Segregation of the Pathways Leading to Cortical Reaction and Cell Cycle Activation in the Rat Egg1

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Abstract
At fertilization of the mammalian egg, resumption of the cell cycle and the cortical reaction are two events of egg activation, correlated with an increase in intracellular Ca2+ concentration and activation of protein kinase C. To evaluate the pathways leading to both events, rat eggs were parthenogenetically activated by the calcium ionophore ionomycin, or by the protein kinase C activators 12-O-tetradecanoyl phorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG). Cortical granule exudate was visualized by the lectin Lens culinaris and Texas Red streptavidin, using a confocal microscope. Resumption of meiosis was detected by Hoechst dye, and intracellular Ca2+ concentration by fura-2. Ionomycin triggered both a cortical reaction and resumption of meiosis, while chelation of intracellular Ca2+ rise by BAPTA-AM (1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester) revealed a segregation between these two events. A low Ca2+ transient (approximately 150 nM) induced a partial cortical reaction in half of the eggs, but the meiotic status was not affected. TPA triggered a cortical reaction with neither resumption of meiosis nor intracellular Ca2+ rise, while OAG induced both aspects of activation, as well as a significant intracellular Ca2+ rise. We conclude that in the cascade of events leading to egg activation, the initial Ca2+ rise is followed by a segregation in the pathway. A relatively low Ca2+ rise is sufficient to induce a partial cortical reaction. However, a higher level of Ca2+ is required to complete the cortical reaction and resumption of meiosis. The activation of the cell cycle is Ca2+-dependent, but protein kinase C-independent.

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