Guinea pig testicular proacrosin-acrosin system: Preliminary immunological characterization

Journal of Reproductive Immunology, 15 (1989) 241--256

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Elsevier Scientific Publishers Ireland Ltd. JRI 00603

Guinea pig testicular proacrosin-acrosin system: Preliminary immunological characterization Ekundayo A.O. Falase, Adeyemi O. Adekunle*, Heather Neblett and Cory Teuscher Division of Reproductive Biology and Endocrinology, Department of Obstetrics and Gynecology, University of Pennsylvania School o f Medicine, Philadelphia, PA 19104-6080 (U.S.A.)

(Accepted for publication 25 April 1989)

Summary In this study, immunochemical techniques were employed to examine the guinea pig (GP) testicular proacrosin-acrosin system. Monospecific polyclonal antibodies to the highly stable enzymatically active 34,000 molecular weight form of G P testicular acrosin were generated. Western blot analysis o f acid extracts prepared from snap-frozen freshly excised G P testes revealed two major immunoreactive bands with mol. wts o f approximately 62,000 and 48,000 and one minor band with an approximate mol. wt o f 54--56,000. The 62,000 mol. wt molecule identified is in close agreement with the previously reported mol. wt for purified G P testicular proacrosin. Western blot analysis o f different species o f testicular acid extracts demonstrated the evolutionary relatedness of the proacrosin-acrosin systems since immunoreactivity was observed primarily with acid extracts from rodent species (rat, mouse and hamster) and not with extracts from evolutionarily less-related species (goat, ram and bovine). The majority o f the cross-reactivity observed was characterized by immunoreactivity with the 62,000 and 48,000 mol wt molecules. The only species which exhibited cross-reactivity with the 54--56,000 mol. wt protein was rat. In addition, the *Present address: Department of Obstetrics and Gynecology, University College Hospital, lbadan, Nigeria. **Address all correspondence to: Cory Teuscher, Ph.D. Division of Reproductive Biology and Endocrinology, 314 John Morgan Building, 36th and Hamilton Walk, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6080, U.S.A.

0165-0378/89/$03.50 © 1989ElsevierScientific Publishers Ireland Ltd. Published and Printed in Ireland

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iso-immunogenic and aspermatogenic properties of the 34,000 mol. wt form of GP testicular acrosin were examined. One out of five Hartley and one out of seven Strain 2 female GPs immunized and boosted with a total of 200 /ag of purified protein exhibited increased levels of circulating anti-acrosin iso-antibodies. The antigenic specificity of the iso-antibodies observed in the two animals was verified by Western blot analysis. All other female animals, including two strain 13 GPs, exhibited very low or undetectable levels of such antibodies following immunization. One out of three male Hartley GPs immunized with 50 /ag of the purified protein exhibited typical lesions of experimental allergic orchitis while none of a group of three animals developed lesions at a 5 /ag dose. Taken together, these results suggest that the 34,000 mol. wt form of GP testicular acrosin is neither a highly potent iso-immunogen nor aspermatogenic autoantigen. Key words: Proacrosin/acrosin system; Immunochemicai terization; Iso-immunogenicity; Autoimmune orchitis.

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Introduction

Acrosin (EC3.4.21.10) is a trypsin-like proteinase present in the acrosomes o f m a m m a l i a n spermatozoa which is believed to be responsible for enabling the spermatozoon to penetrate the zona pellucida o f the ovum during fertilization (Fritz et al., 1973; McRorie and Williams, 1974). In addition, it has been suggested that it may also play a role in facilitating the acrosome reaction (Draviland et al., 1984; Huang et al., 1985) as well as serving as a sperma zona receptor via its lectin-like activity which is independent o f and separable from the proteolytic properties o f the enzyme (Topfer-Peterson and Henschen, 1988). The majority o f the acrosin that is present in freshly excised testes and epididymides as well as freshly ejaculated sperm exists as the activatable zymogen precursor proacrosin (Parrish and Polakoski, 1979). In the boar proacrosin-acrosin system, proacrosin runs as a characteristic doublet with a mol. wt o f approximately 53,000/55,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Polakoski and Parrish, 1977). Following either autoactivation or activation with trypsin, the zymogen is converted into lower mol. wt forms o f enzymatically active acrosin. The principal forms have been designated alpha-acrosin, beta-acrosin and gamma-acrosin with mol. wts o f 49,000, 34,000 and 25,000, respectively (Polakoski and Parrish, 1977). Recently, H a r d y et al. (1987) purified the various forms o f proacrosin present in both GP testes and epididymides. GP testicular proacrosin was

