The involvement of calcium- or protein kinase C (PKC)-dependent pathways in cortical granule exocytosis (CGE) and pronucleus formation was examined in mouse eggs using the specific PKC stimulator OAG (1-oleyl-2-acetyl-sn-glycerol) at different external calcium concentrations ([Ca2+]e) ranging from 1.7mM to 0.1μM. A 10 min exposure of eggs to 150 μM OAG in the presence of 1.7mM [Ca2+e caused a large calcium influx, cortical granule release and 82% activation. The increased permeability of the egg membrane to Ca2+ ions after OAG treatment lasted 20 min. At [Ca2+]e lower than 1.7 mM, both OAG-induced calcium influx and CGE decreased, reaching a non-detectable level at 0.1 μM and 100 μM [Ca2+]e, respectively. Resumption of meiosis was not affected by [Ca2+]e above 200 μM but it was reduced at any lower [Ca2+]e, with a minimum activation frequency of 46% at 0.1 μM [Ca2+]e. Loading of eggs with ≥3μM of the calcium chelator BAPTA AM (1,2-bis(o-aminophenox- y)ethane-N′,N′,N′,N′tetraacetic acid-acetoxymethyl ester) prior to OAG treatment caused a reduction in meiosis resumption with 50% of eggs forming pronuclei. Potent inhibitors of PKC, such as acridine orange and sphingosine, did not interfere with OAG-induced CGE. Conversely, these compounds prevented OAG-induced pronucleus formation in a dose-dependent manner with an IC50 (inhibiting concentration, 50%) of 5μM and 30 μM for acridine orange and sphingosine, respectively. Microinjection of inositol 1,4,5-trisphosphate into eggs at 0.1 pM elicited Ca2+ release from intracellular stores and the cortical reaction, but failed to stimulate pronucleus formation. These results indicate that, in mouse eggs, CGE is a PKC-independent event, and that the transition from M-phase to interphase may require PKC activity for stimulation.
