A major goal of my research program is to identify molecules and metabolic pathways that participate in the control of oocyte maturation in mammals. In our model for meiotic induction, gonadotropin triggers an increase in cAMP phosphodiesterase (PDE) that induces meiotic resumption by both a loss of cAMP-dependent protein kinase activity and an increase in AMP-activated protein kinase (AMPK) activity. The oocyte-cumulus cell complex, isolated from mice 48 h after hormonal priming, will be used as the principal model system, but oocytes induced to mature in vivo by hCG injection will also be examined. Isoform-specific PDE inhibitors will be used to manipulate meiosis according to the cellular target site, and PDE activity will be measured to test for a cause-and-effect relationship with maturation-promoting factor (MPF; measured as histone H1 kinase activity) and germinal vesicle breakdown (GVB). Western analysis of AMPK (beta, gamma subunits) in complexes will be completed, and the temporal relationship between AMPK activation, MPF activation and GVB during spontaneous maturation or maturation induced by adenosine analogs or hormones will be tested. Oocyte microinjection will determine if active AMPK induces GVB and if antibodies to alpha subunits antagonize meiotic induction. Antisera to alpha subunits of AMPK as well as an anti-phosphoantibody to active AMPK will be employed to localize the enzyme in oocytes by indirect immunofluorescence during meiotic resumption. We will also investigate the potential role of ERK1/2 and p38 mitogen-activated protein kinases in mediating the meiosis-inducing action of AMPK. These results will help delineate specific metabolic pathways involved in meiotic maturation and will have important implications for both fertility and contraception, since each is affected by the ability of the oocyte to successfully initiate and complete meiotic maturation. They may also help in preventing or minimizing erroneous meiotic control that can give rise to aneuploidy and its disastrous consequences. Furthermore, these studies will benefit the development of in vitro systems where either meiotic arrest or completion of meiotic maturation is the desired endpoint.
