Journal ql'Reproductive Immunology, 21 (19921 175-187
175
Elsevier Scientific Publishers Ireland Ltd.
JR1 00754
Monoclonal antibodies affecting sperm-zona binding and/or zona-induced acrosome reaction Chorng-Hwa
Fann
and Chi-Yu
Gregory
Lee
Andrology Laboratory, Dept. Obstetrics and Gynecology, The University of British Columbia, Vancouver. V6T 2B5 (Canada) (Accepted for publication 7 November 1991)
Summary The majority of anti-sperm monoclonal antibodies which were shown to inhibit in vitro fertilization in our laboratory react with antigens in the acrosomal region of spermatozoa. To elucidate the mechanism of human and/or mouse fertilization inhibition, the effects of these antibodies on mouse sperm-zona binding and mouse zona-induced acrosome reaction in vitro were studied. Among these antibodies, MS-4 and MS-7 were shown to react partially with uncapacitated mouse sperm, and to react more with capacitated mouse sperm, whereas HS-9, HS-11 and HS-63 react only with capacitated and acrosome-intact human or mouse sperm. HS-63 and MS-4 were shown to inhibit zona-induced acrosome reaction significantly, but not the sperm-zona binding. On the other hand, HS-9, HS-I 1 and MS-7 were shown to inhibit both sperm-zona binding and zona-induced acrosome reaction. The results of this study suggest that fertilization inhibition caused by these antibodies could result from that of the initial sperm-zona binding and/or zona-induced acrosome reaction.
K e y w o r d s : anti-sperm monoclonal antibodies," in vitro fertilization," spermzona binding," zona-induced acrosome reaction
Correspondence to," Dr. Gregory Lee, FI07, Acute Care Unit, 2211 Wesbrook Mall, Vancouver, B.C. Canada, V6T 2B5. 0165-0378/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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Introduction
In previous communications, generation and characterization of numerous monoclonal antibodies against sperm surface antigens from different mammalian species were reported (Lee et al., 1984a,b,c). They were selected initially based on their high tissue-specificity to sperm and later on their ability to inhibit fertilization of mouse oocytes in vitro and/or human sperm penetration to zona-free hamster ova (Lee et al., 1986a; Menge et al., 1987). Through intensive studies, it was clearly demonstrated that the majority of these inhibitory antibodies generated in this laboratory were found to react with antigens on the sperm acrosome (Lee et al., 1986a,b). Since the acrosomal components are known to be involved in fertilization, some of the selected antibodies might interfere with the following fertilization processes: (a) sperm-zona binding; (b) zona-induced acrosome reaction; (c) spermpenetration to zona pellucida; and/or (d) sperm-egg membrane fusion. Consequently, the sperm antigens which are functionally involved in any of these fertilization processes could possibly be good candidates for the development of sperm antigen-based immunocontraceptive vaccines (Anderson et al., 1987). In this communication, efforts were made to investigate the inhibitory effects of these antibodies on sperm-zona binding and zona-induced acrosome reaction in vitro. Hopefully, through such a study, the mechanism of fertilization inhibition by these antibodies can be better understood. Suitable antigens can then be identified and characterized for their precise functional roles during the fertilization processes. Materials and Methods Chemicals The following chemicals were from Sigma Chemical Co.: pregnant mare serum gonadotrophin (PMSG), human chorionic gonadotropin (HCG), agglutinin Pisum sativum (PSA), Ionophore A 23187, bovine serum albumin (BSA), lacmoid, hyaluronidase and trypsin. FITC-labeled goat anti-mouse IgG+M, tissue culture media and supplies were from GIBCO (Burlington, Ontario, Canada). Animals All the mice used in this study were obtained from Charles River (Constant, Quebec, Canada). Mice of CD-1 strain were used for in vitro fertilization experiments and for preparation of sperm and zona pellucida. BALB/c mice were used for the production of ascites fluid to purify monoclonal antibodies according to published procedures (Lee et al., 1982; Chow et al., 1985).
