添加HA纳米颗粒对牛GV期卵母细胞玻璃化冷冻后发育能力的影响

Effect of HA nanoparticle on the developmental competence of vitrified bovine GV oocytes

为了探究羟基磷灰石(HA)纳米颗粒对牛GV期卵母细胞玻璃化冷冻效果的影响,试验以牛卵巢中GV期卵母细胞作为试验材料,将不同粒径的HA纳米颗粒添加到玻璃化冷冻液中,进行超声分散检测。首先,以卵母细胞存活率和成熟率为指标,将0、0.01%、0.05%、0.1%HA纳米颗粒分别添加到玻璃化冷冻液Ⅰ(VSⅠ)和玻璃化冷冻液Ⅱ(VSⅡ)中,不经冷冻直接进行毒性测试;然后,以形态正常率、成熟率、卵裂率和囊胚率为指标测定卵母细胞玻璃化冷冻后的发育能力;最后检测含有0.05%HA纳米颗粒的VSⅡ对冷冻后卵母细胞活性氧水平和线粒体膜电位水平的影响。结果表明:不同浓度HA纳米颗粒对牛GV期卵母细胞均无明显毒性;VSⅡ+0.05%HA纳米颗粒组卵母细胞冷冻后的形态正常率、成熟率、卵裂率、囊胚率显著高于VSⅠ+0.05%HA纳米颗粒组(P<0.05);VSⅡ+0.05%HA纳米颗粒组卵母细胞的活性氧水平显著低于VSⅡ组(P<0.05);VSⅡ+0.05%HA纳米颗粒组卵母细胞的线粒体膜电位水平显著高于VSⅡ组(P<0.05)。说明在VSⅡ中加入0.05%HA纳米颗粒能显著降低牛GV期卵母细胞玻璃化冷冻后活性氧水平并提高其线粒体膜电位水平,从而可能降低了玻璃化冷冻对细胞的损伤。

In order to investigate the effect of hydroxyl apatite(HA)nanoparticles on the vitrified bovine GV oocytes,the bovine GV oocytes were vitrified with concentrationlevels of HA nanoparticlesfor ultrasonic dispersion detection.The toxicity of HA levels of 0,0.01%,0.05% and 0.1% in vitrification solution Ⅰ(VSⅠ)and vitrification solution Ⅱ(VSⅡ)was tested with the indictors of oocyte survival and maturation rates,respectively,following the determination of developmental competence with the indicators of morphological normalization rate,maturation rate,oocyte cleavage rate and blastocyst rate.Finally,the effects of VSⅡ with level of 0.05%HA nanoparticle on the levels of reactive oxygen species and mitochondrial membrane potential of vitrified oocytes were examined.The results showed that the toxicity was not detected for bovine GV oocytes at three different concentrations of HA nanoparticles.The oocytes in VSⅡ with 0.05%level of HA nanoparticle had significantly higher morphological normalization rate,maturation rate,oocyte cleavage rate and blastocyst rate than those in VSⅠ(P<0.05).The reactive oxygen species level of oocytes in VSⅡ with 0.05%level of HA nanoparticle was significantly lower than that in VSⅡ(P<0.05).The mitochondrial membrane potential of oocytes in VSⅡ with level of 0.05%HA nanoparticle was significantly higher than that in VSⅡ(P<0.05).It could be concluded that the addition of 0.05%HA nanoparticle to VSⅡ could significantly lower the level of reactive oxygen species and increase the level of mitochondrial membrane potential in vitrified bovine GV oocytes,thus possibly reducing the risks of the damage to cells by vitrification.