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Monoclonal antibody against a sperm antigen Mr 95 000 inhibits attachment of human spermatozoa to the zona pellucida
Journal of Reproductive Immunology, 11 (1987) 157-166 Elsevier Scientific Publishers Ireland Ltd.
157
JRI 00478
Monoclonal antibody against a sperm antigen M, 95 000 inhibits attachment of human spermatozoa to the zona pellucida H . D . M . M o o r e , T . D . H a r t m a n , A.P. Bye, P. L u t j e n a, M. D e Witt a and A.O. Trounson a MRCI AFRC Comparative Physiology Research Group, Institute of Zoology, The Zoological Society of London, Regent's Park, London NW1 4R Y (U.K.) and aCentre for Early Human Development, Queen Victoria Medical Centre, Monash University, Melbourne (Australia) (Accepted for publication 30 April 1987)
Snmnu~ A murine monoclonal antibody raised against hamster spermatozoa was found to cross-react with human spermatozoa. By immunofluorescence,~e antigen was visualized over the equatorial segmen~ of human sperm heads. In the presence of antibody, sperm binding to the zona peUucida of salt-stored human oocytes was significantly inhibited (P~< 0.005) compared with other antibodies or control preparations. Using SDS-PAGE of whole spermatozoa and membrane preparations followed by Western blot analysis, the antigen was identified as a determinant with a relative molecular weight of 95 000. Key words: sperm antigen, monoclonal antibody, inhibition of binding,
human oocytes
Introduction Mammalian fertilization involves species specific binding of the spermatozoon to the surface of the zona pellucida. This interaction is an Address correspondence to: H.D.M. Moore. 0165-0378/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
158
essential prerequisite for sperm passage through the zona matrix and fusion with the oolemma and is a potential site for a specific contraceptive mechanism (Moore and Bedford, 1983; Ahuja, 1985). The sperm surface components involved in this process have not been determined but are thought to be glycoproteins first expressed during spermiogenesis and modified during epididymal sperm maturation (Moore et al., 1985; Eddy et al., 1985). Monoclonal antibodies raised against sperm surface components have been shown to block fertilization both in vitro and in vivo in several laboratory species (Naz et al., 1983; Moore and Hartman, 1984; Lee et al., 1985; Saling and Lakoski, 1985) and human serum containing antibodies t o spermatozoa can inhibit human in vitro fertilization or human sperm attachment and fusion to zone-free hamster oocytes (Kamada et al., 1985; Menge and Black, 1979). Here, we report on a monoclonal antibody raised against hamster spermatozoa which crossreacts with human spermatozoa and inhibits their attachment to human zona-pellucida in vitro. This antibody recognized an antigen on acrosomal membranes (including the equatorial segment) in the hamster (Moore et al., 1985) but was confined to the equatorial region of human spermatozoa. The relative molecular weight of the determinant was 95 000. Materials and Methods Monoclonal antibodies Murine monoclonal antibodies, specific to spermatozoa or genital tract tissues, and which cross-reacted with human spermatozoa (as detected by immunofluorescence assay) were selected from a panel raised against hamster cauda epididymal spermatozoa as reported previously (Ellis et al., 1985). Antibodies were purified by eluting a 5 ml Protein A Sepharose column (Pharmacia Ltd) with 200 ml of culture supernatant (Trinchieri, 1979). Fractions containing antibody (IgG~ or IgG2°) were returned to pH 7.2 by dialysis against phosphate buffered saline (PBS) (pH 7.2) centrifuged at 10000 g for 10m in and passed through a 0.22 I~m filter (Millipore Ltd) for storage in sterile tubes at 4°C. Final immunoglobulin concentration was between 57 and 150 ~g/ml as measured by absorbance against standard solutions at a wavelength of 280 nm. In vitro sperm binding assay Human spermatozoa attach only to the zona pellucida of the human and great apes and not other mammalian species (Bedford, 1977). It is important therefore to use human oocytes to assess sperm binding capacity. These were obtained from an in vitro fertilization programme and consisted of oocytes that had failed to fertilize. The oocytes were
159
collected within 24 h of culture and washed free of serum by passage (×3) through isotonic saline before being placed in salt solution containing 0.SM ammonium sulphate, 1.0M MgCI2, 0.1% Dextran and 20mM Hepes adjusted to pH 7.4 at 4°C (Yanagimachi et al., 1979). Ooeytes were preserved in this solution for up to 3 months before use. Prior to an assay, salt-stored oocytes were washed free of salt solution by passage through 5 dishes of BWW medium (Biggers et al., 1971) containing 0.4% human serum albumin (Sigma Ltd) and placed in a 400-1~1 drop of medium under silicone oil with antibody or control preparation (10 I~1). Before their use in a binding assay, spermatozoa from proven fertile men were labelled with monobromobimane (Thiolyte, Calbiochem Ltd). This compound is a thiol alkylating agent which fluorescently labels spermatozoal membranes without affecting fertilizing ability (Cummins et al., 1986; Fleming et al., 1986). Thus, by differential counting with phase contrast and epifluorescence microscopy, those spermatozoa bound to the zona in the presence of antibody could be distinguished from spermatozoa attached during the initial IVF procedure. Spermatozoa were first washed twice by centrifugation in BWW medium and labelled according to the method of Cummins et al. (1986) prior to incubation with oocytes at a concentration of 0.5-1.0 x 106/ml. After 6 h, oocytes were removed from culture, washed by gentle aspiration to remove loosely attached spermatozoa and then examined under phase contrast and epifluorescence microscopy (Nikon, Diaphot) using a filter block of 395-425 nm. Oocytes were scored for fluorescent sperm attachment and total sperm attachment to the zona pellucida.
