79) i s consistent with an important role for c y c l i c GMP at meiosis. 101. After the sharp 10 f o l d decrease i n c y c l i c GMP i n trout t e s t i s at the onset of meiosis, c y c l i c GMP decreased gradu-a l l y another 5 f o l d during the remainder of spermatogenesis (Figure 4). After the 2 f o l d decrease i n c y c l i c AMP i n trout t e s t i s p r i o r to meiosis (Figure 5), c y c l i c AMP did not change s i g n i f i c a n t l y during the following stages of meiotic reduction and spermatid d i f f e r e n t i a t i o n . The c y c l i c nucleotide concentrations i n developing trout t e s t i s can be compared with those i n r a t t e s t i s during matur-ation (79). Elevated i n i t i a l concentrations of both c y c l i c GMP and c y c l i c AMP were also 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 concentration was 40 f o l d higher than the i n i t i a l c y c l i c GMP concentration. Both c y c l i c nucleo-tides decreased about 2 f o l d at the time of the f i r s t reduc-t i v e d i v i s i o n s i n rat t e s t i s . The p a r t i c u l a r l y elevated c y c l i c GMP concentrations observed i n trout t e s t i s during early development and the s t r i k i n g decrease i n these concent-rations at the onset of meiosis appear to be sp e c i a l to th i s system. The large 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 development i n the trout t e s t i s may contibute to these observations. During the remainder of r a t t e s t i c u l a r development,'fluctuations i n c y c l i c GMP were seen, but there was an o v e r a l l decrease i n concent-r a t i o n . There was a larger and: clear trend of decrease i n c y c l i c GMP i n trout t e s t i s during spermatid d i f f e r e n t i a t i o n , which may r e s u l t from the homogeneity of germ c e l l type present i n trout t e s t i s . There-is much greater germ 102. c e l l heterogeneity i n rat testis„(1, 10). C y c l i c AMP decreased steadily i n r a t t e s t i s u n t i l the time of the appearance of spermatids (79). During spermatid d i f f e r -e n t i a t i o n the c y c l i c AMP concentration increased to about half of the immature r a t t e s t i s concentration. C y c l i c AMP did not increase i n trout t e s t i s during spermatid d i f f e r e n t i a t i o n , suggesting that t h i s c y c l i c nucleotide may play a more important r o l e i n spermatids of mammals than o f f i s h . I t would be desirable to combine c y c l i c nucleotide de-terminations i n trout t e s t i s , with immunofluorescent c y c l i c nucleotide binding studies and c e l l separation methods, to determine the predominant s i t e s of the c y c l i c nucleotides and t h e i r receptor proteins. The observation of the large and abrupt decrease i n c y c l i c GMP early i n the development of trout t e s t i s , i n i t -iated an investigation into some of the factors which may be important i n the control of c e l l u l a r c y c l i c GMP concent-rations. In p a r t i c u l a r c y c l i c GMP phosphodiesterase a c t i v -i t i e s and guanylate cyclase a c t i v i t i e s i n developing trout t e s t i s were studied. Extrusion of c y c l i c nucleotides i s another p o t e n t i a l l y important method of c o n t r o l l i n g c y c l i c nucleotide concentrations i n trout t e s t i s , but t h i s was not investigated i n the present research. 103. C y c l i c nucleotide phosphodiesterase a c t i v i t i e s i n trout  t e s t i s during spermatogenesis The fa c t that the t o t a l c y c l i c GMP phosphodiesterase a c t i v -i t y i n trout t e s t i s did not change s i g n i f i c a n t l y throughout spermatogenesis (Table III) indicated no obvious r e l a t i o n between c y c l i c GMP phosphodiesterase a c t i v i t y and c y c l i c GMP concentration. This conclusion was confirmed by the studies on c y c l i c GMP phosphodiesterases measured with micromolar substrate concentration during development ' (Tables IV and V) and the p r o f i l e s of phosphodiesterase a c t i v i t y on DEAE-ce l l u l o s e (Figure 11). These studies showed that there was no induction of a high a f f i n i t y c y c l i c GMP phosphodiesterase at the time of the large decrease i n c y c l i c GMP concentration. EGTA in 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-esterase a c t i v i t i e s i n trout t e s t i s from 10 to 50% at d i f f e r -ent stages of development, except for a 30% stimulation of c y c l i c GMP phosphodiesterases i n immature t e s t i s (Table I I I ) . . . . . ". 2+ The i n h i b i t i o n by EGTA indicates the presence of an active Ca binding activator of c y c l i c nucleotide phosphodiesterase . . . 