Effects of sperm-immobilizing antibodies on sperm-zona pellucida tight binding*

Transcription

1 FERTILITY AND STERIUTY Copyright" 1993 The American Fertility Society Printed on acid-free paper in U. S. A. Effects of sperm-immobilizing antibodies on sperm-zona pellucida tight binding* Hiroaki Shibahara, M.D.t+ Lani J. Burkman, Ph.D. Shinzo Isojima, M.D. II Nancy J. Alexander, Ph.D.V Hyogo Medical College, Nishirwmiya, Hyogo, Japan, and The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia Objective: To evaluate the in vitro effects of sperm-immobilizing antibodies on sperm-zona pellucida (ZP) tight binding. Design: The hemizona assay (HZA) was used to study the inhibitory effects of infertile women's sera with and without sperm-immobilizing antibodies on sperm ZP tight binding. These results were compared with those of monoclonal sperm-immobilizing Setting: The patients were collected from a university hospital infertility clinic. Patients: Sera from 40 infertile women (24 with and 16 without sperm-immobilizing antibodies) and 2 postpartum women as control were used. Results: Of 24 patients' sera with sperm-immobilizing antibodies, 23 (96%) showed significant inhibitory effect, whereas none of 16 patient's sera without sperm-immobilizing antibodies exhibited any inhibitory effect. However, there was no correlation between the antibody titers of spermimmobilizing antibody and the hemizona index. Among four monoclonal sperm-immobilizing antibodies tested, only one showed a significant inhibitory effect on the sperm-zona tight binding. A human monoclonal antibody derived from an infertile woman with sperm-immobilizing antibodies, whose serum showed an inhibitory effect on HZA, did not inhibit the HZA. Conclusions: There are at least two kinds of sperm-immobilizing antibodies, one with both activities of sperm immobilization and blocking of sperm-zona tight binding and another with the former activity alone. The vast majority of sperm-immobilizing antibodies reduce zona binding even without the presence of complement. Fertil Steril 1993;60:533-9 Key Words: Sperm-immobilizing antibodies, monoclonal antibodies, human hemizona assay, sperm-zona tight binding, infertility Received January 12, 1993; revised and accepted June 10, * This work was partially supported by the Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School, Norfolk, Virginia, under a Cooperative Agreement (DPE-2044-A ) with the United States Agency for International Development (A.LD.) and Research Grant from Hyogo Medical College, Nishinomiya, Hyogo, Japan. The views expressed by the authors do not necessarily reflect the views of A.LD. t Department of Obstetrics and Gynecology, Hyogo Medical College. :j: Reprint requests: Shinzo Isojima, M.D., Department of Ob- Sperm Immobilization Test (SIT) (1) is a reliable test to detect of antisperm antibodies related to infertility as compared with other methods (2). Approximately 13% to 15% of unexplained infertile women have circulating sperm-immobilizing antibodies (2). The sperm-immobilizing antibodies not stetrics and Gynecology, Hyogo Medical College, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663, Japan. The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. II Present address: GIBCO BRL Laboratories, Life Technologies, Inc. Grand Island, New York. 11 Present address: National Institute of Child Health and Human Development, Center of Population Research, Bethesda, Maryland. Shibahara et ai. Inhibition of sperm-zona tight binding 533

2 only interfere with sperm migration through female genital tracts (3) but also may interfere with various stages of fertilization process (4-7). Some studies have shown an inhibitory effect of patient's sera with sperm-immobilizing antibodies on sperm penetration through salt-stored human zona pellucida (zp) (4,5), and others have shown inhibition of human sperm penetration into zona-free hamster eggs (6, 7). However, these results not necessarily indicated that the sperm-immobilizing antibodies themselves exert the blocking effect on sperm-zona or sperm-egg interactions. These blocking effects might be due to coexisting antibodies in SIT-positive patients' sera. Recently, Burkman and associates (8) reported a new method for evaluating human sperm-zp tight binding