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Effect of a monoclonal anti-mouse sperm antibody (OBF13) on the interaction of mouse sperm with zona-free mouse and hamster eggs
Journalof Reproductive Immunology, 13 (1988) 21 l-219 Elsevier Scientific Publishers Ireland Ltd.
211
JR1 00544
Effect of a monoclonal anti-mouse sperm antibody (OBF13) on the interaction of mouse sperm with zona-free mouse and hamster eggs
Masaru Okabe, Mitsuro Yagasaki, Hiroshi Oda, Sumio Matzno, Yasuhiro Kohama and Tsutomu Mimura Faculty of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita, Osaka, 565 (Japan) (Accepted for publication 18 April 1988)
Summary Mouse eggs freed from zonae by chymotrypsin were mixed with sperm and pronuclear formation was observed. When anti-mouse sperm monoclonal antibody (OBF 13) from ascites fluid was added to the medium (at a final concentration of O.OSVo),fertilization was significantly inhibited (9.7 k 4.3% compared to control 56.7 f 7.4%, P < 0.01). This was based on the inhibition of sperm binding to the egg. However, when similar experiments were performed using zona-free hamster eggs, addition of the OBF 13 antibody caused no significant reduction in fertilization rate (91-+ 7.1% compared to control 97 -+ 3.2%). It was also observed that binding of mouse sperm to hamster eggs was not inhibited by the antibody. It is therefore suggested that mouse sperm and mouse egg recognize each other in a species-specific manner.
Key words: mouse; hamster; sperm-egg interaction; monoclonal antibody; inhibition of fertilization.
Introduction Since Yanagimachi (1972) and Hanada and Chang (1972) reported interspecies fusion of gametes many more cases have been reported (Yanagimachi 0165-0378/88/$03.50 0 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
212
et al., 1976; Imai et al., 1977; Hanada and Chang, 1978; Pavlok, 1979). The results indicated that the recognition mechanism of sperm for egg plasma membrane is less species-specific than for the zona pellucida. On the other hand, many reports have demonstrated that only acrosome-reacted, and not fresh, uncapacitated sperm are able to fuse with the egg. At the present time, however, little is known about the mechanism of interaction of the sperm with the vitellus compared with that of the sperm to the zona pellucida. We have been studying the changes on the sperm surface during capacitation utilizing anti-mouse sperm monoclonal antibodies and have found that one particular antibody (OBF 13) reacts with the head of zona-penetrated sperm but not with fresh epididymal sperm (Okabe et al., 1986). The reactivity increased as sperm were incubated under conditions in which they could undergo capacitation and the acrosome reaction (Okabe et al., 1987). It was suggested that the antibody might serve as a useful probe to study the mechanism of sperm-egg recognition. In the present report we described the effect of the antibody on sperm-vitellus interaction using mouse sperm and zonafree mouse eggs as well as zona-free hamster eggs.
Materials and methods Preparation of sperm Mature male mice of the ddY strain were sacrificed by cervical dislocation and their epididymides were removed. Several incisions were made near the end of the epididymal ducts and the epididymides were placed in 2 ml of modified Kreb’s Ringer bicarbonate (KRB) buffer (Okabe et al., 1987) and were incubated at 37 OC. After 3 min, 500 ~1 of the upper part of the medium containing 1.0-l 5 x lo6 of motile sperm per ml were taken out and used as sperm suspension. Preparation of zona-free eggs Four-week-old ddY strain female mice were superovulated by injecting 5 IU of PMS and hCG respectively at 48-h intervals. The eggs were collected from the ampullary portion of the oviduct at 14-16 h, after the hCG injection by puncturing the duct with a needle under modified KRB. The eggs were washed once with fresh medium and then treated with 0.01% hyaluronidase (type I, Sigma Chem.) for 5 min at 37OC to disperse the cumulus cells. The cumulus-free eggs were then washed twice by transferring them into fresh medium. The eggs were then collected by a finely drawn Pasteur pipette and tranferred into medium containing 0.1% chymotrypsin (type II, Sigma Chem.). The eggs were gently pipetted in and out throughout the 3-min treatment under a dissecting microscope. Those freed from zonae were then washed with fresh medium 3 times and were used for the experiments.
