Mount Sinai School of Medicine, New York, and Albert Einstein College of Medicine, Bronx, New York

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1 FERTILITY AND STERILITY Copyright ro 1992 The American Fertility Society Printed on acid-free paper in U.S.A. The fertilization antigen-l does not have proteolytic/ acrosin activity, but its monoclonal antibody inhibits sperm capacitation and acrosome reaction * Paul Kaplan, Ph.D. t+ Rajesh K. Naz, Ph.D. Mount Sinai School of Medicine, New York, and Albert Einstein College of Medicine, Bronx, New York Objective: To determine if human sperm surface fertilization antigen exhibits proteolytic or acrosin activity and to investigate the mechanism(s) whereby monoclonal antibody (mab) to fertilization antigen inhibits human sperm penetration of zona-free hamster ova. Design: Proteolytic and acrosin activities of human fertilization antigen were determined. Acrosomal status, acrosin activity, and motion characteristics were evaluated after incubation of human sperm with immunoaffinity-purified mab to fertilization antigen. Setting: Academic research environment. Participants: Fertile donors used as controls for infertile patients for fertility evaluation. Interventions: Human spermatozoa were treated with mab to fertilization antigen and induced to undergo acrosome reaction using calcium ionophore A Main Outcome Measures: Proteolytic and acrosin activities of fertilization antigen. Sperm penetration assay, acrosomal status, and motion parameters. Results: Fertilization antigen does not exhibit proteolytic or acrosin activity; however, its mab completely blocks human sperm penetration of zona-free hamster ova. The mab to fertilization antigen inhibits ionophore-induced acrosome reaction and blocks development of the hyperactivated state of human sperm cells. Conclusions: Monoclonal antibody to fertilization antigen blocks fertilization by inhibiting capacitation and acrosome reaction. Fertil Steril 1992;58: Key Words: Proteolytic activity, acrosin activity, acrosome reaction, sperm antibody, sperm antigen The mammalian spermatozoon is covered with a plasma membrane that contains specific antigens that function in the recognition of the zona pellucida of the egg and participate in events involved in capacitation and acrosome reaction (1). These sperm Received January 17, 1992; revised and accepted April 29, * Supported in part by grant HD from the National Institutes of Health (to R.K.N.), Bethesda, Maryland. t Department of Obstetrics, Gynecology, and Reproductive Science. :j: Reprint requests: Paul Kaplan, Ph.D., Department of Obstetrics, Gynecology, and Reproductive Science, Mount Sinai Medical Center, One Gustave L Levy Place, New York, New York Department of Obstetrics and Gynecology. surface components have not been clearly defined. Fertilization antigen-l (FA-I) is a sperm-specific glycoprotein purified from human and murine male germ cell plasma membranes, antibodies (monoclonal and polyclonal) to which cause inhibition of penetration of murine oocytes by murine sperm and inhibition of fertilization of human spermatozoa of zona-free hamster ova (2-4). Female rabbits actively immunized with FA-l show a reduction in fertility by an inhibition of fertilization (5). Sera from immunoinfertile patients, and not from fertile humans, contain antibodies that are reactive with FA-l (6, 7). Human sperm surface FA-l has been shown to bind to purified zona pellucida (ZP3) of porcine ZP that neutralizes its sperm ligand activity in porcine 396 Kaplan and N az FA -1 in human fertilization Fertility and Sterility