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reported to have a mol. wt of approximately 62,000 whereas two forms of proacrosin with mol. wts of 54,000/56,000 and 43,009 were isolated from GP epididymides. Immunological studies using monospecific polyclonal antibodies to the testicular form of proacrosin (62,000) demonstrated that it is antigenically related to the two forms of proacrosin isolated from epididymal sperm (54,000/56,000 and 43,000). Similarly, antibodies to the 43,000 mol. wt form of epididymal sperm proacrosin cross-reacted with the other two forms, however to a lesser extent. Simultaneously, we reported (Adekunle et al., 1987) the purification and characterization of a 34,000 mol. wt form of enzymatically active GP testicular acrosin which appears to be analogous to beta-acrosin in the boar system. In fact, GP testicular beta-acrosin has an amino acid composition similar to that reported for boar beta-acrosin and cross-reacts with polyclonal monospecific antibodies to the same. However, this enzyme has been shown to differ from other mammalian acrosins in that it exhibits weak activation by Ca 2~, is active in the presence of ethylene glycol bis-(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and does not demonstrate the expected "burst" of product in a single enzyme turnover with the active site titrant 4-methylumbelliferyl-p-guanidinobenzoate (Adekunle et al., 1989). While the functional significance of these differences are not known, the possibility exists that they may arise out of structural differences in the enzyme which are not directly reflected in the mol. wt of the molecule as determined by SDS-PAGE. Recently, Siegel et al. (1987) used immunochemical methods to examine the boar and human proacrosin-acrosin systems and demonstrated that specific structural differences occur in the proteins of the proacrosinacrosin systems of the two species. In this report, polyclonal monospecific antibodies to the 34,000 mol. wt form of GP testicular beta-acrosin were generated and used to examine its relationship to the various molecular weight forms (62,000, 54,000/56,000 and 43,000) of GP testicular proacrosin purified by Hardy et al. (1987). The immunologic cross-reactivity of the various proteins associated with the testicular proacrosin-acrosin systems of different species was also examined. In addition, both the iso-immunogenic and aspermatogenic properties of the protein were examined by immunizing female and male GPs, respectively. Materials and methods

Reagents GP testicular acetone powder was obtained from Rockland Inc. (Gilbertsville, PA); Sephadex G-100, SP-Sephadex and Concanavalin A-

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Sepharose were obtained from Pharmacia Fine Chemicals Inc. (Piscataway, N J). The N-benzyloxycarbonyl-L-arginyl amide (CBZ-Arg) of 7amino-4-trifluoromethylcoumarin (AFC) was obtained from Enzyme System Products (Livermore, CA). N,N-Dimethylformamide (DMF) (photorex spectrophotometric grade) was obtained from J.T. Baker Chemical Co. (Phillipsburg, N J). Complete and incomplete Freund's Adjuvant (CFA and ICFA, respectively), 3-aminobenzamidine and bovine serum albumin (BSA) were obtained from Sigma Chemical Co. (St. Louis, MO). Mycobacterium tuberculosis Type H37Ra was obtained from Difco Laboratories (Detroit, MI). All other reagents were obtained from standard commercial sources.

