A human sperm antigen possibly involved in binding and/ or fusion with zona-free hamster eggs

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1 FERTILITY AND STERILITY Copyright<> 1990 The American Fertility Society Vol. 54, No.6, December 1990 Printed on ocid-free paper in U.S.A. A human sperm antigen possibly involved in binding and/ or fusion with zona-free hamster eggs Masaru Okabe, Ph.D.*t Morio Nagira, B.S.* Yuichi Kawai, M.S.:j: Sumio Matzno, M.S.* Tsutomu Mimura, Ph.D.* Tadanori Mayumi, Ph.D.:j: Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka, and Faculty of Pharmaceutical Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Japan A monoclonal antibody to human sperm (MH61) was established. The antibody did not attach to ejaculated sperm but to a head region of some sperm that were inc~bated in medium for 4 to 12 hours. A23187 treatment significantly increased the number of sperm that were reactive to the antibody. When sperm that bound to the zona-free hamster egg were subjected to the indirect immunofluorescence staining, all the sperm reacted to MH61 with their entire head region. The addition of the antibody to the medium before sperm addition reduced the fusion rate of human sperm to zona-free hamster egg. Fertil Steril54:1121, 1990 The interaction between the outer layer of the egg (zona pellucida) and the sperm has been studied extensively for a variety of animal species. 1-4 However, the molecular mechanism by which the sperm and the plasma membrane of the egg recognize each other remains, for the most part, unexplained. The membrane of the sperm undergoes a series of great structural changes called the acrosome reaction. The sperm and egg plasma membrane inter-recognition occurs between an egg and a sperm that has penetrated the zona pellucida and therefore has undergone the acrosome reaction. When sperm are mixed with eggs from which the zona pellucida has been removed, only sperm that have undergone the acrosome reaction can proceed to fuse, although the sperm that have not undergone the Received December 14, 1989; revised and accepted July 23, *Faculty of Pharmaceutical Sciences, Osaka University. t Reprint requests: Masaru Okabe, Ph.D., Faculty of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita, Osaka 565, Japan. :j: Faculty of Pharmaceutical Sciences, Kobe-Gakuin University. Present address: Faculty of Pharmaceutical Sciences, Osaka University. acrosome reaction can also come into contact with eggs. 5 Yanagimachi et al. 6 found that zona-free hamster eggs could fuse with sperm of other species, including human. Even to fuse with an egg of a different species, sperm seem to have undergone the acrosome reaction before fusion. 7 From the viewpoint of molecular biology, these findings suggest that some molecules that play a role in spermegg binding or fusion appear on the sperm surface after the acrosome reaction. To demonstrate the existence of such molecules, we prepared monoclonal antibodies to human sperm and studied the behavior of the antibody in the indirect immunofluorescence staining and in the mixture of human sperm and zona-free hamster eggs. Consequently, we found a sperm antigen that expresses antigenicity after the sperm undergoes the acrosome reaction and is considered to be involved in sperm-egg binding and/or fusion. The results are described below. MATERIALS AND METHODS Preparation of Sperm Semen from healthy donors, liquefied for 30 to 60 minutes at 37oC, was divided into 0.5 ml ali- Vol. 54, No.6, December 1990 Okabe et al. MA against acrosome-reacted human sperm 1121

2 quots and then gently placed below 2 ml of modified Biggers, Whitten, and Whittingham (BWW) medium 8 containing 3 mg/ml of human serumalbumin (HSA; Sigma Chemical Co., St. Louis, MO). The tubes (16 mm X 100 mm) were inclined to an angle of 30 and incubated at 37 C. After 1 hour, approximately 1.5 ml of the upper part of the medium containing motile sperm was taken out and centrifuged for 5 minutes at 500 Xg. The pellet was washed two times in modified BWW by centrifugation, resuspended in modified BWW containing 35 mg/ml of HSA (1 X 10 6 sperm/ml), and used for experiments (swim-up sperm). Sperm Incubation and Calcium-Ionophore Treatment Swim-up sperm were incubated at 37oC under 5% C0 2 in air for 4 or 24 hours and were then treated with 10 ~M of A23187 (free acid; Sigma Chemical) for 10 minutes. 