lmmunofluorescent localization Washed human spermatozoa were air-dried at room temperature on glass slides, fixed in 100% methanol and incubated with monoclonal antibody and FITC-conjugated rabbit anti-mouse IgG and IgM (Miles Research Ltd) as described previously (Ellis et al., 1985). Spermatozoa were examined by epifluorescence microscopy at the wavelength of 495-520 nm. Cross-reactivity of antibody with the zona pellucida was examined by incubating salt-stored oocytes with monoclonal antibody in culture supernatant followed by washing and incubation in FITC-conjugated rabbit anti-mouse IgG as described for spermatozoa. Enzyme linked immunosorbent assay Washed ejaculated human spermatozoa were air-dried at room temperature on 96-weU microtitre plates (Nunc Ltd) to a density of 105 sperm/well, fixed for 5 rain with 0.5% glutaraldehyde, washed (x5) with PBS (pH 7.4) and blocked overnight with 1% bovine serum albumin in
160
PBS. Triplicate aliquots (100 p.l) of antibody or control were serially diluted with PBS followed by incubation for 3 h at room temperature. Plates were washed (x3) with PBS before incubation for 1 h with rabbit anti-mouse immunoglobulin (Dako Ltd) conjugated with alkaline phosphatase. After three further washes with PBS, plates were incubated with substrate (1001~1 p-nitrophenylphosphate, 2.7mM, in diethanolamine buffer, pH 9.8) for 30 min before the reaction was stopped with 30 I~1 of 3 M NaOH. The plates were read at 405 nm.
SDS-PAGE and Western blot analysis Human spermatozoa from 10 pooled ejaculates were washed twice in BWW medium without protein and prepared for electrophoresis as described previously (Ellis et al., 1985). Human sperm membranes were prepared by resuspending a washed sperm pellet in double distilled water and homogenizing the suspension on ice for 2 rain. The preparation was centrifuged at 700 × g for 10 min, the supernatant removed and subjected to 10 000 x g for 45 rain. The membrane pellet was then treated as for intact spermatozoa. Immunolocalization was carried out using 20nm colloidal gold particles coated with anti-mouse IgG (Auroprobe BL, Janssen Ltd) followed by silver enhancement.
Results
In vitro sperm-binding assay In Table 1, the effects of three monoclonal antibodies on sperm-egg binding is presented. Monoclonal antibody 97.25 significantly inhibited TABLE 1 The effect of monocional antibody" on human sperm-egg interaction. Treatment
Control m A b 97.25 m A b 64 m A b 60.6
No. of zonae peilucidae examined 39 20 8 7
Mean no. of sperm bound (fluorescent) b
Mean no. of sperm bound (visual) b
Fluorescent/ visual ratio b
22.8 ~- 26.5 2.3 ~: 3.3" 18.6+ 11.3 37.5 ± 21.5
41.4 • 36.2 38.4:t:35.1 2 6 . 3 ~ 14.1 57.0 :e 23.4
0.48 -4-0.26 0.12+0.22" 0.71 + 0 . 2 0 0.66 ± 0.17
• Final concentrations in culture; m A b 97.25 = 1.3 p~g/ml; m A b 6 4 - - 1 . 8 p,g/ml; m A b 6 0 . 6 = 2.2 p,g/ml. b + S.D. • Significantly different from remaining values P ~<0.005 (Mann-Whitney).
161
Fig. 1. Preparation of salt-stored oocytes after incubation with fluorescent labelled spermatozoa. Bar = 10 pan. (a)°Oocytes incubated with control preparation (phase contrast); (b) the same oocyte under UV fight, many fluorescent sperm were attached; (c) oocytes incubated with mAb 97.25 (phase contras0, (d) the same oocyte under UV light displayed no fluorescent sperm attachment.
sperm attachment to the zona pellucida compared with the control and remaining antibodies. Since oocytes were stored as pooled batches and were randomly assigned in the experiments, this reduction in sperm-egg binding was most probably due to the action of the antibody on sperm function rather than any ,intrinsic egg factor. There was no significant difference in the mean number of spermatozoa bound to each batch of oocytes by the initial IVF treatment. Nor was there any apparent alteration in sperm motility or sperm agglutination. Typical patterns of sperm binding are shown in Fig. 1. Penetration of the zona peUucida by spermatozoa was more difficult to visualize since the storage treatment resulted in a flattening of the oocytes with the consequence that it was difficult to ascertain with certainty whether spermatozoa resided on the inside or outside of the zona. It was clear, however, that no spermatozoa incubated with mAb 97.25 had penetrated the zona.
lmmunofluorescent localization A high proportion (90%) of human ejaculated spermatozoa exhibited a fluorescent band over their equatorial segment when incubated with mAb
162
% :÷
Fig. 2. Immunofluorescent localization of mAb 97.25 binding to human ejaculated spermatozoa. Spermatozoa exhibited a fluorescent band over their equatorial region. Bar = 5 p.m.
