Cryopreservation of oocytes and embryos by inducing the expression of cryoprotectant channels.

我们试图开发一种玻璃化冷冻方法，通过这种方法可以用一种方法冷冻不同阶段的小鼠卵母细胞和胚胎。首先，我们研究了小鼠卵母细胞和胚胎的膜通透性是否可以平衡，因为通透性在很大程度上影响着玻璃化冷冻的条件。我们试图诱导卵母细胞水/冷冻保护剂通道(AQP)的表达，以增加卵母细胞的低膜通透性。我们还试图降低AQP3的通透性，AQP3在桑寄生和囊胚中有明显的表达。维甲酸使卵母细胞的冷冻保护剂通透性略有增加，但成熟率显着降低。据报道，在低pH条件下，AQP3的通透性显著降低。然而，在低pH条件下，大量表达AQP3的小鼠桑椹胚对乙二醇膜的通透性增加。因此，我们认为很难使卵母细胞/胚胎的膜透过率相等。下一步，我们试图开发一种新的玻璃化冷冻液，其中含有高浓度的蔗糖，以减少卵母细胞/胚胎的膜通透性对玻璃化冷冻条件的影响。用该溶液在相同条件下成功地玻璃化冷冻了从2-细胞期到桑椹胚期的胚胎，并可以用干冰(-79℃)运送玻璃化冷冻的胚胎。该方法可用于不同阶段小鼠胚胎的冷冻保存。

We tried to develop a vitrification method by which mouse oocytes and embryos at various stages can be cryopreserved by a single method. First, we examined whether the membrane-permeability of mouse oocytes and embryos can be equalized, because the permeability largely affects the condition for vitrification. We tried to induce the expression of water/cryoprotectant channels (AQP) in oocytes to increase the low membrane-permeability. We also tried in morulae to decrease the permeability of AQP3 which is markedly expressed in morulae and blastocysts. The cryoprotectant-permeability of oocytes increased slightly by retinoic acid but the maturation rate decreased remarkably. It was reported that the permeability of AQP3 decreased markedly at low pH. However, the permeability to ethylene glycol of mouse morulae, which expressed AQP3 abundantly, increased at low pH. Therefore, we considered that it would be difficult to make the membrane-permeability of oocytes/embryos equal. Next, we tried to develop a new vitrification solution in which a high concentration of sucrose was included to reduce the effect of membrane-permeability of oocytes/embryos on the vitrification condition. With this solution, embryos from the 2-cell to morula stages were successfully vitrified in the same condition, and vitrified embryos could be transported with dry ice (at -79℃). The method would be useful for cryopreservation of mouse embryos at various stages.