called the hemizona assay (HZA). Because two matched zona hemispheres are used in this assay, there are three test advantages: [1] more reliable data can be obtained by using the matched zona halves for test and control experiments; [2] the limited number of recovered human oocytes is amplified by dividing them into halves; [3] ethical objections to possible inadvertent fertilization of a viable oocyte are eliminated by first cutting the egg into half. In the present study, the effects of patients' sera with or without sperm-immobilizing antibodies and monoclonal sperm-immobilizing antibodies derived from human and mice were tested in the HZA to examine the interference of sperm-immobilizing antibodies with sperm tight binding to human zp. Patient' Sera MATERIALS AND METHODS Blood samples were collected from 24 infertile women with sperm-immobilizing antibodies (Code 01 to 24), 16 unexplained infertile women without sperm-immobilizing antibodies (Code 25 to 40), and 2 normal postpartum women (Code 41 and 42) at the hospital of Hyogo Medical College. All sera were heat-treated at 56 C for 30 minutes to inactivate complement and kept frozen at -20 C until use. Human Sperm Semen samples were obtained from fertile healthy donors. After liquefaction, semen was centrifuged and washed twice in Ham's F-10 medium (G IBCO Laboratories, Grand Island, NY) supplemented with 3.5% human serum albumin (HSA). The motile sperm, collected by a standard swim-up technique, was suspended in the same medium at a concentration of 2 X 10 6 motile sperm/ml for sperm-zona binding test. Monoclonal Sperm-Immobilizing Antibodies Two human and two mouse monoclonal spermimmobilizing antibodies for human sperm were used in this study. Human monoclonal spermimmobilizing antibody H6-3C4 (Immunoglobin [lg]m) was produced by cell fusion between peripheral blood lymphocytes of an infertile woman (Code 05) with sperm-immobilizing antibodies and mouse myeloma cells as described previously (9). Monoclonal antibody (mab) En 46 (IgG t ) was a classswitched antibody [from IgM (;\) to IgG t (;\)] with the same variable region of H6-3C4 by recombinant DNA technology (10). Mouse monoclonal sperm-immobilizing antibodies to human sperm were produced by cell fusions between the spleen cells of immunized mice and mouse myeloma cells as follows. Mouse monoclonal antibody (mab) 2H12 (IgM) was derived from a Balb/c mouse immunized with human choriocarcinoma cell line JEG-3 (11). Monoclonal antibody 2C6 (IgM) was derived from a Balb/c mouse immunized with human seminal plasma (12). Purification of mabs The IgM fraction of mab H6-3C4 was purified from hybridoma supernatant cultured in serumfree culture medium (Hybrity-1, Sanko-Junyaku, Japan) by gel filtration through Sephacryl S-300 (Pharmacia, Uppsala, Sweden). The IgG t fraction of mab En 46 was purified by Protein A-Sepharose 4B (Pharmacia) column chromatography. The IgM fractions of mabs 2H12 and 2C6 were prepared from hybridoma supernatant cultured in RPMI 1640 medium containing 10% fetal bovine serum by salt precipitation with 50% saturated ammonium sulfate followed by gel filtration through Sephacryl S-300 (Pharmacia). All mabs purified were tested to determine sperm-immobilizing antibody titers (SI 50 ) and sperm-agglutinating antibody titers. Fab Fragment of mab H6-3C4 Urea and trypsin were used to produce the Fab fragment from the purified IgM fraction of mab H6-3C4 (13). To separate the pentagonal structure of the IgM, 1.5 mg of the IgM was incubated in Tris-HCI buffer containing 4 M urea at 25 C for 16 hours, then 45 JLg oftrypsin (Sigma, St. Louis, MO) 534 Shibahara et al. Inhibition of sperm-zona tight binding Fertility and Sterility

