Study of the cell cycle regulation mechanism in oocyte maturation

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I have succeeded in assaying the changes of intracellular concentrations, synthesis and association state of p34^<cdc2> and cyclin B,and the changes of concentration, activity and its regulation of MAP kinase (MAPK) throughout porcine oocyte maturation. At first, using porcine granulosa cells, I established the methords of western brotting for p34^<cdc2> and cyclin B,immunoprecipitation for examining the association states of p34^<cdc2> and cyclin B and the methords for examining the MPF phosphorylation state which affects MPF activity with anti-phosphotyrosine antibody. Using these methods I studied next about porcine oocytes throughout maturation, and found out that immature porcine oocytes had extremely small amount of cyclin B and the amount increased after germinal vesicle breakdown (GVBD), unlike many other reported speceis. confirming this fact, cyclin B synthesis was not detected in GV oocytes and observed in first and second methaphase oocytes. The amount of cyclin B/p34^<cdc2> complex reflected the amount of cyclin B,being very low during GV stage and high in first metaphase then much higher in second metaphase, although the amount of p34^<cdc2> does not change throughout maturation. Dephsophorylation of phosphotyrosine, which prevents p34^<cdc2> activity, was required for activation of p34^<cdc2>. MAPK activity was low during GV stage. The high MAPK activity was observed after GVBD until second metaphase and the decrease of the activity at first polar body emission, shown in MPF activity, was not observed. The intracellular concentation of MAPK did not change during maturation and the activty was regulated by phosphorylation.