97.25 (Fig. 2). The presence of acrosomes on over 90% of spermatozoa in the same samples was confirmed using a monoclonal antibody to an acrosomal component (Moore et al., 1987). The remaining two antibodies exhibited binding over the midpiece and tail regions. Antibody binding was not detected on the zona pellucida.
Enzyme linked immunosorbent assay The specificity of mAb 97.25 affinity was established by immunosorbent assay (Fig. 3). Culture supernatant containing antibody gave significantly (P < 0.005) higher values than control up to a titre of 1/256. SDS-PAGE and Western blot analysis Analysis by SDS polyacrylamide gel electrophoresis and Western blotting of whole spermatozoa and membrane preparations from human ejaculates identified a major protein band at Mr 95 000 with an additional faint band at M, 90 000 as localised with immunogold particles (Fig. 4). Discussion
Polyclonal antiserum has been shown to inhibit human fertilization in vitro but this is the first report of a monoclonal antibody to a specific determinant blocking sperm-binding to the zona pellucida. Previous investigations have been confined to the use of heterologous in vitro assays involving zona-free hamster eggs (Naz et al., 1984). The use of salt-stored human oocytes has advantages over this technique and over the use of fresh human oocytes. Firstly, human sperm are presented with a biologically active and homologous zona surface at which initial stages of fertilization take place. Secondly, pooled stored oocytes reduce patient and egg variation and are ethically more acceptable for experimentation. Monoclonal antibody 97.25 was generated from a BALB/c mouse im-
163
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......
, .......
t .......
.... * ......
...... II
I
1
2
log filre Fig. 3. Enzyate-linked immunosorbent assay for mAb 97.25 (in culture supernatant) against human e~culated spermatozoa (control: culture supernatant containing non-sperm antibody). ., mAb 97.25; . . . . , control; bars -- S.E.M.
munized against hamster cauda epididymal sperm heads and is effective in blocking hamster fertilization in vitro (Ellis et al., 1985) and in vivo (unpublished observation). Such cross-reactivity would suggest that at least part of sperm/egg interactions in mammals involves a common mechanism. How mAb 97.25 blocks sperm/zona binding is at present unclear. Preliminary ultrastructural studies in the hamster and human (unpublished observations) suggested that the antigen is present on the inside face Of the plasmalemma and/or the outer acrosomal membranes in the equatorial region. If this is correct, antibody would not have access to antigen until after the acrosome reaction. In the human, it remains to be resolved with critical experiments, whether the initial stages of the acrosome reaction occur before, during or after sperm attachment to the zona (see Moore and Bedford, 1983). However, the localization of a determinant recognized by antibody on the equatorial segment of the human spermatozoa is of particular interest. This region of the acrosome is retained foUowing the acrosome reaction and is known to be the initial site of
164
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e
94 ~67 160 qlb
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2
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Fig. 4. Nitrocellulose blotting of determinants recognized by mAb 97.25 following SDS-PAGE and immunolocalization. Lane 1, human spermatozoa. Lane 2, human sperm membrane preparation. Lane 3, Coomassie blue staining of whole human spermatozoa. Lanes 4 and 5, relative molecular weight markers (molecular weights as presented).
