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A Study on the Molecular mechanisms of human embryonic development. Useful as markes of

人类胚胎发育的分子机制研究。

基本信息

批准号：
07557256
负责人：
TAKETANI Yuji
金额：
$ 3.46万
依托单位：
UNIVERSITY of TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

We developed some methods that are very useful to investigate the mechanism of intrinsic activities in mammalian embryos : microculture technique, micro Western blot analysis, quantitative RT-PCR analysis and enzymatic cycling technique. By using these methods, we obtained a result that glucose incorporation is increasing during the development of preimplantation embryos, especially in the period when the embryos develop from two-cell stage to eight-cell stage. This phenomenon is based on the expression of glucose transporter (GLUT) expression, and it was also revealed that increasing of hexokinase activity during embryonic development has a great deal to do with this phenomenon.We also obseived the specificity of the embryos which developed in vitro, and the influence of some biochemical materials such as growth factors to these embryos. Delayd expression of hexokinase and GLUT in vitro causes inappropriate glucose incorporation during preimplantation embryo development which were largely corrected by epidermal growth factor (EGF). The implantation rate in recipient mice of in vitro grown embryos is lower than in vivo grown ones, and treatment of embryos in vitro with EGF effectively prevents a decrease in implantation rate. In addition, we examined the effects of cryopreservation to the embryos. Freezing-thawing procedure of 2-cell embryos decreases not only the implantation rates to the recipients, but also decreases glucose incorporation of the blastocyst due to decreased expression of GLUT.This suggests that cryopreservation may have later consequences on the functional development of embryos.From these facts we conclude that glucose incorporation in embryos based on the expression of hexokinase and GLUT can be a measure of embryo viability.

我们开发了一些对于研究哺乳动物胚胎内在活性机制非常有用的方法：微培养技术、微Western印迹分析、定量RT-PCR分析和酶循环技术。通过使用这些方法，我们得到的结果是，在植入前胚胎的发育过程中，特别是在胚胎从二细胞阶段发育到八细胞阶段期间，葡萄糖掺入不断增加。这种现象是基于葡萄糖转运蛋白（GLUT）的表达，同时也揭示了胚胎发育过程中己糖激酶活性的增加与这种现象有很大关系。我们还观察了体外发育的胚胎的特异性，以及生长因子等一些生化物质对这些胚胎的影响。体外己糖激酶和 GLUT 的延迟表达会导致植入前胚胎发育过程中不适当的葡萄糖掺入，这在很大程度上可以通过表皮生长因子（EGF）来纠正。体外培养的胚胎在受体小鼠体内的着床率低于体内培养的胚胎，而用EGF对体外胚胎进行处理可有效防止着床率的下降。此外，我们还检查了冷冻保存对胚胎的影响。 2 细胞胚胎的冻融过程不仅降低了受体的着床率，而且由于 GLUT 表达减少，也减少了囊胚的葡萄糖掺入。这表明冷冻保存可能对胚胎的功能发育产生后续影响。从这些事实我们得出结论，基于己糖激酶和 GLUT 表达的胚胎中葡萄糖掺入可以作为衡量胚胎活力。