Kopf, G. S., Tubb, D. J., and Garbers, D. L. (1979) J. Biol. Chem. 5. Hansbrough, J. R., Kopf, G. S., and Garbers, D. L. (1980). 2235-2241. 254,8554-8560.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.
Vol. 261, No. 16, Issue of June 5, pp. 7327-7331 1986 Printed in ~ 3 . A .
Evidence for a Guanine Nucleotide-binding Regulatory Protein in Invertebrate andMammalian Sperm IDENTIFICATION BY ISLET-ACTIVATING PROTEIN-CATALYZEDADP-RIBOSYLATION AND IMMUNOCHEMICAL METHODS* (Received for publication, January 21, 1986)
Gregory 5. Kopf@, Marilyn J. WoolkalisT]l,and George L. GertonS From the $Division of Reproductive Biology and Endocrinology, Department of Obstetrics and Gynecology, and the 7Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Spermatozoan function has been shown to be modulated, The abalone sperm adenylate cyclase does not appear in part, by cyclic nucleotides (1, 2). In the sea urchin, it is to be regulated by guanine nucleotides, but hasa M$+supported catalytic activity similar to other hormone- known that biological factors associated with eggs that alter and guanine nucleotide-regulated enzymes (Kopf, G. sperm functions, such as motility (3), metabolism (4-6), and S., and Vacquier, V. D. (1984) J. Biol. Chem. 259, the induction of the acrosome reaction (7), also alter cyclic 7590-7596; Kopf, G. S., and Vacquier, V. D. (1985) nucleotide metabolism. These second messengers may meBiol. Reprod. 33,1094-1104). The present studies diate the response of the sperm to extracellular biological were undertaken to ascertain whether the abalone enfactors in a manner analogous to hormone-receptor-adenylate zyme has associated guanine nucleotide-binding regu- cyclase interaction in somatic cells. In mammals, reproductive latory proteins. Membrane fractions were incubated tract- or egg-associated factors alter sperm function through with either islet-activating protein (IAP) or cholera unknown biochemical processes(8-11). toxin and analyzed by sodium dodecyl sulfate SDSThe regulatory mechanisms controlling CAMPsynthesis in polyacrylamide gel electrophoresis for thepresence of sperm are poorly understood. Invertebrate and mammalian toxin-catalyzed ADP-ribosylated proteins. The supernatant from a Lubrol PX-extracted 48,000 X g pellet sperm display Ca2+-dependentadenylate cyclase activity that fraction contained a M, = 41,000 IAP substrate. This may be controlled, in part, by calmodulin (7,12-17). Enzyme substrate could not be ADP-ribosylated prior to deter- activity has also been shown to be regulated by extracellular gentextraction.Lubrol PX-solubilized fractions of effectors of sperm function which alter sperm Ca2+conductmembrane preparations from mouse, bovine, and hu- ance; such an effectoris the fucose-sulfate glycoconjugate man sperm also contained a M,= 41,000 IAP substrate. from sea urchin egg jelly (7, 18). Hormones, which act via the These proteins co-migratedon sodium dodecyl sulfate- adenylate cyclase in somatic cells,havenoeffecton polyacrylamide gels with the M, = 41,000 wsubunit activity of the sperm enzyme (1).The sperm enzyme is also of the inhibitory guanine nucleotide-binding regula- unaffected by fluoride, guanine nucleotides, choleratoxin plus tory protein (Gi) from transformed chicken embryo NAD’, and forskolin (1, 15, 17,19).The apparent absence of fibroblast and mouse 5-49 lymphoma membrane ex- guanine nucleotide regulation suggests that the sperm enzyme tracts. The sperm IAP substrates displayed similar is either devoid of guanine nucleotide-bindingregulatory proprotease digestpatterns to ai of mouse5-49 lymphoma teins or that, under the experimental conditions utilized, these cells. Sea urchin sperm analyzed ina similar manner proteins are functionally uncoupled fromthe catalytic unit of contained a M, = 39,000 I A P substrate. Cholera toxin- the enzyme complex. Recently, HiIdebrandt et al. (20), have catalyzed ADP-ribosylation of specific sperm mem- suggested that the mammalian sperm adenylate cyclase sysbrane proteins wasnot observed in any of the sperm tem lacks both stimulatory (G:) and inhibitory (GJ guanine preparations tested. The presenceof the 8-subunit com- nucleotide-binding regulatory proteins and is not able to mon to both the stimulatory and inhibitory guanine nucleotide-binding regulatory heterotrimers wascon- interact with G. from exogenous sources. These studies, in firmed in sperm using an antiserum directed against addition to previous investigations, suggest that the sperm the purified &subunit of the guanine nucleotide-bind- enzyme exists as a free catalytic moiety (1,Zl). These findings appear to be consistent for allof the sperm adenylate cyclases ing regulatory proteins from bovine brain. It is concluded that all of the sperm tested, withthe studied thus far, with the exception of the abalone enzyme. possible exception of sea urchin sperm, contain a Gi- This particular enzyme is not stimulated by fluoride, guanine like protein. Additional properties of these proteins nucleotides, cholera toxin, or forskolin (17), but displays a and theirrole(s) in spermfunction are currentlybeing Mg+-supported catalytic activity similar to that reported for examined. hormonally responsive adenylate cyclase and contains both a
* This investigation was supported by National Institutes of Health Grants HD-19096 (to G. S. K.), HD-20736 and HD-06274 (to G. L. G.), and a grant from the Andrew Mellon Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. § To whom correspondence and reprint requests should be addressed. I(Supported by Grant Pol-GM-34781.
