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Enzymes of cyclic nucleotide metabolism in invertebrate and vertebrate sperm
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
0.005
0.035
tus) Scallop Abalone Clam Tube worm Vertebrate sperm Mammals: human, dog Fish: salmon, herring Mammalian lung Rat
a Enzyme derived from 0.1 mg (invertebrates) to 2.0 mg (vertebrates) wet weight of packed sperm were assayed as described in Materials and Methods. Values reported are the averages from two separate preparations of each sperm type, each assayed in triplicate. Sperm were tested from four species of salmon, pink, coho, Chinook and sockeye.
activity was totally recovered in particulate fractions. As shown in Table I, the activities of sea urchin or scallop sperm, when assayed with Triton, were scarcely detectable with Mg2+, being less than 2% of the values with Mn2+. With abalone, clam, or tube worm sperm, activities were lo-20-fold less with MgZ+ than with Mn2+ (Table I). In one experiment (data not shown) lOO,OOOg(1 h) supernatant fluids and particles were prepared from sperm homogenates (no Triton) of sea urchin (L. pictus), scallop, abalone, clam and the four species of salmon. Supernatant solutions were assayed with Mn2+ and Triton and contained less than 2% of guanylate cyclase activities measured in the original homogenates. As measured with Mg2+ and Triton, Mn2+ without Triton, or Mn2+ and Triton, particulate activities were identical to those of the original homogenates. Particulate ac-
tivity was not detected without added divalent cation, either with or without Triton. These results indicate that (a) the enzyme was totally particulate, (b) the failure to detect activity in fish sperm homogenates was not likely due to the presence of a soluble inhibitor, and (c) the low activities with Mg2+ and Triton in abalone or clam sperm required the added Mg2+. In other experiments with sea urchin sperm, no activity was detected when Mn2+ was replaced by Ca2+, 10 mM NaF had no effect using either Mn2+ or Mg2+, and the enzyme was stable to storage at -80°C for at least a year unless repeatedly frozen and thawed. Finally, as shown in Table I, the guanylate cyclase activity of rat lung homogenate, one of the most active mammalian tissue preparations (2), was several hundred-fold less than the activities of invertebrate sperm. Adenylate
Cyclase
The adenylate cyclase activities of invertebrate sperm (Table II) assayed with Mn2+, were several hundred-fold less than their guanylate cyclase activities (Table I). Whereas guanylate cyclase was detected only with invertebrate sperm, adenylate cyclase was found in sperm of invertebrates, fish, and mammals. Activities with Mn2+ were somewhat similar among these sperm types except for the relatively low values obtained from human and clam. The activity of rat brain, one of the most active mammalian tissues, was severalfold greater with Mn2+ than were those of sperm. The sperm enzyme, assayed with Mn2+, was not significantly affected by 10 mM NaF. The activity of human, fish, or invertebrate sperm with Mg2+ was also unaffected by fluoride (data not shown). Sperm activity was at least &lo-fold greater with Mn2+ than with Mg2+ (10 mM), with the exception of tube worm and abalone. With Mn2+, the adenylate cyclase activity of invertebrate sperm was increased, that of mammalian sperm was unaffected, and that of fish sperm was decreased by Triton X-100. With invertebrate or fish sperm, cyclic AMP did not accumulate linearly with time, even for 5 min at 37°C. This was not
CYCLIC NUCLEOTIDE TABLE II ADEN~ATE
CYCLASE IN INVERTEBRATE AND VERTEBRATE SPERM”
Organism
Adenylate cyclase activity (pmol of cyclic AMP/mg wet wff5 min at 37°C) Mg2+ Mn2+ Mn*+
Invertebrate sperm Sea urchin (L,.
NiF
Mn2+ Tr&n
10
115
100
150
130
210 35 180
240 50 205
505 145 230
2.5 15 <15 <15 cc15 15
15 105 125 165 190 370
15 120 155 N.D.b 210 415
15 105 100 100 95 185
250
500
2500
2500
pi&us) Tube worm Clam Abalone Vertebrate sperm Human Dog Pink salmon Sockeye salmon Coho salmon Herring Mammalian brain Rat
LIOnce-thawed sperm homogenates from invertebrates or fish (2 mg of packed sperm) were assayed by assay A. The activities of human or dog sperm were determined by assay B and contained 5-10 mg wet weight of sperm per ml. Values are expressed as picomoles of cyclic AMP accumulated over a 5-min interval because velocities in the case of invertebrate and fish sperm were not linear with time (see text and Fig. 1). b Not determined.
METABOLISM
IN SPERM
25
time courses resembled those in Fig. 1. The reason for this nonlinearity is not known. In one experiment, the effect of GTP was examined. Conditions were as in Fig, 1, except that ATP was 1 mM and Mn2+ was 5 mM. Activity was unaffected by 0.1 or 0.2 mM GTP but was inhibited 20-30% by 1 mM GTP at all time points. The poor ability of GTP to inhibit the enzyme provides evidence that adenylate cyclase is likely a separate enzyme from guanylate cyclase in invertebrate sperm since cyclic GMP formation from GTP proceeded at a far higher rate than did cyclic AMP formation from ATP under these conditions. Additional evidence that these enzymes are separate was provided by the observation that they were oppositely affected by high levels of colchicine. Thus, in two experiments, adenylate cyclase activity of S.purpuratus sperm (with Mn2+) was inhibited by 10 mM colchicine. Inhibition was greater in the presence of 1.0% Triton (equalling 43 and 70% in the two experiments) than in the absence of Triton (where it equalled 17 and 27%). In contrast, the guanylate cyclase activity of these sperm was stimulated by colchi&e, but only at levels greater than 1 mM, especially in the absence of Triton. Thus, despite the fact that both activities were totally particulate, highly dependent on Mn2+ compared to Mg2+, stimulated by Triton, and unaffected by fluoride, it is likely that they represent separate enzymes.