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Production of monoclonal antibodies to sperm antigens Five different monoclonal antibodies, HS-9, HS-11, HS-63, MS-4 and MS-7 were produced by antibody-secreting hybridomas generated in our laboratory as previously reported (Lee et al., 1984a; Menge et al., 1987). Pristane-primed Balb/c mice were used to produce ascites f u i d which contained as much as 2 mg/ml of specific monoclonal antibodies (Lee et al., 1984a, 1986a). None of these antibodies were shown to react with mouse oocytes as judged from the results of indirect immunofluorescent assay. Purification of monoclonal antibodies from ascites fluid. Monoclonal antibodies were purified from mouse ascites fluid either by protein A affinity chromatography (Goding et al., 1978) or by ammonium sulfate fractionation followed by DEAE ion exchange chromatography as previously published (Liu et al., 1989a). Normal mouse IgG was purified from non-immune mouse serum using the same purification method. After purification, antibody-containing fractions were pooled and dialysed overnight against PBS. The antibody concentrations were determined by using a protein assay kit from Bio-Rad Co. The purity of antibodies was analysed by SDS-PAGE (Laemmli, 1970). Determination of antibody titers Indirect immunofluorescent assay was used to determine the titers of monoclonal antibodies (Lee et al., 1984a,b,c). All monoclonal antibodies were adjusted to a final concentration of 1 mg/ml prior to any assay. The titers of these antibodies were determined as follows: HS-9 (1:120,000), HS-II (1:120,000), HS-63 (1:120,000), MS-4 (1:16,000), MS-7 (1:16,000). Localization of antigens on sperm surface In order to localize the antigens which are specifically recognized by these monoclonal antibodies, sperm at different physiological stages (uncapacitated, capacitated, acrosome-reacted) were prepared and analysed by indirect immunofluorescence assays. The freshly prepared uncapacitated sperm in PBS were obtained from those just swimming out from the dissected mouse epididymis. After incubation for 1 h in BWW (Biggers, Whitten and Whittingham) medium (Biggers et al., 1971) at 37°C and 5% CO, most sperm became capacitated. The sperm capacitation was judged mainly based on three empirical criteria, including hyperactivated motility, ability to be induced for acrosome reaction and the ability to bind zona pellucida of unfertilized eggs (Gwatkin et al., 1974, Fraser, 1990). Following sperm capacitation, the acrosome-reacted sperm were prepared by incubation with ionophore A23187 at a final concentration of 10 t~M for 2 additional hours. Sperm at different physiological stages were incubated with the monoclonal
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antibodies for 30 min at a final concentration of l0/zg/ml. Following three washes with PBS-BSA, they were dried and fixed on slides. FITC-labeled goat anti-mouse I g G + M were used as the second antibodies for the indirect immunofluorescence assay as previously described (Lee et al., 1986a). The binding positions of antibodies to spermatozoa were observed using fluorescence microscopy.
