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Molecular regulation of gene expression and development of mammalian embryos

哺乳动物胚胎基因表达和发育的分子调控

基本信息

批准号：
08406019
负责人：
YAMADA Masayasu
金额：
$ 14.59万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

In vitro development of mammalian embryos during preimplantation period from the 1-cell to the blastocyst stages is known to be arrested at a stage characteristics for the species when zygotic gene activation (ZGA) occurs. This developmental arrest might, therefore, reflect a ZGA that is not well-regulated under in vitro culture conditions. Recently, involvement of reactive oxygen species (ROS) in this developmental arrest has been noted. Lowering oxygen tension and the addition of anti-oxidants, such as superoxide dismutase or thioredoxin, to the culture medium has been shown to exert a beneficial effect on in vitro development of embryos. Thus, it is much of interest to define the mechanisms by which early embryonic development is regulated by ROS.Recently, it has become clear that ROS have additional functions besides oxidative stress as second messengers for regulation of cell growth and differentiation. In the present studies, attempts were made to define the redox regulation in i … More n vitro development of early mouse embryos. As the results, it was shown that the mRNA and protein of nuclear factor kappa B (NF-kB), a transcription factor that controls the expression of a number of genes under cellular redox potentials, are expressed in mouseembryos during preimplantation development, and NF-kB is stage-specifically activated at early 1-cell stage in mouse embryos, and moreover that the activation of NF-kB is essential for the development of mouse embryos beyond the 2-cell stage. During the course of experiments on the production of antibodies that recognize specifically activated form of NF-kB, we found NF-kB-related 100 kDa protein. Cloning experiments of the protein revealed that it is a novel pre-mRNA splicing factor in mammals, and immunofluorescense experiments revealed that it is also stage -specifically accumulated in the nuclei of late 1-cell mouse embryos. In the second experiments, we found that nitric oxide, a free radical molecule acts as a regulator of development, especially transition of 2-cell embryos to the 4-cell stage and formation of blastocysts. Thus, it is indicated that the development and gene expression of embryos during preimplantation stages, especially at periods of ZGA and blastocyst formation are regulated under redox controls. Less

哺乳动物胚胎在着床前从1-细胞到囊胚期的体外发育被认为是在合子基因激活(ZGA)发生时受阻的。因此，这种发育停滞可能反映了ZGA在体外培养条件下不受很好的调控。最近，人们注意到活性氧物种(ROS)参与了这种发育停滞。降低氧分压和在培养液中添加抗氧化剂，如超氧化物歧化酶或硫氧还蛋白，已被证明对胚胎的体外发育起到了有利的作用。因此，确定ROS调控早期胚胎发育的机制是非常有意义的。最近，人们已经清楚地看到，ROS除了作为第二信使调节细胞生长和分化外，还具有额外的功能。在本研究中，试图定义I-…中的氧化还原调节更多的小鼠早期胚胎的体外发育。结果表明，在小鼠胚胎着床前发育过程中，核因子-kB的转录因子--核因子-kB在小鼠胚胎中表达，核转录因子-kB在细胞氧化还原电位下控制多个基因的表达，并且核因子-kB在小鼠胚胎的早期1-细胞阶段被阶段特异性激活，并且核因子-kB的激活对小鼠胚胎发育至2-细胞阶段以外的发育是必不可少的。在生产识别核因子-kB特异性激活形式的抗体的实验过程中，我们发现了与核因子-kB相关的100 kDa蛋白。克隆实验表明，该蛋白是一种新的哺乳动物前mRNA剪接因子，免疫荧光实验表明，它也在小鼠胚胎晚期1-细胞的细胞核中阶段性积累。在第二个实验中，我们发现一氧化氮是一种自由基分子，它对胚胎发育，特别是2-细胞胚胎向4-细胞阶段的过渡和囊胚的形成具有调节作用。因此，这表明胚胎在着床前，特别是ZGA和囊胚形成阶段的发育和基因表达受到氧化还原调控。较少