due to hydrolysis of ATP; less than 10% of [14ClATP was degraded in 10 min (determined by paper chromatography (22)). Neither was the failure to achieve linearity with time due to degradation of cyclic AMP by phosphodiesterase. Adenylate cyclase was not inhibited by accumulating product, because similar velocities were obtained in the absence or presence of 2 mM added cyclic AMP. An example of this nonlinearity with time is presented in Fig. 1. Results are from one of six experiments done in an attempt to dilute the enzyme to an extent where linearity with time might be achieved. In additional experiments, activities were measured with sperm homogenates or 30,OOOg(30 min) particles incuFIG. 1. The Mn*+-dependent adenylate cyclase acbated at concentrations ranging from 0.1 tivity of sea urchin sperm. Sperm from S. purpurato 2.0 mg wet weight of sperm per ml. tus were incubated at a concentration of 0.1 mg wet Regardless of enzyme concentration, all weight of packed sperm per ml in assay B.
26
GRAY ET AL.
This conclusion is supported cent studies (23).
by other re-
Cyclic Nucleotide Phosphodiesterase Sperm from a variety of invertebrate and fish species were examined for their ability to degrade 1 PM cyclic AMP or cyclic GMP, and the results are presented in Table III. Invertebrate and fish sperm, all of which contain adenylate cyclase activity, degraded cyclic AMP at rates comparable to, or up to 20-fold geater than, the rate of cyclic AMP degradation by homogenates of rat brain, one of the most active mammalian tissues (17). Invertebrate sperm, which contained high levels of guanylate cyclase activity, also degraded cyclic GMP at high rates. In contrast, fish sperm, which did not contain detectable TABLE
III
CYCLIC NUCLE~TIDE PHOSPHODIESTERASEOF INVERTEBRATE AND VERTEBRATE SPERM Organism
Phosphodiesterase activity (pm01 of cyclic A(G)MP degradedmg wet timin at 30°C) Cyclic AMP
Invertebrate sperm Sea urchin @.purpurutus) Sea urchin (L. pi&us) Clam Scallop Abalone Fish sperm Salmon (pink, chinook, coho, sockeye) Herring Mammalian brain* Rat
67 67 145 23 20
Cyclic GMP
594 470 244 27 19
112
0.9
570
0.1
30
83
a Frozen preparations were thawed, diluted and rehomogenized in GG-Na-K-EDTA-D’lT medium containing 1% bovine serum albumin. Albumin was necessary for the preservation of phosphodiesterase activity in dilute enzyme solutions. Homogenates were assayed by Assay A. Values are the average of each sperm type, except in the case of salmon sperm where one preparation of each of the four species was used. b Values for the activities of rat brain homogenate were taken from the report of Beavo et al. (17).
guanylate cyclase activity, were unable to degrade cyclic GMP at appreciable rates. Thus, there seems to be a correlation between the capability of a given sperm type to form either cyclic AMP or cyclic GMP and its ability to degrade that nucleotide. Sperm of S. purpuratus hydrolyzed 1 PM cyclic GMP about nine times faster than cyclic AMP (Table III). When homogenates of these sperm were centrifuged at 30,0009 for 30 min two-thirds of the activity against cyclic AMP was in the supernatant fraction, whereas three-fourths of the activity against cyclic GMP was in the particles. The effects of several agents on phosphodie&erase activity were examined, and the results are presented in Table IV. Triton X-100 (l.O%), inhibited the hydrolysis of both nucleotides, but the hydrolysis of cyclic GMP was more strongly inhibited than was that of cyclic AMP. Colchicine (30 mM) inhibited the hydrolysis of both nucleotides. The degradation of 1 PM cyclic r3H]AMP was inhibited by 1 PM unlabeled cyclic GMP. On the other hand, unlabeled cyclic AMP only slightly inhibited the degradation of cyclic 13HlGMP. The Cyclic AMP and Cyclic GMP Contents of Sea Urchin Sperm Sea urchin sperm that were acid fixed in different states of motility contained cyclic AMP ranging from 0.1 to 0.7 nmol per g of wet packed sperm (Table V). Sperm that were fixed following 15 min after dilution in aerated artificial sea water in which the sperm were highly motile consistently contained higher concentrations of cyclic AMP than did sperm which were fixed without prior dilution (immotile sperm) or following dilution in 19:l solution which contained 25 mM KC1 and in which sperm are poorly motile (11). The cyclic AMP concentration in very motile sperm was not significantly greater than that in poorly motile sperm as determined by a paired t test (p < 0.1). The results, however, are highly suggestive of a real difference between these groups. A positive correlation between cyclic AMP levels and motility had been reported to exist with mammalian sperm (24). Cyclic GMP levels in these
CYCLIC NUCLEOTIDE
METABOLISM
TABLE
27
IN SPERM
IV
EFFECT OF SEVERAL AGENTS ON PHOSPHODIESTERASEACTIVITY 3H-labeled cyclic nucleotide (1.0 CLM) Cyclic t3HlAMP
Cyclic 13HlGMP
Cell fraction
Phosphodiesterase Control
Homogenate Particles Supernatant Homogenate Particles Supernatant
100 100 100 100 100 100
Triton (1.0%) 92 80 85 14 14 27
activity
(% relative
Colchicine (30 InM) 10 7 <5 <5 -
to control)
cGMP (1.0 /.bM)
CAMP (1.0 /AM)
49 24 -
-b 81 84 -
a Sperm from S. purpurutus were homogenized in nine volumes of GG-Na-K-EDTA-D’l”I’ and centrifuged at 30,OOOgfor 30 min. The supernatant fluid and resuspended pellet were assayed by the method of Beavo et al. (17). * Where dashes (-) occur, activity was not determined. TABLE V CYCLIC AMP AND CYCLIC GMP CONTENT OF SEA URCHIN SPERM Sperm preparation
Immotile Poorly motile Very motile
Cyclic nucleotide (nmol/g wet wt) Cyclic AMP
Cyclic GMP
0.17 2 0.05 0.20 T 0.03 0.43 * 0.11
0.38 + 0.25 0.47 k 017 0.42 f 0.13
a Five samples of semen from S. pzqurotus were strained free of debris, centrifuged 10 min at 12,OOOg (4°C) and the pellets were treated in one of three ways. (i) Immotile sperm: pellets were suspended in acid (containing tracer 25 ml of 0.3 N perchloric cyclic 13H]AMP and cyclic [3HlGMP) and homogenized 1 min in a Waring blendor. (ii) Poorly motile sperm: pellets were resuspended in nine volumes of 25 mM KC1 and 475 mM NaCl(15”). Fifteen minutes after suspension, sperm were acid fixed and homogenized. (iii) Very motile sperm: the pellet was treated as described in (ii) except that the suspension medium was aerated artificial sea water. Following acid fixation, preparations were centrifuged at 30,OOOgfor 30 min. Supernatant solutions were assayed for cyclic nucleotides as described in Materials and Methods. Values are means of five different preparations 2 SEM.