membrane recognition and fusion between the mammalian spermatozoon and vitellus (Moore and Bedford, 1978). In the guinea-pig, the equatorial segment has been implicated in the zona adhesion process (Huang and Yanagimachi, 1984). Yanagimachi (1981) has also described acrosome reacting hamster spermatozoa bound to the zona surface and pivoting about the anterior margin of their equatorial segments. He has suggested that material emanating from the equatorial segment of the acrosome could be responsible for this stickiness. The present result would indicate that the equatorial segment may also have an important role in recognition processes at the zona surface during human fertilization. With regard to the nature of the determinant within the equatorial region, several human sperm antigens that elicit immune sera have been implicated as being significant in the development of infertility. In particular, Lee et al. (1983) characterised an antigen of M, 90000, while Lehmann et al. (1985) detected an antigen of M, 98 000 with sera containing antisperm antibodies. A monoclonal antibody to the equatorial
165
region of human spermatozoa has also been generated (Isahakia and Alexander, 1984) to a Mr, 71 000 antigen. Whether the determinant we have characterised is the same as previously identified sperm components remains to be established. The determination of a sperm specific antigen involved in human sperm-egg recognition events clearly marks an important step in the development of an appropriate contraceptive vaccine. Acknowledgements Aspects of this work were supported by a grant from the Ford Foundation, a MRC/AFRC programme grant and the National Health and Medical Research Council of Australia (No. 860250). HDMM received a travel grant from the Royal Society to complete the study. References Ahuja, K.K. (1985) Carbohydrate determinants involved in mammalian fertilization. Am. J. Anat. 174, 207-223. Bedford, J.M. (1977) Sperm/egg interaction: the specificity of human spermatozoa. Anat. Rec. 188, 477-488. Biggers, J.D., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In: Methods in Mammalian Embryology (Daniel, J.C. Jr., ed.), pp. 86-116. Freeman, San Francisco. Cummins, J.M., Fleming, A.D., Crozet, N., Kosower, W.S., Kuehl, T.J. and Yanagimachi, R. (1986) Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status. J. Exp. Zool. 237, 375-382. Eddy, E.M., Vernon, R.B., Muller, C.H., Hahnel, A.C. and Fenderson, B.A. (1985) lmmunodissection of sperm surface modifications during epididymal maturation. Am. J. Anat. 174, 225-237. Ellis, D.H., Hartman, T.D. and Moore, H.D.M. (1985) Maturation and function of the hamster spermatozoon probed with monoclonal antibodies. J. Reprod. Immunoi. 7, 299-314. Fleming, A.D., Cummins, J.M., Kuehl, T.J., Seidei, G.E. and Yanagimachi, R. (1986) Normal development of hamster and rabbit eggs fertilized by spermatozoa labelled with the fluorescent thiol alkylating agent monobromobimane. J. Exp. Zool. 237, 383-390. Huang, T.T.F. and Yanagimachi, R. (1984) Fucoidin inhibits attachment of guinea-pig spermatozoa to the zona-peUucida through binding to the inner acrosomal membrane and equatorial domains. Exp. Cell Res. 153, 363-373. Isahakia, M. and Alexander, N.J. (1984) lnterspecies cross-reactivity of monoclonal antibodies directed against human sperm antigens. Biol. Reprod. 30, 1015-1026. Kamada, M., Hasebe, H. and Yamano, S. (1985) Blocking of human fertilization in vitro by sera with sperm immobilizing antibodies. Am. J. Obstet. Gynecol. 153, 328-331. Lee, C.Y.G., Lure, V., Wong, E., Menge, A.C. and Huang, Y.S. (1983) Identification of human sperm antigens to antisperm antibodies. Am. J. Reprod. Immunol. 3, 183-187. Lee, C.Y.G., Wong, E., Teh, C-Z. and Nishizawa, Y. (1985) Generation of mouse oocyte monocional isoantibodies. Their effect and those of antisperm monoclonal antibodies on in vitro fertilization. J. Reprod. Immunol. 7, 3-13.
166 Lehmann, D., Temminck, B., Da Rugna, D., Leibundgut, B. and Muller, H. (1985) Blot-immunobinding test for the detection of antisperm antibodies. J. Reprod. Immunol. 8, 329-336. Menge, A.C. and Black, C.S. (1979) Effects of antisera on human sperm penetration of zona-free hamster eggs. Fertil. Steril. 32, 214-218. Moore, H.D.M. and Bedford, J.M. (1978) Ultrastructure of the equatorial segment of hamster spermatozoa during penetration of oocytes. J. Ultrastruct. Res. 62, 110-117. Moore, H.D.M. and Bedford, J.M. (1983) The interaction of mammalian gametes in the female. In: Mechanism and Control of Animal Fertilization (Hartmann, J.F., ed.), pp. 453--497. Academic Press, New York. Moore, H.D.M. and Hartman, T.D. (1984) Localization by monoclonal antibodies of various surface antigens of hamster spermatozoa and the effect of antibody of fertilization in vitro. J. Reprod. Fertil. 70, 175-183. Moore, H.D.M., Hartman, T.D., Brown, A.C., Smith, C.A. and Ellis, D.H. (1985) Expression of sperm antigens during spermatogenesis and maturation detected with monoclonai antibodies. Exp. Clin. Immunogenet. 2, 84-96. Moore, H.D.M., Smith, C.A., Hartman, T.D. and Bye, A.P. (1987) Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody. Gamete Res. 17, 245-259. Naz, R., Saxe, J.M. and Menge, A.C. (1983) Inhibition of fertility in rabbits by monoclonal antibodies against sperm. Biol. Reprod. 28, 249-254. Naz, R., Alexander, N.J., Isahakia, M. and Hamilton, M.S. (1984) Monoclonal antibody to a human germ cell membrane glycoprotein that inhibits fertilization. Science 225, 342-344. Saling, P.M. and Lakoski, K.A. (1985) Mouse sperm antigens that participate in fertilization. I1. Inhibition of sperm penetration through the zona pellueida using monoclonai antibodies. Biol. Reprod. 33, 527-536. Trinchieri, G. (1979) Purification of immunoglobulins produced by the hybrid cells. In: Hybridoma Technology with Special Reference to Parasitic Diseases. UNDO/World Bank/WHO. Yanagimachi, R. (1981) Mechanisms of fertilization in mammals. In: Fertilization and Embryonic Development in vitro (Mastroianni, L. and Biggers J.D., eds.) pp. 81-182. Plenum Press, New York. Yanagimachi, R., Loparta, A., Odom, C.B., Bronson, R.A., Mahi, C.A. and Nicoison, G.L. (1979) Retention of biologic characteristics of zona pellucida in highly concentrated salt solution. The use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. Steril. 31, 562-574.
