In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.
In order to understand the role of the protein zona pellucida 2 in fertilization, an antibody against a central segment of the zona pellucida 2 peptide, segment 190-505 (Z2eH), was prepared. The influence of the antibody on sperm-zona interaction was tested using the sperm-egg binding assay. The effect of the antibody on fertility was evaluated by passive immunization with anti-Z2eH antibody. Immunohistochemical assay showed that an antibody from rabbit reacted specifically with the natural zona pellucida on mouse ovarian sections. Immunofluorescence assay showed that the antibody bound specifically to the zonae pellucidae of the ovulated oocytes and 2-cell embryos after passive immunization. The antibody-treated oocytes bound capacitated sperm as control oocytes, passive immunization did not impede the action of sperm to fertilize the oocyte in vivo. These findings suggest that the central peptide of ZP2 (190-505) is immunogenic and contains zona pellucida-specific epitopes, however the central polypeptide might not be the crucial part from which to construct a functional domain to bind sperm.
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