’ The abbreviations used are: GB,stimulatory guanine nucleotidebinding regulatory protein; G,, inhibitory guanine nucleotide-binding regulatory protein; GO,the M,= 39,000 guanine nucleotide-binding regulatory protein from bovine brain; ai,the a-subunit of Gi; a,,the a-subunit ofG.; IAP, islet-activating protein; CEF/RSV, chicken embryo fibroblasts transformed with Rous sarcoma virus; SDS, sodium dodecyl sulfate; cyc-, a variant line of mouse S-49 lymphoma cells deficient in GB;Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.
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MgATP catalytic site and a separate Mg2’ regulatory site (16, 17). The studiesin the presentcommunication were undertaken to examine the mechanism by which the abaloneenzyme displays high Me-supported catalytic activity. We have probed for Gi and G, in abalone sperm, using toxin-catalyzed ADP-ribosylation. We report that abalone sperm, as well as sperm from mouse, bull, and human, contain a M , = 41,000 IAP substrate. Sea urchin sperm contain a M , = 39,000 IAP substrate. Sperm also contain the @-subunitcommon to the guanine nucleotide-binding regulatory proteins as shown by immunochemical methods. It is concluded that both invertebrate and mammalian sperm contain a Gi-like protein. MATERIALS A N D METHODS~ RESULTS
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Identification of a Sperm Substrate for ZAP-catalyzed ADPribosylation-Sperm fractions were assessed for the presence FIG. 1. SDS-polyacrylamide gel electrophoresis of abalone of specific substrates for IAP-catalyzed ADP-ribosylation by sperm extracts incubated in either the absence or presence of incubation in theabsence or presence of the toxin. All sperm IAP. A 48,000 X g pellet fraction was prepared and extracted with preparations and ADP-ribosylations were carried out in the 1%Lubrol PX. ADP-ribosylation was then carried out in either the absence (lanes a, c, e, and g ) or presence (lanes 6, d , f , h, and i) of presence of protease inhibitors since it has been previously IAP. Lanes a and b, 5 pg of 48,000 X g pellet; lanes c and d , 5 pg of demonstrated using reconstitution assays thatsperm contain Lubrol PX-extracted 48,000 X g supernatant; lanes e and f, 9.5 pg of a trypsin-like protease which interferes with the inhibitory Lubrol PX-extracted 48,000 X g supernatant; lanes g and h, 5 pg of modulation (presumably through Gi)of hormonally sensitive Lubrol PX-extracted 48,000 X g pellet; lane i, 25pgof CEF/RSV adenylate cyclases (20, 32-35). When 48,000 x g pellet frac- membranes. Numbers on the left side of the figure represent the M, of the protein standards. Arrows indicate the origin and dye tions of abalone spermhomogenates were assayed, no specific (X of the CEF/RSV IAP substrates are front of the gel. The M , ( X IAP substrateswere detected (Fig. 1,lane b). However, if these shown on the right side of the gel. pellet fractions were subsequently extracted with Buffer A detergent-soluble containing 1%Lubrol PX and the resultant a b c d e f and particulate fractions were assayed, a M , = 41,000 IAP substrate was observed in the detergent-soluble fraction (Fig. 1 r41 1, lanes d and f). The pellet after detergent extraction displayed little, if any, M , = 41,000 IAP substrate (Fig. 1, lane L39 h).The M , = 41,000 detergent-soluble protein co-migrated on FIG. 2. The presence of IAP substrates in Lubrol PX exone-dimensional SDS-polyacrylamide gels with ai of CEF/ tracts of sperm from different species. Lane a, 5 pg of abalone RSV membrane preparations (Fig. 1, lane i). extract; lane b, 5 pgof sea urchin extract; lane c, 5 pgof mouse Our ability to detecta M , = 41,000 ai-like protein in abaloneextract; lane d , 3 pg of human extract; lane e, 5 pg of bovine extract; of the CEF/ sperm encouragedus toexamine other invertebrate and mam- lane f, 25 pgof CEF/RSV membranes. The M , ( X maliansperm for the presence of thisprotein. Recently, RSV substrates are indicated in the figure. Hildebrandt et al. (ZO), suggested that mammalian spermlack Gi. However, the membrane fractions that they analyzed had the sperm membrane fractions