2 sperm-zona binding bioassay (8). Interestingly, in a recent collaborative study (9), it was found that antibodies to FA-1 also inhibit human sperm humanzona interaction. Because the monoclonal antibody (mab) to FA-1 also inhibits human sperm penetration of zona-free hamster ova (4), the question was raised whether FA -1 is also involved in human sperm capacitation and/or acrosome reaction. Because proteolytic and/or acrosin activity may be associated with sperm capacitation/acrosome reaction and binding/penetration through ZP, it remains to be determined whether FA-1 has any of these activities. These questions were addressed by studying the proteolytic and acrosin activities of FA-1 and by examining the effect of immunoaffinity-purified mab to F A-Ion capacitation and acrosome reaction of human spermatozoa. MATERIALS AND METHODS Fertilization Antigen-I Fertilization antigen-1 was purified from lithium diiodosalicylate solubilized human testes by immunoaffinity chromatography using germ-cell specific but species-cross-reactive mab (2, 4). Only those batches that showed a specific band of 49,000 ± 2,000 d in sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique were used in this study. Proteolytic Activity of F A-I Proteolytic activity of FA-1 was examined using BioRad's substrate gel technique (cat. no ; BioRad, Richmond, CA). Aliquots of trypsin solution (15 ill, 2 Ilg/mL), partially purified human acrosin (15 ill, 0.3 Ilg/mL) and FA-1 (15 ill, 64 Ilg/100 ill) in phosphate-buffered saline (PBS) were incubated for 24 hours at 22 C; protease diffusion into the gel is accompanied by digestion of the casein. After this time, the gel was overlayed with 3% acetic acid for 10 minutes and the plate rinsed with distilled water. Trypsin (Sigma Chemical, St. Louis, MO) and partially purified human acrosin served as controls. Acrosin was purified using the method reported previously (10). This method involved the extraction of human spermatozoa with 0.3 M acetic acid in 50 mm N acl, followed by gel filtration chromatography on Sephadex G-100 (Sigma Chemical), and chromatography on benzamidine-sepharose (Pharmacia Fine Chemicals, Piscataway, NJ). Acrosin Activity of F A-I To examine the acrosin activity of FA-I, an aliquot of FA-1 (15 ill, 64 Ilg/100 ul) and partially purified human acrosin (0.3 Ilg/mL, which served as a control) were tested. Acrosin activity was measured using the method previously reported (11). To each sample, 1 ml of reaction mixture (23 mm N-benzoyl-dl-arginine para-nitroanilide-hcl in 0.01% Triton X-100 [Sigma Chemical], M N-[2-hydroxyethy l]piperazine-n' -[ethanesulfonic acid] [Hepes], and M NaCl, ph 8.0) was added, and the specimen was incubated for a minimum of 3 hours at room temperature (RT). The reaction was stopped by the addition of 100 ill of 0.5 M benzamidine and the absorbance measured at 405 nm with a Beckman BD-G spectrophotometer. Acrosin activity was expressed as Ilg/mL. The daily variability of the reaction mixture was determined using a cryopreserved extract of human acrosin purified as described above. Immunoaffinity Purification of F A-I MAb The ascites fluid was raised in pristane-sensitized BALB/c female mice (Jackson Laboratories, Bar Harbor, ME) against the FA-1 hybridomas, and the mab was isolated by first precipitating with 18% Na2S04, and then by diethylaminoethyl cellulose chromatography, followed by immunopurification on FA-I-coupled Sepharose-4B (activated by cyanogen bromide) column as described elsewhere (12). The immunoaffinity-purified antibodies were primarily ofthe immunoglobulin (Ig)G2a subclass. The affinitypurified antibody of the IgG2a subclass from control mouse myeloma ascites fluid (Mouse IgG2a (k) UPClO, cat no ; Cappel Laboratories, Organon Teknika Corporation, Durham, NC) was used as a control antibody to eliminate the nonspecific effects of the murine IgG2a in the assays. Effect of F A-I Antibody on Acrosin Activity To investigate the effect of FA-1 antibody on human acrosin activity, an aliquot of the antibody (10 Ilg/lO ill) was added to 90 ill of partially purified human acrosin (0.3 Ilg/mL). The aliquots treated with 10 Ilg soybean trypsin inhibitor (SBTI; Sigma Chemical) and not treated (10 ill PBS) served as controls. After 1 hour of incubation at 37 C, acrosin activity was determined as described above. Effect of F A-I Antibody on Sperm Penetration Assay (SPA) Semen specimens were obtained from healthy fertile (n = 3) men by masturbation and analyzed Kaplan and N az FA -1 in human fertilization 397

