HUMAN SPERM ACROSOMAL PROTEINASE: ANTIBODY INHIBITION AND IMMUNOLOGIC DISSIMILARITY TO HUMAN PANCREATIC TRYPSIN*

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1 FERTILITY AND STERILITY Copyright 1973 by The Williams & Wilkins Co. Vol. 24, No.6, June 1973 Printed in U.S.A. HUMAN SPERM ACROSOMAL PROTEINASE: ANTIBODY INHIBITION AND IMMUNOLOGIC DISSIMILARITY TO HUMAN PANCREATIC TRYPSIN* L. J. D. ZANEVELD, D.V.M., PH.D., G. F. B. SCHUMACHER, M.D., AND J. TRAVIS, PH.D. Section of Reproductive Biology, Department of Obstetrics and Gynecology, University of Chicago, Pritzker School of Medicine, Chicago, Illinois 60637, and Department of Biochemistry, University of Georgia, Athens, Georgia Mammalian spermatozoa possess an acrosomal proteinase that appears essential for the fertilization process. 1-6 Recently human sperm acrosomal proteinase was partially purified and characterized AI. though the acrosomal enzyme differs biochemically from other known mammalian proteinases,7 it is so similar to pancreatic trypsin that it was initially called "sperm trypsin" or "trypsin-like enzyme." 2. 3, 13 Both enzymes have an optimum ph of 8.0, are stable at ph 3.0, hydrolyze benzoylarginine ethyl ester (BAEE) and tosyl-arginine methyl ester (TAME) but not benzoyl-tyrosine ethyl ester (BTEE), and are inhibited by tosyl-iysine chloromethyl ketone (TLCK) and diisopropyl fluorophosphate (DFP) but not by tosylamidephenylethyl chloromethyl ketone (TPCK). An effort was therefore made to establish whether these enzymes differ immunobiochemically. Independent research by Metz 14, 15 with rabbit and human sperm and by this laboratory with bull sperm 16, 17 showed that acrosomal hyaluronidase activity can be inhibited by incubation of this enzyme with heteroantisera produced against spermatozoa 14, 15 or sperm acrosomal (detergent) extracts. 16, 17 These results indicate the presence of at least one highly antigenic substance in the acrosomes of Received December 4, * Supported by United States Public Health Service Grant NIH-HD-06315, Ford Foundation Grant for research and training in reproductive biology, and Biomedical Center for Population Research Grant NIH-HD spermatozoa whose antibody can conceivably prevent sperm penetration of the cumulus oophorus and, hence, conception. The inhibitory effect of antibodies on the sperm acrosomal proteinase was therefore also evaluated, since inactivation of this enzyme undoubtedly prevents fertilization. 4-6 MATERIALS AND METHODS Preparation of Sperm Extracts and Proteinase. Spermatozoa from healthy donors were washed three times in 0.15 M NaCI (saline), resuspended in the same solution, and treated with equal volumes of 0.075% Hyamine 2389 (Rohm and Haas, Philadelphia, Pennsylvania) and 0.075% Triton X-lOO (Rohm and Haas, Philadelphia, Pennsylvania) for 90 min. at 37 0 C.7, 18 Transmission electron microscopy of human spermatozoa reveals that such detergent treatment removes the acrosome, 19 confirming previous evidence obtained by the lighp8, 20 and scanning electron microscope 21 using ejaculated ram and rabbit spermatozoa. It is possible, however, that during treatment some enzymes leak from other parts of the spermatozoon. This was demonstrated for epididymal rabbit spermatozoa,22 although only negligible amounts of such enzymes could be found in detergent extracts from ejaculated ram and rabbit spermatozoa. 20, 21 Since the extracts structurally contain mostly acrosomal material, they will be referred to as "acrosomal extracts." After incubation, the mixtures were centrifuged, the precipitated spermatozoa 479

2 480 ZANEVELD ET AL. Vol. 24 washed with saline, and the combined supernate treated with 80% ethanol. The precipitated material was dialyzed against three changes of 1000 ml. of M HCI over a 24-hr. period and lyophilized. The human acrosomal proteinase preparation (specific activity 50 mu./mg.) was further purified by Sephadex G-50 gel filtration using 0.1 M KCI-HCI buffer, ph 2.2 as eluent.7 This results in the removal of a proteinase inhibitor that is also present in the acrosomal extracts The proteinasecontaining fraction that was used for the experiments possessed a specific activity of 720 mu./mg. Pure human pancreatic trypsin (17,600 mu./mg.) was obtained by salt fractionation, Sephadex G-75 gel filtration, and ion-exchange chromatography on SE Sephadex C-25, as described by Travis and Roberts. 