3 and 0.05 M of calcium chloride were added to digest the disulfide bonds of the IgM to the Fab and Fc fragments at 25 C for 8 hours. The fragments were gel filtrated through Superose 6 (Pharmacia), and the aliquots were tested to determine both the binding activity to human sperm by the immunofluorescent staining and the biological activities by SIT and sperm agglutination tests. Sodium dodecyl sulfate-polyarylamide Gel (SDS PAGE) Blot Analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed under reducing conditions by using 12% gel in a minigel electrophoretic apparatus (TEFCO Inc., Tokyo, Japan) (14). Prestained size markers (Bio-Rad, Richmond, CA) were used. For protein staining, Coomassie brilliant blue was used. The amounts of applied samples of H6-3C4, En46, and Fab fragment were 30 ILg, 20 ILg, and 13 ILg, respectively. Immunofluorescence Staining and Flow Cytometry For testing the Fab fragment of H6-3C4 bound to human sperm, fluorescent immunostaining was performed. Fresh ejaculated sperm were washed three times in Menezo's B2 medium containing 1 % bovine serum albumin (BSA), and the washed human sperm (15 X 10 4 ) were incubated with the Fab fragment (0.59 mg/ml) for 1 hour at room temperature. Intact IgM ofmab H6-3C4 (0.03 mg/ml) was used as a positive control and buffer (PBS with 0.1 % BSA) was used as a negative control. After washing once with buffer, sperm were then incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-human F(ab)~ antibody (1:100 dilution; Cappel, Durham, NC) for 30 minutes at 4 C. After washing an additional three times with buffer, sperm were analyzed on a F ACSCAN (Becton Dickinson, San Jose, CA). Antibody binding activity offab fragment or IgM ofh6 3C4 with sperm were demonstrated by staining pattern and mean FITC unit. Hemizona Assay Human oocytes were obtained from excised ovarian tissues and stored at -70 C in a 2.0 M dimethylsulphoxide solution in phosphate-buffered saline. The frozen oocytes were thawed and rinsed in Biggers, Whitten, and Whittingham medium supplemented with 0.35% BSA 1 day before use. The 00- cytes were cut almost in half using N arishige micromanipulators (Narishige, Tokyo, Japan) mounted on a phase-contrast microscope (Nikon, Garden City, NY). After discarding the degenerated ooplasma, the two matched hemizona were placed overnight at 4 C in a droplet of medium under mineral oil. Swim-up human sperm were incubated with patient's serum or test solution containing monoclonal sperm-immobilizing antibodies before exposure to hemizona. One hemizona was placed in a 100-ILL drop of swim-up sperm suspension with test sample while another matched hemizona was placed in a drop of control serum or diluent of mabs. Control serum was chosen on the basis of noninhibitory effect on sperm-zona binding. After 4 hours of coincubation, each hemizona was removed and rinsed vigorously to detach loosely associated sperm. Then the number of sperm tightly bound to the outer hemizona surface was counted. Each zona was tested twice. The hemizona index (HZ!) was calculated according to Mahony et al. (15, 16) and Shibahara et al. (17) as follows: number of bound sperm in the patient's serum HZI = X 100 number of bound sperm in the control's serum When the HZI was 50% or less, the sperm binding to zona was considered to be significantly inhibited in the test serum as compared in the control serum (15, 16). Sperm Agglutination Test and SIT The SIT as previously described by Isojima et al. (1) was used. Briefly, 0.25 ml of inactivated patient's serum, ml of human sperm suspension (40 X 10 6 sperm/ml) and 0.05 ml of guinea pig serum (10 C'H50 unit as complement) were mixed in a small test tube and incubated at 32 C for 60 minutes. The sperm immobilization value was calculated by dividing the sperm motility of the control serum by that of the test serum. A sperm immobilization value of 2 or more was considered as positive. The sperm agglutination assay was performed as described by Friberg (18), and a titer of ~20 was considered as positive. Aliquots with and without complement were checked for nonspecific (nonimmune) cytotoxicity. All sera with sperm-immobilizing antibodies were tested to determine the antibody titers by a quantitative SIT (SI 50 : the 50% sperm immobilization units) as described by Isojima et al. (19, 20). Absorption of a Patient Serum With Sperm Fresh ejaculated sperm were washed three times and suspended at a concentration of 4 X 10 7 /ml in Shibahara et al. Inhibition of sperm-zona tight binding 535

