玻璃化冻存对驴卵母细胞超微结构的影响

Effects of Vitrification Freezing on Oocytes Ultrastructure in Equus asinus

试验旨在探究玻璃化冷冻对驴卵母细胞发育的影响,寻求驴卵母细胞冷冻的最佳条件。通过对不同发育时期的驴卵母细胞进行玻璃化冷冻,冷冻复苏后分别进行成熟培养和孤雌激活,并对GV期未冷冻组(对照组)、GV期冷冻组、IVM-MⅡ冷冻组卵母细胞微丝和线粒体超微结构进行免疫荧光标记,统计冷冻复苏后卵母细胞形态正常率、成熟率、孤雌激活卵裂率、超微结构正常率。结果表明,GV期冷冻组卵母细胞的形态正常率与GV期未冷冻组(对照组)间无显著差异(P>0.05),成熟率和卵裂率均显著低于对照组(P<0.05);IVM-MⅡ冷冻组的卵裂率显著低于对照组(P<0.05),且卵裂后细胞发育受到阻滞。冷冻组微丝在皮质区分布明显减少的卵母细胞数目增多,冷冻组卵母细胞的线粒体数量明显低于对照组,由此可以说明冷冻对卵母细胞超微结构有损伤,从而导致复苏后成熟率下降,影响卵母细胞的受精和体外发育,且GV期冷冻组较IVM-MⅡ冷冻组在微丝与线粒体结构上有较小损伤,发育状态较好。

This study was aimed to evaluate the effect of vitrification freezing on the development of oocytes in Equus asinus,and seek the best freezing conditions of oocytes in Equus asinus.The oocytes of Equus asinus in different development stages were vitrified and frozen,after the resuscitation,the mature culture and parthenogenetic activation were performed,respectively.The oocytes were stained with microfilaments and mitochondrial ultrastructure,and the oocytes were divided into GV stage without freezing group（control group）,GV stage freezing group,IVM-MⅡfreezing group.The normal rate of oocyte morphology,maturation rate,the cleavage rate of partial activation,the normal rate of ultrastructural were counted.The results showed that there was no significant difference in the normal rate of morphology between GV stage freezing group and GV stage without freezing（P〈0.05）,and the maturation rate and cleavage rate were significantly lower than those in control group（P〈0.05）,the cleavage rate of IVM-MⅡfreezing group was significantly lower than that of control group（P〈0.05）,and the cell development was blocked after cleavage.The distribution of microfilaments in the cortex of most oocytes in freezing group was significantly reduced,and the number of mitochondria in freezing group wassignificantly lower than that in control group,so it could be explained that freezing caused damage to the oocyte＇s ultrastructure,resulting in decreased resuscitation after the maturity rate,affecting the oocyte fertilization and in vitro development,and the GV stage freezing group had less damage to microfilaments and mitochondrial structure than IVM-MⅡfreezing group,and the developmental status was better.