213
Preparation of zona-free hamster eggs was performed according to the method of Yanagimachi et al. (1976). Six-week-old female golden hamsters received 30 IU PMS and hCG injections in their peritoneal cavity 48 h apart. The eggs were collected from the oviduct at 17 h after the hCG injection. After 3-min treatment with 0.1% hyaluronidase, the eggs were treated with 0.1% trypsin (type III, Sigma Chem.) for 2 min. The eggs were then washed with fresh medium and used for the experiment. Assessment of sperm binding and fusion In order to study the effect of the OBF 13 antibody, eggs were placed in the medium containing the acites fluid at a final concentration of 0.05% and fresh uncapacitated sperm were then introduced to the eggs. After 1,2 and 4 h of incubation with sperm, eggs were transferred to a watch glass and collected in a few microliters of medium. Care was taken to handle the eggs gently throughout this procedure. A Vaseline and paraffin mixture (9: 1) were spotted on the glass slide and the eggs, collected in a minimal volume (mO.3 fi), were placed in the middle of the spots. 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 X 400 magnification. In the mouse, eggs with male and female pronuclei were scored as fertilized. In the hamster, eggs with swollen mouse sperm heads were scored as fused. Indirect immunofluorescence staining After 2h of incubation without the antibody, test tubes containing the eggs and sperm were cooled down in an ice bath and the OBF 13 from ascites fluid was added to make a concentration of 0.05 % , After 15 min of incubation the eggs were washed by transferring them twice to new medium. Ten microliters of FITC-conjugated goat anti-mouse Igs (IgM + IgA + IgG) (Cappel), previously diluted 16 times with phosphate buffered saline containing 1% bovine serum albumin (Sigma Chem. Fr. V), were placed under paraffin oil and the collected eggs were deposited in this droplet. After 15 min of incubation the sperm on the eggs were washed with fresh medium and then assessed for reactivity with the antibody under a fluorescent microscope. Preparation of monoclonal antibody Briefly, hybridoma OBF 13 was derived from fusion of the myeloma P3Ul cell line and spleen cells from C57BL/6 mice which were immunized with syngeneic sperm. The hybridoma was injected into CBFl (BALB/c x C57BL/6) mice pretreated with pristane (Sigma Chem.). Ascitic fluid was removed when the mouse showed sufficient abdominal swelling (for details, see Okabe et al., 1987).
214 TABLE 1 Effect of OBF 13 antibody on fertilization of zona-free mouse eggs. Ascites”
No. of experiments
No. of eggs examined
Eggs with pronuclei (Q)
P3Ulb OBF 13
I 7
121 119
56.7 + 7.4 9.7 f 4.3*
aFinal 0.05% in the medium. bParent myeloma cell line. *Significantly different from control, P-C 0.01.
Results Effect of OBF13 antibody on mouse sperm fusion with zona-free mouse eggs Mouse epididymal sperm were introduced to zona-free mouse eggs and pronuclear formation was observed 4 h after addition of sperm. Ascites fluid from P3Ul injected animals was added to a control group. Fusion was significantly inhibited (9.7 + 4.3%) when 0BF13 from ascites fluid was added to the medium, compared to the control group, 56.7 & 7.4% (Table 1). The inhibition seemed to depend on the blocking of sperm-egg interaction. As shown in Fig. 1, the mean number of sperm bound to the zona-free mouse egg was significantly reduced by the addition of the OBF 13 ascitic fluid. In the control group, it was also noted that after 2 h fewer sperm were
Time
after
insemination
Ihr)
Fig. 1. Effect of OBF 13 antibody on binding of mouse sperm to zona-free mouse eggs. P3Ul (control) or OBF 13 ascites were added to the medium at a final dilution of 0.05%. Significantly different from OBF 13 added group, ***PC 0.001.