2+ . a c t i v i t i e s i n trout t e s t i s . A Ca -binding protein capable of a c t i v a t i n g c y c l i c nucleotide phosphodiesterases has been iso l a t e d from sea urchin sperm (133) but t h i s protein a c t i v -ator had no e f f e c t on endogenous sea urchin sperm c y c l i c nucleotide phosphodiesterases i s o l a t e d on DEAE-cellulose. 2 + I t i s apparent i n several tissues that the Ca -binding prot-ein may be involved with other metabolic functions, such as 104. protein phosphorylation (134), besides phosphodiesterase 2+ a c t i v a t i o n . The p o s s i b i l i t y of the induction of a Ca dependent high a f f i n i t y c y c l i c GMP phosphodiesterase a c t i v i t y associated with the large decrease i n c y c l i c GMP i n devel-oping trout t e s t i s was investigated (Table V). No change 2+ . . . i n the Ca -dependent c y c l i c GMP phosphodiesterase a c t i v i t y was found at the" onset of meiosis.. 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 at micromolar substrate concentrations were higher i n immature trout t e s t i s than i n t e s t i s at the spermatocyte stage of development (Figure 10B). In a study of changes i n c y c l i c AMP and c y c l i c GMP phosphodiesterase a c t i v i t e s i n ra t l i v e r and lung tissues, higher a c t i v i t i e s were found i n f e t a l than i n neonatal tissues, and the a c t i v i t i e s i n neonatal tissues were i n turn higher than those i n adult tissues (135). A reverse pattern was observed i n developing brain (135). In a l l these tissues the magnitude of c y c l i c GMP phosphodi-esterase a c t i v i t i e s at d i f f e r e n t stages of development, correlated well with the magnitude of cyclic.GMP protein kinase a c t i v i t i e s (136). On the basis of high c y c l i c GMP phosphodiesterase and protein kinase a c t i v i t i e s i n f e t a l lung and adult brain, i t was hypothesized that actions mod-ulated by c y c l i c GMP were important i n these tissues (13 5). An investigation of c y c l i c GMP protein kinase a c t i v i t i e s i n trout t e s t i s c e l l s during development might reveal the s i t e of action of c y c l i c GMP at the onset of meiosis. 105. The most s t r i k i n g change i n c y c l i c nucleotide phosphodi-esterase a c t i v i t i e s i n developing trout t e s t i s i s the induc-t i o n of a c y c l i c AMP phosphodiesterase with high substrate a f f i n i t y , which appears at about week 5 of spermatogenesis (Table IV and Figures 10B, 11C and 11D). A high a f f i n i t y c y c l i c AMP phosphodiesterase a c t i v i t y associated with the onset of maturity has been found i n rat and rabbit t e s t i s (93) and i n ram sperm (94). DEAE-cellulose chromatography of trout t e s t i s homogenates at d i f f e r e n t stages of development i l l u s t r a t e d two peaks of c y c l i c AMP phosphodiesterase a c t i v i t y , the f i r s t of which also hydrolysed c y c l i c GMP (Figure 11). During spermatid development the induced high a f f i n t y c y c l i c AMP phosphodi-esterase a c t i v i t y was observed to coelute with the f i r s t of the c y c l i c AMP peaks (Figures 11C and 11D). I t i s notable that there was no co-induction of a high a f f i n i t y c y c l i c GMP phosphodiesterase a c t i v i t y . Kinetic analyses of DEAE-cellulose peak c y c l i c AMP phosphodiesterase a c t i v -i t i e s from mature trout t e s t i s homogenate (Figure 11D) confirmed the presence of high a f f i n i t y a c t i v i t i e s i n both peaks (Figures 15 and 16). The non-linearity of these k i n e t i c plots may r e s u l t from multiple enzyme species i n one peak, from negative co-operativity of a single enzyme species, or from interconvertible forms of phosphodiesterase with d i f f e r i n g a c t i v i t i e s (60, 90). Although ho l o w - a f f i n i t y c y c l i c AMP phosphodiesterase was observed i n mature trout t e s t i s (Figure 8), such an 106. a c t i v i t y i s obviously present 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 at least 4 0 f o l d higher when measured with millimolar c y c l i c AMP than with micromolar substrate (Tables III and IV). There may 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 phosphodiesterase a c t i v i t y i n mature t e s t i s , where a c t i v i t i e s measured with millimolar c y c l i c AMP were only about 2 0% higher than those measured with micromolar substrate (Tables III and IV). This indicates that the induced high a f f i n i t y c y c l i c AMP phosphodiesterase i s the predominant c y c l i c AMP phosphodi-esterase a c t i v i t y present i n mature t e s t i s . Both a low and high a f f i n i t y c y c l i c GMP phosphodiesterase a c t i v i t y were observed i n mature trout t e s t i s (Figure 9). C y c l i c GMP phosphodiesterase a c t i v i t i e s throughout develop-ment were about 4 0 f o l d higher when measured with millimolar c y c l i c GMP, than when measured with micromolar c y c l i c GMP (Tables III and IV). This indicates that 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 . Soluble .