4 Ham's F-10 medium containing 3.5% HSA. An equal volume (50 ~L) of heat-inactivated serum sample (Code 05) and washed sperm suspension were mixed and incubated at 4 C overnight. After centrifugation at 3,000 X g for 10 minutes, the supernatants were subjected to SIT and HZA. RESULTS Effects of Patients' Sera on Sperm-Zona Tight Binding Patients' sera from 24 infertile women with sperm-immobilizing antibodies, 16 unexplained infertile women without sperm-immobilizing antibodies, and 2 postpartum women without sperm-immobilizing antibodies were tested for their effects on sperm-zona tight binding by HZA, and the HZI was calculated in each test sample. The patients' sera with sperm-agglutinating and sperm-immobilizing activities were used for HZA after diluting sera so that no more than 10% of motile sperm were agglutinated. In Table 1, the results of HZA were summarized together with quantitative sperm-immobilizing antibody titers (SI 50 units) in the infertile patients with sperm-immobilizing An HZI of 50 or less was considered a significant inhibition in HZA according to the criteria described by Mahony et al. (15, 16). Of patients' sera with sperm-immobilizing antibodies, 96% (23/24) were positive without complement for inhibition of sperm binding to zp, whereas all 16 patients' sera without sperm-immobilizing antibodies were negative for the same inhibitory effect (Table 1). Two puerperal women's sera were also negative (Table 1). However, there was no correlation between the HZI and the SI 50 titers of sperm-immobilizing The correlation coefficient of the data was calculated to be (P> 0.05). When the results of HZA were compared between the two groups of patients with sperm-immobilizing antibodies and without sperm-immobilizing antibodies, there was no overlap between the groups (Fig. 1). The range of HZI for the infertile patients with sperm-immobilizing antibodies was between to 53, whereas that for the infertile patients without sperm-immobilizing antibodies was between 55 to 113. A significant difference (P < ) was found in the average HZI of the groups. The average HZI (mean ± SD) was 17.3 ± 12.5 and 81.1 ± 16.1, respectively. Because we used the diluted sera containing both sperm-immobilizing and sperm-agglutinating activities for the HZA, the inhibitory effect of sperm-agglutinating antibodies in the patients' Table 1 Comparison of the HZI With Quantitative Sperm- Immobilizing Antibody Titers (SI 50) in Patients' Sera Sperm Patients agglutination code no.*t:j: SI 50 test HZI : : : : : : : : : : : : : : : : : : : : : : : : <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1: <1.0 1:10 86 * Code 01 to 24, infertile women with sperm-immobilizing t Code 25 to 40, unexplained infertile women without spermimmobilizing :j: Code 41 and 42, postpartum patients without sperm-immobilizing Serum samples were diluted according to the test results of sperm agglutination test so that no more than 10% of motile sperm were agglutinated. sera on sperm-zona tight binding is a cansocending factor. Purification and Characterization of Fab Fragment of Monoclonal Sperm-Immobilizing Antibodies H6-3C4 Both human monoclonal sperm-immobilizing antibody H6-3C4 and its class-switched antibody 536 Shibahara et al. Inhibition of sperm-zona tight binding Fertility and Sterility

5 SI positive sera 100.SI negative sera 90 >< ~ " IV c: 60 N 50 E ~ 40 ::I: 30 0.lIt : ± 12.5 ~t81.1 ±18.1 Figure 1 Comparison of the HZI in patients with and without sperm-immobilizing The average HZI (mean ± SD) w~re 17.3 ±.12.5 and 81.1 ± 16.1, respectively, in those patients with and without sperm-immobilizing These mean values were significantly different (P < ). En 46 have strong sperm-agglutinating activity. To test the biological effects ofthese sperm-immobilizing antibodies on the sperm-zona binding at higher concentration of the antibody, a large number of sperm agglutinate during incubation with the antibody and the HZ so that a few number of sperm can bind to the HZ. While at lower concentration of the antibody to prevent the sperm agglutination, inhibitory effects of the sperm-immobilizing antibody on the sperm-zona binding may decline. To increase the amount of mab H6-3C4 for binding to sperm and avoid the sperm-agglutinating activity, we prepared the Fab fragment of mab H6-3C4. Purified mab H6-3C4 (1.5 mg) were treated with trypsin and fractionated through gel filtration by using Superose 6 column. Fab fragment of mab H6-3C4 were collected and applied to SDS-PAGE bolt analysis for testing its purity. The result of SDS-PAGE showed two clear bands that are fragments of heavy chain (VH + CJLi) and light