215
found on the surface of the zona-free fertilized eggs than the unfertilized eggs. The significant differenee in sperm numbers on fertilized and unfertilized eggs may argue for the existence of a plasma membrane block of the mouse egg.
Effect of OBF 13 antibody on mouse sperm fusion with zona-free hamster eggs Similar experiments were performed using zona-free hamster eggs. Unlike the experiment with mouse eggs, the addition of the antibody did not suppress the fusion of the mouse sperm to the zona-free hamster eggs (91 -+ 7.1% compared to control 97 +: 3.2%). Although the number of fused sperm per egg decreased slightly, the change was not significant. When the number of sperm bound to the eggs was counted, the effect of the ascitic fluid was not seen (Table 2).
Indirect immunofluorescence staining of sperm bound to zona-free eggs. After a 2-h incubation of eggs with sperm, OBF 13 was added to the medium and incubated for 15 min at OOC. The eggs were then washed and treated with anti-mouse Igs. Several sperm remained on the surface of the egg throughout the procedure. As shown in Fig. 2, all of the mouse sperm bound to the mouse or hamster eggs were stained over their entire head. Discussion Although a few exceptional cases of zona penetration by foreign sperm have been reported (Hanada and Chang, 1978), the zona pellucida is one of the most prominent sites of species specificity. On the other hand, sperm-egg fusion appears to be less species-specific than sperm-zona interaction. In particular, the zona-free hamster egg is known to be able to bind sperm of various species (see review, Yanagimachi, 1981). However, unlike the hamster egg, the mouse egg is not so readily able to bind foreign sperm (Hanada TABLE 2 Effect of OBF 13 antibody on fertilization of zona-free hamster eggs” Ascite@
No. of eggs examined
Eggs with swollen sperm heads (070)
No. of sperm penetrated/egg
No. of sperm bound to eggs
P3Ulb OBF 13
99 98
97 + 3.2 91 -c 7.1
6.9 f 2.2 5.7 + 1.0
11.6 f 1.2 14.0 + 1.4
“Eight separate experiments in each group. bFinal 0.05% in the medium. ‘Parent myeloma cell line.
216
Fig. 2. Indirect immunofluorescence staining of mouse sperm bound to zona-free eggs. A mouse egg observed under phase contrast (A) and fluorescence (B) microscopy. A hamster egg observed under phase contrast (C) and fluorescence (D) microscopy ( x 400 magnification).
218
and Chang, 1978; Pavlok, 1979; Quinn, 1979). Mouse sperm and mouse egg therefore seem to have a mutual recognition signal. Our findings that mouse sperm treated with the OBF 13 antibody can fuse with hamster eggs but not with mouse eggs support this idea. It may be possible to interpret the data in another way: perhaps mouse sperm have a stronger affinity for the hamster egg than for the mouse egg, consequently inhibiting only the fusion of mouse sperm to mouse egg. However, Yanagimachi stated in his review (1981) that more hamster sperm fused with the hamster egg than did foreign (guinea pig and human) sperm. Therefore, the assumption of a stronger affinity of mouse sperm for hamster eggs is unlikely. It should be noted that the present results also suggest that a different, non-species-specific, recognition mechanism exists because OBF 13-treated mouse sperm were able to fuse with the hamster egg. Even in the cases of inter-species fusion of gametes, the eggs are not able to fuse with sperm from various species without following a particular recognition protocol: the sperm must be acrosome reacted before fusion takes place (Yanagimachi and Noda, 1970; Wolf et al., 1976). It would be interesting to know whether or not acrosome reacted sperm of various species have a common structure recognizable by the hamster egg. Primakoff and Hyatt (1986) reported that anti-guinea pig sperm monoclonal antibody PH20 inhibited the fusion of guinea pig sperm to hamster eggs but not to homologous