Activation of ribosomal S6 kinase (RSK) during porcine oocyte maturation.

The normal kinetics of ribosomal S6 kinase (RSK) during the meiotic maturation of porcine oocytes were examined. The phosphorylation states of RSK and extracellular signal-regulated kinase (ERK), major mitogen-activated protein (MAP) kinases in maturating porcine oocytes, were detected by Western blotting analysis. The S6 protein kinase activity was assayed using a specific substrate peptide which contained the major phosphorylation sites of S6 kinase. Full phosphorylation of RSK was correlated with ERK phosphorylation and was observed before germinal vesicle breakdown. S6 kinase activity was low in both freshly isolated and 20 h cultured oocytes. S6 kinase activity was significantly elevated in matured oocytes to a level about 6 times higher than that in freshly isolated oocytes. Furthermore, full phosphorylation of RSK was inhibited when oocytes were treated with U0126, a specific MAP kinase kinase inhibitor, in dose-dependent manner, indicating that RSK is one of the substrates of MAP kinase. These results suggest that the activation of RSK is involved in the regulation of meiotic maturation of porcine oocytes.

Germinal vesicle materials are not required for the activation of MAP kinase in porcine oocyte maturation.

The requirement of the germinal vesicle (GV) for the normal kinetics of mitogen-activated protein (MAP) kinase activity during porcine oocyte maturation was investigated. Porcine follicular oocytes were enucleated, and the locations of their extracellular signal-regulated kinases 1 and 2 (ERK1/2), major MAP kinases in maturating porcine oocytes, were detected by indirect immunofluorescent microscopy. The MAP kinase activity was assayed as myelin basic protein (MBP) kinase activity, and the phosphorylation states of ERK1/2 were detected by immunoblotting analyses. Translocation of MAP kinase into the GV and association with the spindle were observed in intact oocytes, while MAP kinase in enucleated oocytes was distributed almost uniformly in cytoplasm throughout the culturing period. The phosphorylation and the activation of MAP kinase were induced, and the activity was comparable with that of control denuded oocytes. The high level of activity was maintained through maturation, even in the absence of spindle formation. These results indicate that the presence of nuclear material and translocation into the GV are dispensable for the activation of MAP kinase and that associating with the spindle is not required for maintenance of its activity though porcine oocyte maturation.

Phosphorylation of mitogen-activated protein kinase cascade during early embryo development in the mouse.

The mitogen-activated protein kinase (MAPK) cascade is one of the most important signal transduction pathways that regulate the cell cycle in somatic cells. The present study examined the phosphorylation states of components in the MAPK cascade, Raf-1, MEK-1, and extracellular signal regulated kinases (ERKs), which are activated by mitogens, throughout early mouse embryo development and in cultured somatic cells generally. In somatic cells, Raf-1 and MEK-1 were phosphorylated at M-phase and dephosphorylated during interphase. ERKs were not phosphorylated at any stage during the cell cycle. These results were similar to previous findings for the first and second cell cycles of early mouse embryos. In contrast, after the four-cell stage, not only ERKs, but also Raf-1 and MEK-1, were not phosphorylated at any stage during the cell cycle in mouse early embryos. These results suggest that the MAPK cascade in mouse embryos is regulated by the same mechanism as in somatic cells before the two-cell stage, and that regulation is changed to an embryo-specific mechanism after the four-cell stage.