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 at micromolar substrate concentrat-ions were about 85% and 80%, respectively, of 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 f r a c t i o n from trout t e s t i s homogenate contained both peaks of c y c l i c nucleotide phosphodiesterase a c t i v i t y eluted from DEAE-c e l l u l o s e (Figure 13A). The 2.5 f o l d increase i n the r a t i o 107. o f P e a k I I t o P e a k I c y c l i c TAMP a c t i v i t y i n t h e s o l u b l e p r o f i l e a s c o m p a r e d w i t h t h e same r a t i o i n t h e t o t a l homog-e n a t e p r o f i l e f r o m t h e same t e s t i s ( F i g u r e 11B), may r e f l e c t p a r t i a l c o n v e r s i o n o f P e a k I t o P e a k I I o n s u p e r n a t a n t p r e p -a r a t i o n o r s t o r a g e a t -20°. S u c h a n i n c r e a s e i n t h e r a t i o o f P e a k I I t o P e a k I 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 y o n D E A E - c e l l u l o s e p r o f i l e s h a s b e e n o b s e r v e d i n r a t l i v e r p r e p a r a t i o n s a f t e r s t o r a g e o f h o m o g e n a t e s a t 4° o r a f t e r m i l d t r e a t m e n t w i t h t r y p s i n ( 6 0 ) . The 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 p h o s p h o d i -e s t e r a s e a c t i v i t y i n t h e 1 0 0 , 0 0 0 x g p e l l e t f r a c t i o n f r o m t r o u t t e s t i s h o m o g e n a t e , c o n t a i n e d m a i n l y a c t i v i t y i n t h e s e c o n d p e a k e l u t e d f r o m D E A E - c e l l u l o s e ( F i g u r e 1 3 B ) . I n f r a c t i o n a t i o n s t u d i e s o f r a t l i v e r p h o s p h o d i e s t e r a s e s (60) t h e s e c o n d c y c l i c AMP p h o s p h o d i e s t e r a s e p e a k o n DEAE-c e l l u l o s e p r o f i l e s was i s o l a t e d e x c l u s i v e l y f r o m t h e 1 0 0 , 0 0 0 x g p e l l e t f r a c t i o n . D E A E - c e l l u l o s e p r o f i l e s o f m a l e c r i c k e t r e p r o d u c t i v e a c c e s s o r y g l a n d h o m o g e n a t e s w e r e s i m i l a r t o t h o s e o b s e r v e d i n t h e p r e s e n t s t u d y on t r o u t t e s t i s 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 s , i . e . a f i r s t p e a k h y d r o l y z i n g b o t h c y c l i c AMP a n d c y c l i c GMP and a s e c o n d p e a k s p e c i f i c f o r c y c l i c AMP ( 1 3 7 ) . The o b s e r v a t i o n o f no p h o s p h o d i e s t e r a s e w i t h d i s t i n c t s p e c i f i c i t y f o r c y c l i c GMP, i n t h e t r o u t t e s t i s , e v e n when c y c l i c GMP c o n c e n t r a t i o n s w e r e h i g h i s a l s o o b s e r v e d i n t h e r e p r o d u c t i v e a c c e s s o r y g l a n d o f m a l e c r i c k e t s . 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 a c t i v i t y o r k i n e t i c 108. properties of accessory gland c y c l i c GMP phosphodiesterases were seen during a developmental period over which c y c l i c GMP concentrations rose more than 500 f o l d (137). The c y c l i c nucleotide phosphodiesterase studies i n trout t e s t i s do not indicate a s e l e c t i v e role for phosphodiesterases i n modulating c y c l i c GMP concentrations during spermatogenesis. Guanylate cyclase a c t i v i t i e s i n trout t e s t i s during  spe rmatogene s i s Guanylate cyclase a c t i v i t i e s i n trout t e s t i s were invest-igated to determine t h e i r role i n regulating c y c l i c GMP conc-entrations during spermatogenesis. A d i r e c t c o r r e l a t i o n was found between guanylate cyclase a c t i v i t i e s and c y c l i c GMP con-centrations i n developing trout t e s t i s , as i s shown i n Figure 18. Surveys of guanylate eye lase a c t i v i t i e s i n r a t tissues have demonstrated a c o r r e l a t i o n between guanylate cyclase a c t i v i t y and c y c l i c GMP concentration (82, 138, 139). In immature and mature trout t e s t i s , a p a r t i c u l a t e to soluble guanylate cyclase a c t i v i t y r a t i o of 1.9 was observed (Tables VI and VII). A s i m i l a r p a r t i c u l a t e to soluble r a t i o was observed i n a study of guanylate cyclase a c t i v i t i e s i n immature and adult rat t e s t i s (82). Both p a r t i c u l a t e and soluble guanylate cyclase s p e c i f i c a c t i v i t i e s decreased during"trout t e s t i s maturation (Table VI), but the p a r t i c -ulate enzyme showed a t o t a l a c t i v i t y increase just p r i o r to meiosis (Table VII). This could be related to a s p e c i f i c role for c y c l i c GMP i n the nucleus at meiosis. However, a more thorough investigation of guanylate cyclase a c t i