【作者】 白莉雅；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 哺乳动物在胚胎时期有数十万个原始卵母细胞,但一生只能生产几个到几百个后代,卵母细胞利用率极低。迄今,有关有腔卵泡发育的调控机制基本清楚,有腔卵泡体外成熟的技术相对成熟。然而,有关腔前卵泡发育的调节机制虽然报道很多,但尚无定论,以致腔前卵泡体外培养与体成熟的技术还不完善,成为世界级研究热点和难点。为探讨腔前卵泡发育的调节机制,促进腔前卵泡体外培养、体外成熟、幼畜超排等技术的发展,提高卵泡利用率,本研究应用Q-PCR、West-blot、CHIP、BSP和基因免疫等技术,从DNA甲基化和组蛋白乙酰化角度分析了牛卵泡发育的表观遗传修饰作用；从基因表达模式和表观修饰调控两方面分析了生长因子GDF9(生长分化因子-9)和BMP15(骨形态发生蛋白-15)对卵泡发育的调控规律；从基因表达模式、DNA甲基化和组蛋白乙酰化等方面系统研究了抑制素对牛卵泡发育的调控及其分子机制；另从基因免疫角度通过体内、体外两种途径分析了生长抑素(SS)对牛卵泡发育的调控作用。主要研究内容和结果如下：1.DNA甲基化和组蛋白乙酰化表观修饰对牛卵泡发育的调控作用研究应用Q-PCR方法分析表观修饰相关酶类(DNMTs、HDACs、HAT1)在卵泡发育过程的变化规律,揭示在卵泡发育不同时期的表观修饰调节作用；通过体外添加甲基化转移酶抑制剂5-氮脱氧胞苷(5-aza-dc)及去乙酰化酶抑制剂丁酸钠(NaB),分析这两种抑制剂对卵母细胞和颗粒细胞相关表观修饰酶类的调控及卵母细胞成熟率的影响。研究结果如下(1)DNA甲基化和组蛋白乙酰化作用参与调节牛卵泡发育。DNA甲基化转移酶基因DNMT1、DNMT3a和DNMT3b的表达量随着卵泡的发育逐渐增高(P<0.05)；去乙酰化酶HDAC1和乙酰化转移酶HAT1的表达量也随着卵泡发育逐渐增高(P<0.05),而HDAC2仅在中卵泡阶段显著表达(P<0.05)；所有表观修饰酶类的表达量在即将排卵前卵泡都有所回落。表观修饰酶类的时空特异性表达揭示牛卵泡发育不同阶段的调控作用有差异。(2)DNA甲基化和组蛋白乙酰化作用参与卵母细胞成熟的调控。抑制剂5-aza-dc和NaB通过下调DNMT1、DNMT3b、HDAC1和HDAC2的表达(P<0.05),抑制DNA甲基化和组蛋白去乙酰化作用的发生,显著提高卵母细胞的成熟率(P<0.05)。2.GDF9和BMP15对牛卵泡发育的调控作用研究将牛卵泡按直径大小分为5个级别(<0.2mm；0.2-2mm；2-5mm；5-8mm；>8mm),即腔前卵泡、小卵泡、中卵泡、大卵泡及排卵前卵泡,应用Q-PCR及West-blot方法从mRNA及蛋白两个水平阐述不同时期卵泡中GDF9和BMP15基因的表达规律；分析经抑制剂5-aza-dc和NaB处理后COCs中GDF9和BMP15基因的表达变化。研究结果如下：(1)GDF9和BMP15在腔前卵泡、小卵泡、中卵泡、大卵泡和排卵前卵泡呈差异转录。GDF9和BMP15在小卵泡和大卵泡时期表达量显著增高(P<0.05)这种时空特异性表达揭示其参与调控牛的卵泡发育,主要在腔前卵泡期、排卵和黄体形成时发挥重要作用。(2)GDF9和BMP15蛋白在腔前卵泡、小卵泡、中卵泡、大卵泡和排卵前卵泡呈差异表达。GDF9和BMP15的表达量随着卵泡的发育不断增高(P<0.05),但在即将排卵前又有所下降。(3)抑制剂5-aza-dc和NaB通过下调DNMT1、DNMT3b、HDAC1和HDAC2的表达量(P<0.05),抑制DNA甲基化和组蛋白去乙酰化作用的发生,从而提高COCs中GDF9和BMP15基因的转录水平(P<0.05)。3.INHα对牛卵泡发育的调控作用及机制研究应用Q-PCR及、Vest-blot方法从mRNA及蛋白两个水平阐述不同时期卵泡中INHa基因的表达规律；分析抑制剂5-aza-dc和NaB处理后GCs中INHa基因的表达变化；利用活性分析筛选INHa的核心启动子；构建完全甲基化的启动子载体和P300结合位点突变载体,分析启动子活性,确定DNA甲基化和P300乙酰化的调节作用；用CHIP方法验证乙酰化作用对启动子的调控机制；用BSP法分析4个级别牛卵泡(0.2-2mm；2-5mm；5-8mm；>8mm)中CpG岛区的甲基化状态,最终阐明表观修饰作用对INHα启动子的调控机制。研究结果如下：(1)INHa在腔前卵泡、小卵泡、中卵泡、大卵泡和排卵前卵泡呈差异转录和表达(P<0.05)。INHa的转录水平在中卵泡和排卵前卵泡中显著增高(P<0.05),其蛋白表达随着卵泡的发育持续增高(P<0..05)。INHα在卵泡发育过程中的差异表达揭示其对FSH刺激产生应答,并负反馈调控GDF9和BMP15的表达,对优势卵泡形成和排卵有重要作用。(2)DNA甲基化和组蛋白乙酰化作用通过启动子调控INHα基因的转录。牛颗粒细胞INHa的核心启动子长度为1294bp(-1017bp-+277bp),抑制剂5-aza-dc和NaB抑制DNA甲基化和组蛋白去乙酰化作用的发生,能显著增强INHα的启动子活性(P<0.05),提高INHα基因的转录水平(P<0.05)。启动子区CpG位点完全甲基化可以显著降低INHa的启动子活性(P<0.05)。(3)DNA甲基化作用抑制转录因子AP2a对INHa启动子-907--804和-345--197区段的激活作用。抑制DNA甲基化作用,能够显著提高AP2a转录因子同-907--804和-345--197两区段的结合率(P<0.05)。(4)组蛋白乙酰化作用介导P300对INHα启动子-345--197区段的调控作用。抑制组蛋白去乙酰化作用,可以显著提高INHα启动子-907--804和-345--197区段组蛋白H3的乙酰化水平(P<0.05)；同时提高P300转录因子与-345--197区段的结合率(P<0.05)。