Purification o f GP testicular acrosin and testicular acid extracts All procedures were carried out at 0 - - 4 ° C , unless stated otherwise. GP acrosin was purified from testicular acetone tissue powder, and fluorimetric assays carried out as previously described by Adekunle et al., 1987. Briefly, GP acrosin was purified following acid extraction, pH precipitation of the soluble extract, gel filtration on Sephadex G-100, ion exchange chromatography on SP-Sephadex, and affinity chromatography on Concanavalin A-Sepharose followed by re-chromatography on Sephadex G-100. The enzyme was concentrated and stored in aliquots of 1 ml at pH 3.0 in 1 mM HC1 at - 70°C. Freshly collected snap-frozen testicular tissue was homogenized at high speed in a Waxing blender in 25 volumes (w/v) of distilled deionized water contained 1 mM 3-aminobenzamidine, adjusted to pH 3.0 by the dropwise addition of concentrated HC1. The acid extracts were maintained at pH 3.0 and stirred slowly at 4°C for 12 h. The mixture was centrifuged at 10,000 × g for 30 mins, the supernatant collected and dialysed against 1 mM HC1. Protein concentrations throughout the study were determined spectrophotometricaUy as described by Layne (1957). Production o f anti-acrosin antibody Five female New Zealand white rabbits (2--3 kg) were used to produce polyclonal monospecific antisera to the purified 34,000 mol. wt form of GP testicular beta-acrosin. Prior to immunization each rabbit was bled, the serum collected, pooled and used as the source of preimmune control sera throughout the study. Each rabbit was immunized with 50/~g of purified GP testicular beta-acrosin emulsified in CFA supplement with M. tuberculosis H37Ra at a concentration of 9 m g / m l . The emulsion was distributed i.d. among 10--12 sites on the back of each rabbit. All animals were boosted s.c. with 50 /~g of antigen in ICFA 20 days later and screened for the production of anti-acrosin antibodies using a solid phase radioimmunoassay (Teuscher et al., 1985) 30

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days after the initial injection. All five animals were found to be producing equivalent high titers of anti-acrosin antibody and were immediately killed by exsanguination under anaesthesia. The immune sera was collected, pooled and stored along with the preimmune control serum pool in appropriate aliquots at - 7 0 ° C .

Electrophoresis Aliquots of the various antigen preparations were lyophilized in the presence of 1 mM 3-aminobenzamidine and redissolved in 0.0625 M Tris --HCI (pH 6.8), 1001o glycerol, 0.001070 bromophenol blue containing l mM 3-aminobenzamidine, 10 ~g/ml leupeptin and 10 /~g/ml aprotinin. Analytical SDS-PAGE was carried out according to Laemmli (1970) in gels containing 1 mM 3-aminobenzamidine. Immunoblotting was carried out according to the method of Towbin et al. (1979), with the following modifications. The transfer buffer was 0.025 M ethanolamine adjusted to pH 9.5 with glycine and contained 20070 (v/v) methanol (Szewczyk and Kozloff, 1985). Transfer took place from 16 to 18 h at 10°C at 30 V (Hoefer Transphor, San Francisco, CA). The nitrocellulose sheets were washed in TBS (0.5 M NaCl, 20 mM Tris--HCl, pH 7.5) for l0 min (Woolkalis et al., 1986). Non-specific sites were blocked by incubating the blots in 5070 Carnation non-fat dry milk dissolved in TBS (Johnson et al., 1984) for 30 min. The blots were then incubated in primary serum (pre-immune serum or antiserum) diluted in 5°70 milk in TBS. After 2.5 h, the blots were washed twice in TBS (10 min each) and then incubated in affinity-purified horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) or peroxidase-conjugated goat anti-GP IgG diluted in 5°7o milk in TBS. After 2.5 h, the blots were washed 2 x 10 min in TBS. The bound peroxidase was then detected with 4-chloro-l-naphtol as substrate. Molecular weights were estimated using prestained proteins standards with a molecular weight range of 14,30(0-200,000 (Bethesda Research Labs, Inc., Bethesda, MD).