157
JRI 00478
Monoclonal antibody against a sperm antigen M, 95 000 inhibits attachment of human spermatozoa to the zona pellucida H . D . M . M o o r e , T . D . H a r t m a n , A.P. Bye, P. L u t j e n a, M. D e Witt a and A.O. Trounson a MRCI AFRC Comparative Physiology Research Group, Institute of Zoology, The Zoological Society of London, Regent's Park, London NW1 4R Y (U.K.) and aCentre for Early Human Development, Queen Victoria Medical Centre, Monash University, Melbourne (Australia) (Accepted for publication 30 April 1987)
Snmnu~ A murine monoclonal antibody raised against hamster spermatozoa was found to cross-react with human spermatozoa. By immunofluorescence,~e antigen was visualized over the equatorial segmen~ of human sperm heads. In the presence of antibody, sperm binding to the zona peUucida of salt-stored human oocytes was significantly inhibited (P~< 0.005) compared with other antibodies or control preparations. Using SDS-PAGE of whole spermatozoa and membrane preparations followed by Western blot analysis, the antigen was identified as a determinant with a relative molecular weight of 95 000. Key words: sperm antigen, monoclonal antibody, inhibition of binding,
human oocytes
Introduction Mammalian fertilization involves species specific binding of the spermatozoon to the surface of the zona pellucida. This interaction is an Address correspondence to: H.D.M. Moore. 0165-0378/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
158
essential prerequisite for sperm passage through the zona matrix and fusion with the oolemma and is a potential site for a specific contraceptive mechanism (Moore and Bedford, 1983; Ahuja, 1985). The sperm surface components involved in this process have not been determined but are thought to be glycoproteins first expressed during spermiogenesis and modified during epididymal sperm maturation (Moore et al., 1985; Eddy et al., 1985). Monoclonal antibodies raised against sperm surface components have been shown to block fertilization both in vitro and in vivo in several laboratory species (Naz et al., 1983; Moore and Hartman, 1984; Lee et al., 1985; Saling and Lakoski, 1985) and human serum containing antibodies t o spermatozoa can inhibit human in vitro fertilization or human sperm attachment and fusion to zone-free hamster oocytes (Kamada et al., 1985; Menge and Black, 1979). Here, we report on a monoclonal antibody raised against hamster spermatozoa which crossreacts with human spermatozoa and inhibits their attachment to human zona-pellucida in vitro. This antibody recognized an antigen on acrosomal membranes (including the equatorial segment) in the hamster (Moore et al., 1985) but was confined to the equatorial region of human spermatozoa. The relative molecular weight of the determinant was 95 000. Materials and Methods Monoclonal antibodies Murine monoclonal antibodies, specific to spermatozoa or genital tract tissues, and which cross-reacted with human spermatozoa (as detected by immunofluorescence assay) were selected from a panel raised against hamster cauda epididymal spermatozoa as reported previously (Ellis et al., 1985). Antibodies were purified by eluting a 5 ml Protein A Sepharose column (Pharmacia Ltd) with 200 ml of culture supernatant (Trinchieri, 1979). Fractions containing antibody (IgG~ or IgG2°) were returned to pH 7.2 by dialysis against phosphate buffered saline (PBS) (pH 7.2) centrifuged at 10000 g for 10m in and passed through a 0.22 I~m filter (Millipore Ltd) for storage in sterile tubes at 4°C. Final immunoglobulin concentration was between 57 and 150 ~g/ml as measured by absorbance against standard solutions at a wavelength of 280 nm. In vitro sperm binding assay Human spermatozoa attach only to the zona pellucida of the human and great apes and not other mammalian species (Bedford, 1977). It is important therefore to use human oocytes to assess sperm binding capacity. These were obtained from an in vitro fertilization programme and consisted of oocytes that had failed to fertilize. The oocytes were
159
collected within 24 h of culture and washed free of serum by passage (×3) through isotonic saline before being placed in salt solution containing 0.SM ammonium sulphate, 1.0M MgCI2, 0.1% Dextran and 20mM Hepes adjusted to pH 7.4 at 4°C (Yanagimachi et al., 1979). Ooeytes were preserved in this solution for up to 3 months before use. Prior to an assay, salt-stored oocytes were washed free of salt solution by passage through 5 dishes of BWW medium (Biggers et al., 1971) containing 0.4% human serum albumin (Sigma Ltd) and placed in a 400-1~1 drop of medium under silicone oil with antibody or control preparation (10 I~1). Before their use in a binding assay, spermatozoa from proven fertile men were labelled with monobromobimane (Thiolyte, Calbiochem Ltd). This compound is a thiol alkylating agent which fluorescently labels spermatozoal membranes without affecting fertilizing ability (Cummins et al., 1986; Fleming et al., 1986). Thus, by differential counting with phase contrast and epifluorescence microscopy, those spermatozoa bound to the zona in the presence of antibody could be distinguished from spermatozoa attached during the initial IVF procedure. Spermatozoa were first washed twice by centrifugation in BWW medium and labelled according to the method of Cummins et al. (1986) prior to incubation with oocytes at a concentration of 0.5-1.0 x 106/ml. After 6 h, oocytes were removed from culture, washed by gentle aspiration to remove loosely attached spermatozoa and then examined under phase contrast and epifluorescence microscopy (Nikon, Diaphot) using a filter block of 395-425 nm. Oocytes were scored for fluorescent sperm attachment and total sperm attachment to the zona pellucida.
