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Analysis of the microenvironment and cryopreservation of embryos on their development

胚胎发育的微环境和冷冻保存分析

基本信息

批准号：
08671870
负责人：
TSUTSUMI Osamu
金额：
$ 1.41万
依托单位：
Tokyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671870/
关键词：
GLUT embryo glucose EGF cryopreservation activin development embnyo glucose transporter epidermal growth factor cryopreservation clevelopment implantation

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 preferende of the embryo from pyruvate to glycose during preimplantation development may be explained by increases in the activity of hexokinase and expression of glycose 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 develomental 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.

采用显微技术和高度敏感的方法，如酶循环、Micro-Western分析、逆转录聚合酶链式反应等对这些过程进行了研究。低己糖激酶活性和高活性的磷酸途径酶是未成熟卵的特征。在成熟过程中，己糖激酶和磷酸果糖激酶的活性显著增加。这些变化被用来分析表皮生长因子(EGF)和前列腺素(PG)在卵母细胞成熟中的作用。EGF通过增加颗粒细胞中PG的产生来刺激成熟。在电生理方面，卵母细胞在成熟过程中对三磷酸肌醇的敏感性增加，并形成了钙释放系统。卵母细胞和胚胎的孕酮产生通过酶循环和其他方法使用放射测量法来显示。这种由胚胎本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。小鼠从卵母细胞到囊胚的葡萄糖摄取量增加了100倍。胚胎在着床前发育过程中的底物偏好从丙酮酸向葡萄糖的转变可能是通过己糖激酶活性和葡萄糖转运蛋白GLUT1表达的增加来解释的。NADP循环检测己糖激酶活性增加20倍，而Micro-Western方法检测GLUT1表达增加10倍。RT-PCR分析Glut1的表达，表明Glut1的表达受转录水平的调控。当胚胎在体外发育时，葡萄糖摄取、己糖激酶活性和GLUT1表达的发育变化是延迟的。值得注意的是，EGF在体外刺激了这些发育变化。总之，扩大卵母细胞成熟和胚胎发育过程中的知识不仅对体外受精技术具有重要的临床意义，而且是妇科的突破。