miR-449b对绵羊早期胚胎发育的影响  

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

作　　者：任秀美奥 姚旭东 蒙亚琦 郭延华[2] 唐红[2] 张译元[2] 赵兴旺 王立民[2] 周平[1,2] REN Xiumeiao;YAO Xudong;MENG Yaqi;GUO Yanhua;TANG Hong;ZHANG Yiyuan;ZHAO Xingwang;WANG Limin;ZHOU Ping(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;State Key Laboratory of Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural Reclamation Sciences,Shihezi 832000, China;Agricultural Service Development Center of Shihezi General Farm, Shihezi 832000, China)

机构地区：[1]石河子大学动物科技学院,石河子832000 [2]新疆农垦科学院,省部共建绵羊遗传改良与健康养殖国家重点实验室,石河子832000 [3]石河子总场农业服务发展中心,石河子832000

出　　处：《中国畜牧兽医》2022年第7期2661-2668,共8页China Animal Husbandry & Veterinary Medicine

基　　金：兵团应用基础研究计划(2015AG011);兵团中青年科技创新人才计划(2019CB002);转基因重大专项(2016ZX08008001)。

摘　　要：【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<0,05);miR-449b mimic组胚胎囊胚率提高,但差异不显著(P>0.05);IVF、Con、NC mimic与miR-449b mimic组2-细胞胚胎均表达组蛋白H3K9me3,且都在细胞核表达。与Con组相比,NC mimic组2-细胞胚胎组蛋白H3K9me3的表达水平显著增加(P<0.05),miR-449b mimic组显著降低(P<0.05);NC mimic组2-细胞胚胎组蛋白H3K9me3的表达水平显著高于miR-449b mimic组(P<0.05),miR-449b mimic与IVF组无显著差异(P>0.05)。【结论】miR-449b能显著提高绵羊早期胚胎的发育率,且降低早期胚胎中H3K9me3的表达水平。【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-

关 键 词：miR-449b 早期胚胎 H3K9me3 精子 

分 类 号：S813.1[农业科学—畜牧学]