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

Authors

  • Yinglun Sheng,

    1. Ottawa Health Research Institute (OHRI), Ottawa Hospital, 1053 Carling Avenue, Ottawa, Canada
    2. Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Canada
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  • Véronique Montplaisir,

    1. Ottawa Health Research Institute (OHRI), Ottawa Hospital, 1053 Carling Avenue, Ottawa, Canada
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  • X. Johné Liu

    Corresponding author
    1. Ottawa Health Research Institute (OHRI), Ottawa Hospital, 1053 Carling Avenue, Ottawa, Canada
    2. Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Canada
    3. Department of Obstetrics & Gynecology; University of Ottawa, Ottawa, Canada
    • Ottawa Health Research Institute, Ottawa Hospital, Civic Campus, 1053 Carling Avenue, Ottawa, K1Y 4E9, Canada.
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Abstract

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 βγ complex induces hormone-independent oocyte maturation. In contrast, over-expression of Xenopus Gβ1 (xGβ1), alone or together with bovine Gγ2, elevates oocyte cAMP and inhibits progesterone-induced oocyte maturation. To further investigate the mechanism of Gβγ-induced oocyte maturation, we generated a mutant xGβ1, substituting Asp-228 for Gly (D228G). An equivalent mutation in the mammalian Gβ1 results in the loss of its ability to activate adenylyl cyclases. Indeed, co-injection of xGβ1D228G with Gγ2 failed to increase oocyte cAMP or inhibit progesterone-induced oocyte maturation. To directly demonstrate that oocytes contained a Gβγ-regulated adenylyl cyclase, we analyzed cAMP formation in vitro by using oocyte membrane preparations. Purified brain Gβγ 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 G2 arrest mechanism in Xenopus oocytes. © 2005 Wiley-Liss, Inc.

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