3 within 60 minutes. All samples met the following criteria: >20 X 10 6 /ml; >50% motile spermatozoa; and >50% normal forms. Each sample was tested for the presence of antisperm antibodies using the immunobead test (13). Those samples that showed no evidence of antisperm antibodies were used in the present investigation. Specimens were allowed to liquefy for 30 minutes and washed at RT by the addition of three volumes of Biggers, Whitten, and Whittingham (BWW) medium (14) and centrifugation at 200 X g for 5 minutes. The supernatant was discarded, and the pellet was carefully overlaid with a volume of fresh medium equivalent to that of the original volume. The samples were incubated for 2 hours at 37 C in 5% CO 2 in air, in a 20 angled rack. The upper quarter of the supernatant was removed, spun down at 500 X g for 10 minutes, and washed twice in BWW. The final pellet was resuspended in BWW to a final concentration of 5 to 10 X 10 6 motile cells/ml. The sperm samples were incubated for 4.5 hours at 37 C in 5% CO 2 in air. Although capacitation usually requires overnight incubation, the three donors used undergo rapid capacitation and therefore were capacitated for only 4.5 hours in these studies. Adult female golden hamsters (Charles River, Wilmington, MA) were induced to superovulate, and oocytes were recovered using standard methods (15). After removing the cumulus cells using hyaluronidase (300 U/mL; Sigma Chemical), ova were placed in 0.01 % pronase (Sigma Chemical) to remove the ZP and washed twice in fresh medium. A sample containing 0.1 ml of pre incubated sperm was placed in a Petri dish, and 40 to 50 zona-free hamster 00- cytes were transferred to each drop and coincubated for 3 to 4 hours in 5% CO 2 in air. The eggs were removed and washed thoroughly with two changes offresh medium, fixed with 3% glutaraldehyde, and stained with 0.25% acetocarmine solution. The ova were assessed using phase-contrast microscopy, and penetrations were scored only when ova contained a decondensing sperm head with accompanying tail. The assay was also performed using a cryopreserved pool of spermatozoa with a known level of penetration. Any assay in which the value obtained for the standard preparation was more than two SD away from the mean was rejected. For investigating the effect of FA -1 antibody on the sperm penetration assay, 10 J.LL of the antibody was incubated with 90 J.LL of the sperm suspension (final concentration 10 J.Lg/100 J.LL) at 37 C for 4.5 hours. The zona-free oocytes were added and percent penetration determined as described above. The as- says were repeated using three different donors, and each sample was tested with at least 40 to 50 oocytes. The untreated sperm and sperm treated with the same amount of affinity-purified mouse IgG served as controls. Effect of Acrosin Distribution and Acrosomal Loss The effect of FA-1 antibody on acrosin distribution and acrosomalloss after ionophore-induced acrosome reaction was investigated. Aliquots (90 J.LL) of sperm suspension containing approximately 5 to 10 X 10 6 motile sperm/ml collected, as described above, were incubated with FA-1 antibody (10 J.Lg/10 J.LL), and affinity-purified mouse IgG (10 J.Lg/10 J.LL), or BWW (10 J.LL). The aliquots incubated with affinity-purified mouse IgG and not treated with any factors (BWW only) served as controls. The sperm suspension was incubated for 4.5 hours at 37 C; after this time, the sperm cells were induced to undergo the acrosome reaction by the addition of 10 J.Lm calcium ionophore A23187 (16). After 1 hour, the suspension was centrifuged at 500 X g for 10 minutes, and the supernatant (90 J.LL) was saved. The sperm cells were then washed in PBS, and the sperm pellet resuspended in 90 J.LL of PBS. Acrosin activity was determined in both the supernatant and cells as described above, and the activity was expressed as J.LIU /10 6 spermatozoa. The acrosomal status of the cells was also determined using the triple stain procedure of Talbot and Charcon (17) with a modification by Jager et al. (18). This method discriminates between normal and degenerative acrosome reactions using the vital stain, Trypan Blue. Trypan Blue stains dead sperm cells blue; live cells are always