GPR3 IS A CONSTITUTIVELY ACTIVE CELL SURFACE G PROTEIN-COUPLED RECEPTOR THAT MAINTAINS MEIOTIC ARREST OF Xenopus laevis OOCYTES

Abstract

Normal female fertility requires precise regulation of oocyte meiosis. Females of all animals are born with a full set of oocytes, but these immature oocytes are held in prophase I of meiosis. Just prior to ovulation, gonadotropin-induced signals trigger a subpopulation of oocytes to resume meiosis and progress to metaphase II, where they arrest again until after ovulation and fertilization. Several laboratories have provided evidence that oocytes are actively held in meiotic arrest through a mechanism whereby constitutive cAMP production exceeds phosphodiesterase-mediated degradation, leading to elevations in intracellular cAMP levels, activation of PKA, and inhibition of meiosis. Recent studies have implicated a novel Gαs-coupled receptor, GPR3, as one of the molecules responsible for promoting cAMP production during meiotic arrest of mouse oocytes. To begin characterization of GPR3 in the more manageable meiosis model of Xenopus laevis, we utilized nested PCR techniques to clone a cDNA encoding the Xenopus laevis isoform of GPR3 from a pool of total oocyte RNA. We then engineered a FLAG tag at the amino-terminus of GPR3, allowing for detection of this clone. Transfection of this tagged cDNA into COS cells showed significant cell surface expression and detectable increases in intracellular cAMP. Injection of cRNA encoding the same protein into Xenopus oocytes similarly resulted in substantial cell surface expression of GPR3 and increased cAMP levels. Importantly, expression of very small amounts of GPR3 in the oocytes almost completely abrogated steroid-triggered germinal vesicle breakdown, as well as activation of MAPK and CDK1. Thus, GPR3 activity appears to be sufficient to promote cAMP production and hold oocytes in meiotic arrest in the Xenopus laevis model. Future knock-down studies utilizing RNA interference and antisense oligonucleotides will be required to ascertain whether GPR3 signaling is necessary for physiologic meiotic inhibition in Xenopus oocytes. (platform)