Study of the cell cycle regulation mechanism in oocyte maturation

卵母细胞成熟过程中细胞周期调控机制的研究

基本信息

批准号：
06660350
负责人：
NAITO Kunihiko
金额：
$ 1.34万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660350/
关键词：
oocyte maturation MPF p34^<cdc2>cyclin B MAP kinase

项目摘要

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.

我成功地分析了p34^<cdc2>和细胞周期蛋白B的细胞内浓度，合成和关联状态的变化，以及整个猪卵母细胞成熟的浓度，活性及其调节MAP激酶（MAPK）的变化。首先，使用猪颗粒细胞，我建立了p34^<cdc2>和细胞周期蛋白B的西部制作方法，免疫沉淀，用于检查p34^<cdc2>和cyclin b的关联状态，以及用于检查MPF磷酸化状态的p34^<cdc2>和甲基甲基，这些状态影响了MPF phosphoration cathi-i-phopsi-phosphosposine phossine phosphosposine。使用这些方法，我接下来研究了整个成熟过程中有关猪卵母细胞的研究，发现未成熟的猪卵母细胞的细胞周期蛋白B量极少，并且在生发囊泡分解（GVBD）后的量增加，这与许多其他报道的Speceis不同。确认这一事实，在GV卵母细胞中未检测到细胞周期蛋白B的合成，并在第一和第二甲氧化酶卵母细胞中观察到。 Cyclin B/p34^<cdc2>复合物的数量反映了细胞周期蛋白B的量，在GV期间非常低，在第一中期中，p34^<cdc2>的量在整个成熟过程中没有变化，但在第一个中期中的数量很高。 p34^<cdc2>激活所必需的磷酸酪氨酸的磷酸化磷酸化。在GV阶段，MAPK活动较低。在GVBD之后观察到高的MAPK活性直到第二中期，并且在MPF活性中显示的第一极体发射时的活性降低。 MAPK的细胞内浓度在成熟过程中没有变化，并且激活受磷酸化调节。