不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响

目的:探讨不同冷冻方法对小鼠成熟期(MⅡ期)及生发泡期(GV期)卵母细胞的纺锤体及胚胎发育的影响。方法:收集GV期和有纺锤体的MⅡ期小鼠卵母细胞,随机分为3组:慢速冷冻-快速复温组、超高速玻璃化冷冻组和对照组(未冷冻组)。Polscope观察解冻0、3、6h后存活的MⅡ期及体外培养成熟GV期卵母细胞的纺锤体,有明显纺锤体的行卵胞浆内单精子显微注射受精,评价胚胎发育。结果:(1)超高速玻璃化GV组的存活率、卵裂率均显著高于慢冻GV组(P<0.05);(2)两冷冻MⅡ组解冻后0、3及6h纺锤体出现率和优质胚胎率均显著低于对照MⅡ组(P<0.05);(3)超高速玻璃化GV组体外成熟后纺锤体的出现率、优质胚胎率均显著高于超高速玻璃化MⅡ组(P<0.05)。结论:慢速冷冻-快速复温法对小鼠不同成熟时期卵母细胞的纺锤体损伤较大;超高速玻璃化冷冻对小鼠生发泡期卵母细胞纺锤体的影响则较小,是一种简便、快捷、高效的冷冻方法。

Effects of different cooling and rewarming procedure on meiotic spindle of mature and germinal vesicle mouse oocytes

Objective:To explore the effects of different freezing procedure on meiotic spindle of the mature and in-vitro matured germinal vesicle(GV) mouse oocytes.Methods:The GV and mature mouse oocytes with spindle were randomly divided into three groups respectively:slow freezing group,ultra-rapid vitrification group and control group.Following cryopreservation and in-vitro maturation,the GV oocytes extrusing the polar body I or after thawed 0,3 and 6 hour of mature oocytes were visualizated with polscope.The oocytes with visible spindle were fertilizated by intracytoplasmic sperm injection,the results were assessed.Results:(1)A statistically significant increase was observed in the survival rate and cleavage rate in ultra-rapid vitrification GV group and compared to slow freezing GV group(P<0.05);(2) A statistically significant increase was observed in the exhibiting spindle rate after thawed 0,3,6 hour and the good quality embryo rate in the two freezing MⅡ groups compared to control MⅡ group(P<0.05);(3)A statistically significant increase were observed in the spindle exhibiting rate,high quality embryo rate in vitrification GV group compared to ultra-rapid vitrification MⅡ group(P<0.05).Conclusion:The slow freezing procedure has a deleterious effect on the meiotic spindle of MⅡ and GV stage mouse oocytes.Ultra-rapid vitrification is an ideal,rapid and efficacy method for the GV mouse oocytes.