Effects of protein kinase C stimulation and free Ca2+ rise in mammalian egg activation

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Abstract
Protein phosphorylation activity, chromosome segregation, and cortical granule exocytosis (CGE) have been studied in mouse eggs activated parthenogeneticaliy by specific PKC stimulators such as 4β-phorbol 12-myristate 13-acetate (PMA) and l-oleyl-2-acetylglycerol (OAG), or by agents inducing an immediate increase in cytosolic calcium concentration ([Ca2+]i) such as ethanol and Ca-ionophore A23187. When protein phosphorylation activity of mouse eggs was analyzed 10 min after different activation treatments, the phosphorylation of a 32 kDa polypeptide was a feature common to all different parthenogenetic agents used. The appearance of such labeling was independent of an increasing [Ca2+]i, as indicated by direct measurements of i) cytosolic Ca2+ concentration with fura-2 and 2) exogenous Ca2+ entrance into activated eggs. Emission of the second polar body was blocked in PMA-elicited partheno-genones, whereas it was apparently normal in OAG-treated eggs, unless the eggs were continuously exposed to OAG. CGE was almost immediate in ethanol-activated eggs, but in PMA-treated cells, it occurred significantly later, with a timing corresponding to that found for the appearance of sustained Ca2+ oscillations in this system. Here, we propose that in mammalian eggs 1) PKC stimulation represents an early regulatory step in egg activation; 2) this kinase activity is turned off before the second meiotic cleavage; and 3) cytosolic free Ca2+ rise is essential for CGE occurrence.