ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
172, 20-30 (1976)
Enzymes of Cyclic Nucleotide Metabolism Vertebrate Sperm1
in Invertebrate
and
J. PATRICK GRAY,2 GEORGE I. DRUMMOND,3 DAVID W. T. LUK Department
of Pharmacology,
School of Medicine,
The University V6T 1 w5
of British
Columbia,
Vancouver,
Can&a
AND
JOEL G. HARDMAN, Department
of Physiology,
AND
EARL W. SUTHERLAND4
School of Medicine, Vanderbilt University, Received May 14, 1975
Nashville,
Tennessee 37212
Sperm from several invertebrates contained guanylate cyclase activity severalhundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn2+ was up to several hundred-fold greater than with Mg*+ and was increased 3-lo-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mng+-dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than lo-fold more active with Mn*+ than with Mg*+. Invertebrate sperm contained phosphodiesterase activities against 1.0 pM cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by T&m. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1-l nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive tc stimulation by cyclic AMP than by cyclic GMP.5
It has been reported preliminarily (1) fold greater than the most active mammathat sea urchin sperm possess guanylate lian tissues such as lung (2). In contrast, cyclase activity that is several hundred- human and dog sperm did not contain detectable guanylate cyclase activity (3). All 1This work was surwt~ by grants from the three of these sperm types contained adenNational Institutes of Health (No. GM 16811, AM07462 and HE 08332) and from the Medical Research Council of Canada. 2 Portions of this work were taken from the dissertation submitted by J.P.G. in partial fulfillment of the requirements of the Ph.D. degree, Vanderbilt University, 1971. In Canada, recipient of a Medical Research Council of Canada postdoctoral fellowship. 3 Present address: Biochemistry Group, Department of Chemistry, The University of Calgary, Calgary, Alberta, T2N lN4, Canada. Inquiries should be sent to G.I.D. at this address.
4 Deceased, March 9, 1974. J Abbreviations used: SC 2964, 1-methyl,3-isobutylxanthine; 19:l solution, mixture of 19 volumes of 0.5 M NaCl and 1 volume of 0.5 M KCl; GG-Na-KEDTA-D’M’, 2 mM glycylglycine (pH 7.5), 10 mM NaCl, 10 mM KCl, 5 PM EDTA, and 0.1 mM dithiothreitol. 1% bovine serum albumin included when preparations were assayed for phosphodiesterase activities; toluene-POPOP-PPO, 4 g of 2,5-diphenyloxazole and 50 mg of 1,4-bis[2-(5-phenyloxazolyl)lbenxene dissolved in 1 liter of toluene. 20
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
CYCLIC
NUCLEOTIDE
ylate cyclase activity but in amounts that were several hundred-fold less than guanylate cyclase ‘of sea urchin sperm. Both the guanylate cyclase and adenylate cyclase of sperm were totally particulate, highly dependent on Mn2+ compared with W2+, and not stimulated by 10 mM NaF. In most mammalian tissues, guanylate cyclase is found in both soluble and particulate fractions C&4,5). Preparations of adenylate cyclase from most tissues usually show no dramatic difference in activity with Mn2+ compared to Mg2+ (6-3, and fluoride stimulation of the enzyme has been observed in most tissues with the exception of some adenylate cyclases from several bacteria and from Neurospora 6% 9). Studies on the occurrence of sperm guanylate and adenylate cyclase have been extended to include a variety of invertebra& and vertebrate species and constitute the substance of this paper. Studies are also included on cyclic nucleotide phosphodiesterase, cyclic nucleotide-dependent protein kinase, and the content of cyclic AMP and cyclic GMP in sea urchin sperm. Some of these findings have been presented in preliminary form (1, 3, 10). MATERIALS
AND
METHODS
The medium generally used for suspension and washing of intact sperm is designated in the text as “19:l” solution. It is a mixture of 19 volumes of 0.5 M NaCl and 1 volume of 0.5 M KCl. Where it is referred to as “buffered,” it contained 1 mM Tris-HCl, pH 8.0. This medium is isotonic with sea water, but sperm arc only wea‘kly motile in it due to its high K+ content (11). “GG-Na-K-EDTA-D’IT” is the hypotonic homogenizing medium used in all these studies. It contained 2 mrw glycylgylcine (pH 7.5), 10 mM NaCl, 10 mM KCl, 5 pM EDTA, and 0.1 mM dithiothreitol. It also included I.% bovine serum albumin when preparations were iassayed for phosphodiesterase activities. Scintillation media used included “toluene-POPOP-PPG” (4 g of 2,5-diphenyloxazole and 50 mg of 1,4-bis[2-(5-phenyloxazolyl)]benzene dissolved in 1 liter of toluene) (7), Aquasol (from New England Nuclear), and a Bray’s solution as modified by Butcher et al. (12). The phosphodiesterase inhibitor, I-methyl,3-isobutylxanthine (SC 2964) (13) was a gift from G. D. Searle Co. Calf thymus histone (type IIa) and dithiothreitol were purchased from Sigma. Pyruvate kinase (rabbit muscle) and 2-phosphoenolpyruvic acid were from Calbiochem. Uniformly labeled [14ClATP (tetrasodium salt, 435 mCi/mmol),
METABOLISM
IN SPERM
21
[Y-~~P]ATP (ttra(triethylammonium) salt, 24.3 Cilmmol), cyclic [G-3H1cyclic AMP (ammonium salt, 24.1 Ci/mmol), and cyclic [G-3H]GMP (ammonium salt, 4.47 Ci/mmol) were purchased from New England Nuclear; uniformly [14C1GTP (lithium salt, 40 mCi/mmol) from Schwarz/Mann. Sea urchins and tube worms were purchased from Pacific Biomarine Supply Co., Venice, Calif. Various species of mollusts and fish were obtained locally.
