Molecular Mechanisms of the Inititaiton of Protein Translation during Oocyte Maturation

本研究旨在探讨卵母细胞成熟过程中细胞周期蛋白β mRNA翻译启动的分子机制，特别涉及以下三个主题。1)细胞周期蛋白β翻译在卵母细胞成熟过程中的意义我们之前的研究发现，细胞周期蛋白β mRNA翻译是启动金鱼卵母细胞成熟的必要条件，这一发现在其他物种中也得到了证实，包括除金鱼以外的鱼类和除非洲爪蟾以外的两栖动物（青蛙、蟾蜍和蝾螈），这表明这种机制在低等脊椎动物中普遍采用。2)细胞周期蛋白β mrna结合蛋白的鉴定及激素对翻译的影响。用生物素标记的RNA亲和层析纯化细胞周期蛋白β mrna结合蛋白，由于产率低，未能成功。为了研究激素刺激对翻译控制的影响，我们将细胞周期蛋白β mRNA注射到经激素处理的卵母细胞中。我们发现，注射周期蛋白β mRNA诱导的卵母细胞成熟进程比激素诱导的早，但比注射周期蛋白β诱导的晚。这些结果表明激素处理促进了细胞周期蛋白β蛋白的合成。3)已知蛋白参与细胞周期蛋白β mRNA翻译起始为了研究在其他系统中已被认为是翻译调节因子的蛋白在卵母细胞成熟过程中的功能，我们分离了cDNA克隆并产生了针对其产物的抗体。这些包括细胞质聚腺苷化元件结合蛋白（CPEB）、Nanos、Pumilio、聚(A)聚合酶、聚(A)结合蛋白、Y-box蛋白。我们发现Y-box蛋白和Pumilio蛋白是细胞周期蛋白B mrna结合蛋白，这表明这些蛋白参与了卵母细胞成熟过程中的翻译控制。

This study was undertaken to investigate molecular mechanisms of the initiation of cyclin BETA mRNA translation during oocyte maturation, with special reference to the following three subjects.1) Significance of cyclin BETA translation during oocyte maturationOur previous finding that cyclin BETA mRNA translation is necessary for initiating goldfish oocyte maturation has been also confirmed for other species, including fishes other than goldfish and amphibians (frog, toad and newt) except for Xenopus laevis, suggesting the general adoption of this mechanism in lower vertebrates.2) Identification of cyclin BETA mRNA-binding proteins and the effect of hormone on translation.An attempt to identify cyclin BETA mRNA-binding protein purified with a biotin-labeled RNA affinity chromatography was unsuccessful because of the poor yield. To examine the effect of hormonal stimulation on the translational control, we injected cyclin BETA mRNA into honnone-treated oocytes. We found that the progression of oocyte maturation induced by the injection of cyclin BETA mRNA occurred earlier than that induced by the hormone, but later than that induced by the injection cyclin BETA protein. These results indicate that the hormonal treatment enhances the synthesis of cyclin BETA protein from the injected mRNA.3) Involvement of known proteins in cyclin BETA mRNA translational initiationTo investigate the functions during oocyte maturation of proteins that have been already suggested to be translational regulators in other systems, we have isolated cDNA clones and produced antibodies against their products. Those include cytoplasmic polyadenylation element-binding-protein (CPEB), Nanos, Pumilio, poly(A) polymerase, poly(A) binding proteins, Y-box proteins. We have shown that Y-box proteins and Pumilio are a cyclin B mRNA-binding protein, suggesting the involvement of these proteins in the translational control during oocyte maturation.