display IAP substrateactivity not been detergent-extracted and it is clear from our data prior to detergent extraction. The presence of the M , = 41,000 (Fig. 1) that the M , = 41,000 proteincannot be detected IAP substrates in mouse and human sperm is not due to the without prior Lubrol P X extraction. Furthermore, the inclu- contamination of the sperm preparations with somatic cells, sion of protease inhibitors in thesperm extraction andADP- since these substrates were observed in detergent extracts of ribosylation procedurescould be critical in preserving Gi, since both mouse sperm purified through PercollO gradients and this protein may be a substrate for sperm trypsin-like prohuman sperm prepared by swim-up methods. In both cases, teases (20, 32-35). contamination of these sperm preparations by somatic cells When sea urchin, mouse, human, or bovine sperm memwas less than 0.5%. The bovine sperm preparations were brane fractions were assayed for IAP substrates, it was ob- weakly labeled by IAP, as demonstrated by the faintexposures served that the Lubrol PX-extracted 48,000 X g supernatant of the autoradiograms (Fig. 2, lane e). The low level of ADPfractions from these sperm contained a substrate specific for ribosylation of the M , = 41,000 IAP substrate in bovine sperm this toxin (Fig. 2, lanes b-e). The sperm IAP substrates from may be due to the fact that these cells were obtained 1-2 h all of these species, with the exception of the sea urchin,co- postejaculation and limitedproteolysismayhaveoccurred migrated with the M , = 41,000 protein of abalone sperm and prior to the washing and extraction of the cells. The sea ai of CEF/RSV (Fig. 2, lanes a and f). In no case did any of urchin sperm IAP substrate migrated with an apparent molecular weight of 39,000 (Fig. 2, lane b). This lower molecular The “Materials and Methods” are presented in miniprint a t the weight IAP substrate has been observed in a number of sea end of this paper. Miniprint is easily read with the aid of a standard urchin sperm preparations and also is present in sea urchin magnifying glass. Full size photocopies are available from the Journal eggs (data not shown). It can be concluded that both inverof Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-179, cite the authors, and include a tebrate and mammalian sperm contain a single protein that check or money order for$2.00 per set of photocopies. Full size is a specific substrate for IAP-catalyzed ADP-ribosylation. Peptide Mapping of the ZAP Substrates from Sperm and photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. Mouse S-49 Lymphoma Cells-The datain Figs. 1 and 2
S p e r m Guanine Nucleotide Regulatory Proteins
7329
suggest that sperm contain an ai-like protein. To confirm the A B identity of these sperm proteins, limited proteolytic digestion 2 0 s patterns of the sperm IAP substrates were compared with those of ai from mouse S-49 lymphoma cell membrane prep94arations. Fig. 3 demonstrates that the a-chymotrypsin, V-8 protease, and trypsindigest patterns of the human spermM , 66= 41,000 IAP substratewere similar to patterns obtained from aiof mouse S-49 lymphoma cells. Digests of the IAP sub45strates from abalone and sea urchin sperm also yielded peptides similar to S-49 % (data not shown). These dataprovide additional evidence that the sperm IAP substrate %-like. is Identification in Sperm Membrane Preparations of the pSubunit Common to the Guanine Nucleotide-binding Regula26tory Proteins-Other investigators havesuggested that thepsubunit of Gi is required for the ADP-ribosylation of aiby 1% IAP (36). T o confirm the presence of the P-subunit, mouse sperm membrane proteinswere prepared and analyzed by the method of Towbin et al. (28) using an antiserum thatrecogFIG. 