卵母细胞转录组的调节及其在卵泡存活和早期胚胎发育中的作用
发布时间：2018-11-27 14:12
【摘要】：卵泡是卵巢结构和功能的基本单位,能够产生成熟的卵子并合成雌激素维持正常的生理周期。其中,原始卵泡作为雌性生殖细胞的储存库,在成年个体中不再增加,只会随着生殖活动不断减少。所以卵巢中原始卵泡的规模直接决定了雌性生育年龄,过早消耗将导致卵巢早衰的发生。但是卵泡维持的分子机理和卵巢早衰的发病机制的研究还有很多未知内容。本研究中,我们利用卵母细胞特异性的基因敲除模型来研究卵母细胞发育、卵母细胞减数分裂和早期胚胎发育过程中转录组的变化以及表观遗传调节因子在这些生理过程中的作用,阐明雌性生殖力维持的重要机制,为临床上不孕不育的病例提供新的科学解释和科学依据。因此,本课题分别研究了E3泛素化连接酶CRL4复合体、CpG结合蛋白CFP1和转录辅因子YAP在雌性生殖中的作用,系统地分析了转录组调节在卵母细胞维持和合子基因组激活中的作用。我们的研究发现CRL4通过激活TET家族DNA去甲基化酶的作用,调节卵母细胞中基因的表达和受精后雄原核的去甲基化,从而维持卵母细胞的存活和促进受精卵的发育,在雌性生殖力维持中发挥至关重要的作用。如果CRL4的功能缺失,就会导致卵母细胞死亡,发生卵巢早衰和胚胎受精后不发育。在这些敲除的卵母细胞中,很多卵母细胞里高表达基因的表达量显著降低,而这些基因启动子中CpG岛的甲基化水平明显升高。而且DDB1 (damaged DNA binding protein 1)敲除的卵母细胞受精后大部分不能发育到四细胞,早期胚胎发育所必需的基因的甲基化水平大幅升高,从而导致基因沉默。进一步的机理研究发现,CRL4通过调节TET家族DNA去甲基化酶的作用,调节卵母细胞中基因的表达和受精后雄原核的去甲基化,从而维持雌性生殖力。DNA结合蛋白CFP1是组蛋白甲基化复合体SET1中的重要亚基,识别基因组中处于非甲基化状态的CpG,介导特定染色体区域组蛋白H3K4的三甲基化,从而调控大量基因的转录。我们的研究发现CFP1敲除的雌鼠完全不育。CFP1在卵母细胞中调节下游基因转录,敲除CFP1造成母源mRNA不能积累,导致卵母细胞不能维持以及卵母细胞受精后不能发育。这些结果说明CRLA活性缺失造成的DNA高甲基化和CFP1敲除导致的mRNA转录下降都严重地阻碍了卵母细胞的维持和重编程能力,造成卵巢早衰和雌性不育,阐明了母源mRNA积累对于雌性生殖能力的重要作用。另外一方面,我们还研究了转录辅因子YAP在合子基因组激活中的功能。YAP在卵母细胞和早期胚胎中高表达,但是在卵母细胞中却定位在细胞质中,不能与转录因子TEAD结合发挥转录活性。但是在受精后,YAP进入细胞核,暗示了YAP-TEAD在早期胚胎发育中的作用。因此我们特异性地在卵母细胞中敲除了Yap1基因(编码YAP蛋白)。结果发现,Yap1敲除的卵母细胞虽然能够正常成熟和受精,但是形成的早期胚胎却发育迟缓,并在三四天内死亡。追踪这些胚胎的死亡原因,发现是许多对于早期胚胎发育至关重要的基因不能及时被表达出来,其中包括两个分别参与核酸和蛋白质合成的两个重要基因Rrm2 (ribonucleotide reductase 2)和Rpll3 (ribosomal protein like 13).通过这个研究,我们发现母体在卵细胞中储存了转录辅因子YAP,又利用输卵管液中的LPA在胚胎发育早期激活了它们,从而在MZT过程中及时启动了胚胎基因转录。这些结果为辅助生殖和胚胎工程技术的进一步改进提供了新的思路。
[Abstract]:The follicle is the basic unit of the structure and function of the ovary, capable of producing a mature ovum and synthesizing an estrogen for maintaining a normal physiological cycle. In this case, the original follicle, as a repository for female germ cells, no longer increases in the adult population and will only decrease with the increasing number of reproductive activities. The size of the primary follicle in the ovary directly determines the age of the female, and premature consumption will lead to premature ovarian failure. But the molecular mechanism of the maintenance of the follicle and the pathogenesis of the premature ovarian failure have many unknown contents. In this study, we used the oocyte-specific gene knock-out model to study the changes of the transcription group in the development of the oocyte, the meiosis of the oocyte and the early embryonic development, and the role of the epigenetic adjustment factor in these physiological processes, The important mechanism of the maintenance of female fertility is to provide a new scientific explanation and scientific basis for the clinical infertility cases. Therefore, the role of the E3 ubiquitin ligase CRL4 complex, the CpG-binding protein CFP1 and the transcription factor YAP in the female reproduction is studied, and the role of the transcription group in the maintenance of the oocyte and the activation of the zygote genome is systematically analyzed. Our study found that CRL4 regulates the expression of the gene in the oocyte and the demethylation of the male pronucleus after fertilization by activating the DNA of the TET family, so as to maintain the survival of the oocyte and promote the development of the fertilized egg, and play a vital role in the maintenance of female fertility. The absence of the function of CRL4 leads to the