lmmunofluorescent localization Washed human spermatozoa were air-dried at room temperature on glass slides, fixed in 100% methanol and incubated with monoclonal antibody and FITC-conjugated rabbit anti-mouse IgG and IgM (Miles Research Ltd) as described previously (Ellis et al., 1985). Spermatozoa were examined by epifluorescence microscopy at the wavelength of 495-520 nm. Cross-reactivity of antibody with the zona pellucida was examined by incubating salt-stored oocytes with monoclonal antibody in culture supernatant followed by washing and incubation in FITC-conjugated rabbit anti-mouse IgG as described for spermatozoa. Enzyme linked immunosorbent assay Washed ejaculated human spermatozoa were air-dried at room temperature on 96-weU microtitre plates (Nunc Ltd) to a density of 105 sperm/well, fixed for 5 rain with 0.5% glutaraldehyde, washed (x5) with PBS (pH 7.4) and blocked overnight with 1% bovine serum albumin in
160
PBS. Triplicate aliquots (100 p.l) of antibody or control were serially diluted with PBS followed by incubation for 3 h at room temperature. Plates were washed (x3) with PBS before incubation for 1 h with rabbit anti-mouse immunoglobulin (Dako Ltd) conjugated with alkaline phosphatase. After three further washes with PBS, plates were incubated with substrate (1001~1 p-nitrophenylphosphate, 2.7mM, in diethanolamine buffer, pH 9.8) for 30 min before the reaction was stopped with 30 I~1 of 3 M NaOH. The plates were read at 405 nm.
SDS-PAGE and Western blot analysis Human spermatozoa from 10 pooled ejaculates were washed twice in BWW medium without protein and prepared for electrophoresis as described previously (Ellis et al., 1985). Human sperm membranes were prepared by resuspending a washed sperm pellet in double distilled water and homogenizing the suspension on ice for 2 rain. The preparation was centrifuged at 700 × g for 10 min, the supernatant removed and subjected to 10 000 x g for 45 rain. The membrane pellet was then treated as for intact spermatozoa. Immunolocalization was carried out using 20nm colloidal gold particles coated with anti-mouse IgG (Auroprobe BL, Janssen Ltd) followed by silver enhancement.
Results
In vitro sperm-binding assay In Table 1, the effects of three monoclonal antibodies on sperm-egg binding is presented. Monoclonal antibody 97.25 significantly inhibited TABLE 1 The effect of monocional antibody" on human sperm-egg interaction. Treatment
Control m A b 97.25 m A b 64 m A b 60.6
No. of zonae peilucidae examined 39 20 8 7
Mean no. of sperm bound (fluorescent) b
Mean no. of sperm bound (visual) b
Fluorescent/ visual ratio b
22.8 ~- 26.5 2.3 ~: 3.3" 18.6+ 11.3 37.5 ± 21.5
41.4 • 36.2 38.4:t:35.1 2 6 . 3 ~ 14.1 57.0 :e 23.4
0.48 -4-0.26 0.12+0.22" 0.71 + 0 . 2 0 0.66 ± 0.17
• Final concentrations in culture; m A b 97.25 = 1.3 p~g/ml; m A b 6 4 - - 1 . 8 p,g/ml; m A b 6 0 . 6 = 2.2 p,g/ml. b + S.D. • Significantly different from remaining values P ~<0.005 (Mann-Whitney).
161
Fig. 1. Preparation of salt-stored oocytes after incubation with fluorescent labelled spermatozoa. Bar = 10 pan. (a)°Oocytes incubated with control preparation (phase contrast); (b) the same oocyte under UV fight, many fluorescent sperm were attached; (c) oocytes incubated with mAb 97.25 (phase contras0, (d) the same oocyte under UV light displayed no fluorescent sperm attachment.
sperm attachment to the zona pellucida compared with the control and remaining antibodies. Since oocytes were stored as pooled batches and were randomly assigned in the experiments, this reduction in sperm-egg binding was most probably due to the action of the antibody on sperm function rather than any ,intrinsic egg factor. There was no significant difference in the mean number of spermatozoa bound to each batch of oocytes by the initial IVF treatment. Nor was there any apparent alteration in sperm motility or sperm agglutination. Typical patterns of sperm binding are shown in Fig. 1. Penetration of the zona peUucida by spermatozoa was more difficult to visualize since the storage treatment resulted in a flattening of the oocytes with the consequence that it was difficult to ascertain with certainty whether spermatozoa resided on the inside or outside of the zona. It was clear, however, that no spermatozoa incubated with mAb 97.25 had penetrated the zona.
lmmunofluorescent localization A high proportion (90%) of human ejaculated spermatozoa exhibited a fluorescent band over their equatorial segment when incubated with mAb
162
% :÷
Fig. 2. Immunofluorescent localization of mAb 97.25 binding to human ejaculated spermatozoa. Spermatozoa exhibited a fluorescent band over their equatorial region. Bar = 5 p.m.