sperm were more variable, ranging from 0.04 to 0.89 nmol per g of sperm (Table V>. A relationship between motility state and cyclic GMP concentration could not be discerned. Protein Kinase The ability of cyclic AMP or cyclic GMP, in concentrations ranging from 10 nM to 10
pM, to stimulate histone kinase activity of either whole homogenate or supernatant fractions (lOO,OOOg,1 h) of sea urchin sperm was examined (Fig. 2). All values reported in the figure were obtained in the presence of histone, Mg2+, and Triton X100. In the homogenate, detectable stimulation was observed with concentrations of cyclic AMP and/or cyclic GMP as low as 0.1 PM and 1 PM, respectively. Activity was stimulated at least fivefold by maximally effective levels of cyclic AMP (1 PM). Apparent K, for cyclic AMP was about 0.5 PM. Homogenate activity with maximally effective cyclic AMP equalled about 3 nmol of 32P transferred to protein per mg of
FIG. 2. Protein kinase activity of sea urchin sperm. Homogenates of S. purpurutus sperm were centrifuged for 1 h at 100,OOOgfor preparation of the supernatant fraction. Enzyme from homogenate (25 pg of protein) or supernatant solution (5-50 pg of protein) was assayed for protein kinase activity as described in Materials and Methods. Histone was phosphorylated linearly with incubation time. Values are means 2 SEM of values obtained in four or five separate experiments.
28
GRAY ET AL.
sperm protein per min at 30°C. In the supernatant fractions, detectable stimulation was again observed with concentrations of cyclic AMP as low as 0.1 PM. In contrast to the results obtained with homogenate, however, detectable stimulation of the supernatant activity required very high levels of cyclic GMP, approaching 10 PM. Maximally effective levels of cyclic AMP (1 PM) increased the activity at least 25fold, yielding a specific activity of about 25 nmol of 32Pper mg protein per min. Thus, the specific activity of protein kinase, determined with maximally effective cyclic AMP, was increased about lofold in the supernatant solution relative to that in the original sperm homogenate. With soluble activity, the apparent K, for cyclic AMP was similar to that obtained in the homogenate (about 0.5 PM). The K, for cyclic GMP was not estimatable but was apparently much higher even than in the homogenate, being perhaps as high as 10 PM. A fresh homogenate of tube worm sperm, assayed with histone, Triton, and Mg2+, had protein kinase activity of 0.7 nmol of [32Pl per mg of protein per min without cyclic nucleotide and 8.4 with 10 PM cyclic AMP. When histone was omitted, values with and without cyclic AMP were 0.5 and 0.4, respectively. When sea urchin sperm homogenates were separated into supernatant and particulate fractions (lOO,OOOg, 1 h), 83% of the recovered activity was in the supernatant, fluid as determined with either 10 PM cyclic AMP or cyclic GMP. The studies show that sea urchin sperm contained an active soluble histone kinase which was sensitive to stimulation by levels of cyclic AMP that were found to be present in these cells (Table V). The enzyme was stimulated by cyclic GMP also but only at levels which were far higher than the levels of this cyclic nucleotide found in these cells.