(5)P300以桥梁或支架的形式发挥其乙酰化作用,参与-345--197区段的调控。-345--197区段结合位点突变能够显著增强INHa的启动子活性(P<0.05),显著增强P300同该区段的结合率(P<0.05),提高该区段组蛋白H3的乙酰化水平(P<0.05)。(6)INHα基因在卵泡发育中的表达变化不受近端启动子CpG岛DNA甲基化的影响。INHα启动子区CpG岛(大小131bp,位于-970bp到-840bp之间)在整个卵泡发育阶段的甲基化水平都极低(P>0.05),卵泡发育阶段INHα基因的表达变化同近端启动子CpG岛甲基化水平无关。4.SS对卵泡发育的调控作用研究从个体和细胞两个水平,分别用含生长抑素SS的pGM-CSF/SS质粒免疫雌鼠和转染颗粒细胞,分析SS对动物卵泡发育的调控作用。结果如下：(1)SS促进颗粒细胞凋亡,抑制卵泡发育。牛颗粒细胞过表达SS,可以诱导促凋亡基因Bax和P53的表达(P<0.05),下调凋亡抑制基因Bcl-2的表达(P<0.05),和雌激素分泌水平(P<0.05),促进颗粒细胞凋亡(P<0.05),抑制卵泡发育。(2)SS基因免疫可改善雌鼠的繁殖性能。小鼠免疫SS基因疫苗可促进雌激素分泌(P<0.05),缩短雌鼠的发情周期(P<0.05)。更多还原

【Abstract】 Although several hundred thousand original oocytes exist in embryonic period of mammals, there is only a few to hundreds of generations, and it is very low in utilization of oocytes. In antral follicle, the mechanism of development and technology of maturation in vitro are well studied. However, the technology of maturation of preantral follicle in vitro is difficulty and focus in the world, because it is inconclusive of the mechanism of development. In order to clarify the mechanism of preantral follicular development, promote the process of developmental biology, improve the techniques of maturation of preantral follicle in vitro and superovulation of young animals, and improve the utilization of follicles. In present study, Q-PCR, West-blot, CHIP, BSP and gene immunization were used, to elucidate whether epigenetic modifications such as DNA methylation and histone acetylation were involved in follicular development; to study the regulation of GDF9 and BMP15 in follicular development via the expression profile during folliculogenesis and epigenetic modifications; to explore the regulation and mechanism of INHa in follicular development via the expression profile during folliculogenesis and epigenetic modifications such as DNA methylation and histone acetylation; to investigate the regulation of SS in follicular development via transfection bGCs and immunization female mice with SS plasmid. The main results are as follows:1. Study the epigenetic regulation of DNA methylation and histone acetylation in bovine follicular developmentTo evaluate whether epigenetic modifications including DNA methylation and histone acetylation were involved in regulating bovine follicular development. Q-PCR analysis was utilized to determine expressions of several enzymes of DNA methylation and histone acetylation; to test expressions of DNMTs, HDACs, and