97.25 (Fig. 2). The presence of acrosomes on over 90% of spermatozoa in the same samples was confirmed using a monoclonal antibody to an acrosomal component (Moore et al., 1987). The remaining two antibodies exhibited binding over the midpiece and tail regions. Antibody binding was not detected on the zona pellucida.
Enzyme linked immunosorbent assay The specificity of mAb 97.25 affinity was established by immunosorbent assay (Fig. 3). Culture supernatant containing antibody gave significantly (P < 0.005) higher values than control up to a titre of 1/256. SDS-PAGE and Western blot analysis Analysis by SDS polyacrylamide gel electrophoresis and Western blotting of whole spermatozoa and membrane preparations from human ejaculates identified a major protein band at Mr 95 000 with an additional faint band at M, 90 000 as localised with immunogold particles (Fig. 4). Discussion
Polyclonal antiserum has been shown to inhibit human fertilization in vitro but this is the first report of a monoclonal antibody to a specific determinant blocking sperm-binding to the zona pellucida. Previous investigations have been confined to the use of heterologous in vitro assays involving zona-free hamster eggs (Naz et al., 1984). The use of salt-stored human oocytes has advantages over this technique and over the use of fresh human oocytes. Firstly, human sperm are presented with a biologically active and homologous zona surface at which initial stages of fertilization take place. Secondly, pooled stored oocytes reduce patient and egg variation and are ethically more acceptable for experimentation. Monoclonal antibody 97.25 was generated from a BALB/c mouse im-
163
0-5-
......
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munized against hamster cauda epididymal sperm heads and is effective in blocking hamster fertilization in vitro (Ellis et al., 1985) and in vivo (unpublished observation). Such cross-reactivity would suggest that at least part of sperm/egg interactions in mammals involves a common mechanism. How mAb 97.25 blocks sperm/zona binding is at present unclear. Preliminary ultrastructural studies in the hamster and human (unpublished observations) suggested that the antigen is present on the inside face Of the plasmalemma and/or the outer acrosomal membranes in the equatorial region. If this is correct, antibody would not have access to antigen until after the acrosome reaction. In the human, it remains to be resolved with critical experiments, whether the initial stages of the acrosome reaction occur before, during or after sperm attachment to the zona (see Moore and Bedford, 1983). However, the localization of a determinant recognized by antibody on the equatorial segment of the human spermatozoa is of particular interest. This region of the acrosome is retained foUowing the acrosome reaction and is known to be the initial site of
164
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Fig. 4. Nitrocellulose blotting of determinants recognized by mAb 97.25 following SDS-PAGE and immunolocalization. Lane 1, human spermatozoa. Lane 2, human sperm membrane preparation. Lane 3, Coomassie blue staining of whole human spermatozoa. Lanes 4 and 5, relative molecular weight markers (molecular weights as presented).
membrane recognition and fusion between the mammalian spermatozoon and vitellus (Moore and Bedford, 1978). In the guinea-pig, the equatorial segment has been implicated in the zona adhesion process (Huang and Yanagimachi, 1984). Yanagimachi (1981) has also described acrosome reacting hamster spermatozoa bound to the zona surface and pivoting about the anterior margin of their equatorial segments. He has suggested that material emanating from the equatorial segment of the acrosome could be responsible for this stickiness. The present result would indicate that the equatorial segment may also have an important role in recognition processes at the zona surface during human fertilization. With regard to the nature of the determinant within the equatorial region, several human sperm antigens that elicit immune sera have been implicated as being significant in the development of infertility. In particular, Lee et al. (1983) characterised an antigen of M, 90000, while Lehmann et al. (1985) detected an antigen of M, 98 000 with sera containing antisperm antibodies. A monoclonal antibody to the equatorial
165