Assessment of iso-immunogenicity and aspermatogenic activity Adult female Hartley, Strain 13 and Strain 2 GPs were immunized subcutaneously in multiple sites on the back with 100/~g of purified testicular beta-acrosin in CFA on day 0 and 100/~g in ICFA 4 weeks later. Ten days after the second injection, all animals were killed by exsanguination under anaesthesia. Prior to immunization each GP was bled, the serum collected, pooled and used as the source of pre-immune control serum. The post-immunization sera was collected, pooled and stored along with the preimmune control serum pool in the appropriate aliquots

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at - 7 0 ° C . Circulating anti-acrosin iso-antibody levels were assayed using a solid phase radioimmunoassay (Teuscher et al., 1985). Regression analysis and statistical comparisons of the slopes were performed using the computer program Statsworks (Heyden and Sons, Philadelphia, PA). A value of P < 0.05 was taken as statistically significant. Adult male Hartley GPs were immunized subcutaneously in both hind footpads with either 50.0/ag or 5.0/ag of purified testicular beta-acrosin or 0.5 /ag of purified AP3 (Teuscher et al., 1983) in CFA. To prepare

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the injection emulsion, the various immunogens were dissolved in PBS and emulsified with an equal volume of CFA supplemented with a standard amount (/ag dry weight) of M. tuberculosis H37Ra. Each GP received the dose of immunogen in 0.10 ml containing 100/ag M. tuberculosis. Control animals received CFA alone. Thirty days after immunization, the animals were killed, the testes fixed in Bouins' fixative and embedded in paraffin; 5 /am thick sections were stained with hematoxylin-eosin. The testes were examined for the presence of autoimmune orchitis, aspermatogenesis, epididymitis and/or vasitis, the classical lesions of EAO (Tung and Alexander, 1977). Results

The titration curve of the solid phase radioimmunoassay of the pooled pre-immune and anti-testicular beta-acrosin antiserum pool can be seen in Fig. 1. The results demonstrate that the anti-beta-acrosin antiserum pool is capable of detecting antigen (1 /ag/well) in a dilution range of 1 : 10 to 1 : 10,000 whereas the pre-immune control serum pool does not exhibit antigen specific immunoreactivity. Preimmune sera were screened against GP testicular acrosin and the irrelevant antigen, p-guanidinobenzoylated cytochrome c. Similar levels of non-specific binding were observed with both antigens. The utility of the antiserum pool in Western blot analysis of the GP testicular proacrosin-acrosin system under non-reducing conditions was verified by using partially purified (Concanavalin-A Sepharose binding fraction) GP testicular beta-acrosin. The results of Western blot analysis under non-reducing conditions can be seen in Fig. 2a. The preimmune control pool failed to demonstrate immunoreactivity in Western blots with partially purified testicular betaacrosin when analysed at the same dilution and concentration of antigen as tested with the antiserum pool. (Fig. 2b). Western blot analysis under non-reducing conditions of GP testicular acid extracts obtained from fresh frozen tissue revealed two major immunoreactive bands with mol. wts of approximately 62,000 and 48,000 and one minor band with an approximate mol. wt of 54--56,000 (Fig. 3). Similarly, the 62,000 and the 48,000 molecular weight range bands were also the two major immunoreactive bands observed in Western blots of acid extracts of rat, mouse and hamster testes. Dog and rat testes acid extracts also exhibited minor bands in the 54m56,000 mol. wt range while the rat singularly demonstrated cross-reactivity with a 38,000 tool. wt range protein. In contrast, goat and ram testes acid extracts failed to demonstrate the presence of any cross-reactive antigens (Fig. 3). However, Western blots with bovine testes acid extracts

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revealed cross reactivity with a single band with a mol. wt less than 18,000. The same patterns o f antigens with similar relative intensities o f immunoreactivity among the bands were seen with all testes acid extracts when studied at antigen concentrations as high as 100 /ag/lane. Western

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blots analyzed with the pre-immune control serum pool exhibited weak immunoreactivity with an unrelated minor band with a molecular weight of 68,000 in rat, mouse and hamster testes acid extracts but not in GP, goat, ram, bovine or dog testes extracts (data not shown). One out of five Hartley and one out of seven Strain 2 female GPs immunized with testicular beta-acrosin exhibited significant levels of circulating iso-antibodies to the enzyme (Fig. 4). The antigenic specificity was confirmed by Western blot analysis which revealed immunoreactivity with the purified immunogen (Fig. 5). All other female GPs exhibited very low or undetectable levels of such antibodies.

