Development of an efficient system for cryopreservation of oocytes and embryos by use of ultrarapid vitrification

利用超快速玻璃化冷冻技术开发有效的卵母细胞和胚胎冷冻保存系统

基本信息

批准号：
12460126
负责人：
KASAI Magosaburo
金额：
$ 6.78万
依托单位：
Kochi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460126/
关键词：
embryo oocyte cryopreservation vitrification ovary channel cryoprotectant 耐凍剤凍結保存透過性水チャンネル

项目摘要

The present study aimed to examine the efficacy of ultrarapid vitrification and to develop new strategies for the cryopreservation of mammalian oocytes and embryos, based on the understanding of fundamental cryobiology.Ultrarapid vitrification using the cryoloop was effective for human blastocysts, in which conventional vitrification does not yield consistent results. For mouse oocytes, the post-warming survival after vitrification using cryoloops was not different from that after vitrification using conventional straws. For rat embryos, in which the efficacy of vitrification has not reached a level for practical use, conventional straw vitrification using EFS solution was quite effective, on the condition that embryos at suitable stages of development are used. As a tool to identify the mechanism of injury, we specified morphological characteristics of embryos that is typical to specific types of injuries, such as intracellular ice formation and cryoprotectant toxicity. As a potential … More strategy for cryopreservation, we tried to increase the permeability of the cell membrane artificially. We injected cRNA of a certain type of aquaporins, a water channel that can transport not only water but also cryoprotectants, into mouse oocytes. In oocytes injected with cRNA of aquaporin 3, both water permeability and glycerol permeability increased significantly. These oocytes survived after vitrification in a solution based on glycerol, which scarcely permeates into intact oocytes, whereas none of oocytes injected with water survived after vitrification in the solution. The oocytes in which the plasma membrane was temporarily 'altered' retained the ability to be fertilized and to develop to term.The results obtained in this study would be valuable for the cryopreservation of embryos and oocytes in domestic animals, laboratory animals and humans. In addition, a new strategy found in this study would have potential advantage for the cryopreservation of various genetic resources that need to be preserved. Less

本研究旨在基于对基本冷冻生物学的理解，检验超快速玻璃化的功效，并制定哺乳动物卵母细胞和胚胎冷冻保存的新策略。使用cryoloop的超快速玻璃化对人类囊胚有效，而传统玻璃化则无效。对于小鼠卵母细胞，使用冷冻环玻璃化冷冻后的升温后存活率与使用传统吸管玻璃化冷冻后的存活率没有差异。对于大鼠胚胎，玻璃化冷冻的效果尚未达到实用水平，使用 EFS 溶液的传统秸秆玻璃化冷冻是相当有效的，条件是使用处于适当发育阶段的胚胎作为鉴定损伤机制的工具。，我们指定了特定类型损伤的典型胚胎形态特征，例如细胞内冰形成和冷冻保护剂毒性。作为一种潜在的冷冻保存策略，我们尝试人为地增加细胞膜的渗透性。将某种类型的水通道蛋白的cRNA（一种不仅可以运输水还可以运输冷冻保护剂的水通道）注射到小鼠卵母细胞中，在注射了水通道蛋白3的cRNA的卵母细胞中，水渗透性和甘油渗透性均显着增加，这些卵母细胞在玻璃化冷冻后存活。一种基于甘油的溶液，几乎不能渗透到完整的卵母细胞中，而注射了水的卵母细胞在该溶液中玻璃化后没有一个能存活。其中质膜被暂时“改变”，保留了受精和发育至足月的能力。这项研究获得的结果对于家畜、实验动物和人类的胚胎和卵母细胞的冷冻保存是有价值的。这项研究中发现的一种新策略对于冷冻保存需要保存的各种遗传资源具有潜在的优势。