23 Preparation of Antisera and Antibodies, and Immunodiffusion Tests. Antisera against lyophilized human acrosomal extract material were prepared by mixing 10 mg. with 2.0 ml. of complete Freund's adjuvant (Hyland Laboratories, Los Angeles, California) using a tissue microhomogenizer and injecting the mixture subcutaneously into the inguinal and axillar region of rabbits. Three weeks later, lyophilized acrosomal extract (15 mg.) was administered in a series of six booster injections every 2 days for 12 days. The material was dissolved in saline, centrifuged, and the supernatant solution injected intravenously during the first five injections in increasing portions. The combined precipitate was administered intraperitoneally together with the final intravenous injection. The animals were bled 9 days later. Antisera against pure human pancreatic trypsin were obtained by multiple intradermal injections into rabbits of the enzyme suspended in complete Freund's adjuvant. Seven milngrams of enzyme were injected initially. After 3 weeks a booster injection of 3 mg. of trypsin in incomplete Freund's adjuvant was administered. The animals were bled 7 days later. Human plasminogen antiserum (rabbit) was bought from Behring Diagnostics Inc. (Woodbury, New York). Whole serum contains several proteinase inhibitors2 that would interfere with the inhibition studies.8, 9, 12, 25 The antisera were therefore fractionated by dropwise adding one part saturated (NH.) 2S0. solution to two parts antiserum. 26 After 2-3 hr. incubation at room temperature, the mixture was centrifuged at 1400 g for 30 min. and the precipitated material resuspended to its original volume with saline. The (NH.)2S0. precipitation was repeated two more times. The final precipitate was resuspended in a small volume of saline and dialyzed against three changes of saline, The resultant immunoglobulin preparations were approximately five times more concentrated than the original antisera. These preparations were used throughout the enzyme-inhibition tests. All further dilutions were made with saline. Hyland gel diffusion plates (immunoplates, Travenol Laboratories, Costa Mesa, California) were used in the double immunodiffusion experiments. 27 Enzyme Tests. The esterase activities of the proteinases were measured using benzoyl-arginine ethyl ester (BAEE, Sigma Chemical Co., St. Louis, Missouri, 0.2 mg./ml.) as substrate dissolved in 0.1 M borate buffer, ph 8.0 containing 0.05 M CaCl2 according to the method of Schwert and Takenake. 28 One milliunit was defined as that amount of enzyme that caused a change in absorbance of O.OOl/min. at a wavelength of 253 nm. To test the inhibitory effect of an antibody preparation, 0.1 ml. was incubated with 0.1 ml. of enzyme for 15 min. at room temperature and 0.1 ml. of the mixture added to 3.0 ml. of the BAEE-borate buffer solution. Enzyme concentrations of mu./ml. were used. The rate of change in absorption was measured for 10 min. at a wave length of 1

3 June 1973 HUMAN SPERM ACROSOMAL PROTEINASE ANTIBODIES nm. and compared with that of enzyme incubated with saline. The proteinase activities of the enzymes were evaluated using gelatin as substrate. 25, 29 Kodak projector slide plates (3 1/~ x 4 inches, contrast, with hardened fine grain emulsion and gelatin overcoat) were exposed, developed, fixed, extensively washed, and dried. Dilutions (25 J.Ll.) of the antibody preparation or saline as control were incubated with 25 J.Ll. of enzyme (human acrosomal proteinase, 600 J.Lg./ml.; human pancreatic trypsin, 25 J.Lg./ml.) in 0.1 M borate buffer, ph 8.0, containing 0.05 M CaC1 2 at room temperature for 15 min. and 30-J.Ll. drops added to the plate. After incubation for either 3 or 24 hr., at 37 C. in a moist chamber, the slides were rinsed under tap water and dried. Proteinase activity was indicated by the presence of a transparent hole, whereas undigested gelatin areas remained black. The amount of acrosomal proteinase preparation used possessed essentially the same esterase activity as the pancreatic trypsin and both enzyme preparations just completely digested the gelatin in 3 hr. Specific activity measurements were based on the esterase activity of the enzyme and absorption readings at 280 nm., assuming that 1 mg. of acrosomal protein/ ml. has an optical density of 1.0 using a l-cm. light path, whereas the extinction coefficient (E 1 %) of human pancreatic trypsin is RESULTS Using BAEE as substrate, addition of the immunoglobulin preparations never caused more than a 10-20% decrease in enzyme activity as compared with the controls (Table 1). This weak inhibition was nonspecific, since both pancreatic trypsin and acrosomal proteinase were inhibited to the same extent by their own and each other's antibody preparations in the cross-over experiments. It is therefore justified to assume that traces of rabbit serum proteinase inhibitors were present in the immunoglobulin preparations. Strong and specific inhibition occurred with gelatin as substrate (Fig. 1). Antibodies prepared against human acrosomal extracts only inhibited the acrosomal proteinase and not pancreatic trypsin. Similarly, human pancreatic trypsin antibodies inhibited human pancreatic trypsin but not the human acrosomal proteinase. After 3 hr. of incubation, the inhibition was very effective, even in the 1: 8 dilution (Fig. 1). Some digestion had occurred after 24 hr. of incubation in the spots where enzyme mixed with 1: 8 and 1: 4 dilutions of the immunoglobulin preparation was applied, although the other concentrations of antibody still completely inhibited proteinase activity. The results obtained in double immunodiffusion experiments were analogous (Fig. 2). No signs of immunochemical cross-reactivity were found between human pancreatic trypsin and human TABLE 1. Effect of Undiluted, (NH')2S0, Precipitated Antibodies on Human Proteinase Activity using BAEE as Substrate' Source of proteinase Spermatozoa Pancreas Incubated with antibody preparations produced against the following Acrosomal extracts Pancreatic trypsin Acrosomal extracts Pancreatic trypsin, See text for experimental details. Activity compared to buffer controls % FIG. 1. Effect of (NH')2S0, precipitated antibody preparations on human acrosomal proteinase and human pancreatic trypsin using gelatin as substrate. H.A.E., human acrosomal extracts, H,P.T., human pancreatic trypsin. Time of incubation, 3 hr. See text for further experimental details.

4 482 Vol. 24 ZANEVELD ET AL..' FIG. 2. Ouchterlony plate test of human sperm acrosomal extract and human pancreatic trypsin vs. their antisera. 1 and 11, sperm acrosomal extracts 10 mg./ml.; 2 and 7, pancreatic trypsin 500 /Lg./ml.; 3 and 9, pancreatic trypsin 250 /Lg./ml.; 4 and 8, sperm acrosomal extracts 5 mg./ml.; 5 and 10, partially purified acrosomal proteinase 600 /Lg./ml.; ahpt, antipancreatic trypsin serum; ahae, antisperm acrosomal extract serum. acrosomal proteinase preparations as judged by the precipitin band formation with both types of antisera, even if equivalent amounts (BAEE activity) of human pancreatic trypsin (25 JLg./ml.) and unpurified human acrosomal extract (10 mg./ml.) were applied. The antiserum against the human pancreatic trypsin formed one very strong and, at high concentrations, one very weak band with its homologous antigen preparation, but neither precipitin lines with the acrosomal extracts nor deviations of the precipitin bands in the diffusion zone of the acrosomal extract could be observed. -The antiserum against human acrosomal extract formed at least four different precipitin bands with its homologous antigen solution, but no precipitation occurred with the human pancreatic trypsin. None of the four precipitin lines showed any deviation in the diffusion zone of the pancreatic trypsin that would indicate the presence of immunochemically, related constituents. Neither human pancreatic trypsin nor human acrosomal extracts formed precipitin lines with a monospecific antiserum against human plasminogen. Control experiments showed that the detergents hyamine and triton alone at the concentrations used to prepare the acrosomal extracts cause the formation of a precipitin band with normal sera or antisera. Immunodiffusion experiments with acrosomal extracts and normal rabbit sera were therefore made to ensure that the precipitin bands formed between the acrosomal extracts and their antisera were indeed due to antigen-antibody interactions and not to a nonspecific precipitation by residual detergents in the acrosomal extracts. No precipitin band occurred, showing that the detergents were completely removed from the acrosomal extracts during alcohol precipitation and dialysis. DISCUSSION Based on the specificity of inhibition and lack of cross-reactivity, the results clearly show the immunochemical dissimilarity between human pancreatic trypsin and

