PCBP1蛋白在小鼠卵母细胞成熟过程中调控大规模转录的研究

发布时间：2018-10-25 14:14

【摘要】：哺乳动物自胚胎形成时,初级卵母细胞便停滞在双线期(Diplotene stage),被一层前颗粒细胞密切围绕形成静止的始基卵泡(Primordial follicle);青春期后分批被激活和征募,进入发育的轨道。随着卵母细胞的发育,染色质逐渐浓集并向核仁周围迁移,在生发泡破裂(GVBD)前完成核型结构的改变。与核成熟相关的GVBD标志着卵母细胞减数分裂的恢复和开始。同时,卵母细胞基因组转录活性逐渐抑制,最终于排卵前期呈现整体水平的转录静止。这种大规模转录沉默储存了丰富的母源性物质而成为排卵前卵母细胞获得第一次减数分裂及其后继发育潜能的前提条件。因此,染色质的重塑(Chromatin Remolding)和大规模转录沉默的启动,正是排卵前卵母细胞能够恢复减数分裂从而获得进一步成熟的核心基础。在本实验室前期工作中,通过蛋白质组学的方法从MII期母源蛋白谱系中鉴定出PCBP1蛋白。已经证实,在哺乳动物中,PCBP1蛋白在转录水平上发挥多重而复杂的调节功能;同时有研究显示,与MII期小鼠卵母细胞相比该蛋白在未成熟的生发泡(Germinal Vesicle,GV)期表达更为丰富,且本研究中荧光定位结果显示,PCBP1蛋白于GV期卵母细胞胞核中特异性表达。上述结果使我们将注意力集中到PCBP1蛋白在GV期卵母细胞第一次减数分裂成熟过程中的功能。我们分别运用两对siRNAs分别特异性阻断PCBP1蛋白在小鼠GV期卵母细胞减数分裂成熟过程中的功能。结果显示,PCBP1蛋白的功能缺失显著导致了卵母细胞减数分裂成熟进程的显著障碍;以及NSN/SN核型比例的失衡提示我们染色质重塑过程明显受到干扰。进一步应用转录标记物及表达谱芯片综合分析了排卵前卵母细胞基因组整体水平的转录状态,结果显示这些发育阻滞的卵母细胞转录活性呈现出异于常态的活跃。而通过对PCBP1与核纤层蛋白LaminA/C相互作用的深入探索,最终揭示了这种由PCBP1蛋白功能缺失所导致的卵母细胞成熟障碍,是通过LaminA/C为介导,通过干扰其参与的核型重塑事件进而影响排卵前卵细胞基因组整体转录水平,造成转录抑制无法启动;与正常生理机制相悖的大规模转录本的转录活跃,直接致使母源性效应建立的失败,从而最终导致卵母细胞减数分裂进程的障碍。综上,本研究证实了PCBP1蛋白作为一个初级卵母细胞核内特异表达的母源蛋白,通过参与卵母细胞发育过程中的染色质构型重塑及宏观转录调控,从而推进了GV期卵母细胞减数分裂成熟。这一研究为我们深刻理解小鼠卵母细胞减数分裂的恢复,切入了伴随成熟而启动的基因组整体转录沉默事件这一独特视角。我们相信在哺乳动物雌性配子的发育相关研究中,PCBP1蛋白将作为正性调控卵母细胞减数分裂成熟的关键因子之一,而被人们重新认识。
[Abstract]:In mammalian self-embryogenesis primary oocytes stagnate in the two-line phase (Diplotene stage), is closely surrounded by a layer of pre-granulosa cells around the formation of static primordial follicle (Primordial follicle); after puberty by batch activation and recruitment into the development of the orbit. With the development of oocytes, chromatin gradually accumulated and migrated around the nucleoli, and the karyotype structure was changed before the blastocyst rupture of (GVBD). GVBD associated with nuclear maturation marks the recovery and commencement of meiosis in oocytes. At the same time, the genomic transcriptional activity of oocytes was inhibited gradually, and at the preovulation stage, the transcriptional activity remained at a global level. This massive transcriptional silencing is a prerequisite for the first meiosis and subsequent developmental potential of pre-ovulation oocytes. Therefore, the initiation of chromatin remodeling (Chromatin Remolding) and large-scale transcriptional silencing is the core basis for preovulation oocytes to resume meiosis and gain further maturation. The PCBP1 protein was identified from the maternal protein lineage of MII by proteomics. It has been demonstrated that PCBP1 protein plays multiple and complex regulatory functions at the transcriptional level in mammals, and studies have shown that the protein is more expressed in immature Germinal Vesicle,GV stage than in MII phase mouse oocytes. The results of fluorescence localization showed that PCBP1 protein was specifically expressed in the nucleus of GV oocytes. These results focus our attention on the function of PCBP1 protein during the first meiotic maturation of GV oocytes. We used two pairs of siRNAs to specifically block the function of PCBP1 protein during meiotic maturation of mouse GV oocytes. The results showed that the functional loss of PCBP1 protein resulted in a significant obstacle in the process of meiosis and maturation of oocytes, and the imbalance of NSN/SN karyotype indicated that the process of chromatin remodeling was obviously interfered. Transcriptional markers and expression microarray were used to analyze the transcriptional status of the whole genome of preovulation oocytes. The results showed that the transcriptional activity of these developmental arrested oocytes showed abnormal activity. Through the deep exploration of the interaction between PCBP1 and nuclear laminin LaminA/C, it was revealed that the oocyte maturation disorder caused by the absence of PCBP1 protein was mediated by LaminA/C. By interfering with the karyotype remodeling events in which they are involved, the overall transcriptional level of the pre-ovulation oocyte genome is affected, resulting in the transcription inhibition being unable to be initiated; the transcription of large-scale transcripts, which are contrary to normal physiological mechanisms, is active. The failure of the establishment of maternal effect directly leads to the obstacle of oocyte meiosis. In conclusion, this study confirmed that PCBP1 protein, as a mother protein specifically expressed in the nucleus of primary oocytes, participates in chromatin remodeling and macro-transcriptional regulation during oocyte development. Thus, the meiotic maturation of GV oocytes was promoted. This study provides us with a profound understanding of the recovery of meiosis in mouse oocytes and cuts into the unique perspective of the global transcriptional silencing events that are initiated with maturation. We believe that in the development of mammalian female gametes, PCBP1 protein will be one of the key factors in positive regulation of meiosis and maturation of oocytes.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R321

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