In vitro fertilization experiments Mouse in vitro fertilization experiments were performed as reported previously (Biggers et al., 1971; Lee et al., 1985) with some minor modifications. Briefly, mouse oocytes were obtained from the superovulated CD-I female mice which had been injected with P M S G followed by H C G according to described protocols (Lee et al., 1985). Following retrieval, cumulus cells were removed from oocytes with 0.1% hyaluronidase. Capacitated mouse sperm (final concentration 2 × 105 motile sperm/100 /zl) were incubated with different antibodies of known concentrations. Twenty to forty cumulus-free oocytes were added to the antibody-treated sperm and incubated for 5 h in a CO2 incubator at 37°C. Following fixation with 2% glutaraldehyde, the oocytes were stained with lacmoid in acetic acid. Fertilization rates in each experiment were determined according to the reported criteria (Biggers et al., 1971). Sperm-zona binding assay The epididymis of the mature male mice were removed and dissected to allow motile sperm to swim out in B W W medium. Motile sperm were incubated with antibodies of known concentrations for 1 h at a final sperm concentration of 2 × 106/ml. Cumulus-free oocytes (20-40 eggs/experiment) were added to a solution containing antibody-treated sperm. The sperm-egg mixture was incubated for 30-40 min at 37°C to reach a maximum binding of sperm to the zona pellucida. At the end of incubation, oocytes were vigorously pipetted five times to remove loosely bound sperm (Archibong et al., unpublished). The number o f tightly bound sperm on the zona surface of oocytes was determined either by direct counting under microscope, or by fixing the oocytes with 2% glutaraldehyde, prior to counting (Liu et al., 1989b; Jeffrey et al., 1980). As a control to this experiment, none of the antibodies used in this study were shown to react with zona pellucida of mouse oocytes as judged by the results of indirect immunofluorescent assay (data not shown). Zona-induced acrosome reaction Zona pellucida were extracted and solubilized from mouse oocytes according to the procedures of Lakoski et al. (1989). A b o u t 400 or more oocytes
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were retrieved from superovulated female mice. Cumulus cells were removed with 0.1% hyaluronidase in 0.9(¼, NaC1. Following three washes, the zona pellucida were dissolved by adding 5/xl 20 mM sodium phosphate buffer, pH 2.5. After centrifugation, the supernatant was collected and neutralized with 2 M NaOH. The purity o f solubilized zona was analysed by SDS-PAGE based on the known molecular weight o f three zona proteins (Jeffrey et al., 1980). To induce sperm acrosome reaction, 1 /~l of zona solution from the above preparation was added to 9/x] solution of capacitated sperm which had been incubated with antibodies for 30 min. The final concentration was 40 zona-equivalents per 2 x 105 motile sperm (Lakoski et al., 1988). After incubation for an additional 30 min, sperm were fixed on slides by the air-dry method, and then stained with FITC-labeled Pisum sativum agglutinin (PSA) to determine the percentage o f acrosome-reacted sperm (Cross et al., 1986). At the end of the incubation period, the viability and motility of the incubated sperm were determined. The experimental results were valid only when no significant changes in sperm viability and motility were observed during the course of this assay.
Statistical analysis To analyse the data obtained from the present studies, a statistical analysis ~ioo
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70
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INCUBATION T I M E ( M I N I )
Fig. I. Percentage of mouse sperm with immunofluorescent staining upon incubation with five different anti-sperm monoclonal antibodies for different incubation times. A, staining with FITC-labeled PSA was used to assess the percentage of acrosome-intact sperm at different incubation times: O, incubation with HS-9; O, incubation with HS-I 1; r'l, incubation with HS-63; I incubation with MS-4: ,L incubation with MS-7. Mouse sperm was removed from the epididymis in PBS at time = 0. Arrows A and B indicate the time (in minutes) at which the sperm were transferred from PBS and incubated in BWW medium to induce capacitation and the time at which calcium ionophore, A 23187 (10 ,aM) was added to the sperm solution to induce acrosome reaction, respectively. During the course of this experiment, no significant changes in sperm motility and viability were observed except on the addition of calcium ionophore which was known to inactivate sperm,
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using two sample t-test was performed to assess the significance of the experimental results. Results
Binding between monoclonal antibodies and mouse and~or human spermatozoa The binding of monoclonal antibodies to mouse and human spermatozoa was studied under different experimental conditions by indirect immunofluorescence assay. When sperm were fixed on slides with methanol, all of the antibodies revealed strong staining on the acrosomal region of mouse and/or human sperm. HS-9, HS-I 1 and HS-63 were shown to react with the acrosome of both mouse and human sperm (Menge et al., 1987); MS-4 and MS-7 reacted only with that of mouse sperm (Lee et al., 1984a). On the other hand, when the epididymal mouse sperm were freshly prepared, only MS-4 and MS-7 showed partial immunofluorescent staining (26.0% and 30.6%, respectively). When the sperm were prepared in a capacitated medium to
A
C
D
! •,
t
.