occur widely in nature (2, 16, 18, 19, 25, 26). The finding of high guanylate cyclase activity in invertebrate sperm suggests that some metabolic system involving cyclic GMP is concentrated there. The most active nonsperm preparation of guanylate cyclase found has been the outer rod segments from retina (27), where the enzyme is only an order of magnitude less active than that in invertebrate sperm. A large percentage of sperm structure and function is related to its flagellum, and one might consider the possibility that cyclic GMP may be involved in flagellar motility. However, neither cyclic GMP nor cyclic AMP affects motility of demembranated sperm (28). Solubilization and removal of guanylate cyclase from sperm is effected by treatment with Triton X-100. However, such treatment does not render sperm immotile in medium containing ATP and Mg2+ (28). Thus, the cyclic GMP system seems to have no direct role in the motility mechanism. What metabolic properties shared by invertebrate sperm, with high guanylate cyclase, are absent in the vertebrate sperm, with no guanylate cyclase? Guanylate cyclase is not present in all sperm types that are dependent on oxidation of endogenous substrate, since these include both invertebrate and fish sperm which are shed into environments where exogenous glycolyzable substrates are virtually absent (14, 29). Neither do all sperm that are shed into sea water, with its high salt content, contain guanylate cyclase, since herring sperm dwell in sea water and would likely require guanylate cyclase if cyclic GMP were vital to ionic balance mechanisms in the sperm cell membrane. Sperm of most lower life forms are designated as “9 + 2” type since the only longitudinal fibers within their flagella are those in the microtubule set (axoneme) that are common to almost all flagella and cilia (29,30). Sperm of mammals, snakes, and of some birds, DISCUSSION molluscs, and insects contain (in addition No specific metabolic roles for cyclic to the axoneme) nine outer y fibers, comGMP in any cell type have been defined, posed of the protein “spermosin” which is although guanylate cyclase, cyclic GMP, not microtubular, and are designated as “9 cyclic GMP-degrading phosphodiesterase, + 9 + 2” type (29, 30). It has been specuand cyclic GMP-activated protein kinases lated that the axoneme may be somewhat
CYCLIC
NUCLEOTIDE
vestigial in “9 + 9 + 2” sperm, not serving directly to generate the flagellar propulsive force (30). Thus, it would be significant if only “9 + 9 + 2” sperm lacked guanylate cyclase. This relationship does not hold, however, since sperm of fish, without guanylate cyclase, as well as those of the tested invertebrates, with guanylate cyclase, are of the simple “9 + 2” type. Thus metabolic properties which are exclusively common to all sperm types having guanylate cyclase are as yet unidentified. The results presented here for protein kinase activity in sea urchin sperm soluble fractions are similar to those reported by Lee and Iverson (31). Our activities, however, are lo-fold higher. Although neither guanylate cyclase nor cyclic GMP-sensitive protein kinases have been found in mammalian sperm, cyclic GMP has been detected in bovine epididymal sperm (32). Cyclic GMP has been measured in sea urchin sperm (33) in amounts similar to those reported here. With sperm from various mammalian species, exogenously added phosphodiesterase inhibitors and/or cyclic nucleotides or their analogs have been reported to stimulate sperm motility, respiration, and the utilization of certain substrates (32, 3440). Cyclic AMP-sensitive protein kinases (41, 42), cycl:ic AMP (32, 36, 37, 39,43), and cyclic AMP,-degrading phosphodiesterase (44) have been measured in sperm from various mammalian species. An understanding of the metabolic or functional significance of cyclic nucleotide in these cells will, hopefully, be forthcoming. REFERENCES 1. GRAY, J. P., HARDMAN, J. G., BIBRING, T., AND SUTHERLAND, E. W. (1970) Fed. Proc. 29, 608 (abstract). 2. HARDMAN, J. G., AND SUTHERLAND, E. W. (1969) J. Viol. Chem. 244, 6363-6370. 3. GRAY, J. F’., HARDMAN, J. G., HAMMER, J. L., Hoes, R. T., AND SUTHERLAND, E. W. (1971) Fed. Proc. 30, 1267 (abstract). 4. CHRISMAN,, T. D., GARBERS, D. L., PARKS, M. A., AND HA~MAN, J. G. (1974) J. BioZ. Chem. 250, 374-381. 5. KIMURA, H., AND MURAD, F. (1975) J. Biol. Chem. 2,49, 6910-6916. 6. BIRNBAUMER, L., POHL, S. L., AND RODBELL., M.
METABOLISM
29
IN SPERM
(1969) J. Biol. Chem. 244, 3468-3476. 7. DRUMMOND, G. I., AND DUNCAN, L. (1970) J.
Biol. Chem. 245,976-983. 8. TAO, M., AND LIPMANN,
F. (1969) Proc. Nut.
Acad. Sci. USA 63,86-92. 9. FLAWIA, M. M., AND TORRES, H. N. (1972) J.
Biol. Chem. 247,6873-6879 10. GRAY, J. P., AND DRUMMOND, G. I. (1973) Proc. Can. Fed. Biol. Sot. 16, 79. 11. NELSON, L. (1972) Biol. Reprod. 6.319-324. 12. BUTCHER, R. W., Ho, R. J., MENG, H. C., AND SUTHERLAND, E. W. (1965) J. Biol. Chem. 240, 4515-4523. 13. BEAVO, J. A., ROGERS, N. L., CROFFORD, 0. B., HARDMAN, J. G., SUTHERLAND, E. W., AND NEWMAN, E. V. (1970) Mol. Pharmacol. 6,
597-603. of Semen and 14. MANN, T. (1964) The Biochemistry of the Male Reproductive Tract, Methuen, London. 15. CHAN, P. S., BLACK, C. T., AND WILLIAMS, B. J. (1973) Anal. B&hem. 55, 16-25. 16. ISHIKAWA, E., ISHIKAWA, S., DAVIS, J. W., AND SUTHERLAND, E. W. (1969) J. Biol. Chem. 244, 6371-6376. 17. BEAVO, J. A., HARDMAN, J. G., AND SUTHERLAND, E. W. (1970) J. Biol. Chem. 245, 56495655. 18. Kuo, J. F., AND GREENGARD, P. (1970) J. BioZ.
Chem. 245, 2493-2498. 19. HARDMAN, J. G., DAVIS, J. W., AND SUTHERLAND, E. W. (1969) J. Biol. Chem. 244, 63546362. 20. BROADUS, A. E., KAMINSKY, N. I., HARDMAN, J. G., SUTHERLAND, E. W., AND LIDDLE, G. W. (1970) J. Clin. Invest. 49, 2222-2236. 21. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioZ. Chem. 193, 265-275. 22. IVES, D. H., MORSE, P. A., AND POTTER, V. R. (1963) J. Biol. Chem. 238,1467-1474. 23. GARBERS, D. L., SUDDATH, J., AND HARDMAN, J. (1975) Biochim. Biophys. Actu 377,186-196. 24. TASH, J. S., AND MANN, T. (1973) Proc. Roy. Sot. Lond. B 184.109-114. 25. GOLDBERG, N. D., DIETZ, S. B., AND O’TOOLE, A. G. (1969) J. Biol. Chem. 244, 4458-4466. 26. Kuo, J. F., WYATT, G. R., AND GREENGARD, P. (1971) J. Biol. Chem. 246, 7159-7167. 27. GRIDIS, C., VIRMAUX, N., URBAN, P. F., AND MANDEL, P. (1973) FEBS Lett. 30, 163-166. 28. GIBBONS, B. H., AND GIBBONS, I. R. (1972) J. Cell
Biol. 54, 75-97. (Metz, C. B., 29. MANN, T. (1967) in Fertilization and Monroy, A., eds.), Vol. 1, Academic Press, New York. (Metz, C. B., 30. NELSON, L. (1967) in Fertilization and Monroy, A., eds.), Vol. 1, Academic
GRAY ET AL.