eggs. Their antibody might react to a non-species-specific sperm-egg recognition site. In order to obtain more concrete evidence about the sperm-egg recognition mechanism, purification and characterization of the receptor is essential. However, as far as we tested, the antigen which reacts with the OBF 13 antibody was not detected on the western blotted membrane after SDS-electrophoresis. Considering this result together with the generalized localization of the antigen on the sperm head, the epitope recognized by the antibody could be a glycolipid rather than a peptide. We reported previously that the OBF 13 antibody does not detect a change which occurs only in the acrosomal region after the acrosome reaction, but one which spreads even to the post-acrosomal region. We further noted that reactivity to the antibody reflects the fertilizing ability of the mouse sperm (Okabe et al., 1986, 1987). Therefore, it was not surprising to find that all the sperm bound to the mouse eggs had antigen exposed over the entire head. However, it was noteworthy that the mouse sperm bound to hamster eggs also showed the same antigen distribution. Whatever the nature of the substance recognized by the hamster eggs, the results indicated the concomitant appearance of the substance and the OBF 13 antigen, which underlined the previous reports that only acrosome reacted (foreign) sperm can fuse with the hamster egg (see review, Yanagimachi, 198 1).
219
Acknowledgements This work is supported by a Grant-in-Aid from the Tokyo Biochemical Research Foundation. We are grateful to Ms. Stephanie L. Cook for her help in the preparation of the manuscript. References Fukuda, Y., Maddock, M.B. and Chang, M.C. (1979) In vitro fertilization of two species of deer mouse eggs by homologous or heterologous sperm and penetration of laboratory mouse eggs by deer mouse sperm. J. Exp. Zool. 207,481-490. Hanada, A. and Chang, M.C. (1972) Penetration of zona-free eggs by spermatozoa of different species. Biol. Reprod. 6,300-309. Hanada, A. and Chang, M.C. (1978) Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro. J. Exp. Zool. 203,277-286. Imai, H., Nlwa, K. and Iritani, A. (1977) Penetration in vitro of zona-free hamster eggs by ejaculated boar spermatozoa. J. Reprod. Fertil. 54,379-383. Okabe, M., Takada, K., Adachi, T., Kohama, Y. and Mimura, T. (1986) Inconstant reactivity of antisperm monoclonal antibody and its relationship to sperm capacitation. J. Reprod. Immunol. 9, 67-70. Okabe, M., Adachi, T., Takada, K., Oda, H., Yagasaki, M., Kohama, Y. and Mimura, T. (1987) Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro. J. Reprod. Immunol. 11,91-100. Pavlok, A. (1979) Interspecies interaction of zona-free ova with spermatozoa in mouse, rat and hamster. Anim. Reprod. Sci. 2,395-402. Primakoff, P. and Hyatt, H. (1986) An antisperm monoclonal antibody inhibits sperm fusion with zonafree hamster eggs but not homologous eggs. Fertil. Steril. 46,489-493. Quinn, P. (1979) Failure of human spermatozoa to penetrate zona-free mouse and rat ova in vitro. J. Exp. Zool. 210,479-506. Yanagimachi, R. and Noda, Y.D. (1970) Physiological changes in the post-nuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between sperm and egg cells. J. Ultrastruct. Res. 31,486-493. Yanagimachi, R. (1972) Penetration of guinea pig spermatozoa into hamster eggs in vitro. J. Reprod. Fertil. 28,477-480. Yanagimachi, R., Yanagimachi, H. and Rogers, B.J. (1976) The use of zone-free ova as a test system for the assessment of the fertilizing capacity of spermatozoa. Biol. Reprod. 15,471-476. Yanagimachi, R. (1981) Mechanism of fertilization in mammals. In: Fertilization and Embryonic Development In Vitro. (L. Mastroianni, Jr. and J.D. Biggers eds.), pp. 82-182. Plenum Press, New York. Wolf, D.P., Inoue, M. and Stark, R.A. (1976) Penetration of zona-free mouse ova. Biol. Reprod. 15, 213-221.