v i t i e s during spermatogenesis i s needed to substantiate t h i s idea. FIGURE 18 Comparison of guanylate cyclase a c t i v i t i e s and c y c l i c  GMP concentrations i n trout t e s t i s during hormonally- induced spermatogenesis. Guanylate cyclase was assayed, i n trout t e s t i s at d i f f e r -ent stages of development, as described i n Materials and Methods. Data from Table VI. Units of a c t i v i t y are pmol c y c l i c GMP formed/min/mg protein. C y c l i c GMP con-centrations assayed, i n trout t e s t i s at d i f f e r e n t stages of development, as described i n Materials and Methods. Data from Table IA. 110. Spermatogonia Spermatocytes Spermatids 2 4 6 8 10 Week of Hormonal Induction 111. T r i t o n X-100 (a non-ionic detergent) activated p a r t i c u l a t e guanylate cyclase a c t i v i t i e s i n trout t e s t i s , at 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. Soluble guanylate cyclase a c t i v i t y i n immature trout t e s t i s was activated by T r i t o n X-100 to the same extent as the p a r t i c u l a t e enzyme. This suggests that there may be a close r e l a t i o n i n form between the soluble and p a r t i c u l a t e enzyme. I t has been suggested by Goldberg and others that the soluble form of guanylate cyclase may originate from c e l l membranes (54). Discussion on the importance of the two forms of guanylate cyclase, i n mamm-al i a n tissues, has stressed the possible p h y s i o l o g i c a l regulation of the two forms (140). Increased p a r t i c u l a t e and decreased soluble guanylate cyclase a c t i v i t i e s have been found i n regenerating r a t l i v e r , f e t a l r at l i v e r , and hepa-tomas (140). These observations led to the speculation that p a r t i c u l a t e guanylate cyclase a c t i v i t y may be associated with tissue growth and that soluble a c t i v i t y may be assoc-iated with acquired functions of d i f f e r e n t i a t i o n . The 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 soluble guanyl-ate cyclase a c t i v i t y during trout t e s t i s development does not support t h i s 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 cyclase a c t i v i t y has been convincingly demonstrated. The finding of an apparent stimulation of both p a r t i c u l a t e and soluble guanylate cyclase a c t i v i t i e s i n immature trout 112. t e s t i s , by a salmon p i t u i t a r y gonadotropin extract (Figure 16), was shown to be due to endogenous a c t i v i t y i n the hormone preparation. The soluble t e s t i s guanylate cyclase, presum-ably not the physiological form which would respond to gonadotropin stimulation, was apparently stimulated to a greater extent than the p a r t i c u l a t e enzyme. Hormone stim-ul a t i o n of guanylate cyclases i n other tissues has been reported to be suspect due to the impurities of the prepar-ations used (54). In these cases also, the soluble guanylate cyclase was apparently stimulated to a greater extent than the p a r t i c u l a t e enzyme. Conclusions This study of trout spermatogenesis has revealed that during t e s t i s development there are three s t r i k i n g and pr e c i s e l y timed changes related to c y c l i c nucleotide meta-bolism. These are a decrease i n c y c l i c GMP concentration, a decrease i n guanylate cyclase a c t i v i t i e s and the induct-ion of a high a f f i n i t y c y c l i c AMP phosphodiesterase. It i s reasonable to assume that there i s a connection between the decrease in c y c l i c GMP concentration, the decrease i n guanylate cyclase a c t i v i t y and the onset of meiosis (Figure 18). C l e a r l y further studies should be directed towards the i d e n t i f i c a t i o n of the c e l l s i n which the decrease i n c y c l i c GMP concentration occurs and an investigation of the guanylate cyclase a c t i v i t i e s i n those c e l l s . If these experiments confirm the presumed 113. r e l a t i o n s h i p , then i t w i l l be of great i n t e r e s t to define the nature of the decreased a c t i v i t y of the guanylate cyclases, and the source and nature of the signal which induces the change. Also of importance w i l l be an investigation of the target of the c y c l i c GMP i n the c e l l s where the concentration changes. A detailed study of the c y c l i c GMP-binding proteins and c y c l i c GMP-dependent protein kinase a c t i v i t i e s i n trout t e s t i s during spermatogenesis may reveal exciting r e s u l t s . The induction of 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 to the developing trout t e s t i s ( 9 3 , 94). I d e n t i f i c a t i o n of^ and studies on, the c e l l type i n trout t e s t i s i n which the induction takes place could provide relevant insights into c y c l i c nucleotide reg-u l a t i o n during spermatogenesis. Investigations of thi s type, should be possible i n trout t e s t i s , but i t may be that studies on the mammalian t e s t i s , with better defined endocrinology and cytology may be more f r u i t f u l at t h i s time; i d e a l l y , d e t a i l e d studies on both systems should be c a r r i e d out. I t seems clear from t h i s introductory study that a detailed study of the involvement of c y c l i c nucleotides i n t e s t i s development w i l l provide both surprising and inte r e s t i n g r e s u l t s . 