Collection and Treatment of Gametes Sea urchins (Strongylocentrotus purpuratus and Lytechinus pictis). Shedding of gametes was induced by injecting 1 ml of 0.5 M KC1 into the perivisceral cavity. Sperm from several urchins were pooled, diluted several-fold with buffered 19:l medium and filtered through a silk screen (pore size, 20 pm) to remove broken spines and other debris. The sperm were washed three times in 10 to 20 volumes of buffered 19:l medium by centrifugation at 2OOOgfor 10 min. The pellet was homogenized in 10 to 20 volumes of GG-Na-K-EDTA-D’IT medium by using a Ten Broeck homogenizer, and aliquots were stored at -80°C. About 0.5 g wet packed sperm were recovered from each sea urchin. Homogenization and storage of aliquots at -80°C was done in identical fashion with sperm from all species. One gram of wet packed sperm represented about 0.5-1.0 x 10” cells (14) and contained 100-125 mg of protein. Tube worms (Chuetopterus uariopedutus). Worms were removed from their tubes and the posterior segments were cut into small pieces and placed in 500 ml of ice-cold buffered 19:l medium. Usually five male worms were needed for 1 g of wet packed sperm. After mixing and settling, the top portion of the suspension was poured off and filtered through six layers of cheesecloth. The remaining portion (with minced segments) was diluted to about 200 ml, and the mince was repeatedly pressed with the base of a 500-ml Florence flask to release more sperm. This suspension was then diluted to 500 ml and allowed to settle, and the top portion was filtered as above. This process was repeated one more time. The three filtrates were combined and passed through silk cloth (100 pm, pore size). Filtrates were centrifuged in 300-ml buckets at 650.6 for 5 min. The upper 75% of the supernatant fluid was removed as the final sperm suspension and was devoid of nonsperm debris as determined by phase-contrast microscopy. Sperm were then sedimented at 10,OOOgfor 30 min and washed once in buffered 19:l solution. Final pellets were obtained by centrifuging at 5OOOg for 10 min. Mollusca. (a) Butter clams (Saxidomus giganteus, Deshuyes). Shells were opened and numerous shallow incisions were made with a scalpel along the lateral surface of the body. Gametes oozed out slowly and were washed into a petri dish with small amounts of 19:l solution and recovered with a Pas-
22
GRAY ET AL.
teur pipet. About 7 ml of semen were collected from 10 male clams, yielding finally about 4 g of wet packed sperm. The semen, diluted in several volumes of 19:l medium, was filtered twice through surgical gauze, and sperm pellets were obtained by centrifugation for 10 min at 5OOOg (in a portable centrifuge at the collection site). Pellets were stored on ice for several hours until being washed twice in 19:l solution. (b) Abalones Vlaliotus Kamtschatkana, Jonas). Organisms were extracted from their shells with a wooden wedge, and the gonads were exised. Numerous incisions were made along the long axis of each gonad, and semen was collected either with a Pasteur pipet or by mincing the gonads in 19:l solution. Further treatment was as described for clam sperm. Each male abalone yielded about 1.5 g of wet packed sperm. (c) Spiny scallops (Chlamys hericius, Gould). Testes were excised, minced in 19:l solution, the suspension was filtered, and the sperm were further treated as described for clam and abalone. About 10 male scallops were required for 1.0 g of wet packed sperm. Fish. Sperm were collected from live fish (pink, coho, Chinook, and sockeye salmon and herring) by the technique of “stripping,” i.e., the application of manual pressure from the anterior abdomen toward the posteriorly located genital-anal opening. Sperm were pelleted immediately by centrifugation at 5OOOgfor 10 min in a portable centrifuge at the site of collection. Pellets were stored on ice and transported to the laboratory. They were gently suspended in lo-20 volumes of 19:l solution; the suspension was filtered through surgical gauze and centrifuged. The packed sperm were similarly washed one more time. Humans. Ejaculated semen samples from lo-13 donors were pooled after storage for 0.5-2 h at room temperature to allow liquefaction. Volumes averaged about 2.5 ml per donor, and samples contained about 108 cells (10 mg wet weight or about 1 mg of protein) per ml. In addition to sperm, semen contains various other cells and particulate debri -(14). Pooled semen was centrifuged 30 min at 30,OOQg (4°C). The pellet was washed twice with 0.9% NaCl (original semen volume), centrifuging as above. Dogs. Epididymides were dissected from testes from four dogs. They were minced in a petri dish containing 0.9% NaCl, the mince was repeatedly pressed with the base of a small beaker to release sperm. The upper portion of the suspension was filtered through glass wool and centrifuged 2 min at 12Og (4°C) to remove large cells and other debri. The supernatant fluid was removed and centrifuged at 4°C for 30 min at SOOQg.The pellet (about 300 mg of sperm) was suspended in 40 ml of 0.9% NaCl and again centrifuged at 6OOOg.
