Katsu Y, Yamashita M, Kajiura H, Nagahama Y
Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki, Japan.
Dev Biol. 1993 Nov;160(1):99-107. doi: 10.1006/dbio.1993.1289.
We examined the changes that occurred in the two components of maturation-promoting factor (MPF), cdc2 kinase and cyclin B, during oocyte maturation in goldfish, using monoclonal antibodies against the C-terminal sequence of goldfish cdc2 kinase and Escherichia coli-produced full-length goldfish cyclin B. Immature oocytes contained a 35-kDa inactive cdc2 kinase. In addition to the 35-kDa form, a 34-kDa active cdc2 kinase was detected in oocytes undergoing germinal vesicle breakdown (GVBD). Cyclin B was absent in immature oocytes and appeared just before GVBD, coinciding exactly with the appearance of the 34-kDa active cdc2 kinase. Precipitation with p13suc1 beads and anticyclin B antibody revealed that cyclin B formed a complex with cdc2 kinase as soon as it appeared. MPF activation was induced by 1 ng cyclin B after introduction into immature oocytes or oocyte extracts. This corresponds to the amount of cyclin B found in mature oocytes (the concentration in the oocyte is 2 micrograms/ml). These results suggest that MPF activation in fish oocytes is induced by complex formation with preexisting cdc2 kinase and newly synthesized cyclin B during oocyte maturation, a situation differing from that in Xenopus and starfish, in which the cdc2 kinase-cyclin B complex is already present in immature oocytes. Unlike that in Xenopus, an inhibition of protein synthesis in unfertilized mature goldfish oocytes caused a decrease in the cdc2 kinase activity/cyclin B protein level and led to a progression from meiotic metaphase to meiotic anaphase. This result indicates that the mechanisms of maintaining MPF activity in mature goldfish oocytes differ from those in Xenopus.
我们使用针对金鱼cdc2激酶C末端序列的单克隆抗体和大肠杆菌产生的全长金鱼细胞周期蛋白B，研究了金鱼卵母细胞成熟过程中成熟促进因子（MPF）的两个组成部分cdc2激酶和细胞周期蛋白B的变化。未成熟卵母细胞含有一种35 kDa的无活性cdc2激酶。除了35 kDa的形式外，在经历生发泡破裂（GVBD）的卵母细胞中还检测到一种34 kDa的活性cdc2激酶。细胞周期蛋白B在未成熟卵母细胞中不存在，在GVBD之前出现，与34 kDa活性cdc2激酶的出现恰好同时发生。用p13suc1珠子和抗细胞周期蛋白B抗体沉淀表明，细胞周期蛋白B一出现就与cdc2激酶形成复合物。将1 ng细胞周期蛋白B引入未成熟卵母细胞或卵母细胞提取物后可诱导MPF激活。这与成熟卵母细胞中发现的细胞周期蛋白B的量相对应（卵母细胞中的浓度为2微克/毫升）。这些结果表明，鱼类卵母细胞中的MPF激活是在卵母细胞成熟过程中由与预先存在的cdc2激酶和新合成的细胞周期蛋白B形成复合物诱导的，这种情况不同于非洲爪蟾和海星，在它们的未成熟卵母细胞中已经存在cdc2激酶-细胞周期蛋白B复合物。与非洲爪蟾不同，未受精的成熟金鱼卵母细胞中蛋白质合成的抑制导致cdc2激酶活性/细胞周期蛋白B蛋白水平下降，并导致从减数分裂中期向减数分裂后期的进展。这一结果表明，成熟金鱼卵母细胞中维持MPF活性的机制与非洲爪蟾不同。
