冷冻环玻璃化法冷冻小鼠卵母细胞的效果评价

Assessment of a new cryoloop vitrification protocol in the cryopreservation of mouse mature oocytes

目的评价ED15(15%ethylene glycol,EG+15%dimethylsulphoxide,DMSO)冷冻环玻璃化法冻存小鼠成熟卵母细胞的效果,以及对其纺锤体形态、染色体分布和发育潜能的影响。方法分别以小鼠新鲜成熟卵母细胞为对照组,玻璃化冷冻复苏卵母细胞为冷冻组。玻璃化冷冻以冷冻环为载体,以乙二醇(EG)及二甲亚砜(DMSO)联合作冷冻保护剂。解冻后观察细胞存活情况并对解冻后培养0、1、2h的卵母细胞行纺锤体及染色体免疫荧光染色,激光共聚焦扫描显微镜观察;其余卵母细胞行体外受精培养,计算受精率、囊胚形成率。结果冷冻组成熟卵母细胞存活率为98.2%,复苏后卵母细胞培养0、1、2h的纺锤体形态正常率及染色体分布正常率与对照组比较无显著性差异(分别为87.0%、90.9%、90.3%vs95%,P>0.05;91.3%、95.4%、93.5%vs90%,P>0.05);冷冻组受精率及囊胚形成率与对照组比较均无显著性差异(81.3%vs85.2%,P>0.05;76.1%vs75.4%,P>0.05)。结论ED15冷冻环玻璃化法冻存小鼠成熟卵母细胞复苏存活率高,对其纺锤体形态、染色体分布及发育潜能无明显影响。

Objective： To assess the outcome of a new cryoloop vitrification protocol in the cryopreservation of mouse MII oocytes. Methods： Frozen-thawed oocytes were investigated against the fresh mouse MII oocyte as control. With the cryoloop carrier, mouse MII oocytes were vitrified in vitrification solution consisting of 15% ethylene glycol （EG）, 15% dimethylsulphoxide （DMSO）, 5. 8 mg/ml Ficoll 400 and 0. 58 mol/L sucrose （ED 15 protocol）. After thawing, the survival rates were recorded. Some thawed oocytes and fresh oocytes were fixed, and the microtubules of the spindles and chromosomes were then stained by immunofluorescent method. Confocal microscopy was used to reveal the configurations of the spindles and chromosomes. Most of the other survived oocytes were inseminated in vitro, then the fertilization rates and blastocyst formation rates were compared with those of the control group. Results： The oocytes vitrified by this protocol survived very well. The number of thawed oocytes with normal spindles and chromosomes was similar to that of the fresh ones. The fertilization rates and blastocyst formation rates of the thawed oocytes were also high, similar to those of the control group. Conclusion： The ED15 vitrification protocol has little deleterious affect on the MII mouse oocytes＇ survival rate, spindles and chromosomes.