Hirai T, Yamashita M, Yoshikuni M, Lou Y H, Nagahama Y
Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Japan.
Mol Reprod Dev. 1992 Oct;33(2):131-40. doi: 10.1002/mrd.1080330204.
Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [35S]methionine, indicating its de novo synthesis. Introduction of E. coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17 alpha, 20 beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.
在卵泡细胞分泌的成熟诱导激素（MIH）的影响下，卵母细胞成熟最终由成熟促进因子（MPF）触发，MPF由裂殖酵母的cdc2 +基因产物（p34cdc2）的同源物和细胞周期蛋白B组成。从成熟金鱼卵母细胞构建的cDNA文库中分离出两种细胞周期蛋白B克隆。序列比较显示这两个克隆高度同源（95%），并且发现它们与非洲爪蟾细胞周期蛋白B1相似。使用针对大肠杆菌产生的金鱼细胞周期蛋白B和p34cdc2的PSTAIR序列的单克隆抗体，我们检测了在17α，20β - 二羟基 - 4 - 孕烯 - 3 - 酮（17α，20β - DP）诱导的金鱼卵母细胞体外成熟过程中细胞周期蛋白B和p34cdc2蛋白的水平，17α，20β - DP是鱼类中的一种天然MIH。在未成熟卵母细胞提取物中发现了蛋白p34cdc2，并且在卵母细胞成熟过程中没有明显变化。在未成熟卵母细胞提取物中未检测到细胞周期蛋白B，当卵母细胞发生生发泡破裂时出现。在卵母细胞成熟过程中出现的细胞周期蛋白B用[35S]甲硫氨酸标记，表明其从头合成。将大肠杆菌产生的细胞周期蛋白B引入未成熟卵母细胞提取物中诱导了p34cdc2（MPF）的激活。虽然不完全排除未成熟金鱼卵母细胞含有不溶性细胞周期蛋白B的可能性，但这些结果强烈表明17α，20β - DP诱导卵母细胞合成细胞周期蛋白B，进而激活预先存在的p34cdc2，形成活性MPF。