nonblue white. The absence or presence of the acrosome on the sperm cells was determined by phase-contrast microscopy (X1,000). Two hundred live spermatozoa per slide were examined and classified as acrosome intact. Effect on Sperm Motion Parameters The sperm suspension was divided into 90-J.LL aliquots, and 10 J.LL of FA-1 antibody or affinity-purified mouse IgG was added to the test solutions, giving a final concentration of 10 J.Lg/100 J.LL. After 4.5 hours, an aliquot (7 J.LL) of sample was placed into a Makler Chamber (Sefi-Medical Instruments, Rehovot, Israel), and motion parameters were determined using a computerized semen analyzer (CellSoft; Cryo Resources Ltd., New York, NY). The following parameter settings were used throughout 398 Kaplan and N az FA -1 in human fertilization Fertility and Sterility

4 Table 1 Acrosin Activity offa-1 and the Effect offa-1 Antibody on Acrosin Activity Acrosin activity FA-l Acrosin Acrosin + control IgG Acrosin + FA-l antibody Acrosin + SBTI JI./J/mL 0* t 0.027t 0.003* * P < compared with control. t Not significant versus control. Figure 1 Proteolytic activity of FA-I. (A), Aliquots of trypsin (2Itg/mL, (B) FA-1 (64Itg/mL), and (C) partially purified human acrosin (0.3 Itg/mL) in PBS were incubated for 24 hours at 22 C. The gel was overlayed with 3% acetic acid for 10 minutes and washed with distilled water to highlight the digested areas. the study: 30 frames analyzed at an image frequency of 30 Hz; 3 frames minimum sampling for percent of motile sperm (sperm motility); 15 frames minimum sampling for both velocity and amplitude of the lateral head movement (ALH) measurements; 10 Jlm/sec threshold velocity; minimum linearity of 2.5 for ALH measurement; and cell size range of 4 to 40 pixels with a magnification calibration of Jlm/pixel (19). Statistical Analysis The unpaired Student's t-test was used to analyze the data obtained from the effect of FA-Ion the SPA, ionophore-induced acrosome reaction, distribution of acrosin activity, and motion parameters. Pearson's product-moment correlation coefficient was used to analyze the relationship between spermassociated acrosin, acrosin in the medium, and the percent of acrosome-reacted spermatozoa. RESULTS Proteolytic Activity and Acrosin Activity of F A-I Fertilization antigen-l was evaluated for proteolytic and acrosin activity. Fertilization antigen-l did not display any proteolytic activity under the given conditions. As illustrated in Figure 1, trypsin (A), partially purified human acrosin (control groups) (C) showed proteolytic activity as evident by digestion of the gel, whereas FA-l did not display proteolytic activity (B). In addition, FA-l did not display acrosin activity (Table 1). Effect of FA-I Antibody on Acrosin Activity The addition of the FA-l antibody (10 Jlg/100 JlL) to an aliquot of partially purified human acrosin at the given concentration did not result in the loss of enzyme activity (Table 1). In contrast, the addition of SBTI (10 Jlg/I00 JlL) to the same preparation resulted in the inhibition of >90% of the acrosin activity. Effect of FA-I Antibody on SPA The effect of FA-l antibody on the ability of human spermatozoa to penetrate zona-free hamster ova is shown in Table 2. Total inhibition of penetration was observed when sperm were incubated in the presence of FA-l antibody (10 Jlg). The FA-l antibody did not induce sperm agglutination under the conditions of the assay, nor did it result in a significant decrease in percent motile spermatozoa. Effect of F A-I Antibody on Ionophore-Induced Acrosomal Loss and Acrosin Distribution Human sperm undergo acrosomalloss after exposure to the calcium ionophore A The results are presented as the percent of unreacted (percent of the live cell population that had not undergone acrosomal loss) when treated with calcium iono- Table 2 Effect of FA-l MAb on the Penetration of Zona-Free Hamster Ova by Human Sperm* Control ControllgG FA-1 antibody No. of ova examinedt No. of ova penetrated o Penetration % O:j: * Data are the sum of values (n = 3). t 5 to 10 X 10 6 motile cells/ml used for each experiment. :j: P < compared with controls; others were not significant. Kaplan and Naz FA-l in human fertilization 399

