Development of Pig Oocytes Activated by Stimulation of an Exogenous G Protein-Coupled Receptor¹

^a Department of Animal Sciences, University of Missouri-Columbia, Missouri 65211

This study determined whether stimulation of a G protein-coupled receptor could initiate the events that occur at fertilization in pig oocytes and, if so, whether the activated oocytes were competent to form blastocysts. 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 additional 15 h to complete maturation of oocytes and translation of microinjected mRNA, and they were subsequently cultured in the presence of ACh. 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 characterized by the appearance of the 22-kDa polypeptide derived from dephosphorylation of the 25-kDa precursor. Furthermore, after being cultured in a ligated pig oviduct for 6 days, 17.4% of treated oocytes developed 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 fibrillar and granular components. In addition, mitochondria in the blastocysts were dispersed throughout the cytoplasm and contained numerous transverse cristae. These results show that pig oocyte activation mediated by a G protein-coupled signal transduction system can signal a series of intracellular changes that lead to activation events associated with fertilization. Furthermore, oocytes activated through this pathway showed preimplantation development consistent with normal development.

¹ This material is based upon work supported by Food for the 21st Century and the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture, under agreement No. 95-37203-2073. The manuscript is a contribution from the Missouri Agriculture Experiment Station Journal Series No. 12,727.

² Correspondence: Randall S. Prather, 162 Animal Science Research Center, University of Missouri-Columbia, Columbia, MO 65211. FAX: (573) 882-6827; randall_prather{at}muccmail.missouri.edu

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