Enzyme Assays Guanylate cyclase. Assay tubes kept in an ice bath contained 50 mM Tris-HCl, pH 7.5,2 mM dithio-
threitol, 1 mM SC 2964,0.1% bovine serum albumin, 0.5 mM [‘*ClGTP (0.2 &i), 5.0 mM MgCl, or MnCl*, and 1% Triton X-100 as indicated in a final volume of 150 ~1. The reaction was started by adding enzyme derived from 0.1-2.0 mg wet weight of packed sperm, and the tubes were incubated up to 20 min at 37°C. Control tubes either received homogenizing medium rather than enzyme or reactions were stopped immediately after adding enzyme. Other tubes received cyclic [3H]GMP (0.25 &i) with 0.5 mM unlabeled GTP for the estimation of cyclic GMP recoveries, which ranged from 47 to 53% through the entire isolation procedure described below. Reactions were stopped by the addition of 30 ~1 of a solution consisting of one-third volume of 60 mM unlabeled cyclic GMP and two-thirds volume of 0.31 M Z&O,. To each was added 20 ~1 of 0.31 M Na$O,. The ZnCO, formed was removed by centrifugation at 3000g for 10 min. More than 90% of the labeled GTP, but only 25-30% of the labeled cyclic GMP, was removed from solution with ZnCO,. Supernatant liquids were removed from the ZnCO, within an hour, otherwise variable recoveries were obtained. The general method of salt precipitation in cyclase activity determinations was developed by others (15). From each assay tube, after ZnCOB precipitation, 150 ~1 of supernatant fluid was spotted on Whatman 3 MM paper, evaporation being facilitated with a stream of warm air. Chromatograms were developed (descending) for 24 h (room temperature) with 1 M ammonium acetate/95% ethanol (3:7) as the solvent (7). Papers were dried, areas containing the cyclic nucleotide were cut out, and the 4.0 x 5.8~cm strips were folded in an accordion fashion with five creases each parallel to the short axis of the paper and to the vertical walls of the counting vial. Papers were placed in counting vials containing 20 ml of toluene-POPOP-PPO scintillation fluid, and radioactivity was determined in a Nuclear Chicago Isocap 300 scintillation spectrometer. Counting efficiencies for 14C ranged from 65 to 70%. For the calculation of activity, experimental counts (minus controls and corrected for recovery) were compared to the counts obtained when an aliquot of the lL4C1GTP substrate (no salt precipitation step) was chromatographed and counted. Adenylate cyclase (assay A). Sperm preparations were incubated for up to 10 min at 37°C in a 150~~1 system containing 0.4 mM [‘QATP (0.5 &i), 50 mM Tris-HCl (pH 7.51, 6 mM KCl, 2 mM dithiothreitol, 2 mM unlabeled cyclic AMP, 1 mM SC 2964, 20 mM phosphoenolpyruvate, 5 units of pyruvate kinase, and, as indicated, 10 mM MgCIZ or MnC&, 1.0% Triton X-100, or 10 mM NaF. Some tubes contained cyclic 13HlAMP (0.25 &i) with 0.4 mM unlabeled ATP for estimation of cyclic AMP recovery, which ranged from 70 to 75% through the entire isolation procedure. Control vessels received homogenizing medium rather than enzyme. Reactions were started with substrate and stopped by adding 25 ~1
CYCLIC
NUCLEOTIDE
METABOLISM
of 0.25 M ZnSO,. The tubes then received 25 ~1 of 0.25 M Na,CO,, were centrifuged 10 min at 3OO@g, and 150 ~1 of each supernatant fraction was spotted on ‘Whatman 3 MM paper. Cyclic li4CIAMP was isolated by descending paper chromatography for 18 h with 1 M ammonium acetate/95% ethanol (3:7) as solvent, and radioactivity was determined by scintillation spectrometry (7). The salt precipitation step removed more than 95% of labeled ATP but less than 9% of labeled cyclic AMP from the incubation mixtures. Adenylatc cyclase (assay B). Reaction mixture (2 ml), in triplicate, contained 50 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol, 10 mM theophylline, 5 mM ATP, and 10 mM MgCl, or MnCl,, 1.0% Triton X-100, or 10 mM NaF, as indicated. Reactions were started with enzyme and stopped after incubating for up to 10 min at 37°C by adding 200 ~1 of 1.5 N HCI. Control vessels either received acid before enzyme or were incubated without ATP. Cyclic 13H]AMP (5 rnM) was included during the incubation of one vessel out of each triplicate set, and a similar amount was added to the other two after stopping the reaction for purposes of calculating recovery. Samples were then fractionated by Dowex 50 column chromatography (see below) and appropriate fractions were assayed for cyclic AMP by an enzymatic cycling technique