Fig. 2. Indirect immunofluorescent staining of capaeitated mouse sperm by HS-11 (A and B} and MS-4 (C and D) monoclonal antibody, respectively. The sperm preparation in BWW medium was incubated with HS-11 or MS-4 monoclonal antibody for 30 rain followed by removal of the sperm for indirect immunofluorescent assay as described in the text. (A) and (C) are mouse sperm under UV light following the indirect immunofluorescent staining. (B) and (D) are those under visible light (magnification 400 x ). The arrows in (B) and (D) indicate mouse sperm which revealed immunofluorescent staining.
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allow capacitation to take place, all o f the monoclonal antibodies showed positive staining to sperm (60-70% staining) (Fig. 1). When calcium ionophore A23187 was added to the sperm preparation to induce acrosome reaction, the acrosome-reacted sperm (as judged by PSA-FITC staining) no longer reacted with any o f the five monoclonal antibodies ( < 10% staining). The results of this study are summarized in Fig. 1. Typical indirect immunofluorescent stainings o f capacitated mouse spermatozoa with HS-I 1 and MS-4 monoclonal antibodies are presented in Fig. 2.
Inhibitory effects of monoclonal antibodies on in vitro fertilization of
mouse
oocytes
All of the monoclonal antibodies selected in this study react with mouse sperm. Based on the results of indirect immunofluorescent assay using methanol-fixed sperm, all o f the antibodies employed in this study were shown to stain more than 95% of the sperm on the slides at a concentration of 0.01 mg/ml. Therefore, the mouse in vitro fertilization experiments were employed to evaluate the inhibitory effect o f these antibodies on the fertilization o f mouse oocytes. Purified monoclonal antibodies o f different concentrations were added to an incubation mixture containing mouse sperm and oocytes. Unrelated monoclonal antibodies were used as controls. The results o f this analysis are presented in Table 1. All of these antibodies showed sig-
TABLE 1 Inhibitory effect of five different purified monoclonal antibodies on the in vitro fertilization of mouse oocytes. Antibody
Number of assays
Ova examined
Ova fertilized
Fertilization rate (%)
Percent inhibition (P value)
HS-9 a (Control) b HS-I 1 (Control) HS-63 (Control) MS-4 (Control) MS-7 (Control)
2 (2) 2 (2) 2 (2) 2 (2) 2 (2)
57 (61) 53 (61) 52 (6l) 66 (62) 65 (62)
13 (30) 14 (30) 18 (30) 27 (38) 30 (38)
22.8 /49.2) 26.4 (49.2) 34.6 (49.2) 40.9 (61.3) 46.2 (61.3)
53.7 (P < 46.3 (P < 29.7 (P < 33.3 (P < 24.6 (P
0.50) 45.2 4- 21.9 (P < 0.12)
aNormal mouse lgG or RP 215 monoclonal antibody of the same concentration served as the control. bin the control experiments, approximately the same number of mouse oocytes (of the same concentration) and sperm were incubated in the presence of purified RP 215 monoclonal antibody or normal mouse lgG.
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of 0.2 mg/ml antibody (P < 0.1). The results of this study are summarized in Table 2. Consistent with their inhibition on in vitro fertilization, HS-9 and HS-I1 also gave the stronger inhibition (56.7% and 53.5%, respectively) to the binding between capacitated sperm and oocytes. In contrast, MS-4 and HS-63 showed little inhibition (9.5% and 18.2%, respectively). In control experiments, none of the antibodies employed in this study were found to affect the sperm motility or to agglutinate sperm even at an antibody concentration o f 1 mg/ml. Effect o f antisperm monoclonal antibodies on zona-induced acrosome reaction o f mouse spermatozoa The effect o f different antisperm monoclonal antibodies on sperm acrosome reaction induced by acid-solubilized mouse zona was studied and compared. FITC-labeled PSA (Pisum sativum lectin) was employed as a tool to assess the acrosomal status of sperm following induction by solubilized zona in the presence o f different antibodies. As shown in Table 3, all of the five antisperm monoclonal antibodies employed in this study were shown to inhibit the sperm acrosome reaction induced by solubilized zona preparation in vitro as compared with that o f the control. Among these antibodies, HS-63 showed a higher degree of inhibition (55.1% + 15.0%, P < 0.20) to the zonainduced acrosome reaction than any other antibodies under the same experimental conditions.
