Analysis of zona pellucida modifications due to cortical granule exocytosis in single porcine oocytes, using enhanced chemiluminescence
1999
Tatemoto, H. | Terada, T.
The present study was carried out to determine whether modification of zona pellucida (ZP) of a single oocyte following the cortical granule (CG) exocytosis induced by electrical stimulation could be analyzed using enhanced chemiluminescent (ECL) detection of the biotinylated ZP in a porcine oocyte. When a biotinylated ZP derived from a single oocyte matured in vitro was subjected to SDS-PAGE, 3 major bands (ZP1, ZP2 and ZP3) were observed following ECL detection. In these oocytes, CGs staining with fluorescein isothiocyanate (FITC)-labeled peanut agglutinin (FITC-PNA) had formed a monolayer underlying the plasma membrane. Electrical stimulation to induce artificial activation caused a decline in the fluorescent intensity of the CGs 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 1,22-bis(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid tetraxis(acetoxymethyl) ester (BAPTA/AM). In addition, when a time-dependent decrease in amounts of ZP1 and ZP2 bands on SDS-PAGE was observed in a single oocyte during activation, a maximum decrease in these bands was detected in occytes incubated for at least 3.5 h after electrical stimulation. These results show that the method employed, ECL detection of the biotinylated ZP of a single oocyte, is a valuable tool 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 the prolonged period after activation is required for complete ZP modification in porcine oocytes.
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