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One out of three male Hartley GPs immunized with 50.0/ag of purified testicular beta-acrosin demonstrated the presence of inflammatory infiltrate of the testes accompanied by extensive aspermatogenesis, i.e. autoimmune orchitis (Fig. 6). All other GPs at both the 50.0/ag and 5.0 /ag doses failed to exhibit such lesions. Three out of three positive con-

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trol animals immunized with 0.5/~g of purified AP3 demonstrated extensive inflammatory infiltrates of the testis, epididymis and vas deferens while no such lesion were observed in the negative controls, i.e. animals immunized with CFA alone.

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Fig. 6. Histopathology of the testis from a GP immunized with 0.5 gg of purified AP3 in CFA showing severeorchitis, desquamationoftesticutar cells and aspermat°genesis" (Haematoxylin-eosin; x 200).

Discussion We have generated monospecific polyclonal antibodies to highly purified GP testicular beta-acrosin and have demonstrated that the immunogen appears to be antigenically related to the 62,000 mol. wt form of proacrosin purified from GP testes by Hardy et al., (1987). This conclusion is based on the results of Western blot analysis, under nonreducing condition, of GP testicular acid extracts prepared from fresh snap-frozen testes. Such immunoblots revealed cross-reactivity with a major band corresponding to a mol. wt of approximately 62,000. Hardy et al., (1987) reported that the predominant form of proacrosin present in GP testes was 62,000 whereas 54,000/56,000 and 43,000 mol. wt forms were predominant in epididymal sperm. In addition to the 62,000 mol. wt form of proacrosin, the anti-beta acrosin also demonstrated sig-

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nificant immunoreactivity with a 48,000 mol. wt molecule as well as minor reactivity with a molecule in the 54--56,000 mol. wt range. The exact relationship between the 62,000 mol. wt form of proacrosin and the other two immunoreactive molecules observed in our study is unknown. They may however be analogous to the 54,000/56,000 and 43,000 mol. wt forms of proacrosin purified by Hardy et al., (1987) from GP epididymal sperm. Hardy et al., (1987) suggested that the differences in the molecular weight forms of proacrosin observed between the testes and epididymis may be a function of epididymal maturation. If the two immunologically cross-reactive molecules observed in Western blots of crude acid extracts of GP testes are molecular weight variants of proacrosin, then the modifications associated with the appearance of these forms presumably occur during spermatogenesis rather than during epididymal maturation. In this regard, Arboleda and Gerton (1988) using Western blot analysis with anti-boar acrosin antisera demonstrated that a 54,000/56,000 mol. wt form of proacrosin was the predominant form present in round spermatids whereas a 51,000/55,000 molecular weight species appears to be the major form present in the more mature condensing spermatids. The discrepancy in mol. wts between our results and Hardy et al., (1987) and those of Arboleda and Gerton (1988) may be due, in part, to the fact that Arboleda and Gerton determined the mol. wts for the various forms of proacrosin seen in Western blots under non-reducing conditions relative to mol. wt standards run under reducing conditions. Alternatively, the major immunologically cross-reactive molecule with a mol. wt of 48,000 identified in this study may be an activated form of proacrosin analogous to alpha-acrosin in the boar system (Polakoski and Parrish, 1977). However, acrosin activity was not detected in dialysed samples of any of the acid extracts used for Western blot analysis suggesting that the 48,000 mol. wt molecule may not be a highly enzymatically active form of acrosin. Both the 54--56,000 and 48,000 mol. wt molecule may represent degraded forms of non-enzymatically active proacrosin which have not been appropriately activated due to the extraction procedures used. i.e. low pH and p-aminobenzamidine. Both forms may have been generated via proteolysis by acid proteases which would more than likely be active under these conditions. In point of fact, such a protease has been identified in extracts of GP sperm (Arboleda and Gerton, 1987) and would presumably be present in the acid extracts of GP testes. The possibility also exists that the three cross-reactive antigens identified may not all represent proacrosin-acrosin size variants particularly if the antisera is not truly monospecific, i.e. antisera raised against a single