TABLE 3
Inhibitory effect of five different purified monoclonal antibodies on zona-induced acrosome reaction. Antibody a
Number of assays
Percentage of acrosome-reacted sperm (experimental/control) b
Average percent inhibition (P value)
HS-9
2
50.9/85.4, 44.5/60.3
HS-I1
3
HS-63
3
MS-4
2
25.7/68.4, 58.3/85.4, 35.0/60.3 10.7/38.8, 48.4/85.4, 30. I/60.3 42.2/50.9, 50.9/57.0
MS-7
2
45,0/50.9, 47.9/57.0
33.3 + 10.0 (P < 0.20) 45.3 + 15.7 (P < 0.10) 55.1 ± 15.0 (P < 0.20) 13.9 + 4.5 (P < 0.50) 13.8 ± 3.1 (P < 0.20)
aFinal antibody concentration = 0.1 mg/ml. bControl: 0.1 mg normal mouse IgG or RP 215 monoclonal antibody.
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Discussion In this communication, attempts were made to elucidate the possible mechanisms by which the selected antisperm monoclonal antibodies inhibit fertilization processes. All of the selected antibodies were shown to react with capacitated and acrosome-intact sperm. Following induction of acrosome reaction either by calcium ionophore, A23187 or by zona pellucida, none of these antibodies showed any significant binding to the acrosome-reacted sperm. These observations indicated that the cognate antigens recognized by these monoclonal antibodies are acrosomal components and can be shed after completion o f the sperm acrosome reaction. Therefore, it is reasonable to assume that the acrosome antigens recognized by these antibodies might only be involved in the initial stages of the fertilization process including the sperm-zona binding and zona-induced acrosome reaction, but not in the late stages such as sperm penetration to zona pellucida and sperm-egg membrane fusion, when acrosome-reacted spermatozoa are required. If this is the case for these antibodies, one would expect some correlations between the inhibition o f in vitro fertilization and that caused by sperm-zona binding and/or zona-induced acrosome reaction. Since the relative contributions of these two sperm-zona interaction steps to the overall fertilization process are not known at this moment, we are not able to draw a quantitative correlation between the inhibitory effects of these antibodies to fertilization and those in any of the steps. The results of our present study seem to be consistent with such an explanation. It is worth noting that during this study none of these antibodies showed quantitative inhibition to any of the initial stages of the fertilization process, at relatively high antibody concentrations. There are two possible explanations for this observation. We suspect that none of these antibodies has a sufficiently high affinity to block the fertilization process totally. It is also possible that the initial stages of the fertilization process are much more complicated than the two basic steps proposed in this study. The antibodies may not inhibit any of the critical initial steps during the fertilization process. Species-specific recognition between sperm and eggs during the initial stages of sperm-zona binding has been well documented (O'Rand et al., 1988). The so called 'sperm receptors' on the zona surface usually have sufficiently high binding affinity to sperm (K,; < I × 10-~3M) so that epitopespecific monoclonal antibodies are unable to block this process, but only serve to interfere with some of the interactions. Therefore, one would expect that a combination o f these monoclonal antibodies or polyclonal antibodies which react with multiple sites on sperm surface antigens might have a much stronger inhibition on the fertilization process (Saling et al., 1986; Biggers, 1986). This has been an important concern when designing an effective sperm