chain (data not shown). To test the binding activity of Fab fragment of mab H6-3C4 to the human sperm, immunofluorescence staining was performed. Ejaculated human sperm were washed, and motile fraction were collected and stained with Fab fragment and F1TC-Iabeled antihuman F(ab')2 antibody for the second antibody. Fluorescence intensity of 1 X 10 4 sperm were analyzed on a FACSCAN. Mean F1TC units of washed sperm stained with stain buffer and second antibody only, Fab fragment and second antibody, mab H6-3C4 and second antibody were 59.9, 272.4, and 856.9, respectively (Fig. 2). Fab fragment clearly bound to washed motile sperm but showed no agglutination (data not shown)..8 E ::J C a; u (a) 100 (b) 100 (c) FITC unit Figure 2 Flow cytometric analysis of sperm with the Fab fragment ofmab H6-3C4 by FACSCAN. Staining patterns were demonstrated by a two-dimensional figure as FITC unit at horizontal axis and cell number at vertical axis. Mean FITC units of washed sperm stained with (A) stain buffer: negative; (B) Fab fragment ofh6-3c4; (C) IgM ofh6-3c4: positive, were (A) 59.9, (B) 272.4, and (C) 856.9, respectively. It was confirmed the Fab fragment had binding activity to washed sperm. Effects of Monoclonal Sperm-Immobilizing Antibodies on Sperm-Zona Tight Binding Two human (H6-3C4, En 46) and two mouse (2HI2, 2C6) monoclonal sperm immobilizing antibodies were subjected to HZA, and the results were summarized in Table 2. Monoclonal antibody 2H12, which reacted with sulfated galactose residue, had a strong sperm-immobilizing activity (S1 50 > 15,000) but did not show any inhibitory effect on HZA (HZ1 = 111). Because three other mabs (2C6, H6-3C4, En 46) had both sperm-immobilizing and sperm-agglutinating activities, they must be appropriately diluted to avoid the sperm agglutination so that >90% sperm did not agglutinate. Monoclonal antibody 2C6 showed a significant inhibition that indicated 9 as HZ1, whereas human mabs H6-3C4 and En 46 did not show any inhibitory effect on sperm-zona tight binding. No correlation was Table 2 Comparison ofthe HZI With Quantitative Sperm Immobilizing Antibody Titers of MAbs Monoclonal sperm-immobilizing antibodies Ig class SI50* HZI 2H12 mouse M 15000< 111 2C6 mouse M H6-3C4 humanm En46 human G, Human Fab of H6-3C4 NDt 97 * Quantitative sperm-immobilizing antibody titers. t ND, not detectable because of no complement fixation of Fab fragment. Protein concentration of Fab was 0.59 mg/ml. Shibahara et al. Inhibition of sperm-zona tight binding 537

6 observed between the HZI and 8150 titers of monoclonal sperm-immobilizing The dilution of mab H6-3C4 to avoid the sperm agglutination may diminish the inhibitory effect on sperm-zona binding; therefore, the Fab fragment of mab H6-3C4 was produced by digestion of the pentagonal structure with urea and trypsin because it had binding activity to human sperm (Fig. 2) but had no sperm-immobilizing and sperm -agglutinating activities (Table 2). Though an excess amount ofthe Fab fragment (0.59 mg/ml) ofmab H6-3C4 was used to test the inhibition of sperm binding, the HZI was 97. This finding suggests that there was no inhibitory effect of mab H6-3C4 on the HZA after elimination of the sperm-agglutinating activity. One patient's serum showed a strong inhibitory effect on the HZA (HZI:O), though the human monoclonal sperm-immobilizing antibody H6-3C4, which was produced from a same patient (Code No. 05), possessed no inhibitory effect on sperm-zona tight binding. Though her serum was diluted 1:80 and did not show any sperm-immobilizing activity, it still caused a significant inhibitory effect on spermzona tight binding (HZI:29). When this serum was absorbed with human sperm, both sperm-immobilizing activity and the inhibitory effect on HZA were significantly decreased. Before absorption of this serum with sperm, the 8150 was 50, and no sperm binding was observed in the HZA, whereas sperm-immobilizing activity was not detectable, and 55 sperm could bind to the hemizona tightly after absorption with washed sperm (data not shown). DISCUSSION The normal fertilization process includes sperm capacitation, acrosome reaction, sperm-zona binding and penetration, sperm-egg fusion and postfusion events. Previous investigations using saltstored human zp (4, 5), zona-free