DEVELOPMENT OF PIG OOCYTES ACTIVATED BY STIMULATION OF AN EXOGENOUS G-PROTEIN-COUPLED RECEPTOR

This study determined whether stimulation of a G protein-coupled recep tor could initiate the events that occur at fertilization in pig oocyt es and, if so, whether the activated oocytes were competent to form bl astocysts. After maturation for 30 h, oocytes received microinjections of mRNA encoding the rat M1 muscarinic receptor, a G protein-coupled acetylcholine (ACh) receptor. Oocytes were then incubated for an addit ional 15 h to complete maturation of oocytes and translation of microi njected mRNA, and they were subsequently cultured in the presence of A Ch, ACh treatment of these oocytes triggered pronuclear formation (50. 4%) as well as cortical granule exocytosis, SDS-PAGE showed that mRNA- microinjected oocytes treated with ACh were activated (61.1%), as char acterized by the appearance of the 22-kDa polypeptide derived from dep hosphorylation of the 25-kDa precursor, Furthermore, after being cultu red in a ligated pig oviduct for 6 days, 17.4% of treated oocytes deve loped to the compact morula or blastocyst stage, Transmission electron microscopy revealed that blastocysts recovered from ligated oviducts contained reticulated nucleoli with fibrillar cores surrounded by fibr illar and granular components, In addition, mitochondria in the blasto cysts were dispersed throughout the cytoplasm and contained numerous t ransverse cristae. These results show that pig oocyte activation media ted by a G protein-coupled signal transduction system can signal a ser ies of intracellular changes that lead to activation events associated with fertilization. Furthermore, oocytes activated through this pathw ay showed preimplantation development consistent with normal developme nt.