The zona pellucida, a transparent envelope surrounding the mammalian oocyte, comprises three glycoproteins, ZPA, ZPB and ZPC, and plays important roles in fertilization. We have previously reported that apparent relative molecular masses of bovine zona glycoproteins on SDS/PAGE under nonreducing conditions after removal of poly N-acetyllactosamine at the nonreducing portion of sugar chains with endo-b-galactosidase are 72 000, 58 000 and 45 000 [Noguchi, S., Yonezawa, N., Katsumata, T., Hashizume, K.,Kuwayama, M., Hamano, S., Watanabe, S. & Nakano, M. (1994) Biochim. Biophys. Acta 1201, 7±14]. The N-terminal amino-acid sequences and crossreactivity to antibodies specific to each porcine zona component show that the bovine components correspond to porcine ZPA, ZPB and ZPC, respectively. In this study, we deduced amino-acid sequences of bovine ZPA and ZPB by cDNA cloning and sequencing. Identities in amino-acid sequences between bovine and porcine counterparts were 77% for ZPA and 75% for ZPB, whereas between bovine and murine counterparts identities were 57% for ZPA and 37% for ZPB. The positions of Cys were completely conserved in bovine ZPA and ZPB compared with counterparts of other mammalian species. Bovine ZPA was processed between Ala and Asp on fertilization, suggesting that the consensus motif for the processing is Ala-Asp-Asp/Glu. We purified bovine zona components and examined their sperm-binding activity with an in vitro competition assay and sperm-bead-binding assay. As a result, ZPB showed the strongest sperm-binding activity among the components. ZPC also showed sperm-binding activity and the activity per molecule was about one-sixth that of ZPB according to the result of the sperm-bead-binding assay. We could not determine if ZPA has significant sperm-binding activity, but the activity may be much lower than that of ZPB even if ZPA has significant activity. Thus, ZPB may play a major role in sperm binding in bovine zona.

alpha-Mannosidase and beta-galactosidase were released from boar sperm into the medium by treatment with calcium ionophore A23187 or by 0.2% Brij-35/2% acetic acid. About half as much alpha-mannosidase activity as that in the acid extract was recovered by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the liberation rate of beta-galactosidase treated with PI-PLC was low. These results suggest that some alpha-mannosidase is anchored in the plasma membrane of the acrosomal region by attachment to the lipid phosphatidylinositol and that beta-galactosidase is localized mainly in the acrosome or integrated in the plasma membrane by a spanning stretch of hydrophobic peptides. beta-Galactosidase, which is present as an oligomers in the acid extract of sperm, dissociated into monomers under weakly alkaline conditions; under acidic conditions, the monomers associated again. No pH-sensitive association-dissociation of alpha-mannosidase was observed.