玻璃化冷冻对不同成熟阶段小鼠卵母细胞的影响

目的：通过比较玻璃化冷冻对不同成熟阶段小鼠卵母细胞冷冻复苏、体外成熟、胚胎发育及细胞骨架的影响,寻求最佳的卵子冷冻方案。方法：玻璃化冷冻生殖泡期卵（GV）、成熟中期卵（MⅡ）及体外成熟卵（IVM-MⅡ）,解冻复苏后,分别作体外成熟、体外受精（IVF）、胚胎培养或固定作免疫荧光标记,统计复苏率、成熟率、受精率、囊胚率、细胞骨架正常率。结果：GV冷冻组、MⅡ冷冻组及IVM-MⅡ冷冻组三组复苏率差异无显著性（65%vs 60.92%vs 69.93%,P〉0.05）。GV冷冻组的成熟率显著低于对照组（79.6%vs 96.19%,P〈0.01）。GV冷冻组、MII冷冻组及IVM-MⅡ冷冻组三组受精率均明显低于对照组（P〈0.01）,且IVM-MⅡ冷冻组受精率显著低于MⅡ冷冻组（18.06%vs 31.34%,P〈0.01）。IVM-MⅡ冷冻组囊胚率低于MⅡ冷冻组和对照组,差异均有显著性（28.57%vs 52.38%,P〈0.05;28.57%vs 57.14%,P〈0.01）。GV冷冻组染色体和纺锤体均正常率均高于IVM-MⅡ冷冻组（53.85%vs 26.67%,P〈0.05）。IVM-MⅡ冷冻组细胞骨架三项指标均显著低于对照组,差异均有显著性（P〈0.05,P〈0.01）。结论：GV卵先玻璃化冷冻再体外成熟,其细胞骨架损伤较小,胚胎发育较好。

Effects of cryopreservation on different maturation stage of mouse ocytes

Objective： To evaluate the effects of vitrification on the rates of survival, in vitro maturation （IVM）, development and cytoskeleton of mouse frozen-thawed oocytes at different stages in order to find the most suitable eryopreservation protocol. Methods： M Ⅱ, GV and IVM-M Ⅱ were vitrified. After thawed, they underwent in vitro maturation, in vitro fertilization,embryo cultivation or fixed for immunofluorescence. The rates of survival, maturation, fertilization, blastulation and cytoskeleton with normal configuration were calculated. Results： There were no significant differences of survival rates in GV, M Ⅱ and IVM-M Ⅱ group （65% vs 60.92% vs 69.93%, P〉0.05）. The maturation rate of GV group was significantly lower than those of control group （79.6% vs 96.197o, P〈0.01）. The fertilization rate of controI group was obviously higher than GV, M Ⅱ and IVM-M Ⅱ group （P〈0.01）, and that of IVM-M Ⅱ group was significantly lower than M Ⅱ group （18.06% vs 31.34%, P〈0.01）. The blastulation rate of IVM- M Ⅱ was significantly lower than that of M Ⅱ and control groups, respectively （28.57% vs 52.38%, P〈0.05 and 28.57% vs 57.14%, P〈0.01）. There were significant differences in the rate of normal feature of spindles and chromosomes between GVgroupand IVM-MⅡ group （53.85% vs 26.67%, respectively, P〈0.05）. The rate of cytoskeleton indexe of IVM-M Ⅱ group were much lower, comparing with control group （P〈0.05）. Conclusion： There are less influences of vitrification on cytoskeleton and developmental capacity of GV ooctyes when they are vitrified before IVM.