5 June 1973 HUMAN SPERM ACROSOMAL PROTEINASE ANTIBODIES 483 human sperm acrosomal proteinase. The sperm enzyme is also immunochemically unrelated to plasminogen or plasmin. These data further emphasize the uniqueness of the sperm acrosomal proteinase. Virtually no inhibition of the enzymes was obtained using BAEE as substrate. The inhibition that did occur was nonspecific and was most likely due to the presence of small amounts of residual proteinase inhibitor in the antibody preparations. The small amount of serum proteinase inhibitors did not seem to influence the gelatin plate test, most likely because of the lesser sensitivity in evaluating inhibitor activity by this test system. Metz 14 obtained a similar nonspecific inhibition using TAME as substrate although, interestingly, the bivalent rabbit semen antibody preparations inhibited much stronger than the univalent antibody preparations. The effective specific inhibition of the proteolytic activity of the two enzymes by their respective antibodies in the presence of gelatin as substrate, in contrast to the lack of specific inhibition of the esterolytic activity in the presence of the low molecular weight BAEE (MW = 342) as substrate, sheds light on the interesting structural aspects of the antigen-antibody complex. The active site of the enzyme does not seem to be involved in a reaction with the combining site of the immunoglobulin(s) specific for the determining group(s) that are often located in prominent areas of the protein molecule. The combination of determining groups with the specific immunoglobulin most likely results in a steric hindrance preventing adequate contact of the active site with the macromolecular substrate, while this active site may still be accessible to the low molecular weight ester-substrate. Metz 30 came to the same conclusion based on his data described above. Moreover, the gelatin is not in a colloidal solution under the experimental conditions and actually represents an insoluble membrane (at 37 C.). In this respect it resembles the zona pellucida, a natural substrate for the Bcrosomal proteinase. If specific isoantibodies against this sperm proteinase can be induced in uterine secretions, they will probably also act as effective inhibitors under in vivo conditions and should exert an antifertility effect. SUMMARY Immunoglobulins prepared by (NH.) 2- SO. fractionation of heterologous antisera (rabbit) against human sperm acrosomal (detergent) extracts inhibited human sperm acrosomal proteinase activity using gelatin as substrate. Such antibody preparations did not inhibit the activity of pure human pancreatic trypsin. Similarly prepared human pancreatic trypsin antibody preparations showed the same but opposite specificity, since only pancreatic trypsin was inhibited by these antibodies and not the acrosomal proteinase. Virtually no inhibition of specific character was observed if benzoyl-arginine ethyl ester (BAEE), a low molecular weight substrate, was used. At least four immunoprecipitin bands were formed between human sperm acrosomal extracts and their respective antisera in double immunodiffusion tests. One strong and one weak band occurred between human pancreatic trypsin and the homologous antiserum. No signs of crossreaction were found between human acrosomal extracts and human pancreatic trypsin. Plasminogen or plasmin were not detectable in the acrosomal extracts by means of agar gel diffusion against a nonspecific plasminogen antiserum. The acrosomal proteinase therefore differs immunochemically from pancreatic trypsin, plasminogen, and plasmin. An immunological interference with fertilization by antibodies against acrosomal proteinase appears to be conceivable. Acknowledgments. The authors wish to thank