(16). Phosphodiesterase. Reaction vessels contained 1.0 cyclic lSHIAMP or cyclic 13HlGMP (0.7 PCi), 50 rnM Tris-HCl (pH 7.5), 2 mM MgC&, and homogenate derived from 1 pg to 1 mg of wet packed sperm in a final volume of 150 ~1, and mixtures were incubated for 30 min at 30°C. Reactions were stopped by placing the tubes in a boiling-water bath for 1.5 min, tubes were cooled, and to each was added 10 ~1 of 5 mg/ml lyophilized Crotalus adamanteus venom. After 10 min at 3O”C, reactions were stopped by adding 10 ~1 of 0.25 N HCl. Tubes were centrifuged for 10 min at 3OOOg,and 100 ~1 of each supernatant solution was spotted on Whatman 3 MM paper. Each spot received a mixture containing 0.5 wmol of the appropriate unlabeled cyclic nucleotide, nucleoside 5’-monophosphate, and nucleoside. Activities were determined from counts in each of the above spots after 18 h of descending chromatography with 1 M ammonium acetate/95% ethanol solution as described for the guanylate cyclase assay. Reaction velocities were proportional to time of incubation and to protein concentration. In some experiments, phosphodiesterase activity was determined as described by Beavo et al. (17). Protein kinuse. Enzyme preparations were incubated for 2 min at 30°C in a 200-~1 system containing 50 mM Tris-HCl (pH 7.5), 1.8 mM theophylline, 10 mM NaF, 1.0% Triton X-100, 1.0 mgiml of calf thymus histone (type IIa), 100 pM [y-32P1ATP (2 &i), 10 mM MgCl,, and 10 nM to 10 pM cyclic AMP or cyclic GMP. The assay procedure used was that PM
IN SPERM
23
described by Kuo and Greengard (18). the final protein pellet was dissolved in 0.1 ml of 1 N NaOH, and 0.5 ml of water was added to each tube. A O.&ml aliquot from each was used for determination of 32P in 15 ml of Aquasol.
Determination GMP
of Cyclic AMP and Cyclic
Small tissue samples (less than 100 ~1) containing dispersed cells were diluted and mixed in 1 or 2 ml of 0.1 N HCl. Larger samples of all types were homogenized in 0.15 or 0.3 N perchloric acid in a small metal Waring blendor. All samples received 0.2 &i of cyclic 13H1AMP and/or cyclic 13HlGMP for determination of recoveries. Supernatant fractions from these samples were fractionated for cyclic AMP and/or cyclic GMP by passage over 0.6~cm-diameter columns of Dowex 50 cation-exchange resin. Columns were equilibrated and eluted with 0.1 N HCI, and cyclic AMP and/or cyclic GMP were collected in appropriate fractions (2, 16, 19, 20). All fractions were lyophilized, taken up in several milliliters of 0.1 N HCl, and rechromatographed over a second set of Dowex 50 columns, as before. When tissue samples of more than 20 g were examined, the acid supernatant solutions were treated with charcoal as a concentration step prior to passage over the Dowex 50 columns (19). The fractions from the second column were lyophilized, taken up in 0.5-2.0 ml of 50 mM Tris-HCl (pH 7.4) and adjusted to neutrality if necessary, and samples were stored at -20°C until assay. Cyclic AMP and cyclic GMP were determined by enzymatic cycling techniques (16, 19).
Assay of Protein The method of Lowry et al. was used (21). Samples were predigested for 1 h in 1 N NaOH to solubilize particulate protein. RESULTS
Guanylate
Cyclase
Results with sperm from a variety of species are presented in Table I. Sperm of mammals or fish had no detectable activity, but those from three different invertebrate phyla, Echinodermata, Mollusca and Annelida, had high guanylate cyclase when assayed in the presence of Mn2+. These activities were increased 3-lo-fold by including 1.0% Triton X-100, a nonionic detergent, in the assay. In other experiments, homogenization of sea urchin sperm in medium containing 1.0% Triton caused “solubilization” of the activity, as determined by centrifugation for 1 h at 100,OOOg.Without Triton in the medium,
24
GRAY ET AL.