9 Treated sperm were washed once with fresh modified BWW and resuspended to obtain an adequate concentration for the experiment. Immunization With Sperm and Hybridoma Preparation Swim-up sperm were incubated in modified BWW for 4 hours and treated with A23187 (see above). C57BL/6 (B6) female mice were immunized by intraperitoneal injection of 1 X 10 7 sperm/ mouse with Freund's complete adjuvant on day 0, with Freund's incomplete adjuvant on day 21, and without adjuvant (suspended with phosphatebuffered saline [PBS]) on day 28. Spleens were removed 3 days later and used as a source of immune cells for fusion to the BALB/c myeloma cell line P3-NS/1-Ag-1 (P3U1) by dropwise addition of polyethylene glycol 4,000 at a spleen cell to myeloma cell ratio of 5:1. Hybridomas were selectively proliferated in Dulbecco's modification of Eagle's medium containing 15% fetal calf serum, antibiotics, 2 X 10~ 5 M 2-mercaptoethanol, 10~ 4 M hypoxantine, 4 X 10~ 7 M aminopterine and 1.6 X 10~ 5 M thymidine. An indirect immunofluorescence technique wa,s employed for the screening of hybridomas. Cloning was performed three times by limiting dilution. 10 Preparation of Zona-Free Hamster Eggs Eight to 10-week-old female golden hamsters were superovulated by an intraperitoneal injection of 30 IU of pregnant mare serum gonadotropin followed by an equal amount of human chorionic gonadotropin (hcg) 72 hours later. The eggs were collected from the oviduct 17 hours after the hcg injection by puncturing the duct with a needle under modified BWW. The eggs were washed one time with fresh modified BWW and then treated with 0.1% hyaluronidase (type IV -S; Sigma Chemical) within 5 minutes to disperse the cumulus cells. The cumulus-free eggs were then washed two times by transferring them into fresh modified BWW. The eggs were then collected and transferred into modified BWW containing 0.1% trypsin (type III; Sigma Chemical). After a 2-minute treatment, the eggs, removed of the zona pellucida, were washed three times and used for the experiments. Zona-Free Hamster Egg Sperm Penetration Assay (SPA) Twenty to 25 eggs were placed in 0.4 ml of modified BWW containing 35 mg/ml of HSA, and test sperm were then added at a final concentration of 1 X 10 6 sperm/ml. After 5 hours of incubation with the sperm, eggs were transferred to a watch glass and collected in <1 ~L of the medium and placed in the middle of a spot of vase line and paraffin mixture (9:1) on the slide glass. The eggs were then gently pressed by a cover glass and observed for sperm binding and also for the formation of enlarged heads and/or pronuclei under phase contrast microscopy at 400X magnification. Indirect Immunofluorescence Staining of Sperm The MH61 hybridoma was injected into mice pretreated with 0.5 ml pristane and was proliferated in the peritoneal cavity for 2 to 3 weeks. The ascites fluid from these animals was stored at -sooc and diluted 1,000 times with PBS containing 5% newborn calf serum to make a MH61 antibody solution. Pellets containing 0.5 X 10 6 of sperm were mixed with MH61 antibody (100 ~L) and were incubated for 1 hour at room temperature. The sperm were washed two times with 0.5 to 1 ml of newborn calf serum-phosphate buffered saline. The sperm were incubated with 15 ~L of fluorescein isothiocyanate (FITC) conjugated goat antimouse immunoglobulin (Ig) (G +A+ M) (Cappel, West Chester, PA), which was diluted 100 times with newborn calf serum-phosphate buffered saline for 1 hour at room temperature. Immunostained sperm were washed 1122 Okabe et al. MA against acrosome-reacted human sperm Fertility and Sterility

3 one time with PBS and resuspended in 0.5 ml of PBS. Flow Cytometry Ten minutes before the flow cytometric analysis, the immunostained sperm suspension were mixed with propidium iodide (Fluka, Buchs, Switzerland) stock solution to provide 1 #Lg/mL of propidium iodide. Fluorescence-stained sperm were analyzed on a flow cytometer/cell sorter EPICS-753 (Coulter Corp., Hialeah, FL). Sperm were excited with a 488 nm argon laser oval (16 #LID X 160 #Lm) beam. Red fluorescence from propidium iodide was collected through a 590-nm-long pass filter and judged to contain either a "dead" or a "live" population. 