5 phore. Ionophore-induced acrosomal loss in capacitating medium was significantly higher in control groups compared with antibody-treated spermatozoa (Table 3). A reduction in motility (20%) was observed in A23187-treated sperm, in both controls and antibody-treated groups; the dead cells (20%) were excluded when calculating the percent of acrosome-intact versus acrosome-loss sperm. Significant changes in the distribution of spermassociated acrosin activity were observed in sperm cells incubated in the presence of FA -1 antibody under conditions known to induce an acrosome reaction. Induction of the acrosome reaction of the control groups resulted in a loss of sperm-associated acrosin, with a concomitant increase in the total acrosin recovered in the medium (P < 0.001). In contrast, sperm incubated with FA -1 antibody (10 /lg) and induced to undergo acrosome reaction showed a significantly higher concentration of sperm-associated acrosin activity and a decrease of total acrosin recovered in the medium (Table 3). There was a strong association between the presence of acrosin in the medium and the extent of acrosome reaction (r = , sperm-associated acrosin; r = 0.901, acrosin in the medium, P < 0.001). Effect of F A-I Antibody on Motion Parameters of Human Spermatozoa The effect of FA-1 antibody on human sperm motion parameters was evaluated using computer-assisted digital image analysis system. Sperm motility was not significantly affected after treatment with FA-1 antibody (Table 4). In contrast, significant decreases (P < 0.001) in all other motion parameters, (velocity, ALH, linearity, and beat/cross frequency) were observed in antibody-treated sperm cells (Table 4). Table 3 Effect of FA-1 MAb on Ionophore-Induced Acrosomal Loss and Acrosin Distribution * Control Control IgG FA-1 antibody % Acrosome intact % 29.7 ± 2.9 (85.6 ± 2.1H 39.5 ± 1.4 (80.9 ± 3.6):j: 64.5 ± 7.1 (88.1 ± 3.5) Cells 12.3 ± ± 5.5:j: 78.9 ± 9.8 Acrosin Medium 65.0 ± ± 7.9:j: 11.2 ± 3.3 * Values are means ± SEM of values (n = 3). Acrosin activity is expressed as /liu/10 6 spermatozoa. t Values in parentheses are mean ± SEM percents of total live cells. :j: Not significant. P < versus controls. There was a significant correlation (P < 0.001) between % intact sperm and acrosin activity (r = , sperm-associated acrosin; r = 0.902, acrosin in the medium). Table 4 Effect of FA -1 MAb on Motion Characteristics of Human Sperm Fertilization antigen-1 Control Control antibody IgG Motility (%) 76.9 ± ± 10.1* 75.0 ± 9.3* Velocity 64.4 ± ± 4.4t 64.0 ± 6.6* Linearity 4.3 ± ± 0.7t 4.4 ± 0.4 * ALH 4.1 ± ± 0.8t 4.2 ± 0.6* Beat/cross frequency 13.1 ± ± 3.2t 13.1 ± 3.9* * Not significant; values are means ± SEM of values (n = 3). t p < versus control. DISCUSSION These results indicate the purified FA-1 from human testes does not have acrosin or proteolytic activity. The affinity-purified mab also did not affect the activity of human acrosin. Monoclonal antibody to FA -1 completely blocked human sperm penetration of zona-free hamster ova at the given concentration. These findings are in agreement with our previous report (4). However, in the previous study (4), we examined the effects of FA-1 mab in SPA using whole ascites fluid. In the present study, we used immunoaffinity-purified FA-1 mab of known concentration (10 /lg%) comparing with the control mab of the same concentration and isotype (IgG2a) specificity. The present study conclusively indicates that the FA-1 mab at a concentration of 10 /lg% completely blocks human sperm penetration of zona-free hamster ova. Immunological inhibition of human acrosin activity has been shown to result in the loss of human sperm penetration of zona-free hamster ova (20). Our results demonstrate that FA -1 mab does not have any effect on acrosin activity. Thus, the inhibition of fertilization of zona-free hamster oocytes was not the result of immunological inhibition of acrosin activity. Antisperm antibodies may inhibit preparatory steps that have to do with the acquisition