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antigen-based immunocontraceptive vaccine in the near future (Anderson, 1987). All of the antisperm antibodies employed in this study have been shown to be sperm-specific (Lee et al., 1984a, 1986a). HS-9, HS-I 1 and HS-63 react with species-conserved antigenic epitopes among different mammalian spermatozoa (Menge et al., 1987), whereas MS-4 and MS-7 only recognize mouse-sperm specific antigenic determinants (Lee et al., 1986). The lack of binding of these antibodies to oocytes was demonstrated using indirect immunofluorescent assay (data not shown). Among the cognate sperm antigens recognized by these antibodies, those reactive to HS-63 were selected as 'primary vaccine candidates' by the World Health Organization's Task Force on Contraceptive Vaccines (Anderson et al., 1987). Therefore, in our laboratory, the mouse sperm antigen recognized by HS-63, MSA-63, was purified and characterized at mRNA/protein levels (Liu et al., 1990). cDNA clones expressing MSA-63-related fusion proteins have been isolated through the immunoscreening of a mouse testis cDNA library (Liu et al., 1990). The nucleotide sequence of the longest cDNA insert with an open reading frame of 783 bp was determined and the corresponding sequence of 261 amino acid residues deduced (Liu et al., unpublished). The antisera raised against the MSA-63-related recombinant fusion protein were shown to inhibit mouse in vitro fertilization, similar to that by HS-63 monoclonal antibody (Liu et al., 1990). Recently, the effect of HS-63 monoclonal antibody and the corresponding rabbit antisera against purified mouse antigen, MSA-63 on sperm-zona binding has been studied in primate species by Wolf and his co-workers (Archibong et al., unpublished). It was clearly demonstrated that HS-63 and rabbit anti-MSA-63 significantly inhibited sperm-zona binding dose dependently, using a hemi-zona assay in Rhesus monkeys (Archibong et al., unpublished). However, such an inhibitory effect was not clearly observed in our study of mouse sperm-zona binding (Table 2). This may reflect the species difference between primates and mice in regard to the role of HS-63-reactive sperm antigen in the initial sperm-zona interactions. In view of the significant inhibitory effect of HS-63 on the sperm-zona binding in primate species, it was suggested by Wolf and his associates that the cognate sperm antigen may be a legitimate antifertility vaccine candidate (Archibong et al., unpublished). These encouraging results indicate that it may be possible to employ a similar strategy to mass-produce cognate sperm antigens reactive to other antibodies for the development of immunocontraceptive vaccines.
Acknowledgements The authors wish to thank Dr. Ming-Sun Liu for his technical advice, and
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Mr. Trevor Shew for his assistance in statistical analysis. Support for this project was provided in part by the Contraceptive Research and Development Program (CONRAD-009), Eastern Virginia Medical School, under a cooperative agreement with the U.S. Agency for International Development (PEP-3044-A-00-606300), and by the Medical Research Council of Canada (5-99802) (C.-Y.G.L.) and the Canadian Genetic Diseases Network (5-90490). The views expressed here do not necessarily reflect the views of CONRAD. References Anderson, D.J., Johnson, P.M., Alexander N.J., Jones, M.R. and Griffin, P.D. (1987) Monoclonal antibodies to human trophoblast and sperm antigens: Report of two WHO-sponsored workshops. J. Reprod. immunol. 