Effects Of Protein Kinase On Meiotic Maturation, Fertilization, And Early Embryonic Development Of Mouse Oocytes

The meiosis and fertilization of vertebrate oocyte are extensively regulated by various protein kinases. Recently, a great progress has been achieved in the studies on the molecular mechanisms of oocyte maturation, activation and fertilization. The cooperation of these protein kinases is essential to the development and fertilization of the oocyte. Protein kinase C (PKC) is a family of ser/thr protein kinases widely distributed in eukaryotes. There is evidence that PKC plays key roles in meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we revealed in mouse eggs and early embryos: 1) the effects of PKC modulation on meiotic and mitotic cell cycle progression; 2) the functional importance of classical PKC subclasses in these processes; and 3) the subcellular localization of PKC( isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicated that PKC activator PMA inhibited meiotic resumption of denuded mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after GVBD leads to inhibition of MAPK phosphorylation and cell cycle arrest at the MI stage. PKC activation also caused failure of the second polar body emission after fertilization. PKC( isoform was typically concentrated around the condensed chromosomes before the MI stage, but localized in the pronuclei of zygotes and the nuclei of blastomeres of early embryos. Pronucleus formation in fertilized eggs was inhibited by PKC inhibitor calphostin C, but not by cPKC-specific inhibitor Go 6976. Cleavages of early embryos were blocked after prolonged PKC activation by PMA. All these results suggest that PKC plays multiple functional roles in cell cycle progression of mouse oocytes and embryos.Pig oocytes cultured in vitro for some time were inseminated by frozen–thawed ejaculated sperm. At specified times after insemination, sperm penetration, cell cycle progression and mitogen-activated protein kinase (MAPK) phosphorylation were evaluated. It was shown that: (1) oocytes at various maturational stages could be penetrated by sperm; (2) sperm penetration did not affect meiotic cell cycle progression; (3) sperm penetration of germinal vesicle (GV) oocytes and maturing oocytes did not alter MAPK phosphorylation; and (4) when premetaphase I (pre-MI) and metaphase I (MI) oocytes, in which MAPK was activated, were fertilised, no evident MAPK dephosphorylation was detected as in metaphase II oocytes. The data suggest that sperm penetration before oocyte maturation does not affect MAPK phosphorylation and that the machinery inactivating MAPK upon fertilisation is not developed in maturing (pre-MI to MI) oocytes.