of fertilizing ability by impairing capacitation, acrosome reaction, and attachment to and penetration of the ZP. Capacitated human spermatozoa, induced to undergo acrosome reaction, release significant amounts of acrosin into the medium. In contrast, antibody-treated (fertilization antigen-i) samples show a significant reduction in the acrosin activity released in the medium, with the majority of the activity remaining sperm-associated. The release of acrosin into the medium was highly correlated with the extent to which human spermatozoa had un- 400 Kaplan and N az FA -1 in human fertilization Fertility and Sterility

6 dergone the acrosome reaction (r = , 0.901, sperm-associated acrosin and acrosin in the medium, respectively, P < 0.001). The percentage of sperm with intact acrosomes was approximately 65% in antibody-treated samples; in contrast, the control groups showed only 30% of the cells with intact acrosomes. Changes in the acrosin system during and subsequent to the acrosome reaction have been observed in humans using immunohistochemical techniques (21). In other mammalian species, similar changes in the acrosin system have been observed, with >90% ofthe sperm-associated acrosin released into the medium from guinea pig spermatozoa during nonsynchronous acrosome reaction (22). The data reported in this study also indicate that antibody-treated (FA-I) samples display lower ALH, velocity, and beat/cross frequency values compared with the controls. Capacitation is known to be associated with changes in motion characteristics of human spermatozoa, including velocity (23) and the onset of the hyperactivation, which may be significant in defining the fertilizing potential of a given sample (24). Alterations in motion characteristics such as increased velocity, increased ALH, and beat/ cross frequency is consistent with hyperactivation. The differences in motion characteristics between the groups appear to be consistent with our findings that FA -1 mab inhibits capacitation of human spermatozoa. The ALH and mean velocity are positively correlated with the fertilizing capacity of human sperm (25). In another collaborative study of Naz et al. (9), it was found that the rabbit polyclonal antiserum to FA-1 (tested at 5% concentration) inhibited the human sperm-zp interaction but did not affect the ZPinduced acrosome reaction. The polyclonal antibodies did not affect the percentage of acrosome-reacted sperm bound to the ZP, although it reduced the total number of sperm bound. These findings, along with the present study, indicate the effects of the antibody on spontaneous acrosome reaction rather than ZPinduced acrosome reaction. Also, in the previous collaborative study (9), we did not find the effects of the polyclonal antiserum (5% concentration) on the sperm motility or any other movement characteristics. This discrepancy with the present study may be because of the differences in nature, valency, and concentration of the antibody used in the two studies. These studies may also suggest that the FA- 1 epitope, recognized by the mab, is more involved in the sperm motility function. Thus, the higher concentration of epitope-directed mab is more effective (than the polyclonal antibodies directed against the various epitopes) at least in affecting one parameter of human sperm cell function, the motion characteristics. The effects were specific because (1) we used highly specific immunoaffinitypurified mab and (2) the same concentration ofthe affinity-purified control antibody of the same isotype specificity did not affect the sperm function. In conclusion, our data indicate that human sperm-specific FA-1 does not have proteolytic or acrosin activity, and even its immunoaffinity-purified mab does not inhibit acrosin activity. Fertilization antigen-1 mab completely blocks human sperm penetration of zona-free hamster ova by mechanism(s) involving an inhibition of capacitation and/ or acrosome reaction of the sperm cells. These results indicate that this antigen is relevant to human sperm cell function. The present findings along with earlier reports (4-9, 12) suggest