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水和冷冻保护剂的渗透性对其低温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和水通道是否可以提高它们的水和冷冻保护剂的渗透性，以及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-3 cRNA可显著增加水和甘油的渗透性，以及对甘油基溶液玻璃化的耐受性，因此完整的卵母细胞在玻璃化后不能存活。许多注射了crna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cRNA是否也能提高它们的水渗透性和低温渗透性。在卵母细胞的情况下，注射aquqprin3 cRNA似乎增加了卵母细胞的水和冷冻保护剂的渗透性。然而，在胚胎的情况下，由于注射了水通道蛋白-7 rna的胚胎没有表现出典型的收缩-膨胀体积曲线，因此不能阐明增加的水和冷冻保护剂的渗透性。通过在卵母细胞中人工表达水/冷冻保护剂通道，可以实现卵母细胞的低温保存。水和冷冻保护剂的渗透性是一项重要的低温生物学特性，它影响细胞内结冰和冷冻保护剂的化学毒性。如果能够通过胚胎的外观推断出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。2细胞期和囊胚期小鼠胚胎细胞内结冰损伤后，解冻稀释后在等渗PB1培养基中肿胀。在冷冻保存过程中，当母细胞受到冷冻保护剂的化学毒性损伤时，培养1 h后，母细胞出现分解。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 h后未发育到4细胞期。因此，可以从胚胎在恢复和随后的培养过程中的外观推断出冷冻保存胚胎中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。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