Basic Stydy in the Cryopreservation of Unfertilized and Fertilized Eggs

自Whittingham报道小鼠受精卵的高存活率以来，哺乳动物受精卵的冷冻保存已应用于各种物种。虽然关于影响冻融小鼠胚胎存活的因素已经产生了大量的信息，但对于未受精卵的最佳冷冻和解冻方法却知之甚少。本研究的目的是探讨添加冷冻保护剂和蔗糖稀释剂对未受精卵和受精卵的影响。冷冻培养基中添加1.5M DMSO + 0.25M蔗糖，采用5步蔗糖稀释法，未受精卵存活率最高。该方法的成活率显著高于其他3种方法。冷冻培养基中添加10%甘油+ 0.25M蔗糖，采用一步蔗糖稀释法，8细胞胚胎冻融后存活率最高。所有的动物和植物都有一些酶，即所谓的清道夫，它们保护细胞免受细胞毒性活性氧的侵害。超氧化物歧化酶存在于所有需氧器官中，它能将两种超氧化物自由基（O_2^-）分解为过氧化氢和氧。采用大鼠抗Cu、Zn-SOD和Mn-SOD多克隆抗体，研究大鼠卵巢免疫定位。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，而间质腺组织中酶活性较低。在卵巢中，几乎所有的标本中，卵泡外膜层的Mn-SOD免疫过氧化物酶染色最强烈。

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.