211
JR1 00544
Effect of a monoclonal anti-mouse sperm antibody (OBF13) on the interaction of mouse sperm with zona-free mouse and hamster eggs
Masaru Okabe, Mitsuro Yagasaki, Hiroshi Oda, Sumio Matzno, Yasuhiro Kohama and Tsutomu Mimura Faculty of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita, Osaka, 565 (Japan) (Accepted for publication 18 April 1988)
Summary Mouse eggs freed from zonae by chymotrypsin were mixed with sperm and pronuclear formation was observed. When anti-mouse sperm monoclonal antibody (OBF 13) from ascites fluid was added to the medium (at a final concentration of O.OSVo),fertilization was significantly inhibited (9.7 k 4.3% compared to control 56.7 f 7.4%, P < 0.01). This was based on the inhibition of sperm binding to the egg. However, when similar experiments were performed using zona-free hamster eggs, addition of the OBF 13 antibody caused no significant reduction in fertilization rate (91-+ 7.1% compared to control 97 -+ 3.2%). It was also observed that binding of mouse sperm to hamster eggs was not inhibited by the antibody. It is therefore suggested that mouse sperm and mouse egg recognize each other in a species-specific manner.
Key words: mouse; hamster; sperm-egg interaction; monoclonal antibody; inhibition of fertilization.
Introduction Since Yanagimachi (1972) and Hanada and Chang (1972) reported interspecies fusion of gametes many more cases have been reported (Yanagimachi 0165-0378/88/$03.50 0 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
212
et al., 1976; Imai et al., 1977; Hanada and Chang, 1978; Pavlok, 1979). The results indicated that the recognition mechanism of sperm for egg plasma membrane is less species-specific than for the zona pellucida. On the other hand, many reports have demonstrated that only acrosome-reacted, and not fresh, uncapacitated sperm are able to fuse with the egg. At the present time, however, little is known about the mechanism of interaction of the sperm with the vitellus compared with that of the sperm to the zona pellucida. We have been studying the changes on the sperm surface during capacitation utilizing anti-mouse sperm monoclonal antibodies and have found that one particular antibody (OBF 13) reacts with the head of zona-penetrated sperm but not with fresh epididymal sperm (Okabe et al., 1986). The reactivity increased as sperm were incubated under conditions in which they could undergo capacitation and the acrosome reaction (Okabe et al., 1987). It was suggested that the antibody might serve as a useful probe to study the mechanism of sperm-egg recognition. In the present report we described the effect of the antibody on sperm-vitellus interaction using mouse sperm and zonafree mouse eggs as well as zona-free hamster eggs.
Materials and methods Preparation of sperm Mature male mice of the ddY strain were sacrificed by cervical dislocation and their epididymides were removed. Several incisions were made near the end of the epididymal ducts and the epididymides were placed in 2 ml of modified Kreb’s Ringer bicarbonate (KRB) buffer (Okabe et al., 1987) and were incubated at 37 OC. After 3 min, 500 ~1 of the upper part of the medium containing 1.0-l 5 x lo6 of motile sperm per ml were taken out and used as sperm suspension. Preparation of zona-free eggs Four-week-old ddY strain female mice were superovulated by injecting 5 IU of PMS and hCG respectively at 48-h intervals. The eggs were collected from the ampullary portion of the oviduct at 14-16 h, after the hCG injection by puncturing the duct with a needle under modified KRB. The eggs were washed once with fresh medium and then treated with 0.01% hyaluronidase (type I, Sigma Chem.) for 5 min at 37OC to disperse the cumulus cells. The cumulus-free eggs were then washed twice by transferring them into fresh medium. The eggs were then collected by a finely drawn Pasteur pipette and tranferred into medium containing 0.1% chymotrypsin (type II, Sigma Chem.). The eggs were gently pipetted in and out throughout the 3-min treatment under a dissecting microscope. Those freed from zonae were then washed with fresh medium 3 times and were used for the experiments.