region of human spermatozoa has also been generated (Isahakia and Alexander, 1984) to a Mr, 71 000 antigen. Whether the determinant we have characterised is the same as previously identified sperm components remains to be established. The determination of a sperm specific antigen involved in human sperm-egg recognition events clearly marks an important step in the development of an appropriate contraceptive vaccine. Acknowledgements Aspects of this work were supported by a grant from the Ford Foundation, a MRC/AFRC programme grant and the National Health and Medical Research Council of Australia (No. 860250). HDMM received a travel grant from the Royal Society to complete the study. References Ahuja, K.K. (1985) Carbohydrate determinants involved in mammalian fertilization. Am. J. Anat. 174, 207-223. Bedford, J.M. (1977) Sperm/egg interaction: the specificity of human spermatozoa. Anat. Rec. 188, 477-488. Biggers, J.D., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In: Methods in Mammalian Embryology (Daniel, J.C. Jr., ed.), pp. 86-116. Freeman, San Francisco. Cummins, J.M., Fleming, A.D., Crozet, N., Kosower, W.S., Kuehl, T.J. and Yanagimachi, R. (1986) Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status. J. Exp. Zool. 237, 375-382. Eddy, E.M., Vernon, R.B., Muller, C.H., Hahnel, A.C. and Fenderson, B.A. (1985) lmmunodissection of sperm surface modifications during epididymal maturation. Am. J. Anat. 174, 225-237. Ellis, D.H., Hartman, T.D. and Moore, H.D.M. (1985) Maturation and function of the hamster spermatozoon probed with monoclonal antibodies. J. Reprod. Immunoi. 7, 299-314. Fleming, A.D., Cummins, J.M., Kuehl, T.J., Seidei, G.E. and Yanagimachi, R. (1986) Normal development of hamster and rabbit eggs fertilized by spermatozoa labelled with the fluorescent thiol alkylating agent monobromobimane. J. Exp. Zool. 237, 383-390. Huang, T.T.F. and Yanagimachi, R. (1984) Fucoidin inhibits attachment of guinea-pig spermatozoa to the zona-peUucida through binding to the inner acrosomal membrane and equatorial domains. Exp. Cell Res. 153, 363-373. Isahakia, M. and Alexander, N.J. (1984) lnterspecies cross-reactivity of monoclonal antibodies directed against human sperm antigens. Biol. Reprod. 30, 1015-1026. Kamada, M., Hasebe, H. and Yamano, S. (1985) Blocking of human fertilization in vitro by sera with sperm immobilizing antibodies. Am. J. Obstet. Gynecol. 153, 328-331. Lee, C.Y.G., Lure, V., Wong, E., Menge, A.C. and Huang, Y.S. (1983) Identification of human sperm antigens to antisperm antibodies. Am. J. Reprod. Immunol. 3, 183-187. Lee, C.Y.G., Wong, E., Teh, C-Z. and Nishizawa, Y. (1985) Generation of mouse oocyte monocional isoantibodies. Their effect and those of antisperm monoclonal antibodies on in vitro fertilization. J. Reprod. Immunol. 7, 3-13.
166 Lehmann, D., Temminck, B., Da Rugna, D., Leibundgut, B. and Muller, H. (1985) Blot-immunobinding test for the detection of antisperm antibodies. J. Reprod. Immunol. 8, 329-336. Menge, A.C. and Black, C.S. (1979) Effects of antisera on human sperm penetration of zona-free hamster eggs. Fertil. Steril. 32, 214-218. Moore, H.D.M. and Bedford, J.M. (1978) Ultrastructure of the equatorial segment of hamster spermatozoa during penetration of oocytes. J. Ultrastruct. Res. 62, 110-117. Moore, H.D.M. and Bedford, J.M. (1983) The interaction of mammalian gametes in the female. In: Mechanism and Control of Animal Fertilization (Hartmann, J.F., ed.), pp. 453--497. Academic Press, New York. Moore, H.D.M. and Hartman, T.D. (1984) Localization by monoclonal antibodies of various surface antigens of hamster spermatozoa and the effect of antibody of fertilization in vitro. J. Reprod. Fertil. 70, 175-183. Moore, H.D.M., Hartman, T.D., Brown, A.C., Smith, C.A. and Ellis, D.H. (1985) Expression of sperm antigens during spermatogenesis and maturation detected with monoclonai antibodies. Exp. Clin. Immunogenet. 2, 84-96. Moore, H.D.M., Smith, C.A., Hartman, T.D. and Bye, A.P. (1987) Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody. Gamete Res. 17, 245-259. Naz, R., Saxe, J.M. and Menge, A.C. (1983) Inhibition of fertility in rabbits by monoclonal antibodies against sperm. Biol. Reprod. 28, 249-254. Naz, R., Alexander, N.J., Isahakia, M. and Hamilton, M.S. (1984) Monoclonal antibody to a human germ cell membrane glycoprotein that inhibits fertilization. Science 225, 342-344. Saling, P.M. and Lakoski, K.A. (1985) Mouse sperm antigens that participate in fertilization. I1. Inhibition of sperm penetration through the zona pellueida using monoclonai antibodies. Biol. Reprod. 33, 527-536. Trinchieri, G. (1979) Purification of immunoglobulins produced by the hybrid cells. In: Hybridoma Technology with Special Reference to Parasitic Diseases. UNDO/World Bank/WHO. Yanagimachi, R. (1981) Mechanisms of fertilization in mammals. In: Fertilization and Embryonic Development in vitro (Mastroianni, L. and Biggers J.D., eds.) pp. 81-182. Plenum Press, New York. Yanagimachi, R., Loparta, A., Odom, C.B., Bronson, R.A., Mahi, C.A. and Nicoison, G.L. (1979) Retention of biologic characteristics of zona pellucida in highly concentrated salt solution. The use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil. Steril. 31, 562-574.