TABLE I GUANYLATE CYCLASE IN VARIOUS INVERTEBRATE AND VERTEBRATE SPERM= Organism
Guanylate cyclase activity (nmol of cyclic GMP/mg wet wtfmin at 37°C) Mgz+ + Triton
Invertebrate sperm Sea urchin (S. purpurutus) Sea urchin a. pit-
Mn2+
Mn*+ + Tritan
CO.1
2.2
6.2
CO.1
1.5
15.6
co.1 0.6 0.5 0.9
0.8 2.7 1.7 4.5
6.3 7.8 8.9 18.3
OF
BIOCHEMISTRY
AND
BIOPHYSICS
172, 20-30 (1976)
Enzymes of Cyclic Nucleotide Metabolism Vertebrate Sperm1
in Invertebrate
and
J. PATRICK GRAY,2 GEORGE I. DRUMMOND,3 DAVID W. T. LUK Department
of Pharmacology,
School of Medicine,
The University V6T 1 w5
of British
Columbia,
Vancouver,
Can&a
AND
JOEL G. HARDMAN, Department
of Physiology,
AND
EARL W. SUTHERLAND4
School of Medicine, Vanderbilt University, Received May 14, 1975
Nashville,
Tennessee 37212
Sperm from several invertebrates contained guanylate cyclase activity severalhundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn2+ was up to several hundred-fold greater than with Mg*+ and was increased 3-lo-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mng+-dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than lo-fold more active with Mn*+ than with Mg*+. Invertebrate sperm contained phosphodiesterase activities against 1.0 pM cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by T&m. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1-l nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive tc stimulation by cyclic AMP than by cyclic GMP.5
It has been reported preliminarily (1) fold greater than the most active mammathat sea urchin sperm possess guanylate lian tissues such as lung (2). In contrast, cyclase activity that is several hundred- human and dog sperm did not contain detectable guanylate cyclase activity (3). All 1This work was surwt~ by grants from the three of these sperm types contained adenNational Institutes of Health (No. GM 16811, AM07462 and HE 08332) and from the Medical Research Council of Canada. 2 Portions of this work were taken from the dissertation submitted by J.P.G. in partial fulfillment of the requirements of the Ph.D. degree, Vanderbilt University, 1971. In Canada, recipient of a Medical Research Council of Canada postdoctoral fellowship. 3 Present address: Biochemistry Group, Department of Chemistry, The University of Calgary, Calgary, Alberta, T2N lN4, Canada. Inquiries should be sent to G.I.D. at this address.
4 Deceased, March 9, 1974. J Abbreviations used: SC 2964, 1-methyl,3-isobutylxanthine; 19:l solution, mixture of 19 volumes of 0.5 M NaCl and 1 volume of 0.5 M KCl; GG-Na-KEDTA-D’M’, 2 mM glycylglycine (pH 7.5), 10 mM NaCl, 10 mM KCl, 5 PM EDTA, and 0.1 mM dithiothreitol. 1% bovine serum albumin included when preparations were assayed for phosphodiesterase activities; toluene-POPOP-PPO, 4 g of 2,5-diphenyloxazole and 50 mg of 1,4-bis[2-(5-phenyloxazolyl)lbenxene dissolved in 1 liter of toluene. 20
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
CYCLIC
NUCLEOTIDE
ylate cyclase activity but in amounts that were several hundred-fold less than guanylate cyclase ‘of sea urchin sperm. Both the guanylate cyclase and adenylate cyclase of sperm were totally particulate, highly dependent on Mn2+ compared with W2+, and not stimulated by 10 mM NaF. In most mammalian tissues, guanylate cyclase is found in both soluble and particulate fractions C&4,5). Preparations of adenylate cyclase from most tissues usually show no dramatic difference in activity with Mn2+ compared to Mg2+ (6-3, and fluoride stimulation of the enzyme has been observed in most tissues with the exception of some adenylate cyclases from several bacteria and from Neurospora 6% 9). Studies on the occurrence of sperm guanylate and adenylate cyclase have been extended to include a variety of invertebra& and vertebrate species and constitute the substance of this paper. Studies are also included on cyclic nucleotide phosphodiesterase, cyclic nucleotide-dependent protein kinase, and the content of cyclic AMP and cyclic GMP in sea urchin sperm. Some of these findings have been presented in preliminary form (1, 3, 10). MATERIALS
AND
METHODS
The medium generally used for suspension and washing of intact sperm is designated in the text as “19:l” solution. It is a mixture of 19 volumes of 0.5 M NaCl and 1 volume of 0.5 M KCl. Where it is referred to as “buffered,” it contained 1 mM Tris-HCl, pH 8.0. This medium is isotonic with sea water, but sperm arc only wea‘kly motile in it due to its high K+ content (11). “GG-Na-K-EDTA-D’IT” is the hypotonic homogenizing medium used in all these studies. It contained 2 mrw glycylgylcine (pH 7.5), 10 mM NaCl, 10 mM KCl, 5 pM EDTA, and 0.1 mM dithiothreitol. It also included I.% bovine serum albumin when preparations were iassayed for phosphodiesterase activities. Scintillation media used included “toluene-POPOP-PPG” (4 g of 2,5-diphenyloxazole and 50 mg of 1,4-bis[2-(5-phenyloxazolyl)]benzene dissolved in 1 liter of toluene) (7), Aquasol (from New England Nuclear), and a Bray’s solution as modified by Butcher et al. (12). The phosphodiesterase inhibitor, I-methyl,3-isobutylxanthine (SC 2964) (13) was a gift from G. D. Searle Co. Calf thymus histone (type IIa) and dithiothreitol were purchased from Sigma. Pyruvate kinase (rabbit muscle) and 2-phosphoenolpyruvic acid were from Calbiochem. Uniformly labeled [14ClATP (tetrasodium salt, 435 mCi/mmol),
METABOLISM
IN SPERM
21
[Y-~~P]ATP (ttra(triethylammonium) salt, 24.3 Cilmmol), cyclic [G-3H1cyclic AMP (ammonium salt, 24.1 Ci/mmol), and cyclic [G-3H]GMP (ammonium salt, 4.47 Ci/mmol) were purchased from New England Nuclear; uniformly [14C1GTP (lithium salt, 40 mCi/mmol) from Schwarz/Mann. Sea urchins and tube worms were purchased from Pacific Biomarine Supply Co., Venice, Calif. Various species of mollusts and fish were obtained locally.
