不同冷冻方法对小鼠卵母细胞发育潜能及细胞骨架的影响

目的比较不同冷冻方法对不同成熟阶段小鼠卵母细胞复苏、胚胎发育及细胞骨架的影响,寻求最佳的卵母细胞冷冻方法。方法分别以SOPS玻璃化及慢速法冷冻小鼠中期(MII)和生发泡期(GV)卵母细胞。解冻复苏后,分别作体外培养成熟(IVM)、体外受精(IVF)或固定作免疫荧光标记,统计复苏率、成熟率、受精率、囊胚率及纺锤体等细胞骨架指标。结果玻璃化冷冻组MII卵和GV卵母细胞的复苏率及囊胚率均显著高于慢速冷冻组(P<0.05)。慢速冷冻组中MII卵复苏率显著低于GV卵(40.4%vs 57.1%,P<0.01)。但同一种冷冻方法保存的MII卵和GV卵的受精率及囊胚率相比,均无显著性差异(P>0.05)。两组的GV卵成熟率无显著差异。玻璃化冷冻组GV卵的纺锤体、染色体及纺锤体和染色体正常率均高于慢速冷冻组GV卵但无显著差异。结论玻璃化冷冻能有效改善冻融卵母细胞的复苏及胚胎发育;与MII卵比较,冷冻GV卵对细胞骨架损伤较小;两种冷冻方法均不影响GV卵冻融后成熟。

Effects of cryopreservation on developmental capacity and cytoskeleton of mouse oocytes

Objective: To evaluate the effects of vitrification and slow cooling on the survival,in vitro maturation(IVM),development and cytoskeleton of mouse frozen-thawed oocytes at different stages in order to find the most suitable cryopreservation methods.Methods: MII and GV oocytes were cryopreserved by SOPS vitrification or slow cooling.After thawed,oocytes were matured and fertilized in vitro(IVM/IVF) or fixed for immunofluorescence.Then we calculated the rates of survival,maturation,fertilization,blastulation and cytoskeleton with normal configuration.Results: The rates of survival and blastulation of MII and GV oocytes in vitrification group were significantly higher than those in slow cooling group(P<0.05).The survival rate of MII oocytes was significantly lower than that of GV oocytes in slow-cooling group(40.38% vs.57.14%,P<0.01).With the same cryopreservation method,there were no differences in the rates of fertilization and blastulation between MII and GV oocytes.The maturation rates in two groups were not significantly different(P>0.05).Compared with slow cooling group,the rates of spindle,chromosomes or both with normal configuration in vitrification group were higher although no significant difference was found(70.97% vs.63.64%,61.29% vs.36.36% and 61.29% vs.36.36%,P>0.05). Conclusions: Vitrification could be more favorable than slow cooling in the survival and development of frozen-thawed oocytes.GV oocytes were more tolerable(with less damage of the cytoskeleton) to the cryopreservation than MII oocytes.No difference was found in the maturation capacity of frozen-thawed oocytes in two cryopreservation methods.