4. Immunoblot analysis of a mouse sperm membrane nizes the P-subunit of the guanine nucleotide-binding regu- extract using an antiserum that recognizes the ,%subunit of latory proteins (22). A M , = 35,000 protein of mouse sperm the guanine nucleotide-binding regulatory proteins. Lane A , membranescross-reactedwiththisantiserumand co-mi- 100 pgof CEF/RSV membranes; lane B , 100 pgof mouse sperm extract. The positive immunoreactive M , = 35,000 0-subunit of the grated with the P-subunit from CEF/RSV membranes (Fig. guanine nucleotide-binding regulatory protein heterotrimer of CEF/ 4). No cross-reactivity with preimmune serum was observed RSV membranes is indicated on the right side of the gel. Numbers on (data not shown). the left side of the figure indicate the M , (X of the protein Absence of Sperm Substrates for Cholera Toxin-catalyzed standards. Arrows indicate the origin and dye front of the gel. ADP-ribosylation-Sperm were extracted and analyzed for the presenceof specific substrates for cholera toxin-catalyzed lymphoma cyc- cells do not containa, but do containa factor ADP-ribosylation. No specific substrates were detected in any that promotes ADP-ribosylation in other a,-containing memof the fractions assayed (data not shown). In other experibrane fractions (37, 38). Additional experiments were carried ments, sperm 48,000 X g pellet fractions were assayed in the out with a cytosolic factor frombovine testis which promotes We were unable presence of membrane fractions frommouse S-49 lymphoma cholera toxin-catalyzed ADP-ribo~ylation.~ cyc- cells. Under these conditions,specific cholera toxin sub- to demonstratespecific cholera toxin substrates under anyof strates would be derived from sperm since the mouse S-49 these conditions (data not shown).
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FIG. 3. Peptide maps of the human sperm and mouse S-49 lymphoma cell M, = 41,000 IAP substrates. The supernatant fraction of the Lubrol PX-extracted 48,000 X g pellet of human sperm and mouse S-49 lymphoma cell membrane extracts were incubated with [32P]NADand IAP, and the proteins were separated by SDSpolyacrylamide gel electrophoresis on 10% gels. The bands corresponding to the M , = 41,000 IAP substrates were excised and run on 15% SDS-polyacrylamide gels in eitherthe absence of proteases (lane a) or in presence of 4 pg of a-chymotrypsin (lane b), 4 pg of V-8 protease (lane c ) , or 4 pg of trypsin (lane d ) by the method described by Cleveland et al. (29). The gels were then subjected to autoradiography. Numbers on the left side of the figure indicate the M , ( X of the protein standards. Arrows indicate the origin and dye front of the gel. The M , ( X W 3 ) of the IAP substrate is indicated between the two panels.
DISCUSSION
The data in this report suggest that sperm containa Gi-like protein. This conclusion is supported by experiments demonstrating that: 1) IAP catalyzes the ADP-ribosylation of a M , = 41,000 protein in detergent-soluble extracts of sperm membrane fractions, 2) peptide maps of IAP substrates from sperm and mouse S-49 lymphoma cells are similar, and 3) sperm membrane fractions contain a M , = 35,000 protein which cross-reacts with an antiserum that recognizes the Psubunit common to the guanine nucleotide-binding regulatory proteins. Recently, Hildebrandt et al. (20) concluded that sperm do not contain the inhibitory guanine nucleotide-binding regulatory protein, as evidenced by a n inability to demonstrate the presence of any IAP substrate (20). However, they did not use detergent-extracted fractions in their studies and it is clear from the results presented in this report Lubrol that PX extraction solubilizes the IAP substrate and makes this protein susceptible to IAP-catalyzed ADP-ribosylation. Furthermore, protease inhibitors in the buffers used for sperm fractionation and ADP-ribosylation may be important for preserving Gi since it has been reported that mammalian sperm contain a trypsin-likeprotease(named“ninhibin”) which appears to stimulate somaticcell adenylate cyclase in a manner consistent with attenuating the inhibitory regulation of the enzyme mediated via Gi (32-35, 39). If Gi serves as a substrate for ninhibin, sperm IAP substrate activity could be greatly reduced or even lost when conditions to limit proteolysis are not met. Although a M , = 41,000 IAP substrate in Lubrol P X extracts of bovine sperm is detected (Fig. 2), the M. J. Woolkalis and D. M. Gill, unpublished results.