11 Green fluorescence from FITC was collected through a 525-nm band pass filter and recorded as a measure of the amount of antibody bound to sperm. Five tenths milliliters of the sperm suspension (1 X 10 6 sperm/ml) was required for each analysis. To avoid contamination among samples, the flow cell was rinsed with a sheath buffer (Isoton III; Coulter Corp.) before each run. The first 100 to 200 #LL of the suspension was flushed out to obtain a constant flow of sperm. Routinely, 30,000 sperm were measured for each distribution at a flow rate of <300 cells/s. Analysis was conducted until 30,000 sperm were counted. The data were analyzed by an MDADS II system (multiparameter data acquisition display system; Coulter Corp.). Western Blotting Pellet containing 5 X 10 7 of swim-up sperm was resuspended in 110 #LL of PBS. To this suspension, 120 #LL of2-mercaptoethanol-free 2 X sodium dodecyl sulfate (SDS) sample treatment buffer (0.125 M Tris-HCl, ph 6.8; 4% SDS; 20% glycerol; 0.002% bromophenol blue) was added and thoroughly mixed. The suspension was incubated at 100oC for 3 minutes and rapidly cooled on ice. After 3 minutes, it was returned to room temperature and centrifuged at 8,000 X g (room temperature). The supernatant was used as a sample solution for electrophoresis. Electrophoretic separation was conducted using 10% to 20% gradient SDS-polyacrylamide gel (84 X 90 X 1.0. mm; Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) for approximately 1.5 hours. The separated proteins were electrically transferred on a nitrocellulose membrane filter ( 0.45 #Lm; Advantec TOYO, Tokyo, Japan) by using a transfer blotting cell (TEFCO, Tokyo, Japan). For this transfer, a current of 180 ma/80 cm 2 was applied for 1.5 hours in a transfer buffer (0.025 M Tris, M glycine, 20% methanol, ph 8.3). The nitrocellulose membrane filter was blocked with 5% skim m~lk in PBS at room temperature for 1 hour and rinsed with Tris-buffered saline supplemented with Tween 20 (TBST: 20 mm Tris-HCl, ph 7.6, NaCl137 mm, 0.1% Tween 20) for 3 minutes. The paper was allowed to react with MH61- containing ascites fluid, which had been diluted 1: 200 with newborn calf serum-phosphate buffered saline at room temperature for 1 hour. After being rinsed with TBST, the paper was allowed to react with diluted (1:500) peroxidase-conjugated antimouse IgG (Cappel) at room temperature for 1 hour. The paper was then incubated with a substrate solution (0.05% 4-chloro-1-naphthol; 16.7% methanol; 0.05% H 20 2 in TBS: 20 mm Tris-HCl, ph 7.6, 137 mm NaCl) for approximately 10 minutes. The reaction was stopped by rinsing the mixture with water. RESULTS For easier production of antibodies to acrosomereacted sperm, human sperm were incubated in modified BWW, treated with A23187, and used as an immunogen. Ejaculated sperm were washed with PBS and frozen. They were thawed immediately before being used for antibody screening with the indirect immunofluorescence staining technique. A monoclonal antibody, MH61, is produced by one of 10 clones established through four repetitions of fusion. As we had hoped, MH61 antibody did not react with fresh "live" sperm at all. (The positive antibody-sperm reaction observed at the time of screening with the indirect immunofluorescence staining might have been because of impaired membrane of the frozen -thawed sperm used: they were no longer viable.) After 4 to 24 hours' incubation of fresh "live" sperm, however, some of them became reactive to the antibody. Incubated human sperm were mixed with zonafree hamster eggs, and egg-bound sperm were stained with the indirect immunofluorescence technique. Even if the sperm suspension used contained only a small number of MH61-reactive sperm, all of the sperm bound to eggs were stained with the antibody at their heads (Fig. 1). The shape of the staining patterns and their appearance in sperm suspension were observed under fluorescent microscopy. As a result, we found headstained sperm increased as duration of incubation prolonged. It was also noted that A23187 treatment, which induced acrosome reaction, after 4 VoL 54, No.6, December 1990 Okabe et al. MA against acrosome-reacted human sperm 1123

4 l j Figure 1 Monoclonal antibody MH61 and its reactivity to human sperm. A and C represent an indirect immunofluorescence stained sperm that was classified as "headstained" and "not stained" in Table 1, respectively, observed under fluorescence microscopy. B and D show the same sperm observed under bright field microscopy (Xl,OOO magnification). A hamster egg with human sperm bound to its surface was observed under fluorescence (E) and bright field (F) microscopy (Xl,OOO magnification). Although the staining was spotty, it was obvious that the stained area extended to the postequatorial segment of sperm head. hours and 24 hours of the incubation, significantly increased the ratio of head-stained sperm. The