death of the oocyte, the occurrence of premature ovarian failure, and the non-development of the embryo after the embryo is fertilized. In these knock-out oocytes, the expression of high-expression genes in many of the oocytes is significantly reduced, and the methylation level of CpG islands in these gene promoters is significantly increased. In addition, most of the genes of DDB1 (damed DNA binding protein 1) can not be developed to four cells, and the methylation level of the genes necessary for early embryonic development is greatly increased, leading to gene silencing. Further mechanism study found that CRL4 regulates the expression of the gene in the oocyte and the demethylation of the male pronucleus after fertilization by adjusting the role of the TET family DNA to the methylase, thereby maintaining the female fertility. The DNA binding protein CFP1 is an important subunit in the histone methylation complex SET1, recognizes the CpG in the non-methylated state in the genome, mediates the trimethylation of the histone H3K4 in the specific chromosomal region, thereby regulating the transcription of a large number of genes. Our study found that CFP1 knockout female mice were completely sterile. CFP1 regulates the transcription of the downstream gene in the oocyte, and the knockout of CFP1 leads to the non-accumulation of the mother-source mRNA, which leads to the inability of the oocyte to maintain and not to develop after the oocyte is fertilized. These results indicate that the high methylation of the DNA and the reduction of the mRNA transcription caused by the CFP1 knock-out caused by the loss of the activity of the CRLA severely impede the maintenance and reprogramming of the oocyte, resulting in premature ovarian failure and female infertility, indicating the important role of maternal mRNA accumulation in female reproductive capacity. In addition, we have also studied the function of the transcription factor YAP in the activation of the zygote genome. The YAP is highly expressed in the oocyte and in the early embryo, but in the cytoplasm it is located in the cytoplasm and cannot be combined with the transcription factor TEAD to play a transcriptional activity. However, after fertilization, the YAP entered the nucleus, suggesting the role of YAP-TEAD in early embryonic development. Therefore, we specifically knock the Yap1 gene (coding YAP protein) in the oocyte. As a result, the Yap1 knockout oocytes were found to be mature and fertilized, but the early embryos formed were slow to develop and died within three or four days. To track the cause of the death of these embryos, it was found that a number of genes that were essential for early embryonic development could not be expressed in time, including two important genes, Rrm2 (ribbonotides 2) and Rpll3 (ribnomal protein like 13), which were involved in the synthesis of nucleic acids and proteins, respectively. Through this study, we found that the mother had stored the transcription factor YAP in the egg cell and activated them in the early stage of the development of the embryo by using the LPA in the fallopian tube liquid, so that the transcription of the embryo gene was started in time in the MZT process. These results provide a new way to improve the further improvement of assisted reproduction and embryo engineering.