30
Press, New York. 31. LEE, M. Y. W., AND IVERSON, R. M. (1972) Ewp. Cell Res. 75, 300-304. 32. GARBERS, D. L., LUST, W. D., FIRST, N. L., AND LARDY, H. A. (1971) Biochemistry 10, 1825 1831. 33. BLOMQUIST, C. H., HADDOW, M. K., O’DEA, R. F., HADDEN, J. W., AND GOLDBERG, N. D. (1973) J. Cell Biol. 59, Part 2, 27 (abstract). 34. GARBERS, D. L., FIRST, N. L., SULLIVAN, J. J., AND LARDY, H. A. (1970) Biol. Reprod. 5,336-
339. 35. DREVIUS, L. 0. (1972) J. Reprod. Fert. 28,41-54. 36. GARBERS, D. L., FIRST, N. L., AND LARDY, H. A. (1973) Biol. Reprod. 8, 589-599. 37. GARBERS, D. L., FIRST, N. L., GERMAN, S. K., AND LARDY, H. A. (1973)Biol. Reprod. 8,599-
606. 38. HOSKINS, D. D. (1973) J. B’>l phem. 248, 11351140. 39. MORTON, B., HARRIGAN-LUM, J., ALBAGLI, L., AND Jooss, T. (1974) B&hem. Biophys. Res.
Commun. 56, 372-379. 40. LI, T.-K. (1972) Ltfe Sci. 11, 939-948. 41. GARBERS, D. L., FIRST, N. L., AND LARDY, H. A. (1973) J. Biol. Chem. 248, 875-879. 42. HOSKINS, D. D., CASILLAS, E. R., AND STEPHENS, D. T. (1972) B&hem. Biophys. Res. Commun. 48, 1331-1338. 43. CASIUAS, E. R., AND HOSKINS, D. D. (1971) Arch. B&hem. Biophys. 147, 148-155. 44. CASILLAS, E. R., AND HOSEINS, D. D. (1970) Bio-
them. Biophys. Res. Commun. 40.255-262.
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
0.005
0.035
tus) Scallop Abalone Clam Tube worm Vertebrate sperm Mammals: human, dog Fish: salmon, herring Mammalian lung Rat
a Enzyme derived from 0.1 mg (invertebrates) to 2.0 mg (vertebrates) wet weight of packed sperm were assayed as described in Materials and Methods. Values reported are the averages from two separate preparations of each sperm type, each assayed in triplicate. Sperm were tested from four species of salmon, pink, coho, Chinook and sockeye.
activity was totally recovered in particulate fractions. As shown in Table I, the activities of sea urchin or scallop sperm, when assayed with Triton, were scarcely detectable with Mg2+, being less than 2% of the values with Mn2+. With abalone, clam, or tube worm sperm, activities were lo-20-fold less with MgZ+ than with Mn2+ (Table I). In one experiment (data not shown) lOO,OOOg(1 h) supernatant fluids and particles were prepared from sperm homogenates (no Triton) of sea urchin (L. pictus), scallop, abalone, clam and the four species of salmon. Supernatant solutions were assayed with Mn2+ and Triton and contained less than 2% of guanylate cyclase activities measured in the original homogenates. As measured with Mg2+ and Triton, Mn2+ without Triton, or Mn2+ and Triton, particulate activities were identical to those of the original homogenates. Particulate ac-
tivity was not detected without added divalent cation, either with or without Triton. These results indicate that (a) the enzyme was totally particulate, (b) the failure to detect activity in fish sperm homogenates was not likely due to the presence of a soluble inhibitor, and (c) the low activities with Mg2+ and Triton in abalone or clam sperm required the added Mg2+. In other experiments with sea urchin sperm, no activity was detected when Mn2+ was replaced by Ca2+, 10 mM NaF had no effect using either Mn2+ or Mg2+, and the enzyme was stable to storage at -80°C for at least a year unless repeatedly frozen and thawed. Finally, as shown in Table I, the guanylate cyclase activity of rat lung homogenate, one of the most active mammalian tissue preparations (2), was several hundred-fold less than the activities of invertebrate sperm. Adenylate
Cyclase
The adenylate cyclase activities of invertebrate sperm (Table II) assayed with Mn2+, were several hundred-fold less than their guanylate cyclase activities (Table I). Whereas guanylate cyclase was detected only with invertebrate sperm, adenylate cyclase was found in sperm of invertebrates, fish, and mammals. Activities with Mn2+ were somewhat similar among these sperm types except for the relatively low values obtained from human and clam. The activity of rat brain, one of the most active mammalian tissues, was severalfold greater with Mn2+ than were those of sperm. The sperm enzyme, assayed with Mn2+, was not significantly affected by 10 mM NaF. The activity of human, fish, or invertebrate sperm with Mg2+ was also unaffected by fluoride (data not shown). Sperm activity was at least &lo-fold greater with Mn2+ than with Mg2+ (10 mM), with the exception of tube worm and abalone. With Mn2+, the adenylate cyclase activity of invertebrate sperm was increased, that of mammalian sperm was unaffected, and that of fish sperm was decreased by Triton X-100. With invertebrate or fish sperm, cyclic AMP did not accumulate linearly with time, even for 5 min at 37°C. This was not
CYCLIC NUCLEOTIDE TABLE II ADEN~ATE
CYCLASE IN INVERTEBRATE AND VERTEBRATE SPERM”
Organism
Adenylate cyclase activity (pmol of cyclic AMP/mg wet wff5 min at 37°C) Mg2+ Mn2+ Mn*+
Invertebrate sperm Sea urchin (L,.
