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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.

采用酶循环、微Western分析、逆转录聚合酶链反应等微技术和高灵敏度方法来研究这些过程。低己糖激酶活性和磷酸途径中酶的高活性是未成熟卵母细胞的特征。在成熟过程中，己糖激酶和磷酸果糖激酶的活性显着增加。这些变化用于分析表皮生长因子 (EGF) 和前列腺素 (PG) 在卵母细胞成熟中的参与情况。EGF 通过增加颗粒细胞中 PG 的产生来刺激卵母细胞成熟。电生理学方面，成熟过程中卵母细胞对肌醇三磷酸的敏感性增加，Ca 2+ 释放系统发育。卵母细胞和胚胎的黄体酮产生通过酶循环和其他使用放射测量的方法来显示。这种由胚胎本身产生的激素可能以内分泌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增加了 100 倍。在植入前发育过程中，胚胎的底物偏好从丙酮酸转变为葡萄糖，这可以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。通过 NADP 循环测定的己糖激酶活性增加了 20 倍，而通过 micro-Western 方法评估的 GLUT1 表达增加了 10 倍。 RT-PCR分析GLUT1的表达情况，表明GLUT1的表达在转录水平受到调控。当胚胎在体外发育时，葡萄糖摄取、己糖激酶活性和 GLUT1 表达的发育变化会出现延迟。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的知识不仅可能对体外受精技术具有重要的临床意义，而且可能成为妇科的突破。