213
Preparation of zona-free hamster eggs was performed according to the method of Yanagimachi et al. (1976). Six-week-old female golden hamsters received 30 IU PMS and hCG injections in their peritoneal cavity 48 h apart. The eggs were collected from the oviduct at 17 h after the hCG injection. After 3-min treatment with 0.1% hyaluronidase, the eggs were treated with 0.1% trypsin (type III, Sigma Chem.) for 2 min. The eggs were then washed with fresh medium and used for the experiment. Assessment of sperm binding and fusion In order to study the effect of the OBF 13 antibody, eggs were placed in the medium containing the acites fluid at a final concentration of 0.05% and fresh uncapacitated sperm were then introduced to the eggs. After 1,2 and 4 h of incubation with sperm, eggs were transferred to a watch glass and collected in a few microliters of medium. Care was taken to handle the eggs gently throughout this procedure. A Vaseline and paraffin mixture (9: 1) were spotted on the glass slide and the eggs, collected in a minimal volume (mO.3 fi), were placed in the middle of the spots. 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 X 400 magnification. In the mouse, eggs with male and female pronuclei were scored as fertilized. In the hamster, eggs with swollen mouse sperm heads were scored as fused. Indirect immunofluorescence staining After 2h of incubation without the antibody, test tubes containing the eggs and sperm were cooled down in an ice bath and the OBF 13 from ascites fluid was added to make a concentration of 0.05 % , After 15 min of incubation the eggs were washed by transferring them twice to new medium. Ten microliters of FITC-conjugated goat anti-mouse Igs (IgM + IgA + IgG) (Cappel), previously diluted 16 times with phosphate buffered saline containing 1% bovine serum albumin (Sigma Chem. Fr. V), were placed under paraffin oil and the collected eggs were deposited in this droplet. After 15 min of incubation the sperm on the eggs were washed with fresh medium and then assessed for reactivity with the antibody under a fluorescent microscope. Preparation of monoclonal antibody Briefly, hybridoma OBF 13 was derived from fusion of the myeloma P3Ul cell line and spleen cells from C57BL/6 mice which were immunized with syngeneic sperm. The hybridoma was injected into CBFl (BALB/c x C57BL/6) mice pretreated with pristane (Sigma Chem.). Ascitic fluid was removed when the mouse showed sufficient abdominal swelling (for details, see Okabe et al., 1987).
214 TABLE 1 Effect of OBF 13 antibody on fertilization of zona-free mouse eggs. Ascites”
No. of experiments
No. of eggs examined
Eggs with pronuclei (Q)
P3Ulb OBF 13
I 7
121 119
56.7 + 7.4 9.7 f 4.3*
aFinal 0.05% in the medium. bParent myeloma cell line. *Significantly different from control, P-C 0.01.
Results Effect of OBF13 antibody on mouse sperm fusion with zona-free mouse eggs Mouse epididymal sperm were introduced to zona-free mouse eggs and pronuclear formation was observed 4 h after addition of sperm. Ascites fluid from P3Ul injected animals was added to a control group. Fusion was significantly inhibited (9.7 + 4.3%) when 0BF13 from ascites fluid was added to the medium, compared to the control group, 56.7 & 7.4% (Table 1). The inhibition seemed to depend on the blocking of sperm-egg interaction. As shown in Fig. 1, the mean number of sperm bound to the zona-free mouse egg was significantly reduced by the addition of the OBF 13 ascitic fluid. In the control group, it was also noted that after 2 h fewer sperm were
Time
after
insemination
Ihr)
Fig. 1. Effect of OBF 13 antibody on binding of mouse sperm to zona-free mouse eggs. P3Ul (control) or OBF 13 ascites were added to the medium at a final dilution of 0.05%. Significantly different from OBF 13 added group, ***PC 0.001.