NiF
Mn2+ Tr&n
10
115
100
150
130
210 35 180
240 50 205
505 145 230
2.5 15 <15 <15 cc15 15
15 105 125 165 190 370
15 120 155 N.D.b 210 415
15 105 100 100 95 185
250
500
2500
2500
pi&us) Tube worm Clam Abalone Vertebrate sperm Human Dog Pink salmon Sockeye salmon Coho salmon Herring Mammalian brain Rat
LIOnce-thawed sperm homogenates from invertebrates or fish (2 mg of packed sperm) were assayed by assay A. The activities of human or dog sperm were determined by assay B and contained 5-10 mg wet weight of sperm per ml. Values are expressed as picomoles of cyclic AMP accumulated over a 5-min interval because velocities in the case of invertebrate and fish sperm were not linear with time (see text and Fig. 1). b Not determined.
METABOLISM
IN SPERM
25
time courses resembled those in Fig. 1. The reason for this nonlinearity is not known. In one experiment, the effect of GTP was examined. Conditions were as in Fig, 1, except that ATP was 1 mM and Mn2+ was 5 mM. Activity was unaffected by 0.1 or 0.2 mM GTP but was inhibited 20-30% by 1 mM GTP at all time points. The poor ability of GTP to inhibit the enzyme provides evidence that adenylate cyclase is likely a separate enzyme from guanylate cyclase in invertebrate sperm since cyclic GMP formation from GTP proceeded at a far higher rate than did cyclic AMP formation from ATP under these conditions. Additional evidence that these enzymes are separate was provided by the observation that they were oppositely affected by high levels of colchicine. Thus, in two experiments, adenylate cyclase activity of S.purpuratus sperm (with Mn2+) was inhibited by 10 mM colchicine. Inhibition was greater in the presence of 1.0% Triton (equalling 43 and 70% in the two experiments) than in the absence of Triton (where it equalled 17 and 27%). In contrast, the guanylate cyclase activity of these sperm was stimulated by colchi&e, but only at levels greater than 1 mM, especially in the absence of Triton. Thus, despite the fact that both activities were totally particulate, highly dependent on Mn2+ compared to Mg2+, stimulated by Triton, and unaffected by fluoride, it is likely that they represent separate enzymes.
due to hydrolysis of ATP; less than 10% of [14ClATP was degraded in 10 min (determined by paper chromatography (22)). Neither was the failure to achieve linearity with time due to degradation of cyclic AMP by phosphodiesterase. Adenylate cyclase was not inhibited by accumulating product, because similar velocities were obtained in the absence or presence of 2 mM added cyclic AMP. An example of this nonlinearity with time is presented in Fig. 1. Results are from one of six experiments done in an attempt to dilute the enzyme to an extent where linearity with time might be achieved. In additional experiments, activities were measured with sperm homogenates or 30,OOOg(30 min) particles incuFIG. 1. The Mn*+-dependent adenylate cyclase acbated at concentrations ranging from 0.1 tivity of sea urchin sperm. Sperm from S. purpurato 2.0 mg wet weight of sperm per ml. tus were incubated at a concentration of 0.1 mg wet Regardless of enzyme concentration, all weight of packed sperm per ml in assay B.
26
GRAY ET AL.
This conclusion is supported cent studies (23).
by other re-
Cyclic Nucleotide Phosphodiesterase Sperm from a variety of invertebrate and fish species were examined for their ability to degrade 1 PM cyclic AMP or cyclic GMP, and the results are presented in Table III. Invertebrate and fish sperm, all of which contain adenylate cyclase activity, degraded cyclic AMP at rates comparable to, or up to 20-fold geater than, the rate of cyclic AMP degradation by homogenates of rat brain, one of the most active mammalian tissues (17). Invertebrate sperm, which contained high levels of guanylate cyclase activity, also degraded cyclic GMP at high rates. In contrast, fish sperm, which did not contain detectable TABLE
III
CYCLIC NUCLE~TIDE PHOSPHODIESTERASEOF INVERTEBRATE AND VERTEBRATE SPERM Organism
Phosphodiesterase activity (pm01 of cyclic A(G)MP degradedmg wet timin at 30°C) Cyclic AMP
Invertebrate sperm Sea urchin @.purpurutus) Sea urchin (L. pi&us) Clam Scallop Abalone Fish sperm Salmon (pink, chinook, coho, sockeye) Herring Mammalian brain* Rat
67 67 145 23 20
Cyclic GMP
594 470 244 27 19
112
0.9
570
0.1
30
83
a Frozen preparations were thawed, diluted and rehomogenized in GG-Na-K-EDTA-D’lT medium containing 1% bovine serum albumin. Albumin was necessary for the preservation of phosphodiesterase activity in dilute enzyme solutions. Homogenates were assayed by Assay A. Values are the average of each sperm type, except in the case of salmon sperm where one preparation of each of the four species was used. b Values for the activities of rat brain homogenate were taken from the report of Beavo et al. (17).