hamster oocytes (6, 7), and retrospective analysis of routine human IVF results (21) have suggested that patient's serum with sperm-immobilizing antibodies could impair the fertilizing capability of human sperm. Monoclonal antibodies to sperm were also demonstrated to interfere with fertility in various stages of fertilization process (22-25). The HZA provides the opportunity to examine the effect of antisperm antibodies on sperm-zp tight binding. First, we examined whether human sera from infertile women with or without spermimmobilizing antibodies contain any inhibitory factor(s} on HZA and showed that 96% of 24 pa- tients' sera with sperm-immobilizing antibodies had the inhibitory factor(s}. On the contrary, none of the sera from the patients without sperm-immobilizing antibodies and postpartum patients showed any inhibitory effect. These results indicate that sperm-immobilizing antibodies might be the inhibitory factor or that other blocking antibodies of sperm-zp tight binding coexist with sperm-immobilizing antibodies in the same sera. The latter possibility is shown by the result that there was no correlation between the HZI and the 8150 and also from the result in case of a patient's serum from whom the human mab H6-3C4 was produced. To examine the possibility of heterogeneous nature of sperm-immobilizing antibodies, we examined sperm-immobilizing antibodies for their inhibitory effect on HZA. One (2C6) of the four monoclonal sperm-immobilizing antibodies tested possessed both activities of sperm immobilizing and the inhibitory effect on sperm-zona tight binding, but the human monoclonal sperm-immobilizing antibodies H6-3C4, En46 derived from an infertile woman, whose serum showed both sperm-immobilizing and sperm-zona binding inhibiting activities, had no inhibitory effect on HZA. This supports the heterogeneous nature of sperm-immobilizing A patient from whom the monoclonal sperm-immobilizing antibody H6-3C4 was produced possessed possibly two kinds of antibodies, one of which is sperm-immobilizing antibody and the other is blocking antibody of sperm-zona tight binding. When we made the hybridoma (H6-3C4) from a patient, only the lymphocyte that contributed to produced sperm-immobilizing antibody to the neolactosamin epitope on the sperm surface was fused to mouse myeloma cell line (N8-1), and the lymphocyte that is responsible to produce the antibody to block fertilization seemed not to be selected for cell fusion. Group studies on HZA in the patients with and without sperm-immobilizing antibodies clearly indicated that these patients producing sperm-immobilizing antibodies may also produce the blocking We can assume two possibilities for the antisperm antibodies in women's sera. One is the antibody that indicates only sperm-immobilizing activity with complement, and the other is the activity that expressed sperm-immobilizing activity together with inhibiting activity for sperm tight binding to HZ. The latter example is also proved by the mab 2C6. 80 far, we cannot prove any mono specific active antibody inhibits sperm binding to HZ in the sera of 16 unexplained sterile women without sperm-immobilizing antibody. Therefore sperm- 538 Shibahara et al. Inhibition of sperm-zona tight binding Fertility and Sterility

7 immobilizing antibody in women's sera may also have the inhibiting activity for the sperm binding to HZ itself, but we cannot rule out the existence of antibody that only blocks to sperm binding to zp because we have an example of a patient from whose lymphocytes the mab H6-3C4 was produced. More studies are required to prove whether there is a mono specific antibody to block sperm tight binding to HZ in the sera of unexplained sterile women without sperm-immobilizing and sperm-agglutinating Acknowledgments. We express our sincere thanks to Koji Koyama, M.D., and Minoru Shigeta, M.D., Department of Obstetrics and Gynecology, Hyogo Medical College, Nishinomiya, Hyogo, Japan, and Mary C. Mahony, Ph.D., The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, for their useful suggestions and helps. REFERENCES 1. Isojima S, Li TS, Ashitaka Y. Immunologic analysis of sperm immobilizing factor found in sera of women with unexplained sterility. Am J Obstet GynecoI1968;101: Isojima S. Human sperm antigens corresponding