miR-449b对绵羊早期胚胎发育的影响

【目的】研究miR-449b对绵羊早期胚胎发育的影响。【方法】采用Percoll密度梯度离心法获得纯化绵羊精子，将绵羊卵丘-卵母细胞复合体(COCs)在培养箱中培养16～18 h获得成熟卵母细胞，从1月龄纯种萨福克绵羊耳组织分离成纤维细胞，通过实时荧光定量PCR比较绵羊精子、成熟卵母细胞与成纤维细胞中miR-449b的相对表达量；给成熟卵母细胞分别显微注射0.9%生理盐水(Con组)、miR-449b模拟物对照(NC mimic)与miR-449b模拟物(miR-449b mimic),注射后进行孤雌激活，分别于激活的第2、7天统计胚胎卵裂率与囊胚率；将成熟卵母细胞进行体外受精(IVF),收集IVF、Con、NC mimic与miR-449b mimic组2-细胞胚胎，采用免疫荧光法比较组蛋白H3K9me3的变化。【结果】miR-449b在精子中的表达显著高于成熟卵母细胞和成纤维细胞(P<0.05);与Con组相比，miR-449b mimic和NC mimic组胚胎的卵裂率均显著降低(P<0.05),NC mimic组胚胎卵裂率显著低于miR-449b mimic组(P&...

Effects of miR-449b on Early Embryo Development in Sheep

【Objective】 The study was to investigate the effect of miR-449b on early embryo development in sheep.【Method】 Purified sheep sperm and mature oocytes were obtained by Percoll density gradient centrifugation and in vitro maturation of cumulus-oocyte complexes (COCs) for 16-18 h,respectively.And fibroblasts were isolated from the ear tissues of one-month-old Suffolk sheep.Firstly,the relative expression of miR-449b in sperms,mature oocytes and fibroblasts were compared by Real-time quantitative PCR.Mature oocytes were injected with 0.9% normal saline (Con group),NC mimic (NC mimic group) and miR-449b mimic (miR-449b mimic group) by microinjection,respectively.Then the embryonic cleavage rate and blastocyst rate were counted on the 2nd and 7th day after parthenogenetic activation. Mature oocytes were fertilized in vitro (IVF), 2-cell embryos in IVF, Con, NC mimic and miR-449b mimic groups were collected, and the changes of histone H3K9me3 were compared by immunofluorescence method. 【Result】 The expression of miR-449b in sperm was significantly higher than that in mature oocytes and fibroblasts (P＜0.05). Compared with Con group, the cleavage rates of embryos in miR-449b mimic and NC mimic groups were significantly decreased (P＜0.05), and the cleavage rate of embryos in NC mimic group was significantly lower than that of miR-449b mimic group (P＜0.05). The blastocyst rate of embryos in miR-449b mimic group was increased, but the difference was not significant (P＞0.05). Histone H3K9me3 was expressed in 2-cell embryos in IVF, Con, NC mimic and miR-449b mimic groups, and was expressed in the nucleus. Compared with Con group, the expression level of histone H3K9me3 in 2-cell embryos in NC mimic group was significantly increased (P＜0.05), and was significantly decreased in miR-449b mimic group (P＜0.05). The expression level of histone H3K9me3 in 2-cell embryos in NC mimic group was significantly higher than that in miR-449b mimic group (P＜0.05), but there was no significant difference between miR-449b mimic and IVF groups (P＞0.05). 【Conclusion】 miR-449b could significantly increase the development rate of early sheep embryos and reduce the expression of H3K9me3 in early embryos.