7330
Sperm Guanine Nucleotide Regulatory Proteins
6. Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 1447-1452 7. Kopf, G. S., and Garbers, D.L. (1980) Biol. Reprod. 2 2 , 11181126 8. Bavister, B. D. (1982) J. Androl. 3,365-372 9. Lenz, R.W., A x , R. L., Grimek, H. J., and First, N.L. (1982) Biochem. Biophys. Res. Commun. 106, 1092-1098 10. Bleil, J. D., and Wassarman, P. M. (1983) Deu. Biol. 95,317-324 11. Bradley, M. P., and Garbers, D. L. (1983) Biochem. Biophys. Res. Commun. 115,771-787 12. Braun, T. (1975) J. Cyclic Nucleotide Res. 1, 271-281 13. Watkins, H. D., Kopf,G. S., and Garbers, D. L. (1978) Biol. Reprod. 19,890-894 14. Hyne, R. V., and Garbers, D.L. (1979) Biol. Reprod. 2 1 , 11351142 15. Hyne, R. V., and Lopata, A. (1982) Gamete Res. 6,81-89 16. Kopf, G. S., and Vacquier, V. D. (1984) J. Bwl. Chem. 259,75907596 17. Kopf, G. S., and Vacquier, V. D. (1985) Biol. Reprod. 3 3 , 10941104 18. Garbers, D. L., Kopf, G. S., Tubb, D. J., and Olson, G. (1983) Biol. Reprod. 29, 1211-1220 19. Forte, L. R., Bylund, D.B., and Zahler, W. L. (1983) Mol. Pharmacol. 24,42-47 20. Hildebrandt, J. D., Codina, J., Tash, J. S.,Kirchick, H. J., Lipschultz, L., Sekura, R. D., and Birnbaumer, L. (1985) Endocrinology 116,1357-1366 21. Ross, E. M., and Gilman, A. G. (1980) Annu. Rev. Biochem. 4 9 , 533-564 22. Woolkalis, M. J., Nakada, M. T., and Manning, D. R. (1986) J. Biol. Chem. 261,3408-3413 23. Freedman, M. F., and Kopf, G. S. (1985) Biol. Reprod. 3 2 , 322332 24. Schumacher, M., Schafer, G., Holstein, A. F., and Hilz, H. (1978) FEBS Lett. 91,333-338 25. Sullivan, J. J., Elliot, F. I., Bartlett, D. E., Murphy, D. M., and Kuzdas, C. D. (1966) J. Dairy Sci. 4 9 , 1569-1571 26. Cassel, D., and Pfeuffer, T. (1978) Proc. Natl. Acud. Sci. U. S. A. 75,2669-2673 27. Laemmli, U. K. (1970) Nature 2 2 7 , 680-685 28. Towbin, H., Staehlin, T.,and Gordon, J. (1979) Proc. Nutl. Acad. Sci. U. S. A. 76,4350-4354 29. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252,1102-1106 Acknowledgments-We wish to thankDr. David R. Manning, Dept. 30. Bradford, M. M. (1976) Anal. Biochem. 74,248-254 of Pharmacology, for his valued scientific expertise throughout the 31. Schaffner, W., and Weissman, C. (1973) Anal, Biochern. 56,502course of these studies and for providing us with CEF/RSV and 504 mouse S-49 lymphoma (wild type and cyc-) membranes and the anti- 32. Johnson, R.A., Jakobs, K.H., and Schultz, G. (1985) J. Biol. Chem. 260,114-121 @ antiserum. Wealso wish to thankDr. Robert Kenney (New Bolton Center) and Chuck Allen (Atlantic Breeders Service, Lancaster, PA) 33. Johnson, R. A., Awad, J. A., Jakobs, K. H., and Schultz, G. (1983) FEBS Lett. 1 5 2 , l l - 1 6 for providing the bovine sperm. The excellent technical assistanceof Nancy Schwab and Elana Braz is gratefully acknowledged. We wish 34. Hanski, E., and Garty, N. B. (1983) FEBS Lett. 162,447-452 to thank Dm. Bayard T. Storey and David R. Manning for critically 35. Jakobs, K. H., Johnson, R. A., and Schultz, G. (1983) Biochim. Biophys. Acta 756,369-375 reading the manuscript. 36. Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Bid. Chem. 259,14222-14229 REFERENCES 37. Johnson, G. L., Kaslow, H. R., and Bourne, H. R. (1978) J. Bid. 1. Garbers, D. L.. and KoDf, Chem. 253,7120-7123 _ .G. S. (1980) . . Adu. Cyclic Nucleotide Res. 13,251-306 38. Kahn, R. A., and Gilman, A. G. (1984) J. Biol. Chem. 259,62282. Tash. J. S.. and Means. A. R. (1983) Biol. Revrod. 28. 75-104 6234 3. Hansbrough, J. R., and’aarbers, D. L. (198l)j. Biol. Chem. 2 5 6 , 39. Anand-Srivastava, M. B., Johnson, R. A., Picard, S., and Cantin, 2235-2241 M. (1985) Biochern. Biophys. Res. Commun. 129,171-178 4. Kopf, G. S., Tubb, D. J., and Garbers, D. L. (1979) J. Biol. Chem. 40. Sternweis, P. C., and Robishaw, J. D. (1984) 3. Biol. Chem. 2 5 9 , 254,8554-8560 13806-13813 5. Hansbrough, J. R., Kopf, G. S., and Garbers, D.L. (1980) 41. Audigier, Y.,Pantaloni, C., Bigay, J., Deterre, P., Bockaert. J.. and Homburger, V. (1985) FEBS Lett. 1 8 9 , 1-7 Biochim. Biophys.Acta 630,82-91
weak signal may be due, in part, to the fact that the sperm were 1-2 h old prior to washing and extraction in the buffers containing the protease inhibitors. Experiments using freshly ejaculated bull sperm may resolve this issue. Unlike the other sperm tested, sea urchin sperm appear to have an TAP substrate of M , = 39,000. Further studies willbe required to determine whether this protein represents a proteolytic fragment of ai,the a-subunit of GO(36, 40), or a totally unique protein. In addition to demonstrating that sperm contain an IAP we have also shown that these substrate which is similarto ai, cells contain a M , = 35,000 protein which displays immunoreactivity with an antiserum that recognizes the Mr = 35,000 @subunit common to theguanine nucleotide-binding regulatory proteins (Fig. 4).Although the presence of the @-subunit in spermmay be implied by the fact that thissubunit appears to be required for IAP-catalyzed ADP-ribosylation of aiin other tissues (36), it was necessary to probe for this subunit since a recent report had suggested that ram sperm were devoid of this protein (41). At this time, we have found no compelling evidence for the existence of G, in sperm. This is inagreement with the data of Hildebrandt et al. (20) and is also supported by the conclusions of others who have examined the guanine nucleotide regulation of the sperm enzyme (1).The fact that the sperm adenylate cyclase does not reconstitute with purified G, from an exogenous source implies thatthis enzyme may have unique regulatory properties (20). However, one cannot dismiss the possibility that additional regulatory ligands in sperm are required for efficient coupling to the catalytic moiety of the enzyme. It is concluded that sperm contain a Gi-like protein. The role of this protein in sperm is currently unknown, but its potential importance in the regulation of adenylate cyclase, motility, capacitation, and theacrosome reaction is currently being investigated.
Sperm Guanine Nucleotide Regulatory Proteins SupplememaJ Materialto
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Pharmacology) and were prepared by the appropriate methods: chicken embryo fibroblasts S-49 lymphomacalls (wild type and cy0 transformed with Rous sarcuma v i ~ (CEF/RSV) s and mouse (22). A partially purified cytosolic factor prepared from bovine testis3 was agifl from Dr.D. Michael Gill Vufts University School of Medicine).