guanylate cyclase activity, were unable to degrade cyclic GMP at appreciable rates. Thus, there seems to be a correlation between the capability of a given sperm type to form either cyclic AMP or cyclic GMP and its ability to degrade that nucleotide. Sperm of S. purpuratus hydrolyzed 1 PM cyclic GMP about nine times faster than cyclic AMP (Table III). When homogenates of these sperm were centrifuged at 30,0009 for 30 min two-thirds of the activity against cyclic AMP was in the supernatant fraction, whereas three-fourths of the activity against cyclic GMP was in the particles. The effects of several agents on phosphodie&erase activity were examined, and the results are presented in Table IV. Triton X-100 (l.O%), inhibited the hydrolysis of both nucleotides, but the hydrolysis of cyclic GMP was more strongly inhibited than was that of cyclic AMP. Colchicine (30 mM) inhibited the hydrolysis of both nucleotides. The degradation of 1 PM cyclic r3H]AMP was inhibited by 1 PM unlabeled cyclic GMP. On the other hand, unlabeled cyclic AMP only slightly inhibited the degradation of cyclic 13HlGMP. The Cyclic AMP and Cyclic GMP Contents of Sea Urchin Sperm Sea urchin sperm that were acid fixed in different states of motility contained cyclic AMP ranging from 0.1 to 0.7 nmol per g of wet packed sperm (Table V). Sperm that were fixed following 15 min after dilution in aerated artificial sea water in which the sperm were highly motile consistently contained higher concentrations of cyclic AMP than did sperm which were fixed without prior dilution (immotile sperm) or following dilution in 19:l solution which contained 25 mM KC1 and in which sperm are poorly motile (11). The cyclic AMP concentration in very motile sperm was not significantly greater than that in poorly motile sperm as determined by a paired t test (p < 0.1). The results, however, are highly suggestive of a real difference between these groups. A positive correlation between cyclic AMP levels and motility had been reported to exist with mammalian sperm (24). Cyclic GMP levels in these
CYCLIC NUCLEOTIDE
METABOLISM
TABLE
27
IN SPERM
IV
EFFECT OF SEVERAL AGENTS ON PHOSPHODIESTERASEACTIVITY 3H-labeled cyclic nucleotide (1.0 CLM) Cyclic t3HlAMP
Cyclic 13HlGMP
Cell fraction
Phosphodiesterase Control
Homogenate Particles Supernatant Homogenate Particles Supernatant
100 100 100 100 100 100
Triton (1.0%) 92 80 85 14 14 27
activity
(% relative
Colchicine (30 InM) 10 7 <5 <5 -
to control)
cGMP (1.0 /.bM)
CAMP (1.0 /AM)
49 24 -
-b 81 84 -
a Sperm from S. purpurutus were homogenized in nine volumes of GG-Na-K-EDTA-D’l”I’ and centrifuged at 30,OOOgfor 30 min. The supernatant fluid and resuspended pellet were assayed by the method of Beavo et al. (17). * Where dashes (-) occur, activity was not determined. TABLE V CYCLIC AMP AND CYCLIC GMP CONTENT OF SEA URCHIN SPERM Sperm preparation
Immotile Poorly motile Very motile
Cyclic nucleotide (nmol/g wet wt) Cyclic AMP
Cyclic GMP
0.17 2 0.05 0.20 T 0.03 0.43 * 0.11
0.38 + 0.25 0.47 k 017 0.42 f 0.13
a Five samples of semen from S. pzqurotus were strained free of debris, centrifuged 10 min at 12,OOOg (4°C) and the pellets were treated in one of three ways. (i) Immotile sperm: pellets were suspended in acid (containing tracer 25 ml of 0.3 N perchloric cyclic 13H]AMP and cyclic [3HlGMP) and homogenized 1 min in a Waring blendor. (ii) Poorly motile sperm: pellets were resuspended in nine volumes of 25 mM KC1 and 475 mM NaCl(15”). Fifteen minutes after suspension, sperm were acid fixed and homogenized. (iii) Very motile sperm: the pellet was treated as described in (ii) except that the suspension medium was aerated artificial sea water. Following acid fixation, preparations were centrifuged at 30,OOOgfor 30 min. Supernatant solutions were assayed for cyclic nucleotides as described in Materials and Methods. Values are means of five different preparations 2 SEM.
sperm were more variable, ranging from 0.04 to 0.89 nmol per g of sperm (Table V>. A relationship between motility state and cyclic GMP concentration could not be discerned. Protein Kinase The ability of cyclic AMP or cyclic GMP, in concentrations ranging from 10 nM to 10
pM, to stimulate histone kinase activity of either whole homogenate or supernatant fractions (lOO,OOOg,1 h) of sea urchin sperm was examined (Fig. 2). All values reported in the figure were obtained in the presence of histone, Mg2+, and Triton X100. In the homogenate, detectable stimulation was observed with concentrations of cyclic AMP and/or cyclic GMP as low as 0.1 PM and 1 PM, respectively. Activity was stimulated at least fivefold by maximally effective levels of cyclic AMP (1 PM). Apparent K, for cyclic AMP was about 0.5 PM. Homogenate activity with maximally effective cyclic AMP equalled about 3 nmol of 32P transferred to protein per mg of
FIG. 2. Protein kinase activity of sea urchin sperm. Homogenates of S. purpurutus sperm were centrifuged for 1 h at 100,OOOgfor preparation of the supernatant fraction. Enzyme from homogenate (25 pg of protein) or supernatant solution (5-50 pg of protein) was assayed for protein kinase activity as described in Materials and Methods. Histone was phosphorylated linearly with incubation time. Values are means 2 SEM of values obtained in four or five separate experiments.
