Study of the cell cycle regulation mechanism in oocyte maturation

本研究成功地分析了猪卵母细胞成熟过程中细胞内p34 ~+、cyclin B的浓度、合成和结合状态的变化<cdc2>，以及MAP激酶（MAPK）的浓度、活性及其调控的变化。首先，以猪颗粒细胞为材料，建立了p34 ~（1+）<cdc2>和cyclin B蛋白的Western印迹法、p34 ~（1+）和cyclin B蛋白结合状态的免疫沉淀<cdc2>法以及用抗磷酸酪氨酸抗体检测影响MPF活性的MPF磷酸化状态的方法。利用这些方法，我进一步研究了猪卵母细胞在整个成熟过程中细胞周期蛋白B的表达情况，发现猪未成熟卵母细胞中细胞周期蛋白B的表达量极低，且在成熟囊泡破裂（GVBD）后细胞周期蛋白B的表达量增加。在GV期卵母细胞中没有检测到细胞周期蛋白B合成，而在第一和第二中期卵母细胞中观察到细胞周期蛋白B合成，证实了这一事实。cyclin B/p34复合物的量<cdc2>反映了cyclin B的量，在GV期很低，在第一中期较高，然后在第二中期高得多，尽管p34的量在<cdc2>整个成熟过程中没有变化。磷酸酪氨酸的去磷酸化，阻止p34 ^<cdc2>的活性，是p34^激活所必需的<cdc2>。MAPK活性在GV期较低。在GVBD后至第二分裂中期观察到高的MAPK活性，并且未观察到MPF活性在第一极体发射时的活性降低。MAPK在成熟过程中胞内的含量无明显变化，其活性受磷酸化调节。

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.