Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs

Here, we describe an in vitro assay that has permitted further characterization of a proteinase (called "ZP2-proteinase") that is released upon activation of ovulated mouse eggs and cleaves ZP2, one of three glycoproteins present in mouse zonae pellucidae. Results presented suggest that ZP2-proteinase readily diffuses through the zona pellucida within 5 min of activation of eggs by ionophore A23187 and carries out limited proteolysis of ZP2. Appearance of ZP2-proteinase is completely dependent upon activation of eggs, consistent with it being present in cortical granule exudate. The proteinase is insensitive to a wide variety of proteinase inhibitors, but is inhibited when either an anti-ZP2 monoclonal antibody or an Fab fragment of the antibody is bound to ZP2. Proteolysis occurs near the amino- or carboxy-terminus of ZP2, producing a 23,000 M_r glycopeptide(s) that remains attached to ZP2 by intramolecular disulfide bonds. HPLC fractionation of activated egg exudate suggests that ZP2-proteinase has an apparent M_r between 21,000 and 34,000. Proteolysis of ZP2 correlates with "hardening" of the zona pellucida following egg activation and, thus, may be responsible for one aspect of the zona reaction.