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Biochemical and endocrinological analysis of oocytes and embryos

卵母细胞和胚胎的生化和内分泌分析

基本信息

批准号：
03670771
负责人：
TSUTSUMI Osamu
金额：
$ 1.41万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

Microtechniques and highly sensitive methods such as enzymatic cycling, micro-Western analysis, reverse transcription polymerase chain reaction and so on were employed to study these processes. Low hexokinase activity and high activities of enzymes in the phosphate pathway were characteristic of immature oocytes. During maturation, the activities of hexokinase and phosphofructokinase increased significantly. These changes were used to analyze involvement of epidermal growth factor (EGF) and prostaglandins (PG) in oocyte maturation. EGF is shown to stimulate maturation by increasing PG production in granulosa cells. Electro-physiologically, the sensitivity of oocyte to inositol triphosphate increased and Ca^<2+> release system developed during maturation. Progesterone production of oocyte and embryos are shown by enzymatic cycling and other methods using radiometry. This hormone produced by embryos themselves may play a role in embryonic development in intracrine fashion. There is 100-fold increase in glucose uptake from oocyte to blastocyst in mice. A switch in substrate preference of the embryo from pyruvate to glucose during preimplantation development may be explained by increases in the activity of hexokinase and expression of glucose transporter, GLUT1. Hexokinase activities determined by NADP cycling increased 20-fold while expression of GLUT1 assessed by micro-Western method 10-fold. GLUT1 expression was also analyzed by RT-PCR, which indicated that the expression is regulated at transcription level. There is a delay in the developmental changes in glucose uptake, hexokinase activity and GLUT1 expression when the embryos are developed in vitro. It is of interest to note that EGF stimulates these developmental changes in vitro. In conclusion, expanding knowledge in the process of oocyte maturation and embryonic development may serve not only as significant clinical implications for in vitro fertilization techniques but as breakthroughs in gynecology.

利用酶循环、微量Western分析、逆转录聚合酶链反应等微量技术和高灵敏度的方法对这些过程进行了研究。低己糖激酶活性和高活性的酶在磷酸途径是未成熟卵母细胞的特征。成熟过程中，己糖激酶和磷酸果糖激酶活性显著升高。这些变化被用来分析表皮生长因子（EGF）和前列腺素（PG）在卵母细胞成熟的参与。EGF通过增加颗粒细胞中PG的产生来刺激成熟。在电生理上，卵母细胞成熟过程中对三磷酸肌醇的敏感性增加，Ca^2+释放系统发育。通过酶循环和其他使用放射测量的方法显示卵母细胞和胚胎的前体生产。这种由胚胎自身产生的激素可能以内分泌的方式在胚胎发育中发挥作用。小鼠从卵母细胞到胚泡的葡萄糖摄取增加了100倍。胚胎在着床前发育过程中对丙酮酸盐和葡萄糖的底物选择性的改变可以通过增加己糖激酶的活性和葡萄糖转运蛋白GLUT 1的表达来解释。通过NADP循环测定的己糖激酶活性增加20倍，而通过微量蛋白质方法评估的GLUT 1表达增加10倍。RT-PCR分析GLUT 1的表达，结果表明GLUT 1的表达在转录水平上受到调控。胚胎在体外发育时，葡萄糖摄取、己糖激酶活性和GLUT 1表达的发育变化延迟。值得注意的是，EGF在体外刺激这些发育变化。总之，扩大卵母细胞成熟和胚胎发育过程中的知识，不仅可以作为体外受精技术的重要临床意义，但在妇科学的突破。