H3K4和H3K9三甲基化在猪体外成熟卵母细胞和克隆胚胎中的表达分析
Expression of Histone H3K4 and H3K9 Trimethylation in Porcine in vitro maturation Oocytes and Somatic Nuclear Transfer Embryos

该研究主要运用单细胞免疫荧光技术分析猪卵母细胞和克隆胚胎发育过程中，H3K4和H3K9三甲基化(H3K4me3，H3K9me3) 的表达情况.收集体外成熟0 h，6 h，12 h，24 h，30 h，36 h，42 h的猪卵母细胞；同时通过体细胞核移植操作获得一细胞、四细胞、桑葚胚和囊胚期克隆胚胎，经过免疫荧光染色，在激光共聚焦显微镜下观察H3K4me3和H3K9me3修饰在卵母细胞和胚胎中的变化情况.免疫荧光结果显示，在不同减数分裂阶段的猪卵母细胞内均能观察到H3K4me3的表达，并且呈现出逐渐下降的趋势；在生发泡期(GV)，生发泡晚期(L-GV)和生发泡破裂(GVBD)阶段的卵母细胞中能发现明显的H3K9me3表达，随着减数分裂的进行，H3K9me3表达消失，在第一次减数分裂中期前阶段(Pre-MI)到第二次减数分裂中期(MⅡ)阶段卵母细胞中未发现H3K9me3荧光信号. H3K4me3和H3K9me3在不同发育阶段的猪克隆胚胎中都有表达，其中H3K4me3在不同阶段胚胎中荧光信号强度相近，H3K9me3在四细胞胚胎中荧光强度降低，在桑葚胚和囊胚中荧光强度出现升高的趋势.以上结果说明，H3K4me3参与调控猪卵母细胞体外成熟的整个过程，H3K4me3和H3K9me3在猪体细胞核移植胚胎早期发育过程中可能具有调控作用.
This study was designed to observe the expression of histone H3K4 and H3K9 trimethylation in porcine in vitro maturation oocytes and somatic nuclear transfer (SCNT) embryos by single cell immunofluorescence. Porcine oocytes at different maturation stages (0 h, 6 h, 12 h, 24 h, 30 h, 36 h, 42 h) and the SCNT embryos at 1-cell, 4-cell, morula, blastocyst periods were collected for immunostaining. The expression pattern of histone H3K4 and H3K9 trimethylation was analyzed by laser confocal microscopy. The results showed that there was significant H3K4me3 signal in oocytes of all in vitro maturation stages, decreased from GV to MⅡstages. The H3K9me3 signal was detected in oocytes at the GV, L-GV and GVBD periods, but it was disappeared after meiosis. Both of the H3K4me3 and H3K9 me3 expressed in SCNT embryos at 1-cell, 4-cell, morula and blastocyst stages, but there was only some weak H3K4me3 signal in embryos of different stages. The expression of H3K9me3 was decreased in 4-cell embryos, but increased in morula and blastocyst. Altogether, our study revealed that H3K4me3 participated in the regulation of porcine oocyte in vitro maturation. Both of H3K4me3 and H3K9me3 were contributed to regulate the development of porcine SCNT embyos