Basic Stydy in the Cryopreservation of Unfertilized and Fertilized Eggs

自从Whittingham报道小鼠受精卵冷冻解冻后的高存活率以来，哺乳动物受精卵的冷冻保存已被应用于各种物种。虽然已经产生了大量的信息有关的因素影响冷冻解冻小鼠胚胎的存活，相对较少的是已知的最佳冷冻和解冻方法为未受精的小鼠卵子。本研究的目的是探讨添加冷冻保护剂和蔗糖稀释液对未受精和受精小鼠卵的影响。当冷冻液中含有1.5M DMSO + 0.25M蔗糖，采用蔗糖五步稀释法时，小鼠未受精卵的存活率最高。该方法的成活率明显高于其他三种方法。以10%甘油+0.25M蔗糖为冻存液，采用蔗糖一次稀释法冻存8-细胞胚胎的存活率最高。超氧化物歧化酶存在于所有需氧器官中，能将超氧自由基（O_2^-）的两个分子歧化为过氧化氢和氧气。用抗Cu、Zn-SOD和Mn-SOD的多克隆抗体研究了大鼠卵巢的免疫定位。生长卵泡、排卵卵泡和血管也表现出较高的酶活性。颗粒细胞和黄体中Cu，Zn-SOD含量较高，而间质腺组织中无酶活性。在卵巢，卵泡膜外层表现出最强烈的Mn-SOD免疫过氧化物酶染色几乎所有的标本检查。

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.