【学位授予单位】：浙江大学
【学位级别】：B
【学位授予年份】：2016
【分类号】：R321
本文编号：2361069
[Abstract]:The follicle is the basic unit of the structure and function of the ovary, capable of producing a mature ovum and synthesizing an estrogen for maintaining a normal physiological cycle. In this case, the original follicle, as a repository for female germ cells, no longer increases in the adult population and will only decrease with the increasing number of reproductive activities. The size of the primary follicle in the ovary directly determines the age of the female, and premature consumption will lead to premature ovarian failure. But the molecular mechanism of the maintenance of the follicle and the pathogenesis of the premature ovarian failure have many unknown contents. In this study, we used the oocyte-specific gene knock-out model to study the changes of the transcription group in the development of the oocyte, the meiosis of the oocyte and the early embryonic development, and the role of the epigenetic adjustment factor in these physiological processes, The important mechanism of the maintenance of female fertility is to provide a new scientific explanation and scientific basis for the clinical infertility cases. Therefore, the role of the E3 ubiquitin ligase CRL4 complex, the CpG-binding protein CFP1 and the transcription factor YAP in the female reproduction is studied, and the role of the transcription group in the maintenance of the oocyte and the activation of the zygote genome is systematically analyzed. Our study found that CRL4 regulates the expression of the gene in the oocyte and the demethylation of the male pronucleus after fertilization by activating the DNA of the TET family, so as to maintain the survival of the oocyte and promote the development of the fertilized egg, and play a vital role in the maintenance of female fertility. The absence of the function of CRL4 leads to the death of the oocyte, the occurrence of premature ovarian failure, and the non-development of the embryo after the embryo is fertilized. In these knock-out oocytes, the expression of high-expression genes in many of the oocytes is significantly reduced, and the methylation level of CpG islands in these gene promoters is significantly increased. In addition, most of the genes of DDB1 (damed DNA binding protein 1) can not be developed to four cells, and the methylation level of the genes necessary for early embryonic development is greatly increased, leading to gene silencing. Further mechanism study found that CRL4 regulates the expression of the gene in the oocyte and the demethylation of the male pronucleus after fertilization by adjusting the role of the TET family DNA to the methylase, thereby maintaining the female fertility. The DNA binding protein CFP1 is an important subunit in the histone methylation complex SET1, recognizes the CpG in the non-methylated state in the genome, mediates the trimethylation of the histone H3K4 in the specific chromosomal region, thereby regulating the transcription of a large number of genes. Our study found that CFP1 knockout female mice were completely sterile. CFP1 regulates the transcription of the downstream gene in the oocyte, and the knockout of CFP1 leads to the non-accumulation of the mother-source mRNA, which leads to the inability of the oocyte to maintain and not to develop after the oocyte is fertilized. These results indicate that the high methylation of the DNA and the reduction of the mRNA transcription caused by the CFP1 knock-out caused by the loss of the activity of the CRLA severely impede the maintenance and reprogramming of the oocyte, resulting in premature ovarian failure and female infertility, indicating the important role of maternal mRNA accumulation in female reproductive capacity. In addition, we have also studied the function of the transcription factor YAP in the activation of the zygote genome. The YAP is highly expressed in the oocyte and in the early embryo, but in the cytoplasm it is located in the cytoplasm and cannot be combined with the transcription factor TEAD to play a transcriptional activity. However, after fertilization, the YAP entered the nucleus, suggesting the role of YAP-TEAD in early embryonic development. Therefore, we specifically knock the Yap1 gene (coding YAP protein) in the oocyte. As a result, the Yap1 knockout oocytes were found to be mature and fertilized, but the early embryos formed were slow to develop and died within three or four days. To track the cause of the death of these embryos, it was found that a number of genes that were essential for early embryonic development could not be expressed in time, including two important genes, Rrm2 (ribbonotides 2) and Rpll3 (ribnomal protein like 13), which were involved in the synthesis of nucleic acids and proteins, respectively. Through this study, we found that the mother had stored the transcription factor YAP in the egg cell and activated them in the early stage of the development of the embryo by using the LPA in the fallopian tube liquid, so that the transcription of the embryo gene was started in time in the MZT process. These results provide a new way to improve the further improvement of assisted reproduction and embryo engineering.
【学位授予单位】：浙江大学
【学位级别】：B
【学位授予年份】：2016
【分类号】：R321
【引证文献】
相关硕士学位论文 前1条
1 曾冰冰;中医药治疗未破裂卵泡黄素化综合征致不孕的临床研究[D];南京中医药大学;2017年
，本文编号：2361069
本文链接：https://www.wllwen.com/yixuelunwen/jichuyixue/2361069.html
