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The study for cryopreservation of vertebrate oocytes and embryos by artificially expression of gene products involved in cryo-tolerance

通过人工表达低温耐受基因产物来冷冻保存脊椎动物卵母细胞和胚胎的研究

基本信息

批准号：
14560235
负责人：
EDASHIGE Keisuke
金额：
$ 2.18万
依托单位：
Kochi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Water-and cryoprotectant-permeability of the plasma membrane of oocytes and embryos plays vital roles on their tolerance to cryopreservation. I studied whether artificial expression of aquaporins, water channels, in mouse oocytes and zebrafish oocytes and embryos can improve water-and cryoprotectant-permeability of them and whether the expression can improve the tolerance of them to cry preservation. Injection of aquaporin-3 cRNA in mouse oocytes significantly increased water-and glycerol permeability, and the tolerance to vitrification with glycerol-based solutions, with which intact oocytes did not survive after vitrification. Many of the cRNA-injected and cryopreserved oocytes were fertilized by in vitro fertilization. These results indicate that artificial expression of water/cryoprotectant channels in the cell can improve the tolerance to cryopreservation. I also studied whether injection of aquaporin cRNA into zebrafish oocytes and embryos can also improve their water-and cryopro … More tectant-permeability. In the case of oocytes, injection of aquqprin-3 cRNA appeared to increase water-and cryoprotectant permeability of the oocytes. In the case of embiyos, however, increased water-and cryoprotectant-permeability could not be elucidated because the aquaporin-7 cRNA-injected embryos did not show typical shrink-swell volume curves. Fish oocytes could be cryopreserved by artificial expression of water/cryoprotectant channels in them.Water-and cryoprotectant-permeability is an important cryobiological property that influences on cryoinjuries caused by intracellular ice-formation and chemical toxicity of cryoprotectants. If it were possible to deduce which type of injury had occurred during cryopreservation of embryos by their appearance, it would help to optimize cryopreservation protocols. When mouse embryos at the 2-cell stage and the blastocyste stage were injured by intracellular ice formation, they swelled in isotonic PB1 medium just after being thawed and diluted. When blastocytes were injured by chemical toxicity of cryoprotectants during cryopreservation, they showed decompaction after 1 h of culture. In the case of 2-cell embryos, they looked normal after being thawed and diluted. However, they did not develop to the 4-cell stage after 24 h of culture. Thus, it is possible to deduce which type of injury had occurred in cryopreserved embryos from their appearance at recovery and during subsequent culture. This may help to improve cryopreservation protocols for embryos of many species. Less

卵母细胞和胚胎质膜的水和冷冻保护剂的通透性对其耐受性起着至关重要的作用。我研究了在小鼠卵母细胞和斑马鱼卵母细胞和胚胎中人工表达水通道蛋白是否可以提高它们的水分和冷冻保护剂的渗透性，以及这种表达是否可以提高它们对哭泣保存的耐受性。在小鼠卵母细胞中注射水通道蛋白-3cRNA显著提高了水和甘油的渗透性，以及对以甘油为基础的溶液玻璃化冷冻的耐受性，在玻璃化冷冻后，完整的卵母细胞无法存活。许多注射CRNA的卵母细胞和冷冻保存的卵母细胞通过体外受精受精。这些结果表明，在细胞中人工表达水/低温保护剂通道可以提高细胞对低温保存的耐受性。我还研究了在斑马鱼卵母细胞和胚胎中注射水通道蛋白cna是否也能改善它们的水和低温…。更强的保护剂渗透性。在卵母细胞中，注射Aquqprin-3cna似乎增加了卵母细胞的水和冷冻保护剂的渗透率。然而，在胚胎的情况下，水和冷冻保护剂渗透性的增加无法解释，因为注射水通道蛋白-7cna的胚胎没有表现出典型的收缩-膨胀体积曲线。鱼卵母细胞可以通过人工表达水/低温保护剂通道进行冷冻保存，水和低温保护剂的通透性是影响细胞内结冰和低温保护剂化学毒性引起的冷冻损伤的重要低温生物学特性。如果能够根据胚胎的外观推断胚胎在冷冻保存过程中发生了哪种类型的损伤，将有助于优化冷冻保存方案。小鼠2-细胞期和囊胚期胚胎在细胞内结冰损伤后，解冻稀释后即在等渗PB1培养液中肿胀。当胚泡细胞在冷冻保存过程中受到低温保护剂的化学毒性损伤时，培养1h后，胚泡细胞出现解体现象。在2-细胞胚胎的情况下，它们在解冻和稀释后看起来正常。但培养24小时后，仍未发育到4细胞期。因此，可以从复苏时和随后的培养过程中的外观来推断冷冻胚胎发生了哪种类型的损伤。这可能有助于改进许多物种胚胎的冷冻保存方案。较少