to spermimmobilizing antibodies in the sera of women with infertility of unknown cause: personal review of our recent studies. Hum Reprod 1989;4: Koyama K, Ikuma K, Kubota K, Isojima S. Effect of antisperm antibodies on sperm migration through cervical mucus. Excerpta Med Int Congr Ser 1979;512: Kamada M, Daitoh T, Hasebe H, Irahara M, Yamano S, Mori T. Blocking of human fertilization in vitro by sera with sperm-immobilizing Am J Obstet Gynecol 1985;153: Tsukui S, Noda Y, Fukuda A, Matsumoto H, Tatsumi K, Mori T. Blocking effect of sperm immobilizing antibodies on sperm penetration of human zonae pellucidae. J In Vitro Fert Embryo Transf 1988;5: Dor J, Rudak E, Aitken RJ. Antisperm antibodies: their effect on the process of fertilization studied in vitro. Fertil Steril 1981;35: Alexander NJ. Antibodies to human spermatozoa impede sperm penetration of cervical mucus or hamster eggs. Fertil Steril1984;41: Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GD. The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril1988;49: Isojima S, Kameda K, Tsuji Y, Shigeta M, Ikeda Y, Koyama K. Establishment and characterization of a human hybridoma secreting monoclonal antibody with high titers of sperm immobilizing and agglutinating activities against human seminal plasma. J Reprod ImmunoI1987;10: Komori S, Yamasaki N, Shigeta M, Isojima S, Watanabe T. Production of heavy-chain class-switch variants of human monoclonal antibody by recombinant DNA technology. Clin Exp ImmunoI1988;71: Tsuji Y, Fukuda H, Iuchi A, Ishizuka I, Isojima S. Sperm immobilizing antibodies react to 3-0-sulfated galactose residue of seminolipid on human sperm. J Reprod Immunol 1992;22: Kameda K, Takada Y, Hasegawa A, Tsuji Y, Koyama K, Isojima S. Sperm immobilizing and fertilization-blocking monoclonal antibody 2C6 to human seminal plasma antigen and characterizations of the antigen epitope corresponding to the monoclonal antibody. J Reprod ImmunoI1991;20: Shimizu A, Watanabe S, Yamamura Y, Putnam FW. Tryptic digestion of immunoglobulin M in urea: conformational lability of the middle part of the IgM molecule. Immunochem 1974;11: Kameda K, Tsuji Y, Koyama K, Isojima S. Comparative studies of the antigens recognized by sperm-immobilizing monoclonal BioI Reprod 1992;46: Mahony MC, Fulgham DL, Blackmore PF, Alexander NJ. Evaluation of human sperm -zona pellucida tight binding by presence of monoclonal antibodies to sperm antigens. J Reprod Immunol 1991;19: Mahony MC, Blackmore PF, Bronson RA, Alexander NJ. Inhibition of human sperm-zona pellucida tight binding in the presence of antisperm antibody positive polyclonal patient sera. J Reprod ImmunoI1991;19: Shibahara H, Shigeta M, Koyama K, Burkman LJ, Alexander NJ, Isojima S. Inhibition of sperm-zona pellucida tight binding by sperm immobilizing antibodies as assessed by the hemizona assay (HZA). Acta Obstet Gynecol Jpn 1991;43: Friberg J. A simple and sensitive micro method for demonstration of agglutinating antibodies from infertile men and women. Acta Obstet Gynecol Scand SuppI1974;36: Isojima S, Koyama K. Quantitative estimation of sperm immobilizing antibody in the sera of women with sterility of unknown etiology: the 50% sperm immobilization unit (SI 50). Excerpta Med Int Congr Ser 1974;370: Koyama K, Kubota K, Ikuma K, Shigeta M, Isojima S. Application of the quantitative sperm immobilization test for follow-up study of sperm immobilizing antibody in the sera of sterile women. Int J FertiI1988;33: Yovich JL, Kay D, StrangerJD, Boettcher B. In vitro fertilization of oocytes from women with serum antisperm Lancet 1984;1: Lee CYG, Wong E, Zhang JH. Inhibitory effects of monoclonal sperm antibodies on the fertilization of mouse oocytes in vitro and in vivo. J Reprod ImmunoI1986;9: Saling PM. Mouse sperm antigen that participate in fertilization IV. A monoclonal antibody prevents zona penetration by inhibition of the acrosome reaction. Dev BioI 1986;117: O'Rand MG, Irons GP, Porter JP. Monoclonal antibodies to rabbit sperm autoantigens. I. Inhibition of in vitro fertilization and localization on the egg. BioI Reprod 1984; 30: Primakoff P, Lathrop W, Woolman L, Cowan A, Myles D. Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20. Nature 1988;335: Shibahara et ai. Inhibition of sperm-zona tight binding 539