Collection and Treatment of Gametes Sea urchins (Strongylocentrotus purpuratus and Lytechinus pictis). Shedding of gametes was induced by injecting 1 ml of 0.5 M KC1 into the perivisceral cavity. Sperm from several urchins were pooled, diluted several-fold with buffered 19:l medium and filtered through a silk screen (pore size, 20 pm) to remove broken spines and other debris. The sperm were washed three times in 10 to 20 volumes of buffered 19:l medium by centrifugation at 2OOOgfor 10 min. The pellet was homogenized in 10 to 20 volumes of GG-Na-K-EDTA-D’IT medium by using a Ten Broeck homogenizer, and aliquots were stored at -80°C. About 0.5 g wet packed sperm were recovered from each sea urchin. Homogenization and storage of aliquots at -80°C was done in identical fashion with sperm from all species. One gram of wet packed sperm represented about 0.5-1.0 x 10” cells (14) and contained 100-125 mg of protein. Tube worms (Chuetopterus uariopedutus). Worms were removed from their tubes and the posterior segments were cut into small pieces and placed in 500 ml of ice-cold buffered 19:l medium. Usually five male worms were needed for 1 g of wet packed sperm. After mixing and settling, the top portion of the suspension was poured off and filtered through six layers of cheesecloth. The remaining portion (with minced segments) was diluted to about 200 ml, and the mince was repeatedly pressed with the base of a 500-ml Florence flask to release more sperm. This suspension was then diluted to 500 ml and allowed to settle, and the top portion was filtered as above. This process was repeated one more time. The three filtrates were combined and passed through silk cloth (100 pm, pore size). Filtrates were centrifuged in 300-ml buckets at 650.6 for 5 min. The upper 75% of the supernatant fluid was removed as the final sperm suspension and was devoid of nonsperm debris as determined by phase-contrast microscopy. Sperm were then sedimented at 10,OOOgfor 30 min and washed once in buffered 19:l solution. Final pellets were obtained by centrifuging at 5OOOg for 10 min. Mollusca. (a) Butter clams (Saxidomus giganteus, Deshuyes). Shells were opened and numerous shallow incisions were made with a scalpel along the lateral surface of the body. Gametes oozed out slowly and were washed into a petri dish with small amounts of 19:l solution and recovered with a Pas-
22
GRAY ET AL.
teur pipet. About 7 ml of semen were collected from 10 male clams, yielding finally about 4 g of wet packed sperm. The semen, diluted in several volumes of 19:l medium, was filtered twice through surgical gauze, and sperm pellets were obtained by centrifugation for 10 min at 5OOOg (in a portable centrifuge at the collection site). Pellets were stored on ice for several hours until being washed twice in 19:l solution. (b) Abalones Vlaliotus Kamtschatkana, Jonas). Organisms were extracted from their shells with a wooden wedge, and the gonads were exised. Numerous incisions were made along the long axis of each gonad, and semen was collected either with a Pasteur pipet or by mincing the gonads in 19:l solution. Further treatment was as described for clam sperm. Each male abalone yielded about 1.5 g of wet packed sperm. (c) Spiny scallops (Chlamys hericius, Gould). Testes were excised, minced in 19:l solution, the suspension was filtered, and the sperm were further treated as described for clam and abalone. About 10 male scallops were required for 1.0 g of wet packed sperm. Fish. Sperm were collected from live fish (pink, coho, Chinook, and sockeye salmon and herring) by the technique of “stripping,” i.e., the application of manual pressure from the anterior abdomen toward the posteriorly located genital-anal opening. Sperm were pelleted immediately by centrifugation at 5OOOgfor 10 min in a portable centrifuge at the site of collection. Pellets were stored on ice and transported to the laboratory. They were gently suspended in lo-20 volumes of 19:l solution; the suspension was filtered through surgical gauze and centrifuged. The packed sperm were similarly washed one more time. Humans. Ejaculated semen samples from lo-13 donors were pooled after storage for 0.5-2 h at room temperature to allow liquefaction. Volumes averaged about 2.5 ml per donor, and samples contained about 108 cells (10 mg wet weight or about 1 mg of protein) per ml. In addition to sperm, semen contains various other cells and particulate debri -(14). Pooled semen was centrifuged 30 min at 30,OOQg (4°C). The pellet was washed twice with 0.9% NaCl (original semen volume), centrifuging as above. Dogs. Epididymides were dissected from testes from four dogs. They were minced in a petri dish containing 0.9% NaCl, the mince was repeatedly pressed with the base of a small beaker to release sperm. The upper portion of the suspension was filtered through glass wool and centrifuged 2 min at 12Og (4°C) to remove large cells and other debri. The supernatant fluid was removed and centrifuged at 4°C for 30 min at SOOQg.The pellet (about 300 mg of sperm) was suspended in 40 ml of 0.9% NaCl and again centrifuged at 6OOOg.