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purified immunogen. As far as the purity of the immunogen is concerned, the preparation of GP testicular beta-acrosin used for immunization was 90-95°70 homogeneous as determined by SDS-PAGE and N-terminal sequence analysis (unpublished data). The purity of the immunogen, however, does not rule out the possibility that the antisera contains antibodies which recognize antigenic determinants which are shared by multiple proteins. An intriguing possibility, with potential functional significance, is that the antiserum contains antibodies with specificity for an antigenic determinant or group of determinants shared by all proteins destined for the acrosomal apparatus. Such determinants may serve as signals for trafficking, processing a n d / o r acrosomal packaging during spermatogenesis. In fact they would be analogous to the mannose 6-phosphate residues which serve as recognition markers for lysosomal enzymes (yon Figura and Hasilik., 1986). The results of Western blot analysis of different species of testes acid extracts demonstrates the evolutionary relatedness of the proacrosinacrosin systems. Anti-GP testes acrosin exhibited extensive cross-reactivity with each of the various rodent proacrosin-acrosin systems studied, but failed to demonstrate significant cross-reactivity with any of the evolutionarily more distant species of proacrosin-acrosin systems. The majority of the cross-reactivity among the rodent species was characterized by immunoreactivity with the 62,000 and 48,000 mol. wt molecules. The only species which exhibited cross-reactivity with the 54--56,000 mol. wt form was rat, both mouse and hamster failed to demonstrate such reactivity. This was true even at higher concentrations of the acid extracts (100/ag/lane). Iso-immunization of female Hartley, Strain 2 and Strain 13 GPs with a total of 200.0 /ag of purified testicular beta-acrosin led to significant antibody titers in only 2/14 animals studied. The iso-antibodies detected in the solid phase RIA were shown to be specific for the immunogen by Western blot analysis. The fact that only 2/14 GPs responded indicates that GP testicular beta-acrosin is not a highly iso-immunogenic protein. Similarly, Syner et al., (1979) reported that rabbit testicular acrosin was also not highly iso-immunogenic. Female rabbits immunized with a total of 1.8 mg of purified acrosin generated from activated rabbit testicular proacrosin also failed to exhibit significant increases in anti-acrosin isoantibody titers. The results of these two studies along with those of Morton and McAnulty (1979), who demonstrated that iso-immunization of female sheep with purified ram sperm acrosin did not significantly reduce fertility, indicate that both purified testicular a n d / o r sperm acrosin is probably not a good candidate iso-immunogen/antigen for immunocon-

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traception (Katsh, 1959; Dhindsa and Schumaker, 1980). This is particularly true when compared to the potent immunocontraceptive effects of PH-20 (Primakoff et al., 1988), a GP sperm surface iso-immunogen/antigen with a molecular weight of 64,000. However the fact that acrosin itself is of questionable value as a target antigen for immunocontraception does not rule out the possibility that proacrosin may be a highly iso-immunogenic molecule, especially in light of the immunologic changes associated with the molecules of the proacrosin-acrosin system as a result of activation and conversion to the various forms of the active enzyme (Syner et al., 1979, Siegel et al., 1987). Immunization of male GPs with beta-acrosin lead to significant disease in only one out of three animals at a 50.0/~g and in none of a group of three at a 5.0/~g dose. These results indicate that the molecule is also not a potent aspermatogenic autoantigen, i.e. those sperm and testes specific autoantigens capable of eliciting EAO. This is particularly true in light of the potent aspermatogenic activity seen with AP3 which was biologically active at sub-microgram doses (three out of three animals at 0.5 /~g/animal). However, as with the iso-immunogenic properties discussed above, these results do not rule out the potential aspermatogenic activity of GP proacrosin(s). Detailed analyses of both the iso-immunogenic and aspermatogenic activity of the molecules of the GP proacrosin-acrosin system will best be facilitated by the purification of the various size variants of proacrosin observed in the GP system and analysis of each of the purified forms of acrosin generated following the autoactivation of each.

Acknowledgements This research was supported by NIH Grants HD-21926, HD-06274, Rockefeller Foundation Grant RF-24041 and a grant from the Mellon Foundation. Drs. Adeyemi O. Adekunle and Ekundayo A. Falase are Rockefeller Foundation postdoctoral fellows. We thank Drs. Bayard T. Storey and George L. Gerton for their constructive suggestions and Ms. Debbie Coffin for providing excellent secretarial assistance.

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