Analysis of zona pellucida modifications due to cortical granule exocytosis in single porcine oocytes, using enhanced chemiluminescence

摘要

When a biotinylated zona pellucida (ZP) derived from a single pig oocyte matured in vitro was subjected to SDS-PAGE, 3 major bands (ZP1, ZP2 and ZP3) were observed following enhanced chemiluminescent (ECL) detection. In these oocytes, cortical granule (CG) staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin (FITC-PNA) formed a monolayer underlying the plasma membrane. Electrical stimulation to induce artificial activation deceased the fluorescent intensity of the CG, with a concomitant decrease in the amounts of ZP1 and ZP2 bands. However, the mobility changes of ZP1 and ZP2 on SDS-PAGE were not found under the inhibitory condition of the CG exocytosis in which oocytes were treated with ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA/AM). When a time-dependent decrease in amounts of ZP1 and ZP2 bands on SDS-PAGE was observed in a single oocyte duringactivation, a maximum decrease in these bands was detected in oocytes incubated for at least 3.5 h after electrical stimulation. It is concluded that ECL is a valuable method for the analysis of ZP modification resulting from a decrease in amounts of ZP1 and ZP2 glycoproteins in combination with exocytosis of CGs, and that a prolonged period after activation is required for complete ZP modification in pig oocytes.