10, 231. Biggers, J.D., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In: Mammalian Embryology (Denial, J.C., ed.), p. 86. Freeman and Company, San Francisco. Biggers, J.D. (1986) In: Immunological Approaches to Contraception and Promotion of Fertility (Talmer, G.P., ed.), p. 203, Plenum Press, New York. Bleil, J.D. and Wassarman, P.M. (1983) Sperm-egg interactions in the mouse -- sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev. Biol. 95, 317. Chow, S.N., Ho-Yuen, B., Lee, C.Y.G. (1985) Applications of monoclonal antibodies in immunoassays of human luteinizing hormone. J. Appl. Biochem. 7, 114. Cross, N.L., Morales, P., Overstreet, J.W. and Hanson, F.W. (1986) Two simple methods for detecting acrosome-reacted human sperm. Gamete Res. 15, 213. Fraser, L. (1990) Sperm Capacitation and its Modification in Fertilization in Mammals (Bavister, B.D., Cummins, J. and Roldan, E., eds.), p. 141. Serono Symposia, Norwell, MA. Goding, J.W. (1978) Use of staphylococcal protein A as an immunological reagent. J. Immunol. Methods 20, 241. Gwatkin, R.B.L., Anderson, O.F., Williams, D.T. (1974) Capacitation of mouse spermatozoa in vitro: Involvement of epididymal secretions and cumulus oophorus. J. Reprod. Fertil. 41, 253. Jeffrey, D.B. and Wassarman, W. (1980) Sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 20, 873. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Lakoski, K.~ Carron, C., Cabot, C. and Saling, P. (1988) Epididymal maturation and the acrosome reaction in mouse sperm: Response to zona pellucida develops conincident with modification of M42 antigen. Biol. Reprod. 38, 221. Lee, C.Y.G., Huang, Y.S., Hu, P.C., Gomel, V. and Menge, A.C. (1982) Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method. Anal. Biochem. 123, 14. Lee, C.Y.G., Wong, E., Richter, D.E. and Menge, A.C. (1984a) Monoclonal antibodies to human sperm antigens II. J. Reprod. lmmunol. 6, 227. Lee, C.Y.G., Wong, E. and Teh, C.Z. (1984b) Analysis of mouse sperm isoantigen using specific monoclonal antibodies. Am. J. Reprod. lmmunol. 6, 27. Lee, C.Y.G., Wong, E. and Menge, A,C. (1984c) Monoclonal antibodies to rabbits sperm autoantigen. Fertil. Steril. 41, 131. Lee, C.Y.G., Wong, E., Teh, C.Z. and Nishizawa, Y. (1985) Generation of mouse oocyte monoclonal antibodies: their effects and those ofantisperm monoclonal antibodies on in vitro fertilization. J. Reprod. Immunol. 7, 3. Lee, C.Y.G., Wong, E. and Zhang, J.H. (1986a) Inhibitory effects of monoclonal sperm antibodies on the fertilization of mouse oocytes in vitro and in vivo J. Reprod. lmmunol. 9, 261. Lee, C.Y.G, and Wong, E. (1986b) Developmental studies of sperm surface antigens using sperm-specific monoclonal antibodies. J. Reprod. lmmunol. 9, 275.
187 Lee, C.Y.G., Chen, K.W., Sheu, F.S., Tsang, A., Chao, K.C. and Ng, H.T. (1992) Studies of a tumorassociated antigen, COX-I, recognized by a monoclonal antibody. Cancer Immunol. lmmunother., in press. Liu, M.S., Yang, Y., Pan, J., Liu, H.W., Menge, A.C. and Lee, C.Y.G. (1989a) Purification of an acrosomal antigen recognized by a monoclonal antibody and antifertility effects of isoimmune serum. int. J. Androl. 12, 451. Liu, M.S., Yang, Y. and Lee, C.Y.G. (1989b) Inhibition of acrosome reaction and sperm-zona binding by monoclonal antibodies reactive to sperm acrosome. Mol. Androl. 1, 385. Menge, A.C., Schoultz, G.K., Kelsey, D.E. and Lee, C.Y.G, (1987) Characterization of monoclonal antibodies against human sperm antigens by immunoassays including sperm function assays and epitope evaluation. Am. J. Reprod. lmmunol. Microbiol. 13, 108. O'Rand, M.G. (1988) Sperm-egg recognition and barriers to interspecies fertilization. Gamete Res. 19, 315. Saling, P.M., Morton, P.C. and Waibel, R. (1986) In: Immunological Approaches to Contraception and Promotion of Fertility (Talmar, G.P., ed.), Plenum Press, p. 191.