114. REFERENCES 1. Courot, M., Hocereau-de r e v i e r s , M., and Ortavant,R. (1970) In: The T e s t i s . V o l . I. E d i t e d by A.D. Johnson, W.R. Gomes and N.L. Vandemark. pp.339-432. Academic P r e s s , N.Y. 2. H e i n d e l , J . J . , Rothenberg, R., Robison,G.A., and S t e i n -b e rger, A. (1975) J . C y c l i c N u c l . Res., 1 : 69-79 3. D e s j a r d i n s , C., Z e l e z n i k , A.J., Midgley, A.R.Jr., and R e i c h e r t , L . E . J r . (1974) In: Hormone B i n d i n g and Target C e l l A c t i v a t i o n i n T e s t i s . E d i t e d by M.L. Dufau and A.R. Means, pp.221-235. Plenum, N.Y. 4. D o r r i n g t o n , J.H., and F r i t z , I.B. (1974) E n d o c r i n o l o g y , 94 : 395-403 5. Means, A.R., Fakunding, J.L., and T i n d a l l , D.J. (1976) B i o l . Reprod., 14 : 54-63 6. Moyle, W.R., and Ramachandran, J . (1973) E n d o c r i n o l o g y , 93 : 127-134 7. Hadden, J.W., Hadden, E.M., Haddox, M.K., and Goldberg, N.D. (1972) Proc. Nat. Acad. S c i . USA, 69 : 3024-3027 8. S e i f e r t , W., and Rudland, P.S. (1974) Nature, 248 : 138-140 9. Johnson, L.D., and Hadden, J.W. (1975) Biochem. Biophys. Res. Commun., 66 : 1498-1505 10. L o u i e , A.J. (1972) Ph.D. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia 11. Fawcett, D.W. (1972) In: Ge n e t i c s of the Spermatozoan. E d i t e d by R.A. Beatty and S. Gluecksohn-Waelsch pp.37-68 U 5. Bogtrykkeriet Forum, Copenhagen 12. C h e v a i l l i e r , P.H., and P h i l l i p p e , M. (1976) Exp. C e l l Res., 99 : 237-244 13. Kierszenbaum, A.L., and Tres, L.L. (1975) J. C e l l B i o l . , 65 : 258-270 14. Blackshaw, A.W., and Elkington, J.S.H. (1970) B i o l . Reprod., 2 : 268-274 15. Turkington, R.W., and Majumder, G.C. (1975) J. C e l l Physiol., 85 : 495-508 16. Hoar, W.S. (1965) In: Annu. Rev. Physiol., Vol. 27 : pp51-70 17. Hoar, W.S. (1967) In: Fish Physiology, Vol. 3. Edited by W.S. Hoar and D.J. Randall, pp.1-58. Academic Press, N.Y. 18. Henderson, N.E. (1962) Can. J. Zool., 40 : 631-641 19. Lofts, B. (1968) In: Perspectives i n Endocrinology. Edited by E.J.W. Barrington, J. Barker and C. JjzSrgenson pp.239-304. Academic Press, N.Y. 20. Courot, M. (1967) J. Reprod. F e r t i l i t y , Suppl. 2 : 99 21. French, F.S., and Ritzen, E.M. (1973) J. Reprod. F e r t i l i t y , 32 : 479-483 22. Lacy, D. (1962) B r i t i s h medical B u l l e t i n , 18 : 205-208 23. Steinberger, E., and Wagner, C. (1961) Endocrinology, 69 : 305 24. Barr. W.A. (1963) Gen. Comp. Endocrinol., 3 : 216-225 25. Fevold, H.L., Hisaw, F.L., and Leonard, S.L. (1931) Am. J. Physiol., 97 : 291 11.6. 26. Burzawa-Gerard, E. (1971) Biochimie, 53 : 542-552 27. Donaldson, E.M., Yamazaki, F., Dye, H.M., and P h i l l e o , W.W. (1972) Gen. Comp. Endocrinol., 18 : 469-481 28. Henderson, N.E. (1963) J. Fisheries Res. Board Can., 20 : 859 29. Schmidt, P.J., M i t c h e l l , B.S., Smith, M., and Tsuyuki,H. (1965) Gen. Comp. Endocrinol., 5 : 197-206 30. Ingles, C.J., and Dixon, G.H. (1967) Proc. Nat. Acad. S c i . USA, 58 : 1011-1018 31. Drance, M.G., Hollenberg, M.J., Smith, M., and Wylie,V. (1976) Can. J. Zool., 54 : 1285-1293 32. Louie, A.J., and Dixon, G.H. (1972) J. B i o l . Chem., 247 : 5490-5497 33. Louie, A.J., and Dixon, G.H. (1972) J. B i o l . Chem., 247 : 5498-5505 34. Candido, E.P.M., and Dixon, G.H. (1972) J. B i o l . Chem., 247 : 5506-5510 35. Louie, A.J., and Dixon, G.H. (1972) J. B i o l . Chem., 7962-7968 36. Donaldson, E.M., Funk, J.D., Withler, F.C., and Morley, R.B. (1972) J. Fisheries Res. Board Can., 29 : 13-18 37. Ingles, C.J., Trevithick, J.R., Smith, M., and Dixon, G.H. (1966) Biochem. Biophys. Res. Commun., 22 : 627-634 38. J e r g i l , B., and Dixon, G.H. (1970) J. B i o l . Chem., 245 : 425-434 39. Greengard, P. (1978) Science, 199 : 146-152 40. R a i l , T.W., Sutherland, E.W., and Berthet, J. (1957) J. B i o l . Chem., 232 : 463-475 41. Sutherland, E.W., and R a i l , T.W. (1958) J. B i o l . Chem., 233 : 1077-1091 42. Sutherland, E.W., R a i l , T.W., and Menon, T. (1962) J. B i o l . Chem., 237 : 1220-1227 43. R a i l , T.W., and Sutherland, E.W. (1962) J. B i o l . Chem., 237 : 1228-1232 44. Walsh, D.A., Perkins, J.P., and Krebs, E.G. (1968) J. B i o l . Chem., 243 : 3763-3765 45. Miyamoto, E., Kuo, J.F., and Greengard, P. (1969) J. B