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

Effects of cryopreservation on developmental capacity and cytoskeleton of mouse oocytes

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。

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 significantlyhigher 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.