215
found on the surface of the zona-free fertilized eggs than the unfertilized eggs. The significant differenee in sperm numbers on fertilized and unfertilized eggs may argue for the existence of a plasma membrane block of the mouse egg.
Effect of OBF 13 antibody on mouse sperm fusion with zona-free hamster eggs Similar experiments were performed using zona-free hamster eggs. Unlike the experiment with mouse eggs, the addition of the antibody did not suppress the fusion of the mouse sperm to the zona-free hamster eggs (91 -+ 7.1% compared to control 97 +: 3.2%). Although the number of fused sperm per egg decreased slightly, the change was not significant. When the number of sperm bound to the eggs was counted, the effect of the ascitic fluid was not seen (Table 2).
Indirect immunofluorescence staining of sperm bound to zona-free eggs. After a 2-h incubation of eggs with sperm, OBF 13 was added to the medium and incubated for 15 min at OOC. The eggs were then washed and treated with anti-mouse Igs. Several sperm remained on the surface of the egg throughout the procedure. As shown in Fig. 2, all of the mouse sperm bound to the mouse or hamster eggs were stained over their entire head. Discussion Although a few exceptional cases of zona penetration by foreign sperm have been reported (Hanada and Chang, 1978), the zona pellucida is one of the most prominent sites of species specificity. On the other hand, sperm-egg fusion appears to be less species-specific than sperm-zona interaction. In particular, the zona-free hamster egg is known to be able to bind sperm of various species (see review, Yanagimachi, 1981). However, unlike the hamster egg, the mouse egg is not so readily able to bind foreign sperm (Hanada TABLE 2 Effect of OBF 13 antibody on fertilization of zona-free hamster eggs” Ascite@
No. of eggs examined
Eggs with swollen sperm heads (070)
No. of sperm penetrated/egg
No. of sperm bound to eggs
P3Ulb OBF 13
99 98
97 + 3.2 91 -c 7.1
6.9 f 2.2 5.7 + 1.0
11.6 f 1.2 14.0 + 1.4
“Eight separate experiments in each group. bFinal 0.05% in the medium. ‘Parent myeloma cell line.
216
Fig. 2. Indirect immunofluorescence staining of mouse sperm bound to zona-free eggs. A mouse egg observed under phase contrast (A) and fluorescence (B) microscopy. A hamster egg observed under phase contrast (C) and fluorescence (D) microscopy ( x 400 magnification).
218
and Chang, 1978; Pavlok, 1979; Quinn, 1979). Mouse sperm and mouse egg therefore seem to have a mutual recognition signal. Our findings that mouse sperm treated with the OBF 13 antibody can fuse with hamster eggs but not with mouse eggs support this idea. It may be possible to interpret the data in another way: perhaps mouse sperm have a stronger affinity for the hamster egg than for the mouse egg, consequently inhibiting only the fusion of mouse sperm to mouse egg. However, Yanagimachi stated in his review (1981) that more hamster sperm fused with the hamster egg than did foreign (guinea pig and human) sperm. Therefore, the assumption of a stronger affinity of mouse sperm for hamster eggs is unlikely. It should be noted that the present results also suggest that a different, non-species-specific, recognition mechanism exists because OBF 13-treated mouse sperm were able to fuse with the hamster egg. Even in the cases of inter-species fusion of gametes, the eggs are not able to fuse with sperm from various species without following a particular recognition protocol: the sperm must be acrosome reacted before fusion takes place (Yanagimachi and Noda, 1970; Wolf et al., 1976). It would be interesting to know whether or not acrosome reacted sperm of various species have a common structure recognizable by the hamster egg. Primakoff and Hyatt (1986) reported that anti-guinea pig sperm monoclonal antibody PH20 inhibited the fusion of guinea pig sperm to hamster eggs but not to homologous eggs. Their antibody might react to a non-species-specific sperm-egg recognition site. In order to obtain more concrete evidence about the sperm-egg recognition mechanism, purification and characterization of the receptor is essential. However, as far as we tested, the antigen which reacts with the OBF 13 antibody was not detected on the western blotted membrane after SDS-electrophoresis. Considering this result together with the generalized localization of the antigen on the sperm head, the epitope recognized by the antibody could be a glycolipid rather than a peptide. We reported previously that the OBF 13 antibody does not detect a change which occurs only in the acrosomal region after the acrosome reaction, but one which spreads even to the post-acrosomal region. We further noted that reactivity to the antibody reflects the fertilizing ability of the mouse sperm (Okabe et al., 1986, 1987). Therefore, it was not surprising to find that all the sperm bound to the mouse eggs had antigen exposed over the entire head. However, it was noteworthy that the mouse sperm bound to hamster eggs also showed the same antigen distribution. Whatever the nature of the substance recognized by the hamster eggs, the results indicated the concomitant appearance of the substance and the OBF 13 antigen, which underlined the previous reports that only acrosome reacted (foreign) sperm can fuse with the hamster egg (see review, Yanagimachi, 198 1).
