Title

G-protein regulation of meiosis in the oocyte

Date of Completion

January 2004

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Maintenance of meiotic prophase arrest in vertebrate oocytes depends on an elevated level of cAMP in the oocyte; conversely, the resumption of meiosis in response to hormone depends on a decrease in cAMP. This dissertation concerns the role of heterotrimeric G-proteins in regulating oocyte cAMP levels and meiotic progression, as introduced in chapter one. Results in chapter two show that the reinitiation of meiosis in oocytes of starfish, but not frog, occurs through activation of the Gi family of G-proteins. This is demonstrated by evidence that activation of exogenously expressed Gi-linked 7-transmembrane receptors causes meiosis to resume in starfish oocytes, but not Xenopus oocytes. Results in chapter three show that the Gs family of G-proteins is required to maintain meiotic arrest in vertebrate oocytes. Injection of RNA encoding a dominant negative form of Gs causes Xenopus and mouse oocytes to resume meiosis. Likewise, injection of an inhibitory antibody made against the α subunit of Gs causes meiotic resumption in zebrafish oocytes, as has been reported in Xenopus and mouse oocytes. These results support the hypothesis that there is a Gs-activating receptor in the oocyte membrane that maintains vertebrate oocyte arrest. Chapter four describes a new, high throughput expression cloning method for identifying novel G-protein coupled receptors from cDNA libraries. Pools of RNA transcribed from an arrayed cDNA library are co-injected with RNA encoding a chimeric G-protein into Xenopus laevis oocytes; if the unknown G-protein coupled receptor is present and couples to the exogenously expressed chimeric G-protein, the oocyte pigment will contract in response to agonist application. This is a simple visual screen that can be used to identify G-protein coupled receptors, including receptors involved in the regulation of meiosis in oocytes. ^