共培养对小鼠囊胚质量及其表观遗传修饰的影响

Co-culture of mouse blastocysts and their epigenetic modification

本研究探讨了共培养对小鼠囊胚质量及其表观遗传修饰的影响。将小鼠的受精卵体外随机分别置于含颗粒细胞(试验组I)、输卵管上皮细胞(试验组Ⅱ)、输卵管组织块(试验组Ⅲ)的KSOM培养液中作为试验组进行共培养,同时设立对照组A(体外培养,仅含KSOM)和对照组B(体内培养)。比较各组受精卵的卵裂率和囊胚发育率;并应用碘化丙啶和Hoechest333258对囊胚进行染色,利用ICM/TE值评价各组胚胎体外发育的质量;同时将囊胚进行免疫荧光染色,观察其基因组甲基化和组蛋白乙酰化的水平。结果表明,与对照组A相比,试验组的卵裂率和囊胚发育率均有显著提高(P<0.05);同时其囊胚细胞数目及内细胞团细胞数与滋养层细胞数比值(ICM/TE)值均显著高于对照组A(P<0.05);各试验组囊胚基因组甲基化水平与对照组A差异不显著(P>0.05),但各体外培养组均与对照组B差异显著(P<0.05);试验组组蛋白乙酰化水平与对照组A、B差异均不显著(P>0.05)。共培养能够有效促进小鼠胚胎的体外发育,提高囊胚的发育质量,但是仍不能克服由体外培养造成的基因组甲基化异常。

To discuss the effect of co-culture on the quality of mouse blastocysts and their epigenetic modification. We divided mouse zygotes into three co-culture experiment groups ： with granular cells （group I ）, oviduct epithelium cells （group II） and oviduct tissue （groupIII）. Meanwhile, we set up control A （cultured in vitro, only KSOM （KCl＋ simplex optimized medium）） and control B （cultured in vivo）. Then we compared cleavage rate and blastocyst rate among different groups. After that we evaluated the quality of blastocysts by using ICM/TE （Inner cell mass/Trophectoderm cells） ratio via staining with propidium iodide and Hoechest333258, and analyzed the level of genome methylation and histone acetylation by immunofluorescence. Compared with the control group A, the co-culture groups had increased cleavage rate and blastocyst rate （P〈0.05）, blastocyst cells and the ICM/TE ratio of co-culture groups were higher （P〈0.05）, the level of genome methylation and histone acetylation had no significant difference between groups in vitro （P〉0.05）, but the level of genome methylation in vivo was significantly higher than that of in vitro （P〈0.05）. The co-culture methods can successfully promote the development rate of embryos in vitro, and improve the quality of the blastocyst. However, the methods have drawbacks in changing the abnormal genome methylation with in vitro culture.