number of antibody-reactive sperm varied according to the incubation time and sperm donors. Sperm degradation and membrane alterations induced by capacitation or the acrosome reaction were possible causes of these variations. Therefore, the ratio of the head-stained sperm was measured precisely using flow cytometer. Fine particles, detected in the sperm suspension by side light scattering plotted against the forward light scattering, were eliminated by an electric gating. Sperm that were found to be dead based on PI fluorescence were also removed by gating. These selected sperm were examined for the intensity of FITC fluorescence, which showed a two-peak distribution. The sperm were sorted according to the peaks and observed under the flu~rescent microscope, confirming that one peak corresponded to a "live" and head-stained sperm population and the other not stained population. After this rationalization of fundamental data, sperm samples obtained from healthy volunteers were incubated for 0, 4, and 24 hours and analyzed with a flow cytometer. The typical results were shown in Figure 2A. Head-stained 1124 Okabe et al. sperm appeared after the incubation. These same incubated sperm samples were further treated with A23187 and analyzed by flow cytometer. As shown in Figure 2B, the number of "live" and headstained sperm increased markedly. An aliquot of the ascites fluid containing MH61 antibody was added to SPA mixture containing human sperm and zona-free hamster eggs. The presence of the ascites fluid at 1:1,000 dilution inhibited 0 hr 4 hr 24 hr ADDD:" "DWW"' MA against acrosome-reacted human sperm Intensity of FITC fluorescence Figure 2 Expression of MH61 antigen on human sperm surface during incubation in vitro and A23187 treatment. The antigen expression on sperm from a healthy donor was analyzed by a flow cytometer at 0, 4, and 24 hours of incubation. A significant enhancement of the antigen expression by A23187 was observed in sperm only if they were preincubated for >4 hours. Fertility and Sterility

5 Table 1 Effect of MH61 Antibody on Fusion of Human Sperm to Zona-Free Hamster Egg" No. of eggs examined Penetrated eggs Fertilization index< NQ. of sperm bound/egg Crude ascites fluid % Ascites fluid b Control (P3U1) ± ± ± 2.5 MH ± 6.0d 0.29 ± ± 1.3' Purified F ( ab') fragment MH61 (F[ab'] fragment) OJ.Lg/mL ± ± ± J.Lg/mL ± ± 0.04d 11.5 ± J.Lg/mL ± ± ± 5.7 "All values are means ± SE in four independent experiments. b Final1:1,000 dilution. c Mean number of penetrated sperm/egg. d Significantly different from control; P < Significantly different from control; P < t Significantly different from control; P < the penetration rate by 70% and the fertilization index by 85% (Table 1). To identify the antigen recognized by MH61, western blotting was performed. Electrophoresis, using a 2-mercaptoethanol-reduced SDS-polyacrylamide gel was followed by blotting on nitrocellulose paper, but no band was detected on the paper. For successful antigen detection by western blotting, the use of a nonreduced electrophoretic sample buffer without 2-mercaptoethanol was essential. After this modification, the antigen was detected as a single band at 43 kd of molecular weight (Fig. 3). lane 77k0_ 66kO- 451<0-301<0-17k0 12k0-77k0-66k0-451<0-301<0. f -17k0 I -12k0 Figure 3 Immunoblot analysis ofmh61 antigen. Lanes 1 and 2 show the molecular weight marker and sperm lysate, respectively, both stained with Coomassie brilliant blue R-250. The transferred protein was immunostained with the MH61 ascite fluid (lane 3) and mouse normal serum (lane 4). DISCUSSION The acrosome reaction is a sequence of structural changes: the plasma membrane at the acrosome cap area of the sperm disappears and the inner acrosomal membrane appears. Membrane changes accompanying the reaction, however, occur not only at the tip of the sperm. Myles and Primakoff12 found that PH20, initially obtained as an antibody to the postacrosomal region of guinea pig sperm, react only with the acrosomal cap region when the sperm had undergone the acrosome reaction and suggested that the surface antigen migrates in relation with the acrosome reaction. Using OBF13 monoclonal antibody, we also found that the antigen seemed to have lain initially on the inner acrosomal membrane of the mouse sperm, distributed on entire head after the sperm underwent the acrosome reaction According to Yanagimachi and Noda/ 4 fusion of a sperm and an egg begins at the postacrosomal region, especially at the plasma membrane above the equatorial segment.15 The finding by Myles and Primakoff12 indicates the disappearance of an antigen from this region, whereas our finding indicates an appearance of antigen in this region. MH61 antibody to acrosome-reacted human sperm did not react with ejaculated fresh sperm at all as demonstrated by an indirect immunofluorescence staining, but egg-bound sperm showed positive staining all over the head. We suspect the MH61 antigen might lie on the acrosomal membrane and spread all over after acrosome reaction as we found in OBF13 antigen in mouse. However, Vol. 54, No.6, December 1990 Okabe et al. MA against acrosome-reacted human sperm 1125