28
GRAY ET AL.
sperm protein per min at 30°C. In the supernatant fractions, detectable stimulation was again observed with concentrations of cyclic AMP as low as 0.1 PM. In contrast to the results obtained with homogenate, however, detectable stimulation of the supernatant activity required very high levels of cyclic GMP, approaching 10 PM. Maximally effective levels of cyclic AMP (1 PM) increased the activity at least 25fold, yielding a specific activity of about 25 nmol of 32Pper mg protein per min. Thus, the specific activity of protein kinase, determined with maximally effective cyclic AMP, was increased about lofold in the supernatant solution relative to that in the original sperm homogenate. With soluble activity, the apparent K, for cyclic AMP was similar to that obtained in the homogenate (about 0.5 PM). The K, for cyclic GMP was not estimatable but was apparently much higher even than in the homogenate, being perhaps as high as 10 PM. A fresh homogenate of tube worm sperm, assayed with histone, Triton, and Mg2+, had protein kinase activity of 0.7 nmol of [32Pl per mg of protein per min without cyclic nucleotide and 8.4 with 10 PM cyclic AMP. When histone was omitted, values with and without cyclic AMP were 0.5 and 0.4, respectively. When sea urchin sperm homogenates were separated into supernatant and particulate fractions (lOO,OOOg, 1 h), 83% of the recovered activity was in the supernatant, fluid as determined with either 10 PM cyclic AMP or cyclic GMP. The studies show that sea urchin sperm contained an active soluble histone kinase which was sensitive to stimulation by levels of cyclic AMP that were found to be present in these cells (Table V). The enzyme was stimulated by cyclic GMP also but only at levels which were far higher than the levels of this cyclic nucleotide found in these cells.
occur widely in nature (2, 16, 18, 19, 25, 26). The finding of high guanylate cyclase activity in invertebrate sperm suggests that some metabolic system involving cyclic GMP is concentrated there. The most active nonsperm preparation of guanylate cyclase found has been the outer rod segments from retina (27), where the enzyme is only an order of magnitude less active than that in invertebrate sperm. A large percentage of sperm structure and function is related to its flagellum, and one might consider the possibility that cyclic GMP may be involved in flagellar motility. However, neither cyclic GMP nor cyclic AMP affects motility of demembranated sperm (28). Solubilization and removal of guanylate cyclase from sperm is effected by treatment with Triton X-100. However, such treatment does not render sperm immotile in medium containing ATP and Mg2+ (28). Thus, the cyclic GMP system seems to have no direct role in the motility mechanism. What metabolic properties shared by invertebrate sperm, with high guanylate cyclase, are absent in the vertebrate sperm, with no guanylate cyclase? Guanylate cyclase is not present in all sperm types that are dependent on oxidation of endogenous substrate, since these include both invertebrate and fish sperm which are shed into environments where exogenous glycolyzable substrates are virtually absent (14, 29). Neither do all sperm that are shed into sea water, with its high salt content, contain guanylate cyclase, since herring sperm dwell in sea water and would likely require guanylate cyclase if cyclic GMP were vital to ionic balance mechanisms in the sperm cell membrane. Sperm of most lower life forms are designated as “9 + 2” type since the only longitudinal fibers within their flagella are those in the microtubule set (axoneme) that are common to almost all flagella and cilia (29,30). Sperm of mammals, snakes, and of some birds, DISCUSSION molluscs, and insects contain (in addition No specific metabolic roles for cyclic to the axoneme) nine outer y fibers, comGMP in any cell type have been defined, posed of the protein “spermosin” which is although guanylate cyclase, cyclic GMP, not microtubular, and are designated as “9 cyclic GMP-degrading phosphodiesterase, + 9 + 2” type (29, 30). It has been specuand cyclic GMP-activated protein kinases lated that the axoneme may be somewhat
CYCLIC
NUCLEOTIDE
vestigial in “9 + 9 + 2” sperm, not serving directly to generate the flagellar propulsive force (30). Thus, it would be significant if only “9 + 9 + 2” sperm lacked guanylate cyclase. This relationship does not hold, however, since sperm of fish, without guanylate cyclase, as well as those of the tested invertebrates, with guanylate cyclase, are of the simple “9 + 2” type. Thus metabolic properties which are exclusively common to all sperm types having guanylate cyclase are as yet unidentified. The results presented here for protein kinase activity in sea urchin sperm soluble fractions are similar to those reported by Lee and Iverson (31). Our activities, however, are lo-fold higher. Although neither guanylate cyclase nor cyclic GMP-sensitive protein kinases have been found in mammalian sperm, cyclic GMP has been detected in bovine epididymal sperm (32). Cyclic GMP has been measured in sea urchin sperm (33) in amounts similar to those reported here. With sperm from various mammalian species, exogenously added phosphodiesterase inhibitors and/or cyclic nucleotides or their analogs have been reported to stimulate sperm motility, respiration, and the utilization of certain substrates (32, 3440). Cyclic AMP-sensitive protein kinases (41, 42), cycl:ic AMP (32, 36, 37, 39,43), and cyclic AMP,-degrading phosphodiesterase (44) have been measured in sperm from various mammalian species. An understanding of the metabolic or functional significance of cyclic nucleotide in these cells will, hopefully, be forthcoming. REFERENCES 1. GRAY, J. P., HARDMAN, J. G., BIBRING, T., AND SUTHERLAND, E. W. (1970) Fed. Proc. 29, 608 (abstract). 2. HARDMAN, J. G., AND SUTHERLAND, E. W. (1969) J. Viol. Chem. 244, 6363-6370. 3. GRAY, J. F’., HARDMAN, J. G., HAMMER, J. L., Hoes, R. T., AND SUTHERLAND, E. W. (1971) Fed. Proc. 30, 1267 (abstract). 4. CHRISMAN,, T. D., GARBERS, D. L., PARKS, M. A., AND HA~MAN, J. G. (1974) J. BioZ. Chem. 250, 374-381. 5. KIMURA, H., AND MURAD, F. (1975) J. Biol. Chem. 2,49, 6910-6916. 6. BIRNBAUMER, L., POHL, S. L., AND RODBELL., M.
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