Time Course Analyses of Cortical Granule Exocytosis and Zona Pellucida Modifications after Artificial Activation in In-Vitro Matured Porcine Oocytes.

Abstract

The present study was carried out to determine the time-dependent changes in the cortical granule (CG) distribution and the glycoprotein compositions of zona pellucida (ZP) after egg activation by electrostimulation in porcine oocytes matured in vitro. CG exocytosis was observed by staining with fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) and laser confocal microscopy, and ZP modification was analyzed by using enhanced chemiluminescent (ECL) detection of the biotinylated ZP derived from a single oocyte. In the oocytes matured in vitro, CGs staining with FITC-PNA had formed a continuous monolayer underlying the oolemma, and three major bands (ZP1, ZP2 and ZP3) were observed in a biotinylated ZP subjected to SDS-PAGE followed by ECL detection. Electrostimulation 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, but CG exocytosis according to oocyte activation occurred slowly, and the incidence of oocytes with complete CG exocytosis was first observed 1.0 h after electrostimulation (9%). Similarly, the complete ZP modification, i.e. a maximum decrease in amounts of ZP1 and ZP2 bands, required more than 3.5 h after artificial activation, and the ZP dissolution time caused by 0.1% protease action increased apparently as incubation time was prolonged after electrostimulation. Results of these experiments suggest that the slower ZP modification due to the delayed CG exocytosis in porcine oocytes may reflect the remarkable increase in polyspermy frequency in in-vitro fertilization.