Co-operation of Gsα and Gβγ in maintaining G2 arrest in xenopus oocytes

Progesterone-induced oocyte maturation is thought to involve the inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. Our previous studies demonstrated that injection of inhibitors of G protein betagamma complex induces hormone-independent oocyte maturation. In contrast, over-expression of Xenopus G(beta1) (xG(beta1)), alone or together with bovine G(gamma2), elevates oocyte cAMP and inhibits progesterone-induced oocyte maturation. To further investigate the mechanism of G(betagamma)-induced oocyte maturation, we generated a mutant xG(beta1), substituting Asp-228 for Gly (D228G). An equivalent mutation in the mammalian G(beta1) results in the loss of its ability to activate adenylyl cyclases. indeed, co-injection of xG(beta1D228G) with G(gamma2) failed to increase oocyte cAMP or inhibit progesterone-induced oocyte maturation. To directly demonstrate that oocytes contained a G(betagamma)-regulated adenylyl cyclase, we analyzed cAMP formation in vitro by using oocyte membrane preparations. Purified brain G(betagamma) complexes significantly activated membrane-bound adenylyl cyclase activities. Multiple adenylyl cyclase isoforms were identified in frog oocytes by PCR using degenerate primers corresponding to highly conserved catalytic amino acid sequences. Among these we identified a partial Xenopus adenylyl cyclase 7 (xAC7) that was 65% identical in amino acid sequence to human AC7. A dominant-negative mutant of xAC7 induced hormone-independent oocyte maturation and accelerated progesterone-induced oocyte maturation. Theses findings suggest that xAC7 is a major component of the G(2) arrest mechanism in Xenopus oocytes. (C) 2004 Wiley-Liss, Inc.