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

本研究尝试建立一种玻璃化冷冻方法，使小鼠卵母细胞和不同发育阶段的胚胎可以用一种方法冷冻保存。首先，我们研究了小鼠卵母细胞和胚胎的膜渗透性是否可以均衡，因为渗透性在很大程度上影响玻璃化冷冻的条件。我们试图诱导卵母细胞水/冷冻保护剂通道（AQP）的表达，以增加低膜通透性。我们还尝试在桑椹胚中降低在桑椹胚和囊胚中显著表达的AQP 3的通透性。视黄酸可使卵母细胞的冻融保护膜通透性略有增加，但使卵母细胞的成熟率显著降低。据报道，AQP 3的渗透性显着降低，在低pH值。然而，小鼠桑椹胚，表达AQP 3丰富，乙二醇的渗透性增加，在低pH值。因此，我们认为，这将是困难的，使卵母细胞/胚胎的膜渗透性相等。接下来，我们试图开发一种新的玻璃化溶液，其中包括高浓度的蔗糖，以减少卵母细胞/胚胎的膜渗透性对玻璃化条件的影响。在相同条件下，成功地将2-细胞期至桑椹胚期的胚胎玻璃化冷冻，并且玻璃化冷冻的胚胎可以用干冰（在-79℃下）运输。该方法可用于不同发育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.