Characterization of Anti-Hamster ZP-0 Monoclonal Antibody

Transcription

1 Characterization of Anti-Hamster ZP-0 Monoclonal Antibody K. Ookata (1), K.Takagishi (1), S. Konno (2) and T. Oikawa(1,2) (1) Developmental and Reproductive Biology Center, Yamagata 990, Japan and (2) Bio Science Laboratory, Yamagata 990, Japan. Recentry, Oikawa et al. (1985) discovered that there is a novel zones pellucida glycoprotein (ZP-0) detectable by Bandeiraea simplicifolia-1 lectin (BS-1), which is specific for -D-galactose-like or ƒ -N-acetyl-D-galactosamine-like terminal saccharide residues. ZP-0 is found only on the zona pellucida (ZP) of the oviductal egg (ZP-OVI), it is responsible for biochemical (Oikawa and Sendai, 1984) and immunological (Oikawa et al., 1985) differences between ZP of the ovarian egg (ZP-OVA) and the ZP-OVI. Furthemore, Oikawa et al. (1986, 1987) reported that ZP-0 is Rd component of oviductal origin glycoprotein added to the ZP of egg of the hamster just after ovulation and that ZP-0 has a function to promote speciesspecific sperm-egg interaction. After that, for the purpose of getting probes to analyse the detail physiological function of ZP-0, we prepared some monoclonal antibodies (MoAbs) raised against ZP-O. In this study, as the first step to characterize the MoAbs, we investigated their tissue cross-reactivity, their effect on the in vitro and in vivo fertilization in the hamster and their species cross-reactivity. ZP-0 as the antigen in order to produce the MoAbs was prepared from hamster oviducts as follows; Oviduct extract was salting out with 80 % saturated (NH4)2SO4, dialysed against PBS (ph 7.4), applied on Con A-Sepharose 4B column and the column was equilibrated with PBS (ph 7.4). After that, ZP-0 was eluted

2 from the column with PBS (ph 7.4) containing 0.3 M a -methyl- D-mannoside, fractions which have above 0.02 OD280 nm were pooled and the pooled fractions were dialysed against PBS (ph 7.4) to remove ƒ -methyl-d-mannoside. The dialysed one was concentrated and used as the antigen. Of course, the biological activity of ZP-0 thus obtained was checked by the binding ability of FITC-BS-1 to the ZP-0 treated ovarian egg and it was demonstrated that ZP-0 has a sufficient biological activity. To establish the hybridoma, spleen cells from mice immunized with ZP-0 were fused with NS-1 cells by PEG and the hybridoma thus obtained were selected with the routine method. Culture fluids recovered from the selected hybridoma were tested for their binding activity to ovulated eggs by the indirect immunofluorescence method. An isotype of immunoglobulins which were secreted by the hybridomas was determined by Ouchterlony method. To examine the tissue cross-reactivity of the MoAbs, a routine histological study was done with the method of paraffin section as follows; Various hamster tissues were embedded in paraffin and cut in 5 Atm sections. The sections were stained by the method of enzyme immunoassay and then stained with 1 % Methylgreen solution in order to obtain a clear contrast peparate. The in vitro fertilization was done as follows; Medium used in this study was modified TALP solution (m-talp) (Liebfried and Bavister, 1982). First of all, mature female hamsters were superovulated with PMSG and hcg. Then, denuded eggs were recovered by flushing with Tyrode's solution containing 5 mg/3 ml hyaluronidase. The denuded, cumulus-free eggs were treated with the MoAbs at 37 Ž for 30 min, rinsed three times with m- TALP, transferred into m-talp mounted on the plastic petri dish under liquid paraffin, inseminated with capacitated spermatozoa

3 and incubated at 37 Ž for 7 hrs. To get capacitated spermatozoa, epididymal sperm were preincubated in m-talp at 37 C for 3-4 hrs under 5 % CO 295 % air and the final sperm concentration used for insemination was /ml. To confirm the status of fertilization, the eggs were fixed after the incubation, stained with Lacmoid, and examined for the evidence of fertilization. Eggs having enlarged sperm head or male pronucleous with the corresponding sperm tail in the vitellus were judged to be undergoing fertilization. For preliminary passive immunization to examine whether the MoAbs have physiological effects on fertilization or not, superovulated female hamsters were given i.p. with 1 ml of ascites of C8B11 at the time of PMSG injection and mated. To examine the status of fertilization, eggs were recovered at 20 hrs aftr hcg injection, stained with Lacmoid and observed for the evidence of fertilization. Also, some eggs were examined for binding of antibody by the immunofluorescence method. Species cross-reactivity of these MoAbs were investigated by the indirect immunofluorescence method. Mouse and rat eggs were recovered from ovaries and oviducts of superovulated animals and denuded by hyaluronidase treatment. Cow and porcine eggs were collected by puncturing follicles of the fresh ovaries obtained at a local slaughterhouse and denuded by gentle pipetting. Some porcine eggs were transferred into the porcine oviduct and both ends of the oviduct were ligated. Then, the oviduct was immersed in m-krb and incubated at 37 Ž for 2 hrs. After the incubation, the eggs were recovered by flushing the oviduct with m-krb. Three kinds of MoAbs, socalled C11E8, C8B11, and A3D5, react with ZP-OVI, but not with ZP-OVA. And also, about their isotypes, C11E8 and C8B11 were IgG and A3D5 was IgM. The molecular weight of the antigen corresponding to three MoAbs