EVIDENCE FORA GUANINE NUCLEOTIDE-BINDING REGULATORY PROTEIN IN INVERTEBRATE AND MAMMALIAN SPERM IDENTIFICATIONBY ISLET ACTlVAllNG PROTEINCATALYZED ADP-RIBOSYLATION AND IMMUNOCHEMICAL METHODS
Gregory S. Kopl, Marilyn J. Woolkells, and George L. GeRon
MATERIALS AND METHODS
Materials. GTP, guanylylimidodiphosphate, NAD, NMN, NAD pymphosphorylase and thymidine were from Boehringer-Mannheim. Cholera toxin and islet-activating prolein (IAP Bordetella Penossis toxin) were from Schwarz-Mann and List Biological Laboratories. Inc.. respectively. [u-~~PI-ATP (3000 Ciimmol) was obtainedfrom Amersham-Searle. Lima beant w i n inhibitor and u-chymotrypsin were from Worthington Biochemicals. V-8 protease (S.aureos)was from Miles. Trypsin, molecular weight standard proteins. 4 chloro-1-naphthol and Hz02 were from Sigma. Prestained molecular weight standard pmteins were obtained from Bethesda Research Laboratories. Aflinity-purified, horseradish pemxidaseanjugatedgoat anti-rabbit IgG wasfrom Bio Rad Laboratories. NtroCellUloSS paper was of highest purchased from Schleicher and Schuell. All other reagents were q u a l i and were obtained psubunit the of the guanine either from Sigma or Fsher. Rabbit antiserum that recognizes nucleotide-binding regulatory proteins from bovine brain was a gin from Dr. DavidR. Manning (Dept.of Pharmacology). The reactivityof this antibody has been previously characterized (22).
Extraction of Sperm for ADP-Rlbosyletlon Studles.Sperm, washed as described above, were extracted in the following manrar. All procedures were carried out on im or at 4%. Sperm were 10 Tis-HCI. 1mM dithiothreitol. ( 0 mM resuspendedto 510% (vh or wh) in a buffer containing mM NaCI, 20% (vh) glycerol, 1mM EGTA, 10pM phenylmethylsulfonylliuoride,10 w/ml leupeptin. 10 pg/ml aprotinin. 1 mM pamirmbenzamidine, 1mgml soy bean trypsin inhibitor, pH 7.9 (Buffer A). In wme cases 1 mgmi lima bean trypsin inhibitorwas substituted for thesoy bean trypsin inhibitor. The cell suspension was then homogenized with I O passes of a tight fitting dounce homogenizer. and then soniaed using a BransonicO Sanifier(setling3,5 3;sec blasts). The homogenate was centrifuged at 1000 x g (10 min). p the ellet discarded and the supernatant centrifuged again in the same manner. The then centrifugedat 48.000 x g (1 h), and the supernatant discarded. The resultant supematant was of the suspension placed in pellet was resuspended its to originalvolume with ButferA and ons-third another tube. Both fractions were rmntrifuged at x g (1h) and the supernatants discarded. Buffer Awas a m to the smaller pellet which was resuspended. homogenized and labeled "48 K pellet.. The larger pellet was resuspended in Buffer A containing % Lubrol 1 PX, homogenized. and formin - 1 h. This suspensionwas then centrifugedat allowed tosit on ice with occasional siirring 45 fraction removed and labeled "48 K Lubrol SuperMtant-, and the pellet 48,000 x g(Ih), the supernatant fraction resuspended in Buffer A and labeled' 4 8 K Lubrolpellet.. The tractions were then either used immediatelyor frozen in aliquots at -8oOC.
48.oW
ADP Rlbosylatlon. [32P]- NAD was synthesized essentially by the method of Cassel and Pfeuffer (26). using [U.~~P]-ATP (3000 Ciimmol) in the presence ot an ATP regenerating system ( Wruvate
khase and phosphoenolpyruvate). The radiolabeled produn was purified with a linear formic acid (0 200 mM) gradient and purity was assessed by thin layer chromatography on polyethyleniminezellulow, using 0.15 M NH4HCO3 Toxins (0.3 mgml cholera toxin or 0.12 rfI@mlIAP) were activated for 30 min at 30% in 30 mM Hepes, 24 mM dithiothrebl,l.2 mg/ml bovine serum albumin, 0.15% SDS, p~ 7.5. Sourcesand Preparationof Cells. Abalone (Haliotisrutexens) and sea urchin out in a total volume of 30 PI containing either 50 pglml cholera toxin or ADPribosylations were canied sperm were obtained and washedCa2+-frm in artificial sea waterby 20 pg/ml IAPin 5 mM H e w (pH 7.5). I mM ATP, 100PM guanylylimidodiphospate (with cholera PreViOUSlydescribed methods (7,16). toxin). 100pM GTP (with IAP), 5mM MgC12. ImM EDTA, 5 mM dithiothreitol, 10 mM thymidine. 0.2 Human sperm were obtained from heathy donors with normal semen parameters and washed by mgml bovine serum albumin, 0.025%SDS, 1 mg/ml lima bean trypsin inhibitor.50 pgml aprotinin,50 well established methods (23). Highly purified populations of motile human sperm were obtained by w/ ml leupeptin. 