6 the localization of the antigen needs further investigation. Although the inhibition was not complete, MH61 ascite fluid decreased the fertilization index, suggesting that the corresponding antigen may play a role in the fertilization process. Development of reactivity to MH61 antibody during incubation and ionophore treatment is considered to reflect the fact that the sperm have undergone the acrosome reaction. Flow cytometry analysis found that spontaneous acrosome reaction occurred in approximately 0.5% to 1.4% of the sperm tested after a 4-hour incubation and 2.2% to 10.9% after a 24-hour incubation (data not shown). Treatment of sperm with a calcium ionophore significantly increased the acrosome reaction. The rate of the sperm undergoing the reaction varied among the four donors from 2.6% to 24.3% for 4-hour incubated sperm and 23.0% to 45.8% for 24-hour incubated sperm (data not shown). However, as shown in Figure 2, the ionophore was only effective to incubated sperm. The sperm before preincubation did not respond to A In other words, the sperm were not ready for ionophore-induced acrosome reaction when ejaculated and became ready during incubation in modified BWW medium. lonophore-induced acrosome reaction might be different from the naturally occurring reaction, which is considered to be induced by the zona pellucida.16 However, the present results suggested that there was an individual difference in the time required for acrosome reaction. Acknowledgments. We are grateful to Dr. Takao Hama for helpful discussion throughout the preparation of this manuscript. Thanks also to Ms. Stephanie L. Cook for her assistance in preparing the manuscript. REFERENCES 1. Bleil JD, Wassarman PM: Mammalian sperm egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 20: 873, Bleil JD, Wassarman PM: Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm. J Cell Biol102:1363, Oikawa T, Sendai Y, Kurata S, Y anagimachi R: A glycoprotein of oviductal origin alters biochemical properties of the zona pellucida of hamster eggs. Gamete Res 19:113, Berger T, Davis A, Wardrip NJ, Hedric JL: Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida. J Reprod Fertil 86:559, Tsuiki A, Hoshiai H, Takahashi K, Suzuki M, Hoshi K: Sperm-egg interactions observed by scanning electron microscopy. Arch Androl16:35, Y anagimachi R, Y anagimachi H, Rogers BJ: The use of zona-free animal ova as a test system for the assessment of fertilizing capacity of human spermatozoa. Bioi Reprod 15: 471, Talbot P, Chacon RS: Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucida-free hamster oocytes. Fertil Steril 37:240, Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW: Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 33:534, Green DPL: The induction of the acrosome reaction in guinea-pig sperm by the divalent metal cation ionophore A J Cell Sci 32:137, Okabe M, Adachi T, Takada K, Oda H, Yagasaki M, Kohama Y, Mimura T: Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro. J Reprod Immunol11:91, Krishan A: Rapid flow cytofiuorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Bioi 66:188, Myles DG, Primakoff P: Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization. J Cell Biol99:1634, Okabe M, Yagasaki M, Oda H, Matzno S, Kohama Y, Mimura T: Effect of a monoclonal anti-mouse antibody (OBF13) on the interaction of mouse sperm with zona-free mouse and hamster eggs. J Reprod Immunol13:211, Yanagimachi R, Noda YD: Electron microscope studies of sperm incorporation into the hamster egg. Am J Anat 128: 429, Bedford JM, Moore HDM, Franklin LE: Significance ofthe equatorial segment of the acrosome of the spermatozoa in euthelian mammals. Exp Cell Res 119:119, Wassarman PM: The biology and chemistry offertilization. Science 235:553, Okabe et al. MA against acrosome-reacted human sperm Fertility and Sterility