i o l . Chem., 244 : 6395-6402 46. Corbin, J.D., and Krebs, E.G. (1969) Biochem. Biophys. Res. Commun., 36 : 328-336 47. Robison, G.A., Butcher, R.W., and Sutherland, E.W. (1968) In: Annu. Rev. Biochem., Vol. 37, pp. 149-174 48. Jost, J.P., and Rickenberg, H.V. (1971) In: Annu. Rev. Biochem., Vol. 70, pp. 741-744 49. Perkins, J.P. (1973) In: Adv...in C y c l i c Nucl. Res., Vol. 3. Edited by P. Greengard and G.A. Robison, pp. 1-64. Raven Press, N.Y. 50. Hardman, J.G., Davis, J.W., and Sutherland, E.W. (1966) J. B i o l . Chem., 241 : 4812-4815 51. Steiner, A.L., Parker, C.W., and Kipnis, D.M. (1970) In: Adv. i n Biochemical Psychopharmacology, Vol. 3, Edited by P. Greengard and E. Costa, pp. 89-111. Raven Press, N.Y. 52. Ashman, D.F., Lipton, R., Melicow, M.M., and Price, T.D. (1963) Biochem. Biophys. Res. Commun., 11 : 330-334 53. Goldberg, N.D., O'Dea, R.F., and Haddox, M.K. (1973) In: Adv. i n C y c l i c Nucl. Res., Vol. 3. Edited by P. Greengard and G.A. Robison. pp. 155-22 3. Raven Press, N.Y. 54. Goldberg, N.D., and Haddox, M.K. (1977) In: Annu. Rev. Biochem., Vol. 46, pp. 823-896 55. Matsuzawa, H., and Nirenberg, M. (1975) Proc. Nat. Acad. S c i . USA, 72 : 3472-3476 56. Jimenez de Asua, L., Clingan, D., and Rudland, P.S. (1975) Proc. Nat. Acad. S c i . USA, 72 : 2724-2728 57. Rudland, P.S., Gospodarowicz, D., and S e i f e r t , W.E. (1974) Nature, 250 : 741-742, 773-774 58. White, A.A., and Aurbach, G.D. (1969) Biochim. Biophys. Acta, 191 : 686-697 59. Hardman, J.G., and Sutherland, E.W. (1969) J. B i o l . Chem., 244 : 6363-6370 60. Russell, T.R., Terasaki, W.L., and Appleman, M.M. (1973) J. B i o l . Chem., 248 : 1334-1340 61. Miki, N., Keirns, J.J., Marcus, F.R., Freeman, J., and Bitensky, M.W. (1973) Proc. Nat. Acad. S c i . USA, 70 : 3820-3824 62. Kuo, J.F., and Greengard, P. (1970) J. B i o l . Chem., 245 : 2493-2498 63. Kuo, J.F., Wyatt, G.R., and Greengard, P. (1971) J. B i o l . Chem., 246 : 7159-7167 64. Ishikawa, E., Ishikawa, S., Davis, J.W., and Sutherland, E.W. (1969) J. B i o l . Chem., 244 : 6371-6376 65. Shier, W.T., Baldwin, J.H., Nilsen-Hamilton, M., Ham-i l t o n , R.T., and Thanassi, N.M. (1976) Proc. Nat. Acad. S c i . USA, 73 : 1586-1590 66. Wallach, D., and Pastan, I. (1976) J. B i o l . Chem., 251 : 5802-5809 67. Wallach, D., and Pastan, I. (1976) Biochem. Biophys. Res. Commun., 72 : 859-865 68. DeRubertis, F.R., and Craven, P.A. (1976) Science, 193 : 897-899 69. Kimura, H., M i t t a l , C.K., and Murad, F. (1975) J. B i o l . Chem., 250 : 8016-8022 70. White, A.A., Crawford, K.M., Patt, C.S., and Lad, P.J. (1976) J. B i o l . Chem., 251 : 7304-7312 71. Terasaki, W.L., and Appleman, M.M. (1975) Metabolism, 24 : 311-319 72. Beavo, J.A., Hardman, J.G., and Sutherland, E.W. (1971) J. B i o l . Chem., 246 : 3841-3846 73. Teo, T.S., Wang, T.H., and Wang, J.H. (1973) J. B i o l . . Chem., 248 : 588-596 74. Kakiuchi, S., Yamazaki, R., Teshima, Y., and Uenishi, K. (1973) Proc. Nat. Acad. S c i . USA, 70 : 3526-3530 75. Cheung, W.Y. (1971) J. B i o l . Chem., 246 : 2859-2869 76. G i l l , G.N. (1977) J. C y c l i c Nucl. Res., 3 : 153-162 77. Donnelly, T.E.Jr., Kuo, J.F., Reyes, P.L., L u i , Y., and Greengard, P. (1973) J. B i o l . Chem., 248 : 190-198 78. Cech, S.Y., and Ignarro, L.J. (1977) Science, 198 : 1063-1065 79. S p r u i l l , A., and Steiner, A. (1976) J. C y c l i c Nucl. Res., 2 : 225-239 80. Ong, S.H., Whitley, T.H., Stowe, N.W. , and Steiner, A. (1975) Proc. Nat. Acad. S c i . USA, 72 : 2022-2026 81. Macindoe, J.H., Su l l i v a n , W., and Wray, H.L. (1977) Endocrinology, 101 : 568-576 82. S p r u i l l , W.A., Steiner, A.L., and Earp, H.S. (1977) Fed. Proc., 36 (3) : 347 83. Braun, T., Frank, H., Dods, R., and Sepsenwol, S. (1977) Biochim. Biophys. Acta, 481 : 227-236 84. Braun, T. (1974) In: Current Topics i n Molecular Endocrinology, Vol. 1. Edited by M.L. Dufau and A.R. Means, pp. 2 43-264. Raven Press, N.Y. 85. Catt, K.J., Tsuruhara, T., and Dufau, M.L. (1972) Biochim. Biophys. Acta, 279 : 194-201 86. Braun, T., and Dods, R.F. (1975) Proc. Nat. Acad. S c i . USA, 72 : 1097-1101 87. Yokota, T., and Gots, J.S. (1970) J. B a c t e r i o l . , 103 : 513-516 88. Gray, J.P., Drummond, G.I., and Luk, D.W.T. (1976) Arch. Biochem. Biophys., 172 : 20-30 89. Gray, J.P., and Drummond, G.I. (1976) Arch. Biochem. Biophys., 172 : 31-38 90. Pichard, A., and Cheung, W.Y. (1976) J. B i o l . Chem., 251 : 5726-5737 12 1. 91. Monn, E., Desautal, M., and Christiansen,R.0. (1972) Endocrinology, 91 : 716-720 92. Garbers, P.L., F i r s t , N.L., and Lardy, H.A. (1971) Biochemistry, 10 : 1825-1831 93. Monn, E., and Christiansen, R.O. (1971) Science, . 