论文部分内容阅读
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 investigate the mechanism of large-scale transcriptional silencing during the maturation of mouse green foam oocytes. Methods: The oocytes were isolated from 4 to 6-week-old ICR female mice and the oocytes were isolated from the germinal vesicle (GV) stage. The oocytes were collected and stained with anti-RNA Pol II CTD (clone 4H8) and its carboxy terminal domain (Anti-RNA Pol II CTD Ser2-P, clone H5) and anti-H3K4me3 (anti-H3K9me3) at the 2nd position of the group were used for immunofluorescence staining. To observe the changes of the chromatin configuration of the oocytes in the GV phase from non-surround nucleolus (NSN) to surround nucleolus (SN) Distribution changes. Results: In NSN GV oocytes, RNA Pol Ⅱ CTD Ser2-P showed a characteristic plaque distribution in nucleus pulposus and H3K4me3 also showed a strong positive signal in nucleus pulposus. However, in SN oocytes , RNA Pol Ⅱ CTD Ser2-P loss of characteristic distribution, into a diffuse distribution of nuclear plasma, H3K4me3 nuclear plasma positive signal disappears. Conclusion: The decrease of RNA polymerase Ⅱ activity and the change of histone modification play an important role in the large-scale transcriptional silencing of mouse GV oocyte maturation.