The long-term goal of this research is to elucidate regulatory components of oocyte meiotic maturation in the murine model and develop manipulation strategies which ultimately will result in efficient in vitro maturation (IVM) of human oocytes with the production of developmentally competent embryos. IVM coupled with in vitro fertilization will eliminate the use of exogenous gonadotropin stimulation, and its associated risks, while reducing the cost of infertility treatment. Our current understanding of intracellular mechanisms governing development of germinal vesicle breakdown (GVB) competence and resumption of meiosis are incomplete. Treatment of oocytes with okadaic acid (OA), which inhibits both protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A), stimulates meiotic resumption but causes aberrant spindle formation and abnormal cytoplasmic morphology. Direct identification of PP1 and/or PP2A, and their regulation and function in mammalian oocytes have not been determined. We hypothesize that both PPI and PP2A are present in murine oocytes with distinct roles: PP1 is involved with initiation of maturation and germinal vesicle breakdown and PP2A is associated with microtubule assembly.
Specific aims are proposed to: (1) Characterize PP1 and PP2A intracellular distribution, activity and ultrastructural association in murine oocytes during acquisition of meiotic competence, GVB and metaphase I (MI) development; (2) Identify cytoplasmic and nuclear endogenous regulators of PP1 during acquisition of meiotic competence and resumption of meiosis and (3) elucidate which PP, PP1 or PP2A, regulates MI meiotic microtubule assembly. GVB-incompetent, GVB-competent and MI oocytes and ovaries will be collected from mice. Intracellular PP1 and PP2A will be immuno- localized with confocal fluorescent microscopy in meiotic- incompetent, competent and MI oocytes. Specific PP1 and PP2A activity in the oocyte nucleus and cytoplasm will be analyzed during acquisition of meiotic competence and resumption of meiosis using a [32P]- phosphorylase-a assay. Ultrastructural localization of PP1 and PP2A will be performed by electron microscopy (EM-ICC) to gain insight into structure/function relationships during oogenesis and meiotic resumption. The endogenous regulators governing activity of oocyte cytoplasmic PP1 (glycogen synthase kinase-3, inhibitors 1 and/or 2) and nuclear PP1 (NIPP-1) will be characterized in oocytes of differing meiotic abilities and stages by RT-PCR, Western blotting, modified PP assays and immunohistochemistry. GVB-competent oocytes will be microinjected with neutralizing anti-PP1 or anti-PP2A antibodies and assessed for spindle formation and cytoplasmic morphology to determine which PP is responsible for OA-induced spindle and cytoplasmic abnormalities. These studies will provide novel information concerning control of oocyte maturation and spindle formation that may subsequently influence the developmental competency of resulting embryos.
