The role of protein kinase B/Akt signaling in the resumption of meiosis in oocytes from Xenopus laevis

The serine/threonine kinase Akt mediates many of the phosphoinositide (PI) 3-kinase effects on cell metabolism, cell growth and cell cycle. It has been suggested that PI3-K an is important mediator of resumption of meiosis in oocytes from the African clawed frog Xenopus laevis. Since PI3-K is stimulated by either progesterone or insulin/IGF-1, we have investigated the properties of the Akt expressed in the Xenopus oocyte and its role resumption of meiosis.;The Xenopus cDNA encoding Akt was isolated and its sequence was found to be highly homologous to mammalian Akt. The expression of Akt mRNA and protein in Xenopus oocyte was further confirmed by RT-PCR, Northern Blotting with a Xenopus Akt cDNA, and by immunoprecipitation with at least two distinct antibodies to mammalian Akt. Using phosphospecific antibodies and enzyme assays, activation of the Xenopus Akt was observed upon stimulation of the oocyte with insulin or progesterone.;Injection of mRNA coding for constitutively active Akt stimulates resumption of meiosis in oocytes. Using mutated constructs of Akt it was demonstrated that Akt has to be active and membrane targeted to mediate it effect on meiosis. That this activation is necessary for the resumption of meiosis was demonstrated by a dominant negative approach using a localization deficient Akt mutant. Moreover, a monoclonal antibody, which inhibited the activity of the endogenous Akt, blocked the insulin-stimulated re-entry of the oocyte into the cell cycle, and had some effect on progesterone stimulation.;Akt stimulated resumption of meiosis was dependent on PDE3 activity and Akt induced resumption of meiosis caused a two-fold increase in oocyte PDE activity. Using injection of a PDE4 it was demonstrated that a two-fold increase in PDE activity is sufficient to stimulate resumption of meiosis. That the monoclonal anti Akt antibody is specific for Akt was demonstrated by the finding that the injection of mRNA of an active Akt or the downstream PDE3A could completely overcome the antibody blockade.;These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes and indicates that Akt also has a role in the response to progesterone.