that FA-1 may have applications in diagnosis and treatment of immunoinfertility and in the development of a contraceptive vaccine for humans. Presently, the complementary deoxyribonucleic acid encoding human FA-1 is being cloned and sequenced to investigate the role of recombinant fertilization antigen-1 in immunoregulation of fertility and infertility in humans. REFERENCES 1. Yanagimachi, R. Mammalian fertilization. In: Knobil E, Neill JD, editors. The physiology of reproduction. New York: Raven Press, 1988: Naz RK, Phillips TM, Rosenblum BB. Characterization of the fertilization antigen-1 for the development of a contraceptive vaccine. Proc Natl Acad Sci USA 1986;83: Naz RK. The fertilization antigen (FA-l): applications in immunocontraception and infertility in humans. Am J Reprod Immunol MicrobioI1988;16: Naz RK, Alexander NJ, Isahakia M, Hamilton MS. Monoclonal antibody to a human germ cell membrane glycoprotein that inhibits fertilization. Science 1984;225: Naz RK. The fertilization antigen (FA-1) causes a reduction of fertility in actively immunized female rabbits. J Reprod ImmunoI1987;11: Naz RK. Sperm surface antigens involved in mammalian fertilization. Curr Opin ImmunoI1990;2: Naz RK. Involvement of fertilization antigen (FA-l) in involuntary immunoinfertility in humans. J Clin Invest 1987;80: Naz RK, Sacco AG, Yurewicz EC. Human spermatozoal FA- 1 binds with ZP3 of porcine zona pellucida. J Reprod Immunol 1991;20: Naz RK, Brazil C, Overstreet JW. Effects of antibodies to sperm surface fertilization antigen-1 on human sperm zona pellucida interaction. Fertil SteriI1992;57:1304-1O. 10. Howe SE, Grider SL, Lynch DM, Fink LM. Antisperm antibody binding to human acrosin: a study of patients with unexplained infertility. Fertil Steril 1991;55: Kaplan and N az FA -1 in human fertilization 401

7 11. Kennedy WP, Kaminiski JM, Van der Ven HH, Jeyendran RS, Reid DS, Blackwell J, et al. A simple clinical assay to evaluate the acrosin activity of human spermatozoa. J Androl 1989;10: Naz RK, Deutsch J, Phillips TM, Menge AC, Fisch H. Sperm antibodies in vasectomized men and their effects on fertilization. Bioi Reprod 1989;41: Clarke GN, Elliott PJ, Smaila C. Detection of sperm antibodies in semen using the immunobead test; a survey of 813 consecutive patients. Am J Reprod ImmunoI1985;7: Yanagimachi R, Yanagimachi H, Rogers BJ. The use of zonafree animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Bioi Reprod 1976;15: Biggers JD, Whitten WK, Whittingham DG. The culture of mouse embryos in vitro. In: Daniel JC Jr, editor. Methods of mammalian embryology. San Francisco: WH Freeman, 1971; Byrd W, Tsu J, Wolf D. Kinetics of spontaneous and induced acrosomal loss in human sperm incubated under capacitating and noncapacitating conditions. Gamete Res 1989;22: Talbot P, Charon R. A new procedure for rapidly scoring acrosome reactions of human sperm. Gamete Res 1980;3: Jager S, Kuiken J, Kremer J. Triple staining of human sperm: technical aspects. Arch Androl 1984;12(Suppl): Kaplan P, Neumaier V, Navot D. Cold storage enhances human sperm penetration of zona-free hamster ova without affecting acrosome reaction. Assist Reprod Tech/ AndroI1991;2: Salonen I, Kallajoki M. Monoclonal antibody against human sperm acrosin inhibits sperm penetration of zona-free hamster eggs. Int J AndroI1987;10: Tesarik J, Drahorad J, Peknicova J. Subcellular immunochemical localization of acrosin in human spermatozoa during the acrosome reaction and zona pellucida penetration. Fertil Steril 1988;50: Nuzzo NA, Anderson RA, Zaneveld L. Proacrosin activation and acrosin release during the guinea pig acrosome reaction. Mol Reprod Develop 1990;25: Aitken RJ, Sutton M, Warner P, Richardson DW. Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes. J Reprod Fertil 1985;73: Robertson L, Wolf DP, Tash JS. Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm. Bioi Reprod 1988;39: Aitken RJ, Best FSM, Richardson DW, Djahanbakhch 0, Lees MM. The correlates of fertilizing capacity in normal fertile men. Fertil Steril 1982;38: Kaplan and N az FA -1 in human fertilization Fertility and Sterility