| Popis: |
Xenopus laevis oocytes are physiologically arrested at the first meiotic prophase. Progesterone reinitiates meiosis (maturation) through inhibition of an oocyte adenylyl cyclase (AC) and reduction of intracellular cAMP. However, the mechanism by which progesterone regulates AC activity and cAMP level still remains unclear. In this thesis, I summarize work I conducted that collectively helps elucidate how high levels of cAMP might be achieved in G2 arrested oocytes. In Chapter 2, I describe our finding that inhibiting endogenous G-protein betagamma subunits, through the use of two structurally distinct Gbetagamma scavengers, causes hormone-independent oocyte maturation. In contrast, overexpression of Xenopus Gbeta1, alone or together with bovine Ggamma2, inhibits progesterone-induced oocyte maturation. These results for the first time implicate that an endogenous G protein coupled receptor system releases a Gbetagamma complex as the dominant meiosis inhibitor. Chapter 3 describes my research aiming to reveal the identity of the oocyte AC responsible for generating meiosis-inhibiting cAMP. I provide further evidence here that the ability of Gbetagamma to inhibit meiosis is attributed to the activation of an endogenous AC, rather than other possible Gbetagamma effectors. Through molecular cloning and biochemical characterization, I discovered that the likely AC candidate is Xenopus AC7, an isoform that is activated by Gbetagamma, but only in the presence of GTP-bound Gsalpha. The identification of xAC7 suggests that the maintenance of high levels of cAMP may require the cooperation of Gsalpha and Gbetagamma. Finally, in Chapter 4, I describe our efforts in identifying the GPCR(s) responsible for activating the cAMP signaling in prophase-arrested oocytes. A screening of known antagonists of GPCR(s) led to the identification of ritanserin, a potent antagonist of serotonin receptors, as a potent maturation inducer in Xenopus oocytes. Pharmacological and molecular studies, however, have ruled out the involvement of a known serotonin receptor in meiosis arrest. Instead, the most likely candidate is a "constitutively activated" GPCR that bears structural similarities to Xenopus serotonin receptor 7. |