1mM paminobenzamidine, 5pM [32V-NAD (1.5- 3.0 x IO4 cpnV p m l ) and the resuspending the freshly liquefied ejaculate in 2 MIBiggers. Whitlingham and Whiffen medium, pH appropriate amountof cell prolein. ADP-ribasylations were CamM out in both the absence and 7.56 containing3 mg/ml bovine serum albumin, splitting the suspension in thirds. and centrifuging for 5 I h (30OC). Reactions were terminated by the addition ofpl10 of 4X sample presenceof the loxins for min at200 x g. The supernatant was then removed and the resuitant loose sperm pellets carefully 3 min. for The samples were then subjected to buffer (27). followed by immersion in a boiling water bath pi of the above medium.m e tubes were then placed at a 45O angle in an incubator overlaid with 250 SDS.polyauylamide gel electrophoresis as described below. (5% Cop in air) for h. 1 The supernatants containing the motile sperm that swam outOf the pellet were the for the then removedand the resunant motile sperm suspension ObSewed under micmsmpe immurmblot analysis with antibodies directed against ImmunoblotUngof Sperm Extracts. For presem of non-spermcelk. The sperm suspensions were then combined and centrifuged atx g Mx) the pabun%of the guanine nucleotide-binding reguhiory proteins, purifmd mouse sperm were for 10 min. the supernatant removed, the cells resuspended in 0.9% NaCl. and the spin repeated. After b e d above.4 '8K pellets" were prepared as detailed forthe ADP-ribosylation preparedas this semnd spin the resultant pellets ware extracted with Bufler A dssaibed as below. studies. with the exception that soy bean and lima bean inhibtors trypsin were omined from Buffer A Sperm from Swiss-Wehsler mice (Harian) were obtained by minting the epididymides and The resultant membrane suspensions were analyzed by SDS-polyacrylamide gel electrophoresisas 6 mM allowing the sperm to swim out into Eagle's minimal essential medium supplemented with described below.The electrophoretically-separated proteins were then transfened nlrwllulme to (28) sodium lactate, 1mM sodium pyruvate and 15mM Hepes, pH 7.2. Vas sperm were prepared by and antigen-antibody complexes were visualizedthe by methods usedby Woolkalis af ai. (22). stripping the vasa deferentia into the above medium. Vas and epididymal sperm were combined, pm Nitex0 scfeen and washed once by centrifugation [1,000 x g tor 15 min at finered through a 112 e lelectrophoresis was canied out ElectrophoreticMethods. DisoantinuousSDS POlyaCrylamde g 6OC). Punlieds p e n preparations were obtained by centrifuging the suspended pellets over by the methodof Laemmli (27), using 10%gek. Gelswere then stained with Coomassie R-250 and discontinuous Peroal1° gradients. These gradients contained steps of 25%. 50%,75% and 100% autoradiography was performed using Kodak XRP-1 X-ray film with Dupont CmnexO intensifying Perml1° diluted as desuibedby Schumacher etal(24) using the above medium. Sperm, free of erythrocyte and other somatic cell comamination, were recovered at the 500/.-75% Perml1° interlace weens (-8OOC). Proteolytic digest patterns of the IAP substrates were carried out by the method of Cleveland at d. and then washed twice with the above medium to remove the Percollo. (29). Aner destaining the initial 10% gel,rqlions the of the gelmntainirg the [32P]-ADP.ribosylat& a milk extender Ejaculated bovine sperm were obtained and stored lor a periodof 1-2 h in Skim 41,000 Mr bands were cut out and then run in the absence or presence of either trypsin, a-chymotrypsin 4 MI 01 a buner (25) priorto being washedby the following method. The ejaculate was diluted with on 15%SDS polyacrylamide gels. Digestion of the proteins in the stacking gel was or V-8 protease pgmi soy bean trypsin containing to mM Hepes, 150 mM NaCI. 1 pgiml apmtinin, 1 pg/ml leupeptin.10 canied out lor30 min. inhibitor. 5mM benzamidine, pH 7.0 and centrifugedat 700 x g for10 min (24OC). The resultant supernatant was discarded, the pellet resuspended to the original volume with the above buffer theand centrifugation repeated. The sperm pellets were then stored at -8OOC. (30) Yscellancous Methods. Protein concentrations were determined by the method of Bradford The following crude membrane fraclions were obtained from Dr. David R. Manning (Dept. of as a standard. or Schaffner and Weissman (31). using bovine serum albumin
