Analyses for the regulation mechanisms of protein synthesis and degradation which is aimed for the improvement of the availability in live stock oocyte for the developmental technology

我分析了哺乳动物卵母细胞/早期胚胎正常发育过程中蛋白质合成和降解的分子调控机制。特别是，以成熟的猪卵母细胞为例，我专注于极光A作为蛋白质合成的调节剂和cdc 20和cdh 1，这是后期促进复合物（APC：一种泛素连接酶，在泛素/蛋白酶体系统中工作）的激活剂，作为蛋白质降解的调节剂。2005年，我成功克隆了猪的Aurora A基因，并将其登记在基因库中，猪卵母细胞中Aurora A蛋白的表达水平比体细胞中高出约100倍，表明其在卵母细胞成熟过程中的重要作用。将Aurora A组成型活性突变体的mRNA注射到猪未成熟卵母细胞中，加速了细胞周期蛋白B的合成和随后的减数分裂恢复。相反，注射AuroraA反义RNA显著抑制了减数分裂的恢复， ...更多信息 猪卵母细胞在Aurora A耗竭的条件下进行体外受精。本研究还将cdc 20和cdh 1的反义RNA分别注射到猪未成熟卵母细胞中，观察到cdc 20和cdh 1在猪未成熟卵母细胞中的表达。这些实验表明，抑制cdc 20的表达诱导猪卵母细胞在第一次减数分裂中期的停滞。在这些卵母细胞中，细胞周期蛋白B的降解被阻止，MPF活性维持在高水平。与此相反，抑制cdh 1的表达增加了GV期细胞周期蛋白B的积累，加速了减数分裂的恢复，表明在猪卵母细胞减数分裂停滞期间cdh 1依赖的细胞周期蛋白B降解。这些结果表明cdh 1和cdc 20分别通过降解cyclin B、抑制减数分裂提前恢复和第一次减数分裂/第二次减数分裂转换发挥重要作用。此外，我发现cdh 1的过表达抑制了减数分裂的恢复，cdc 20可能是cdh 1/APC的底物。本研究扩展了我们对哺乳动物卵母细胞蛋白质合成和降解调控的认识。少

I analyzed the molecular mechanisms for regulating the protein synthesis and degradation during normal development in mammalian oocytes/early embryos. Especially, using maturing porcine oocytes as examples, I focused on Aurora A as the regulator of protein synthesis and on cdc20 and cdh1, which were the activator of anaphase promoting complexes (APC: a ubiquitine ligase working in a ubiquitine/proteasome system), as the regulator of protein degradation. During 2005, I succeeded for the cloning of these genes in the pig and registered them in a gene bank.The Aurora A protein level in porcine oocytes was about 100 times higher than that in somatic cells, indicating the importance of its function in the oocyte maturation. The injection of the mRNA for a constitutive active mutant of Aurora A into porcine immature oocytes accelerated the synthesis of cyclin B and subsequent meiotic resumption. Conversely, the injection of the antisense RNA of Aurora A significantly inhibited the meiotic re … More sumption under the condition of Aurora A depletion in porcine oocytes. These are the first results showing the importance of Aurora A on the protein synthesis during porcine oocyte meiosis.I also injected the antisense RNAs of cdc20 and cdh1 into porcine immature oocytes. These experiments revealed that the inhibition of cdc20 expression induced the arrest at the first meiotic metaphase in porcine oocytes. In these oocytes, the degradation of cyclin B was prevented and MPF activity was maintained at a high level. In contrast, the inhibition of cdh1 expression increased the cyclin B accumulation at the GV stage and accelerated the meiotic resumption, indicating the cdh1-dependent cyclin B degradation during meiotic arrest in porcine oocytes. These results suggest the important functions of cdh1 and cdc20 through cyclin B degradation, the inhibition of premature meiotic resumption and the first meiosis/the second meiosis transition, respectively. Furthermore, I found that the cdh1 overexpression inhibited the meiotic resumption and cdc20 might be a substrate of cdh1/APC. The present study extended our understandings in the regulation of protein synthesis and degradation in mammalian oocytes. Less