MS31. MAINTENANCE OF MEIOTIC ARREST IN MOUSE OOCYTES BY GPR3 AND Gs. Mehlmann, Lisa¹, Saeki, Yoshinaga², Tanaka, Shigeru², Brennan, Thomas³, Evsikov, Alexei⁴, Pendola, Frank⁴, Knowles, Barbara ⁴, Eppig, John⁴, Jaffe, Laurinda¹, ¹ University of Connecticut Health Center, Farmington, CT² Ohio State University, Columbus, OH³ Deltagen, Inc, San Carlos, CA⁴ The Jackson Laboratory, Bar Harbor, MA
     Mammalian oocytes are arrested in prophase I until a surge of luteinizing hormone (LH) causes them to re-enter meiosis and mature into fertilizable eggs. The follicle cells surrounding the oocyte are needed to maintain meiotic arrest prior to the LH surge, although the mechanism by which this occurs is not yet understood. Meiotic arrest is maintained by high levels of cAMP in the oocyte prior to the LH surge, and a G_s G-protein in the oocyte is critical for producing cAMP. We have recently found that the orphan G_s-linked receptor GPR3, which is localized in the oocyte, is also essential for the maintenance of meiotic arrest. Oocytes from Gpr3 knockout mice resume meiosis within antral follicles, independently of an increase in luteinizing hormone, and this phenotype can be reversed by injection of Gpr3 RNA into the oocytes. Thus, the GPR3 receptor is a link in the communication between the follicle cells and oocyte of the ovarian follicle, crucial for the regulation of meiosis. It is possible that the role of the follicle cells in maintaining meiotic arrest is to produce a ligand that stimulates GPR3. We are currently examining the fertility and egg quality of oocytes from Gpr3 knockout mice. In addition, we are testing injection of dsRNA into follicle-enclosed oocytes as an approach for further investigating the role of GPR3 and other signalling molecules that may function in this pathway. Understanding the pathway whereby meiotic arrest is maintained provides a framework for examining the signalling pathway by which LH causes meiotic resumption.

MS32. INSIGHT INTO SPERM CAPACITATION: TYROSINE PHOSPHORYLATION OF SP32, A PROACROSIN BINDING PROTEIN. Bailey, Janice ¹, Dubé, Charlotte¹, Leclerc, Pierre ², Guillemette, Christine¹, Beaulieu, Martin¹, ¹ Université Laval, Quebec City, QC, Canada² Centre Hospitalier de l'Université Laval, Quebec City, QC, Canada
     Capacitation refers to the collection of cellular modifications that allow mammalian sperm to bind the zona pellucida of the oocyte and undergo the acrosome reaction. Therefore, it is necessary for successful fertilisation in vivo and in vitro, perhaps with the exception of intracytoplasmic sperm injection. Capacitation is a signal transduction-mediated phenomenon, triggered in the absence of a ligand on the sperm, and is regulated by cAMP, calcium, protein tyrosine phosphorylation and as yet unidentified tyrosine kinase(s). We have reported that in porcine sperm, the appearance of a tyrosine phosphorylated protein, p32, coincides with capacitation. Western blotting of pig sperm proteins separated successively under non-reducing then reducing conditions showed p32 only when sperm were incubated in capacitating conditions. Sequencing p32 by MS/MS revealed it to be sp32, a protein involved in acrosin maturation in pig sperm and binds the Mr 55,000 and 53,000 forms of proacrosin as well as the Mr 49,000 acrosin intermediate (Baba et al J Biol Chem 1994 269:10133-40). Our hypothesis, therefore, is that p32 is a tyrosine phosphorylated form of sp32, the acrosomal protein implicated in acrosin activation. Hybridising the same membranes with anti-sp32 antibody demonstrated that sp32 is present in both non-capacitating and capacitating conditions; anti-sp32 recognised the same spot as p32 in extracts from capacitated sperm. Immunoprecipitation with either anti-phosphotyrosine or anti-sp32 antibody corroborated these results. Including calcium chelators in the media demonstrated that the appearance of sp32 is calcium-dependent, similar to what we have previously established for p32. Indirect immunofluorescence with anti-phosphotyrosine antibody or anti-sp32 antibody show similar labelling of capacitated sperm, supporting the hypothesis that p32 is a tyrosine phosphorylated form of sp32. Both indirect immunofluorescent and electron microscopy showed that sp32 is lost from the sperm following the A23187-induced acrosome reaction. Despite these intriguing data, numerous questions remain regarding the regulation of sp32 tyrosine phosphorylation and its importance to sperm function. This project is funded by NSERC; CD and MB were recipients of FQRNT scholarships.

MS33. RNAi IN THE OOCYTE: A POWERFUL TOOL TO STUDY GENE FUNCTION IN OOCYTE MATURATION AND FERTILIZATION. Stein, Paula¹, ¹ University of Pennsylvania, Philadelphia, PA
     RNA interference (RNAi) is a conserved post-transcriptional gene silencing mechanism present in most eukaryotes. We have demonstrated that RNAi operates in mouse oocytes and early embryos. We further extended the applicability of RNAi by developing a transgenic RNAi approach to study gene function during oocyte development. The approach utilizes the oocyte-specific Zp3 promoter to drive the expression of a long hairpin double-stranded RNA (dsRNA) that contains sequence complementary to the gene of interest. We validated this approach by targeting Mos and were able to recapitulate the Mos null phenotype. This transgenic RNAi approach has been extended to study the function of other genes involved in oocyte maturation, fertilization and early embryo development. Although RNAi is observed from yeast to humans, mammalian cells possess another pathway that responds to long dsRNA by inducing interferon and expression and activating two dsRNA-dependent enzymes, namely, dsRNA-dependent protein kinase (PKR) and 2′,5′-oligoadenylate synthetase (OAS). PKR activation results in a general inhibition of protein synthesis, while OAS activates RNase L, leading to nonspecific degradation of mRNAs. This antiviral pathway is known as the interferon response. Mouse oocytes and preimplantation embryos seem to lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In order to ascertain whether mouse oocytes can mount an interferon response, we assessed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes exposed to long dsRNA. This analysis, confirmed by real-time PCR and Western blot, shows that PKR, RNase L, and the catalytically active isoforms of OAS are absent in mouse oocytes, while catalytically inactive OAS isoforms, believed to act as dominant negative, are present in large amounts. Transgenic oocytes expressing Mos dsRNA show the same pattern of expression as wild-type oocytes. The microarray analysis also provides invaluable information about the target specificity of RNAi in mouse oocytes and demonstrates the complete absence of off-targeting. We conclude that transgenic RNAi is highly specific in mouse oocytes and therefore is a robust and simple method to study gene function during oocyte development.