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Basic Stydy in the Cryopreservation of Unfertilized and Fertilized Eggs

未受精卵和受精卵冷冻保存的基础研究

基本信息

批准号：
01570912
负责人：
ISHIKAWA Mutsuo
金额：
$ 0.58万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570912/
关键词：
Cryopreservation Fertilized egg Unfertilized egg Mn-superoxide dismutase Ovary Mn-SOD

项目摘要

The cryopreservation of fertilized mammalian eggs has been applied to various species since Whittingham reported the high survival rate of frozen-thawed fertilized mouse eggs. While a great deal of information has been generated regarding factors affecting survival of frozen-thawed mouse embryos, relatively little is known about optimal freezing and thawing methods for unfertilized mouse eggs. The aims of this study were to investigate the effect of addition of cryoprotectant and sucrose dilution on unfertilized and fertilized mouse eggs. The highest survival rate of unfertilized mouse eggs was obtained when the freezing medium contained 1.5M DMSO + 0.25M sucrose and 5-step sucrose dilution method was used. The survival rate by this method was significantly higher than the other three methods. The highest survival rate of frozen-thawed 8-cell embryos was obtained when the freezing medium contained 10% glycerol + 0.25M sucrose and one-step sucrose dilution method was used.All animals and plants have some enzymes, so -called scavengers, which protect cell against cytotoxic active oxygen. Superoxide dismutase has been found in all aerobic organs and is capable of dismuting the two molecules of the superoxide radical (O_2^-) to hydrogen peroxide and oxygen. Immunolocalization in rat ovary was investigated using rat polyclonal antibodies against Cu, Zn-SOD and Mn-SOD. Growing follicles, ovulated follicles and blood vessels also exhibited high this enzymic activity. The granulosa cells and corpora lutea showed higher amounts of Cu, Zn-SOD while the interstitial gland tissue was devoid of enzymic activity. In the ovary, theca external layer of grafian follicle demonstrated the most intense immunoperoxidase staining for Mn-SOD in nearly all specimens examined.

自从Whittingham报道冷冻解冻小鼠受精卵的存活率很高以来，哺乳动物受精卵的冷冻保存已经应用于各种物种。虽然已经产生了大量关于影响冷冻解冻小鼠胚胎存活率的因素的信息，但对未受精小鼠卵子的最佳冷冻和解冻方法知之甚少。本研究旨在探讨添加冷冻保护剂和蔗糖稀释液对小鼠未受精卵和受精卵的影响。在含有1.5M二甲基亚砜+0.25M蔗糖的冷冻液中，采用5步蔗糖稀释法，可获得最高的未受精卵存活率。该方法的成活率显著高于其他三种方法。在含有10%甘油+0.25M蔗糖的冷冻液中，采用蔗糖一步稀释法，冻融8-细胞胚胎的存活率最高。所有动植物都有一些酶，即所谓的清除剂，可以保护细胞免受细胞毒性的活性氧的伤害。超氧化物歧化酶存在于所有有氧器官中，它能使超氧自由基(O_2^-)的两个分子歧化为过氧化氢和氧。用大鼠抗铜、锌-超氧化物歧化酶和锰-超氧化物歧化酶多克隆抗体研究了铜锌超氧化物歧化酶在大鼠卵巢的免疫定位。生长中的卵泡、排卵的卵泡和血管也表现出很高的这种酶活性。颗粒细胞和黄体组织中的铜、锌-超氧化物歧化酶含量较高，而间质腺组织中无酶活性。在卵巢中，几乎所有被检测的标本中，卵泡膜外层对Mn-SOD的免疫过氧化物酶染色最强。