4 was about 200 Kd component. During cytological studies of the hamster oviduct usign the MoAbs, it was demonstrated that the ZP-0 located in the oviductal nonciliated epithelial secretory cells, but not in the ciliated epithelial cells (Fig. 1), that endometrium and certain epithelium in some digestive organs reacted with these MoAbs (Table 1) and that the immunoreactivity of all tissues disappeared after the periodate treatment. Perhaps, these facts indicate that the epitopes of ZP-0 these MoAbs can recognize are certain saccharide residues. All three MoAbs artifitially induced formation of precipitin layer on the outer most surface of the ZP of the hamster egg. On the contrary, two of three, C11E8 and A3D5, had no effect on fertilization. One of three, C8B11 which can induce most stable precipitin layer, completely blocked in vitro fertilization at the concentration of 4mg protein/mi. (Table 2). Number of spermatozoa attaching the ZP treated with C8B11 was fewer than that of control and these spermatozoa were readily removed by gentle pipetting. This fact indicates clearly that the MoAb, C8B11, inhibit sperm attachment to the ZP. The blocking mechanism of the fertilization was referred to two categories; that is, 1) Direct masking of sperm receptor (or sperm binding site) by the antibody and 2) Indirect masking of sperm receptor (or sperm binding site) by a certain steric hindrance which was artifitilly induced by binding of the antibody. In this case, it needed relatively large amount of the MoAb to block the in vitro fertilization. This fact indicates that the blocking mechanism of in vitro fertilization may be the category 2) described above. However, it is clear that it needs more investigation to clarify whether the antigen of C8B11 contains sperm receptor (or sperm binding site) or not. In passive immunization test, we could observe the fact that

5 there are C8B11 on the outer most layer of the ZP of ova recovered from the passively immunized animal, but could not observe two phenomena; that is, the formation of the precipitin layer and the block of fertilization. At the present time, we are trying a series of experiments to determine the exact experimental condition to block the in vivo fertilization with the MoAb. In the immunofluorescence study to clarify the species cross-reactivity of the MoAbs, it became clear that three kinds of the MoAbs react neither with ovarian and oviductal eggs of mouse and rat nor with ovarian eggs of cattle and pig and that, when the ovarian eggs were incubated in the porcine oviduct at 37 Ž for 2 hrs, the eggs get a capacity to be able to bind three kinds of the MoAbs (Table 3). This fact indicates clearly that, on the ZP of the porcine eggs passed through the oviduct, certain immunologically detectable changes occur, that these immunological changes can be probably induced by an addition of the oviductal component and that the porcine oviductal component added to the ZP whthin the oviduct can cross-react with the MoAbs raised against the hamster ZP-0. REFERENCES Liebfried, M.L. and Bavister, B.D. (1982) Effect of epinephrine and hypotaurine on the in vitro fertilization inthe golden hamster. J. Reprod. Fert. 66, Oikawa, T. and Sendai, Y. (1984) Biochemical difference between zonae pellucidae of ovarian and oviducal eggs of the golden hamster, Mesocricetus auratus. Zool. Sci. 1, 932. Oikawa,T.Sendai, Y. and Kurata, S. (1985) Detection of a difference of antigenicity between ovarian and oviducal eggs of the hamster, Meoscricetus auratus. Proc. 4th. Ann. Meeting, JAIR, Tokyo, Japan, pp Oikawa, T., Kurata, S. and Sendai,Y. (1986) Discovery of a novel zona glycoprotein(zp-o) having a sperm binding activity. J. Reprod. Immunol. Supple. 81. Oikawa, T., Sendai, Y., Kurata, S. and Yanagimachi, R. (1987) A glycoprotein of oviductal origin alters biochemical properties of the zona pellucida of hamster egg. Gamete R es. in press.

6 Fig. 1 Immunostaining of the Hamster Oviduct Ampulla with Anti-hamster ZP-0 MoAb Table 1. Immunoreactivity of Three Kinds of Anti-ZP-0 Monoclonal Antibodies with Various Hamster Organs

7 Table 2. Effect of Anti-ZP-0 Monoclonal Antibody (C8B11) on the in vitro Fertilization Table 3. Species Cross-reactivity of Three Kinds of Anti-hamster ZP-0 Monoclonal Antibodies ( ) : Porcine ovarian egg cultured within the porcine oviduct at 37 Ž for 2hr. N.D. : not done.