173 : 540 94. Tash, J.S. (1976) J. Reprod. F e r t i l i t y , 47 : 63-72 95. Z e i l i g , C.E., and Goldberg, N.D. (1977) Proc. Nat. Acad. S c i . USA, 74 : 1052-1056 96. Zatz, M., and O'Dea, R.F. (1977) Science, 197 : 174-176 97. Rosen, O.M. , Erlichman, J. , and Rubin, C S . (1975) In: Adv. i n C y c l i c Nucl. Res., Vol. 5. Edited by G.I. Drummond, P. Greengard and G.A. Robison. pp. 253-279. Raven Press, N.Y. 98. Lee, P.C., Radloff, D., Schweppe, J.J., and Jungman, R. (1976) J. B i o l . Chem., 251 : 914-921 99. Bernard, E.A., and Wassermann, G.F. (1977) Biochem. J., 162 : 465-467 100. Walter, U., Uno, I., L i u , A.Y., and Greengard, P. (1977) J. B i o l . Chem., 252 : 6588-6590 101. Byus, CV. , Klimpel, G.R. , Lucas, D.O., and Russell, D.H. (1977) Nature, 268 : 63-64 102. Resko, J.A., Feder, H.H., and Goy, R.W. (1968) J. Endocrinol., 40 : 485-491 103. Boudreau, R.J. (1976) Ph.D. Thesis, University of Calgary 104. Heppel, L.A. (1967) In: Methods i n Enzymology, Vol. 12 Edited by L. Grossman and K. Moldave. pp. 316-317 12 2. 105. Boudreau, R.J., and Drummond, G.I. (1975) A n a l . Biochem., 63 : 388-399 106. K r i s h n a , G., and K r i s h n a n , N. (1975) J . C y c l i c N u c l . Res., 1 : 293-302 107. Lowry, O.H., Rosebrough, N.J., F a r r , A.L., and R a n d a l l , R.J. (1951) J . B i o l . Chem., 193 : 265-275 108. G i l m a n , A.G. (1970) P r o c . Nat. Acad. S c i . USA, 67 : 305-312 109. G o l d b e r g , N.D., D i e t z , S.B., and O'Toole, A.G. (1969) J . B i o l . Chem., 244 : 4458-4466 110. Kuo, J.F., L ee, T.P., Reyes, P.L., Wa l t o n , K.G., D o n n e l l y , T . E . J r . , and Greengard, P. (1972) J . B i o l . Chem., 247 : 16-21 . L .-111. B r o o k e r , G. (1971) J . B i o l . Chem., 246 : 7810 -7816 112. S t e i n e r , A.L., P a r k e r , C.W., and K i p n i s , D.M. (1972) J . B i o l . Chem., 247 : 1106-1113 113. F i l b u r n , C R . , and K a r n , J . (1973) A n a l . Biochem., 52 : 505-516 114. Bohme, E., and S c h u l t z , G. (1974) I n : Methods i n Enzymology, V o l . 38. E d i t e d by J.G. Hardman and B.W. O'Malley. pp. 27-38. Academic P r e s s , N.Y. 115. Chan, P.S., B l a c k , C.T., and W i l l i a m s , B . J. (1970) Fed. P r o c , Fed. Amer. Soc. Exp. B i o l . , 29 : 616 116. S c h u l t z , G., Bohme, E., and Hardman, J.G. (1974) I n : Methods i n Enzymology, V o l . 38. E d i t e d by J.G. Hardman and B.W. O'Malley. pp. 9-13. Academic P r e s s , N.Y. 117. B r o o k e r , G., Thomas, L . J . J r . , and Appleman, M.M. (1968) B i o c h e m i s t r y , 7 : 4177 12 3. 118. Thompson, W.J., Brooker, G., and Appleman, M.M. (1974) In: Methods i n Enzymology, Vol. 38. Edited by J.G. Hardman and B.W. O'Malley. pp. 205-212. Academic Press 119. Beavo, J.A., Hardman, J.G., and Sutherland, E.W. (1970) J. B i o l . Chem. , 245 : 5649-5655 120. Loten, E.G., and Sneyd, J.G. (1970) Biochem. J . , 120 : 187-193 121. T h e r r i a u l t , D.G., and Winters, V.G. (1970) L i f e S c i . , 9 : 1053-1060 122. Lagarde, A., and Colobert, L. (1972) Biochim. Biophys. Acta, 276 : 444-449 123. Thompson, W.J., and Appleman, M.M. (1971) Biochemistry, 10 : 311-316 124. Cheung, W.Y. (1970) In: Adv. i n Biochemical Psycho-pharmacology. Edited by P. Greengard and E. Costa, Vol.3 p51. Raven Press, N.Y. 125. Butcher, R.W. (1974) In: Methods i n Enzymology, Vol. 38. Edited by J.G Hardman and B.W. O'Malley. pp. 218-223 Academic Press, N.Y. 126. Schultz, G. (1974) In: Methods i n Enzymology, Vol. 38. Edited by J,G. Hardman and B.W: O'Malley. pp. 115-125 Academic Press, N.Y. 127. White, A.A., and Zenser, T.V. (1971) Anal. Biochem., 41 : 372 124. 128. Goldberg, N.D., Dietz, S.B., and O'Toole, A.G. (1969) J. B i o l . Chem., 244: 4458-4466 129. Gray, J.P. (1970) Ph.D. Thesis, Vanderbilt University. 130. Fa l l o n , A., and Wyatt, G.R. (1975) Biochim. Biophys. Acta, 411 : 173-185 131. Otten, J . , Johnson, G.S., and Pastan, I. (1972) J . B i o l . Chem., 247 : 7082- 7087 132. S e i f e r t , W., and Paul, D. (1972) Nature New B i o l . , 240: 281-283 133. Wells, J.N., and Garbers, D.L. (1976) B i o l . Reprod., 15 : 46-53 134. Schulman, H., and Greengard, P. (1978) Nature, 271 : 478-480 135. Davis, C.W., and Kuo, J.F. (1976) Biochim. Biophys. Acta, 444 : 554-562 136. Kuo, J.F. (1975) Proc. Nat. Acad. S c i . USA, 72 : 2256-2259 137. Fa l l o n , A., and Wyatt, G.R. (1977) Biochim. Biophys. Acta, 480 : 428-441 138. Kimura, H., and Murad, F. (1974) J. B i o l . Chem., 249 : 6910-6916 139. Craven, P.A., and DeRubertis, F.R. (1976) Biochemistry, 15 : 5131-5137 140. Kimura/ H., and Murad, F. (1975) Proc. Nat. Acad. S c i . USA, 72 : 1965-1969 141. Krishna, G., Krishnan, N., Fletcher, T., and Chader, G. (1975) In: Adv. i n C y c l i c Nucl. Res., Vol. 5. Edited by G.I. Drummond, P. Greengard and G.A. Robison. p823 (Al>s.) Raven Press, N.Y. 125. APPENDIX Various methods for the separation of c y c l i c nucleotides were investigated during t h i s research (Figure 19). The separation of c y c l