219
Acknowledgements This work is supported by a Grant-in-Aid from the Tokyo Biochemical Research Foundation. We are grateful to Ms. Stephanie L. Cook for her help in the preparation of the manuscript. References Fukuda, Y., Maddock, M.B. and Chang, M.C. (1979) In vitro fertilization of two species of deer mouse eggs by homologous or heterologous sperm and penetration of laboratory mouse eggs by deer mouse sperm. J. Exp. Zool. 207,481-490. Hanada, A. and Chang, M.C. (1972) Penetration of zona-free eggs by spermatozoa of different species. Biol. Reprod. 6,300-309. Hanada, A. and Chang, M.C. (1978) Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro. J. Exp. Zool. 203,277-286. Imai, H., Nlwa, K. and Iritani, A. (1977) Penetration in vitro of zona-free hamster eggs by ejaculated boar spermatozoa. J. Reprod. Fertil. 54,379-383. Okabe, M., Takada, K., Adachi, T., Kohama, Y. and Mimura, T. (1986) Inconstant reactivity of antisperm monoclonal antibody and its relationship to sperm capacitation. J. Reprod. Immunol. 9, 67-70. Okabe, M., Adachi, T., Takada, K., Oda, H., Yagasaki, M., Kohama, Y. and Mimura, T. (1987) Capacitation-related changes in antigen distribution on mouse sperm heads and its relation to fertilization rate in vitro. J. Reprod. Immunol. 11,91-100. Pavlok, A. (1979) Interspecies interaction of zona-free ova with spermatozoa in mouse, rat and hamster. Anim. Reprod. Sci. 2,395-402. Primakoff, P. and Hyatt, H. (1986) An antisperm monoclonal antibody inhibits sperm fusion with zonafree hamster eggs but not homologous eggs. Fertil. Steril. 46,489-493. Quinn, P. (1979) Failure of human spermatozoa to penetrate zona-free mouse and rat ova in vitro. J. Exp. Zool. 210,479-506. Yanagimachi, R. and Noda, Y.D. (1970) Physiological changes in the post-nuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between sperm and egg cells. J. Ultrastruct. Res. 31,486-493. Yanagimachi, R. (1972) Penetration of guinea pig spermatozoa into hamster eggs in vitro. J. Reprod. Fertil. 28,477-480. Yanagimachi, R., Yanagimachi, H. and Rogers, B.J. (1976) The use of zone-free ova as a test system for the assessment of the fertilizing capacity of spermatozoa. Biol. Reprod. 15,471-476. Yanagimachi, R. (1981) Mechanism of fertilization in mammals. In: Fertilization and Embryonic Development In Vitro. (L. Mastroianni, Jr. and J.D. Biggers eds.), pp. 82-182. Plenum Press, New York. Wolf, D.P., Inoue, M. and Stark, R.A. (1976) Penetration of zona-free mouse ova. Biol. Reprod. 15, 213-221.