Enzyme Assays Guanylate cyclase. Assay tubes kept in an ice bath contained 50 mM Tris-HCl, pH 7.5,2 mM dithio-
threitol, 1 mM SC 2964,0.1% bovine serum albumin, 0.5 mM [‘*ClGTP (0.2 &i), 5.0 mM MgCl, or MnCl*, and 1% Triton X-100 as indicated in a final volume of 150 ~1. The reaction was started by adding enzyme derived from 0.1-2.0 mg wet weight of packed sperm, and the tubes were incubated up to 20 min at 37°C. Control tubes either received homogenizing medium rather than enzyme or reactions were stopped immediately after adding enzyme. Other tubes received cyclic [3H]GMP (0.25 &i) with 0.5 mM unlabeled GTP for the estimation of cyclic GMP recoveries, which ranged from 47 to 53% through the entire isolation procedure described below. Reactions were stopped by the addition of 30 ~1 of a solution consisting of one-third volume of 60 mM unlabeled cyclic GMP and two-thirds volume of 0.31 M Z&O,. To each was added 20 ~1 of 0.31 M Na$O,. The ZnCO, formed was removed by centrifugation at 3000g for 10 min. More than 90% of the labeled GTP, but only 25-30% of the labeled cyclic GMP, was removed from solution with ZnCO,. Supernatant liquids were removed from the ZnCO, within an hour, otherwise variable recoveries were obtained. The general method of salt precipitation in cyclase activity determinations was developed by others (15). From each assay tube, after ZnCOB precipitation, 150 ~1 of supernatant fluid was spotted on Whatman 3 MM paper, evaporation being facilitated with a stream of warm air. Chromatograms were developed (descending) for 24 h (room temperature) with 1 M ammonium acetate/95% ethanol (3:7) as the solvent (7). Papers were dried, areas containing the cyclic nucleotide were cut out, and the 4.0 x 5.8~cm strips were folded in an accordion fashion with five creases each parallel to the short axis of the paper and to the vertical walls of the counting vial. Papers were placed in counting vials containing 20 ml of toluene-POPOP-PPO scintillation fluid, and radioactivity was determined in a Nuclear Chicago Isocap 300 scintillation spectrometer. Counting efficiencies for 14C ranged from 65 to 70%. For the calculation of activity, experimental counts (minus controls and corrected for recovery) were compared to the counts obtained when an aliquot of the lL4C1GTP substrate (no salt precipitation step) was chromatographed and counted. Adenylate cyclase (assay A). Sperm preparations were incubated for up to 10 min at 37°C in a 150~~1 system containing 0.4 mM [‘QATP (0.5 &i), 50 mM Tris-HCl (pH 7.51, 6 mM KCl, 2 mM dithiothreitol, 2 mM unlabeled cyclic AMP, 1 mM SC 2964, 20 mM phosphoenolpyruvate, 5 units of pyruvate kinase, and, as indicated, 10 mM MgCIZ or MnC&, 1.0% Triton X-100, or 10 mM NaF. Some tubes contained cyclic 13HlAMP (0.25 &i) with 0.4 mM unlabeled ATP for estimation of cyclic AMP recovery, which ranged from 70 to 75% through the entire isolation procedure. Control vessels received homogenizing medium rather than enzyme. Reactions were started with substrate and stopped by adding 25 ~1
CYCLIC
NUCLEOTIDE
METABOLISM
of 0.25 M ZnSO,. The tubes then received 25 ~1 of 0.25 M Na,CO,, were centrifuged 10 min at 3OO@g, and 150 ~1 of each supernatant fraction was spotted on ‘Whatman 3 MM paper. Cyclic li4CIAMP was isolated by descending paper chromatography for 18 h with 1 M ammonium acetate/95% ethanol (3:7) as solvent, and radioactivity was determined by scintillation spectrometry (7). The salt precipitation step removed more than 95% of labeled ATP but less than 9% of labeled cyclic AMP from the incubation mixtures. Adenylatc cyclase (assay B). Reaction mixture (2 ml), in triplicate, contained 50 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol, 10 mM theophylline, 5 mM ATP, and 10 mM MgCl, or MnCl,, 1.0% Triton X-100, or 10 mM NaF, as indicated. Reactions were started with enzyme and stopped after incubating for up to 10 min at 37°C by adding 200 ~1 of 1.5 N HCI. Control vessels either received acid before enzyme or were incubated without ATP. Cyclic 13H]AMP (5 rnM) was included during the incubation of one vessel out of each triplicate set, and a similar amount was added to the other two after stopping the reaction for purposes of calculating recovery. Samples were then fractionated by Dowex 50 column chromatography (see below) and appropriate fractions were assayed for cyclic AMP by an enzymatic cycling technique