6 484 ZANEVELD ET AL. Vol. 24 Mr. Bruno Dragoje for his excellent technical assistance and Miss Diane Johnson for her fine secretarial work. REFERENCES 1. SRIVASTAVA, P. N., ADAMS, C. E., AND HARTREE, E. F. Enzymatic action of acrosomal preparations on the rabbit ovum in vitro. J Reprod Fertil 10:61, STAMBAUGH, R., AND BUCKLEY, J. Zona pellucida dissolution enzymes of the rabbit sperm head. Science 161:585, STAMBAUGH, R., AND BUCKLEY, J. Identification and subcellular localization of the enzymes effect ing penetration of the zona pellucida by rabbit spermatozoa. J Reprod Fertil 19:423, STAMBAUGH, R., BRACKETT, B. G., AND MASTROIANNI, L. Inhibition of in vitro fertilization of rabbit ova by trypsin inhibitors. Bioi Reprod 1 :233, ZANEVELD, L. J. D., ROBERTSON, R. T., AND WILLIAMS, W. L. Synthetic enzyme inhibitors as antifertility agents. Fed Eur Biochem Soc Lett 11 :345, ZANEVELD, L. J. D., ROBERTSON, R. T., KESSLER, M., AND WILLIAMS, W. L. Inhibition of fertilization in vivo by pancreatic and seminal plasma trypsin inhibitors. J Reprod Fertil 25:387, ZANEVELD, L. J. D., DRAGOJE, B. M., AND SCHUMACHER, G. F. B. Acrosomal proteinase and proteinase inhibitor of human spermatozoa. Science 177:702, SCHUMACHER, G. F. B., AND ZANEVELD, L. J. D. Interaction of acrosomal protease from human and rabbit spermatozoa with human protease inhibitors. Fed Proc 31:278, SCHUMACHER, G. F. B., AND ZANEVELD, L. J. D. Inhibition of Rabbit and Human Sperm Acrosomal Protease by Protease Inhibitors of Human Origin." In Proc. VIIth Internat. Congr. on Animal Reprod. and Artif. Insem., 1972, Munich, Germany. In press. 10. FRITZ, H., FORG-BREY, B., FINK, E., MEIER, M., SCHIESSLER, M., AND SCHIRREN, C. Humanakrosin: Gewinnung und Eigenschaften. Hoppe Sevier's Z Physiol Chem 353:1943, FR'ITZ, H., F6RG-BREY, B., MEIER, M., ARNHOLD, M., AND TSCHESCHE, H. Humanakrosin: Hemmbarkeit durch Protein-Proteinase Inhibitoren. Hoppe-Seyler's Z Physiol Chem 353:1950, FRITZ, H., HEIMBURGER, N., MEIER, M., ARNHOLD, M., ZANEVELD, L. J. D., AND SCHUMACHER, G. F. B. Humanakrosin: Zur Kinetik der Hemmung durch Human-Serum inhibitoren. Hoppe-Seyler's Z Physiol Chem 353:1953, ZANEVELD, L. J. D., SRIVASTAVA, P. N., AND WILLIAMS, W. L. Relationship of a trypsin-like enzyme to capacitation. J Reprod Fertil 20:337, METZ, C. B. Effects of antibodies on gametes and fertilization. Bioi Reprod 6:358, METZ, C. B., SEIGUER, A. C., AND CASTRO, A. E. Inhibition of the cumulus dispersing and hyaluronidase activities of sperm by heterologous and isologous antis perm antibodies. Proc Soc Exp Bioi Med 140:776, ZANEVELD, L. J. D., POLAKOSKI, K. L., AND SCHUMACHER, G. F. B. "Characterization of Acrosomal Hyaluronidase from Spermatozoa." In Proc. VIIth Internat. Congr. Animal Reprod. and Artif. Insem., 1972, Munich, Germany. In press. 17. ZANEVELD, L. J. D., POLAKOSKI, K. L., AND SCHUMACHER, G. F. B. Properties of acrosomal hyaluronidase from bull spermatozoa: Evidence for its similarity to testicular hyaluronidase. J Bioi Chem 248:564, HARTREE, E. F., AND SRIVASTAVA, P. N. Chemical composition of the acrosomes of ram spermatozoa. J Reprod Fertil 9:47, CHURG, A., ZANEVELD, L. J. D., AND SCHUMACHER, G. F. B. Fine structure of detergent treated human and rabbit spermatozoa. In preparation. 20. ALLISON, A. C., AND HARTREE, E. F. Lysosomal enzymes in the acrosome and their possible role in fertilization. J Reprod Fertil 21:501, ZANEVELD, L. J. D., GOULD, K. G., HUMPHREYS, W. J., AND WILLIAMS, W. L. Scanning electron microscopy of mammalian spermatozoa. J Reprod Med 6:147, STAMBAUGH, R., AND SMITH, M. An evaluation of several extraction procedures for acrosomal enzymes. Bioi Reprod 7:100, TRAVIS, J., AND ROBERTS, R. C. Human trypsin: isolation and physical-chemical characterization. Biochemistry 8:2884, HEIMBURGER, N., HAUPT, H., AND SCHWICK, H. G. "Proteinase Inhibitors of Human Plasma." In Proc. Int. Conf. Proteinase Inhibitors, Munich, 1970, Fritz, H., and Tschesche, H., Eds. Walter de Gruyter, New York, 1971, p SCHUMACHER, G. F. B. Inhibition of rabbit sperm acrosomal protease by human alpha I-antitrypsin and other protease inhibitors. Contraception 4:67, CAMPBELL, D. H., GARVEY, J. S., CREMER, N. E., AND SUSSDORF, D. H. Methods in Immunology. W. A. Benjamin, New York, 1970, p OUCHTERLONY, O. Progress in Allergy (Vol. VI) Kallos, P., and Walisman, B. H., Eds. Karger, New York, 1962, p SCHWERT, G. W., AND TAKENAKE, Y. A spectrophotometric determination of trypsin and chymotrypsin. Biochim Biophys Acta 16:570, ANDERER, F. A., AND HOERNLE, S. Chemical studies on kallikrein inactivator from bovine lung and parotid gland. Ann NY Acad Sci 146:381, METZ, C. B. Personal communication.