卵母細(xì)胞轉(zhuǎn)錄組的調(diào)節(jié)及其在卵泡存活和早期胚胎發(fā)育中的作用
發(fā)布時(shí)間：2018-11-27 14:12
【摘要】：卵泡是卵巢結(jié)構(gòu)和功能的基本單位,能夠產(chǎn)生成熟的卵子并合成雌激素維持正常的生理周期。其中,原始卵泡作為雌性生殖細(xì)胞的儲(chǔ)存庫(kù),在成年個(gè)體中不再增加,只會(huì)隨著生殖活動(dòng)不斷減少。所以卵巢中原始卵泡的規(guī)模直接決定了雌性生育年齡,過(guò)早消耗將導(dǎo)致卵巢早衰的發(fā)生。但是卵泡維持的分子機(jī)理和卵巢早衰的發(fā)病機(jī)制的研究還有很多未知內(nèi)容。本研究中,我們利用卵母細(xì)胞特異性的基因敲除模型來(lái)研究卵母細(xì)胞發(fā)育、卵母細(xì)胞減數(shù)分裂和早期胚胎發(fā)育過(guò)程中轉(zhuǎn)錄組的變化以及表觀遺傳調(diào)節(jié)因子在這些生理過(guò)程中的作用,闡明雌性生殖力維持的重要機(jī)制,為臨床上不孕不育的病例提供新的科學(xué)解釋和科學(xué)依據(jù)。因此,本課題分別研究了E3泛素化連接酶CRL4復(fù)合體、CpG結(jié)合蛋白CFP1和轉(zhuǎn)錄輔因子YAP在雌性生殖中的作用,系統(tǒng)地分析了轉(zhuǎn)錄組調(diào)節(jié)在卵母細(xì)胞維持和合子基因組激活中的作用。我們的研究發(fā)現(xiàn)CRL4通過(guò)激活TET家族DNA去甲基化酶的作用,調(diào)節(jié)卵母細(xì)胞中基因的表達(dá)和受精后雄原核的去甲基化,從而維持卵母細(xì)胞的存活和促進(jìn)受精卵的發(fā)育,在雌性生殖力維持中發(fā)揮至關(guān)重要的作用。如果CRL4的功能缺失,就會(huì)導(dǎo)致卵母細(xì)胞死亡,發(fā)生卵巢早衰和胚胎受精后不發(fā)育。在這些敲除的卵母細(xì)胞中,很多卵母細(xì)胞里高表達(dá)基因的表達(dá)量顯著降低,而這些基因啟動(dòng)子中CpG島的甲基化水平明顯升高。而且DDB1 (damaged DNA binding protein 1)敲除的卵母細(xì)胞受精后大部分不能發(fā)育到四細(xì)胞,早期胚胎發(fā)育所必需的基因的甲基化水平大幅升高,從而導(dǎo)致基因沉默。進(jìn)一步的機(jī)理研究發(fā)現(xiàn),CRL4通過(guò)調(diào)節(jié)TET家族DNA去甲基化酶的作用,調(diào)節(jié)卵母細(xì)胞中基因的表達(dá)和受精后雄原核的去甲基化,從而維持雌性生殖力。DNA結(jié)合蛋白CFP1是組蛋白甲基化復(fù)合體SET1中的重要亞基,識(shí)別基因組中處于非甲基化狀態(tài)的CpG,介導(dǎo)特定染色體區(qū)域組蛋白H3K4的三甲基化,從而調(diào)控大量基因的轉(zhuǎn)錄。我們的研究發(fā)現(xiàn)CFP1敲除的雌鼠完全不育。CFP1在卵母細(xì)胞中調(diào)節(jié)下游基因轉(zhuǎn)錄,敲除CFP1造成母源mRNA不能積累,導(dǎo)致卵母細(xì)胞不能維持以及卵母細(xì)胞受精后不能發(fā)育。這些結(jié)果說(shuō)明CRLA活性缺失造成的DNA高甲基化和CFP1敲除導(dǎo)致的mRNA轉(zhuǎn)錄下降都嚴(yán)重地阻礙了卵母細(xì)胞的維持和重編程能力,造成卵巢早衰和雌性不育,闡明了母源mRNA積累對(duì)于雌性生殖能力的重要作用。另外一方面,我們還研究了轉(zhuǎn)錄輔因子YAP在合子基因組激活中的功能。YAP在卵母細(xì)胞和早期胚胎中高表達(dá),但是在卵母細(xì)胞中卻定位在細(xì)胞質(zhì)中,不能與轉(zhuǎn)錄因子TEAD結(jié)合發(fā)揮轉(zhuǎn)錄活性。但是在受精后,YAP進(jìn)入細(xì)胞核,暗示了YAP-TEAD在早期胚胎發(fā)育中的作用。因此我們特異性地在卵母細(xì)胞中敲除了Yap1基因(編碼YAP蛋白)。結(jié)果發(fā)現(xiàn),Yap1敲除的卵母細(xì)胞雖然能夠正常成熟和受精,但是形成的早期胚胎卻發(fā)育遲緩,并在三四天內(nèi)死亡。追蹤這些胚胎的死亡原因,發(fā)現(xiàn)是許多對(duì)于早期胚胎發(fā)育至關(guān)重要的基因不能及時(shí)被表達(dá)出來(lái),其中包括兩個(gè)分別參與核酸和蛋白質(zhì)合成的兩個(gè)重要基因Rrm2 (ribonucleotide reductase 2)和Rpll3 (ribosomal protein like 13).通過(guò)這個(gè)研究,我們發(fā)現(xiàn)母體在卵細(xì)胞中儲(chǔ)存了轉(zhuǎn)錄輔因子YAP,又利用輸卵管液中的LPA在胚胎發(fā)育早期激活了它們,從而在MZT過(guò)程中及時(shí)啟動(dòng)了胚胎基因轉(zhuǎn)錄。這些結(jié)果為輔助生殖和胚胎工程技術(shù)的進(jìn)一步改進(jìn)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：B
【學(xué)位授予年份】：2016
【分類號(hào)】：R321
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：B
【學(xué)位授予年份】：2016
【分類號(hào)】：R321
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 曾冰冰;中醫(yī)藥治療未破裂卵泡黃素化綜合征致不孕的臨床研究[D];南京中醫(yī)藥大學(xué);2017年
，本文編號(hào)：2361069
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