i c AMP from c y c l i c GMP, i n trout t e s t i s samples, was routinely performed by elution of AG 1-X8 re s i n columns with formic acid (Figure 19A). Under the conditions detailed i n the legend for Figure 19A, i t was found that 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 of c y c l i c AMP and c y c l i c GMP on DEAE-cellulose columns was also investigated (Figure 19B). While the separation was excellent, t h i s method was too time-consuming for routine 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 , with a li n e a r gradient of 0 - 0.1 M N E U H C O 3 , c y c l i c AMP and c y c l i c GMP separated from 5'AMP (Figure 19B). Cy c l i c AMP, c y c l i c GMP and 5'AMP eluted at 0.02, 0.04, and 0.06 M N H 4 H C O 3 , pH 7.8, respectively. Both c y c l i c AMP and c y c l i c GMP could be eluted by batch e l u t i o n with 0.05 M NHitHCO3, without elu t i o n of 5'AMP. Separation of c y c l i c nucleotides, by high pressure l i q u i d chromatography on P a r t i s i l - 1 0 SAX, was well investigated, (Fig-ure 20)to determine i f t h i s method was sensitive enough to quantitate trout t e s t i s c y c l i c AMP and c y c l i c GMP. Although c y c l i c AMP and c y c l i c GMP separated well on P a r t i s i l - 1 0 SAX, the maximum s e n s i t i v i t y obtained under the operating 12 6. FIGURE 1 9 A. Separation of c y c l i c AMP and c y c l i c GMP on a BioRad AG 1 - X 8 (formate) 2 0 0 - 4 0 0 mesh, re s i n column;. ( 0 . 3 5 x 0 . 7 cm) by e l u t i o n with formic acid. Elution was performed under handpump pressure. Column f r a c t i o n size was 2 ml. [ 3 H ] c y c l i c nucleotides were chromatographed on i d e n t i c a l separate columns and the r e s u l t s graphed as a composite of the two e l u t i o n p r o f i l e s . C y c l i c AMP was eluted with 2 N formic acid ( 1 2 ml; 85% recovery) and c y c l i c GMP was eluted with 5 N formic acid ( 14 ml; 75% recovery). The prewash was with 0 . 1 N formic acid ( 1 0 ml). I I c y c l i c AMP !H c y c l i c GMP B. Separation of c y c l i c AMP, c y c l i c GMP and AMP on a DEAE-ce l l u l o s e (DE 3 2 ) column ( 0 . 5 x 1 0 . 2 cm) by e l u t i o n with a l i n e a r gradient from 0 - 0 . 1 M NHi+HC03, pH 7 . 8 . Column flow rate was 1 4 ml per hr, t o t a l gradient volume was 1 0 0 ml and column f r a c t i o n volume was 2 . 7 ml. Five absorbance units of each compound were applied and t h e i r s p e c i f i c e l u t i o n positions determined by wavelength scans of the peak tubes. Conductivity was measured i n mMHO and con-verted to NH it HCO 3 by comparison with standards. C y c l i c AMP recovery was 9 0 - 1 0 0 % ; c y c l i c GMP recovery was 8 5 -9 0 % . Symbols as above, plus: E3 AMP I 10 20 30 F r a c t i o n N u m b e r FIGURE 20 A. Separation of c y c l i c nucleotides (A,G,C,U) on P a r t i s i l SAX (Reeve-Angel Co., U.S.A). Operating conditions:-Column: PXS - 1025 SAX; 4.6 mm x 25 cm Column Temperature: Ambient Mobile Phase: 0.0 07 M KH 2P0 4, pH 3.2 Flow Rate: 1.1 ml per min Pressure: Pump sett i n g 40; 200 - 840 p s i Detection: 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 total) 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. Separation of c y c l i c nucleotides (A,G,C,U,I) on P a r t i s i l - 1 0 SAX. Operating conditions:-Column: PXS - 1025 SAX; 4.6 x 25 cm Column Temperature: Ambient Mobile Phase: 0.003 M KH 2P0 4, pH 3.2 Flow Rate: 1.1 ml per min Pressure: 200 - 840 p s i Detection: UV A^^nm; 100% s e n s i t i v i t y 0.01 A 2 5 4nm Peaks: a. c y c l i c CMP (0.05 A^^nm total) 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 " conditions used, was 50 pmol/10 y l sample, about 10-100 f o l d higher than the amount of c y c l i c AMP or c y c l i c GMP i n such a sample from trout t e s t i s . Furthermore, c y c l i AMP was shown to overlap with 5'CMP and c y c l i c GMP to overlap with 5'AMP, as shown i n Figures 21A and 21B). Therefore, complete separation of these nucleotides from c y c l i c AMP and c y c l i c GMP would be required before these c y c l i c nucleotides could be quantitated 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 acceptable FIGURE 21 A. Separation of 5 1 CMP, 5'AMP and 5'UMP on P a r t i s i l - 1 0 SAX. Operating conditions:-Column: PXS - 1025 SAX; 4.6 mm x 25 cm Column Temperature: Ambient Mobile Phase: 0.005 M KH 2P0 4, pH 3.2 Flow Rate: 1.1 ml per min Pressure: 200 - 840 p s i Detection: UV A^^nm; 100% s e n s i t i v i t y 0.005 A^^nm Peaks: a. 5 1 CMP (0.13 A 2 5 4nm total) b. 5'AMP (0.05 " ) c. 5'UMP (0.13 " ) B. Separation of 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 conditions as described above. Peaks: a. c y c l i c AMP (0.025 A 2 5 4nm total) b. c y c l i c GMP 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0094622/manifest

Comment

Related Items