【作者】 谢颖颖；

【导师】 马恒东； 李瑞珍；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 本实验旨在探索有效的染色前固定和渗透卵母细胞及早期胚胎的方法,以提高小鼠卵母细胞和早期胚胎免疫荧光染色的效果;同时检测RNA聚合酶Ⅱ(PolⅡ)在小鼠卵母细胞和早期胚胎的表达,探讨PolⅡ在卵母细胞和早期胚胎发育中的作用。为大规模地精确检测小鼠卵母细胞和胚胎中的蛋白质与转录因子打下坚实基础。以小鼠卵母细胞和早期胚胎为试验材料,采用4种不同的固定及渗透方法处理。方案A:先用4%多聚甲醛(PFA)固定1h,再用0.5%Triton X-100穿透10min;方案B:先用0.5%Triton X-100穿透10min,再用4%PFA固定1h;方案C:用1%PFA+0.2%Triton X-100同时固定与穿透1h;方案D:用-20℃预冷甲醇固定15min,再用0.5%Triton X-100孵育10min。分析10种转录因子(TFⅡA、TFⅡB、TAF1、TAF4、PolⅡ、BRF1、MeCP2、MBD2ab、HP1α及HP1β)在小鼠卵母细胞中的免疫荧光染色效果。结果表明,C方案能够完全检测并标记出10种转录因子的荧光信号,其它3种方案都只能检测出部分转录因子的荧光信号;对荧光信号精细分布模式与动态变化最典型的TFⅡB进行仔细分析,表明经C方案处理后10种转录因子抗体标记信号的分布模式很精细,荧光信号的分布清晰,能观察到信号随细胞核成熟而发生的动态变化,卵母细胞的形态完好;其它3种方案处理后卵母细胞中虽然能够观察到核仁,也能观察到信号基本均匀地分布于核区,但不能清楚地显示荧光信号在核仁内是否有分布及信号的动态变化过程,荧光染色效果不理想。在光镜下观察4种方案处理后的卵母细胞,着重比较GV期、MⅠ期、MⅡ期的卵母细胞和2-细胞期胚胎形态,C方案处理后的卵母细胞形态保存较好,GV期细胞核和核仁结构清晰,细胞质均匀分布,形态饱满;MⅠ期透明带紧实清晰,厚薄均匀,胞质的颗粒状突起明显;MⅡ期第一极体结构和2-细胞期的极体结构都保存完好。其它3种方案处理后卵母细胞的形态变形,各发育时期的核仁、透明带、极体等结构受到不同程度的破坏。尤其以D方案对细胞形态的破坏最大,说明-20℃预冷甲醇不适用于小鼠卵母细胞和早期胚胎的固定。对PolⅡ在小鼠卵母细胞和早期胚胎中的表达进行分析:GVBD后直到MⅡ的卵母细胞中和Ana、PN1阶段的胚胎中都没有检测到PolⅡ的信号,而在受精后的PN2、PN3的部分胚胎中检测到信号,PN4、PN5和2-细胞阶段的胚胎全部是阳性。这种表达情况和早期的基因沉默相吻合,支持了关于转录沉默的假说。更多还原

【Abstract】 The aim of this experiment was to study the effective method of fixation and penetration before immunofluorescence staining in oocytes and early embryos to raise the effect of immunofluorescence staining in them.Simultaneously,the aim was to investigate the role of PolⅡduring the implantation and development of mouse oocytes and early embryos by studying the expression of PolⅡin them.Builds the solid foundation for detection the protein and transcription factor accurately in mouse oocytes and early embryos on a large scale.The materials were mouse oocytes and early embryos which treated by four different methods of fixation and penetration.Method A:Oocytes and early embryos were fixed in 4%paraformaldehyde(PFA)/PBS for 1 hour at room temperature, then permeated in 0.5%Triton X-100 for 10 minutes.Method B:Oocytes and early embryos were permeated in 0.5%Triton X-100 for 10 minutes,then fixed in 4% paraformaldehyde(PFA)/PBS.Method C:Oocytes and early embryos were fixed in 1%PFA while permeated in 0.2%Triton X-100 at the same time for 1 hour.Method D: Oocytes and early embryos were fixed in precooled methanol(-20℃) for 15 min,then permeated in 0.5%Triton X-100 for 10 minutes.Ten transcription factors(TFⅡA、TFⅡB、TAF1、TAF4、PolⅡ、BRF1、Mecp2、MBD2ab、HP1αand HP1β)in mouse oocytes were analyzed by immunofluorescence staining.The results indicated that only by using the method C the fluorescence signal of all target transcription factors were detected and marked,the rest three methods can detected the fluorescence signal of the transcription factor partially.To analyse the most typical TFⅡB with fine fluorescence signal distribution pattern and dynamic change carefully,it’s indicated that oocyte’s shape was completely and distribution pattern of ten transcription factors antibody signal were fine by using method C,the fluorescence signal was clear and can observe the signal change dynamic with the cell nucleus mature also.Although the nucleolus can be observed in oocytes treated by other three methods,and also the signal can be beobserved in the nuclear area distribution basic evenly,but not clearly displayed the nucleolus fluorescence signal if there were signals in the distribution and dynamic change.Fluorescence staining didn’t well.The oocytes and early embryos treated by four methods were observed in light microscope and compared sharp with stages of GV、MⅠ、MⅡ、2-cells,it’s showd that oocyte’s shape was completely by using method C.In the oocytes of GV stage, the structure of nucleus and nucleolus were clearly and the distribution of the cytoplasm were evenly,also the sharp were plumply.The granular cytoplasm can be seen in the oocytes of MⅠstage and the ZP were firms,clear and uniform thickness. The first polar body in the oocytes of MⅡstage and the polar body in the embryos of 2-cells stage were keeped well.The oocytes and early embryos treated by other three methods were out of sharp and the structure of nucleolus、ZP and polar body in different stages were damaged.Particularly,the greatest damage of sharp was by using method D indicated that mouse oocytes and early embryos fixed in precooled methanol(-20℃) were inapplicable.Analysis the expression of PolⅡin the mouse oocytes and early embryos:the signal were not detected in the oocytes aider GVBD until MⅡstage and also in the embryos of Ana、PN1 stage.But the signal can be detected in the embryos of PN2 and PN3 stages after fertilization.The embryos of PN4、PN5 and 2-cells stages were all positive.This expression was match of the early gene silencing,which supported the hypothesis on the silence of transcription.更多还原