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

卵母细胞和胚胎质膜的透水性和透冻性对卵母细胞和胚胎的耐冻性起着重要作用。本论文研究了水通道蛋白（aquaporins）在小鼠卵母细胞和斑马鱼卵母细胞及胚胎中的人工表达是否能提高卵母细胞和胚胎的透水性和冷冻保护性，以及表达水通道蛋白是否能提高卵母细胞和胚胎对冷冻保存的耐受性。注射水通道蛋白-3 cRNA的小鼠卵母细胞显着增加水和甘油的渗透性，并与甘油为基础的解决方案，其中完整的卵母细胞玻璃化后不能存活的玻璃化冷冻的耐受性。许多cRNA注射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/冷冻保护剂通道可以提高对冷冻保存的耐受性。我还研究了将水通道蛋白cRNA注射到斑马鱼卵母细胞和胚胎中是否也能改善它们的水和冷冻性能。 ...更多信息 构造渗透性在卵母细胞的情况下，注射aquqprin-3 cRNA似乎增加水和冷冻保护剂的卵母细胞的渗透性。然而，在embiyos的情况下，增加的水和冷冻保护剂的渗透性不能阐明，因为水通道蛋白-7 cRNA注射的胚胎没有显示出典型的收缩-膨胀体积曲线。鱼类卵母细胞可通过人工表达水/冷冻保护剂通道进行冷冻保存，水和冷冻保护剂的通透性是影响细胞内结冰和冷冻保护剂化学毒性所致冷冻损伤的重要低温生物学特性。如果能够通过胚胎的外观来推断在冷冻保存过程中发生了哪种类型的损伤，这将有助于优化冷冻保存方案。当2-细胞期和囊胚期的小鼠胚胎因细胞内结冰而受到损伤时，它们在解冻和稀释后立即在等渗PB 1培养基中膨胀。当胚胎细胞在冷冻保存过程中受到冷冻保护剂的化学毒性损伤时，它们在培养1h后表现出去致密化。在2细胞胚胎的情况下，它们在解冻和稀释后看起来正常。然而，在培养24小时后，它们没有发育到4-细胞阶段。因此，可以从复苏时和随后的培养过程中冷冻保存的胚胎的外观推断出其发生了哪种类型的损伤。这可能有助于改善许多物种胚胎的冷冻保存方案。少

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