(16). Phosphodiesterase. Reaction vessels contained 1.0 cyclic lSHIAMP or cyclic 13HlGMP (0.7 PCi), 50 rnM Tris-HCl (pH 7.5), 2 mM MgC&, and homogenate derived from 1 pg to 1 mg of wet packed sperm in a final volume of 150 ~1, and mixtures were incubated for 30 min at 30°C. Reactions were stopped by placing the tubes in a boiling-water bath for 1.5 min, tubes were cooled, and to each was added 10 ~1 of 5 mg/ml lyophilized Crotalus adamanteus venom. After 10 min at 3O”C, reactions were stopped by adding 10 ~1 of 0.25 N HCl. Tubes were centrifuged for 10 min at 3OOOg,and 100 ~1 of each supernatant solution was spotted on Whatman 3 MM paper. Each spot received a mixture containing 0.5 wmol of the appropriate unlabeled cyclic nucleotide, nucleoside 5’-monophosphate, and nucleoside. Activities were determined from counts in each of the above spots after 18 h of descending chromatography with 1 M ammonium acetate/95% ethanol solution as described for the guanylate cyclase assay. Reaction velocities were proportional to time of incubation and to protein concentration. In some experiments, phosphodiesterase activity was determined as described by Beavo et al. (17). Protein kinuse. Enzyme preparations were incubated for 2 min at 30°C in a 200-~1 system containing 50 mM Tris-HCl (pH 7.5), 1.8 mM theophylline, 10 mM NaF, 1.0% Triton X-100, 1.0 mgiml of calf thymus histone (type IIa), 100 pM [y-32P1ATP (2 &i), 10 mM MgCl,, and 10 nM to 10 pM cyclic AMP or cyclic GMP. The assay procedure used was that PM
IN SPERM
23
described by Kuo and Greengard (18). the final protein pellet was dissolved in 0.1 ml of 1 N NaOH, and 0.5 ml of water was added to each tube. A O.&ml aliquot from each was used for determination of 32P in 15 ml of Aquasol.
Determination GMP
of Cyclic AMP and Cyclic
Small tissue samples (less than 100 ~1) containing dispersed cells were diluted and mixed in 1 or 2 ml of 0.1 N HCl. Larger samples of all types were homogenized in 0.15 or 0.3 N perchloric acid in a small metal Waring blendor. All samples received 0.2 &i of cyclic 13H1AMP and/or cyclic 13HlGMP for determination of recoveries. Supernatant fractions from these samples were fractionated for cyclic AMP and/or cyclic GMP by passage over 0.6~cm-diameter columns of Dowex 50 cation-exchange resin. Columns were equilibrated and eluted with 0.1 N HCI, and cyclic AMP and/or cyclic GMP were collected in appropriate fractions (2, 16, 19, 20). All fractions were lyophilized, taken up in several milliliters of 0.1 N HCl, and rechromatographed over a second set of Dowex 50 columns, as before. When tissue samples of more than 20 g were examined, the acid supernatant solutions were treated with charcoal as a concentration step prior to passage over the Dowex 50 columns (19). The fractions from the second column were lyophilized, taken up in 0.5-2.0 ml of 50 mM Tris-HCl (pH 7.4) and adjusted to neutrality if necessary, and samples were stored at -20°C until assay. Cyclic AMP and cyclic GMP were determined by enzymatic cycling techniques (16, 19).
Assay of Protein The method of Lowry et al. was used (21). Samples were predigested for 1 h in 1 N NaOH to solubilize particulate protein. RESULTS
Guanylate
Cyclase
Results with sperm from a variety of species are presented in Table I. Sperm of mammals or fish had no detectable activity, but those from three different invertebrate phyla, Echinodermata, Mollusca and Annelida, had high guanylate cyclase when assayed in the presence of Mn2+. These activities were increased 3-lo-fold by including 1.0% Triton X-100, a nonionic detergent, in the assay. In other experiments, homogenization of sea urchin sperm in medium containing 1.0% Triton caused “solubilization” of the activity, as determined by centrifugation for 1 h at 100,OOOg.Without Triton in the medium,
24
GRAY ET AL.
TABLE I GUANYLATE CYCLASE IN VARIOUS INVERTEBRATE AND VERTEBRATE SPERM= Organism
Guanylate cyclase activity (nmol of cyclic GMP/mg wet wtfmin at 37°C) Mgz+ + Triton
Invertebrate sperm Sea urchin (S. purpurutus) Sea urchin a. pit-
Mn2+
Mn*+ + Tritan
CO.1
2.2
6.2
CO.1
1.5
15.6
co.1 0.6 0.5 0.9
0.8 2.7 1.7 4.5
6.3 7.8 8.9 18.3
