Segregation of the pathways leading to cortical reaction and cell cycle activation in the rat egg

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 (~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.