小鼠生发泡期卵母细胞成熟过程中转录沉默机制的探讨

The explore of the mechanism of transcriptional silencing during germinal vesicle oocytes maturation in mice

目的:探讨小鼠生发泡卵母细胞成熟过程中发生大规模转录沉默的机制。方法:选用4～6周龄ICR雌性小鼠,分离生发泡(germinal vesicle,GV)期卵母细胞,运用RNA聚合酶Ⅱ抗体(anti-RNA PolⅡCTD,clone 4H8)及其羧基末端结构域(CTD)基团第2位丝氨酸磷酸化的抗体(anti-RNA PolⅡCTD Ser2-P,clone H5)、组蛋白H3K4/H3K9三甲基化的抗体(anti-H3K4me3、anti-H3K9me3)进行免疫荧光染色。观察伴随GV期小鼠卵母细胞染色质构型由非包围核仁(non-surround nucleolus,NSN)型转变为包围核仁(surround nucleolus,SN)型的过程,这两对标记物的表达及分布变化。结果:在NSN型的GV期卵母细胞中,RNA PolⅡCTD Ser2-P于核浆中呈特征性斑块状分布,同时H3K4me3在核浆中也显示强阳性信号;但在SN型卵母细胞中,RNA PolⅡCTD Ser2-P失去特征性分布,转变为核浆中弥漫性分布,H3K4me3的核浆中阳性信号也消失。结论:RNA聚合酶Ⅱ的活性降低及组蛋白修饰状态的改变在小鼠GV期卵母细胞成熟过程的大规模转录沉默中发挥了重要作用。

Objective：To explore the mechanism of large-scale transcriptional silence during germinal vesicle（GV） oocytes maturation in mice.Methods：The separated GV oocytes were obtained from 4～6-week-old ICR female mice.As for the observation about the expressional and distributional changes of both markers in the process of the chromatin configuration from non-surround nucleolus（NSN） type into surround nucleolus（SN） type in mice GV oocytes,these oocytes were immunofluorescence stained with RNA polymeraseⅡ antibody（anti-RNA PolⅡCTD,clone 4H8） and the second serine phosphorylated antibody at RNA polymeraseⅡCTD group（anti-RNA PolⅡCTD Ser2-P,clone H5）;as well as histone H3K4/H3K9 tri-methylation antibodies（anti-H3K4me3,anti-H3K9me3）.Results：In NSN type GV oocytes,RNA CTD PolⅡSer2-P was distributed with characteristic plaques shape in nucleus;meanwhile,H3K4me3 also presented the strong positive signals in nucleus.However,in SN type oocytes,RNA CTD PolⅡSer2-P lost the characteristic distribution and diffused in nucleus;moreover,the positive signal in nucleus showed by H3K4me3 disappeared.Conclusion：The reduction of RNA polymeraseⅡactivity and the change of histone modification status played an important role in the large-scale transcriptional silencing during germinal vesicle oocytes maturation of mice.