HATs after treatments with inhibitors of DNA methylation and histone deacetylation; and oocyte maturing rate was also investigated after treatments with inhibitors. The results are as follows:(1) DNA methylation and histone acetylation were involved in regulating bovine follicular development. DNA methyltransferase DNMT1, DNMT3a, DNMT3b, and histone acetyltransferase HDAC1 and histone deacetylase HAT1 were triggered with follicular development (P<0.05), HDAC2 increased only at 2-5mm stage (P<0.05). However, expression levels of HAT, HDACs and DNMTs reduced in large follicles (diameter>8 mm). Differential expressions of epigenetic modified enzymes suggested that epigenetic switches were involved in regulating folliculogenesis.(2) DNA methylation and histone acetylation were involved in oocyte maturation. Inhibitors of 5-aza-dc and/or NaB enhanced oocyte maturing rate (P<0.05), by down-regulation of transcription of DNMT1, DNMT3b, HDAC1 and HDAC2 (P<0.05).2. Explore the regulation of GDF9 and BMP15 in bovine follicular developmentExpression profiles of GDF9 and BMP 15 genes in different follicular development stages were evaluated by Q-PCR and West-blot, the follicular development stages were divided into five groups according to the follicular diameter (preantral follicle:<0.2mm; small:0.2-2mm; medium:2-5mm; large:5-8mm; ovum:>8mm); Expressions of GDF9 and BMP 15 were investigated after treatments with inhibitors of DNA methylation and histone deacetylation.The results are as follows:(1) Transcriptions of GDF9 and BMP 15 were differential expression during follicular development. GDF9 and BMP 15 expression were up-regulated at 0.2-2 mm and 5-8 mm stage (P<0.05). Differential expressions of GDF9 and BMP 15 suggested the regulation in the non-steroid-dependent phase, ovulation and luteal formation during follicular growth.(2) Proteins of GDF9 and BMP 15 were differential expression during follicular development. The expression of BMP 15 and GDF9 increased with follicular growth (P<0.05), but decreased at>8 mm stage (P<0.05).(3) Inhibitors of 5-aza-dc and/or NaB activated expressions of GDF9 and BMP15 in COCs (P<0.05), by down-regulation of transcriptions of DNMT1, DNMT3b, HDAC1 and HDAC2 (P<0.05). 3. Investigate the regulation and mechanism of INHαin bovine follicular developmentExpression profile of INHa gene in different follicular development stages was evaluated by Q-PCR and West-blot; regulation of inhibitors in INHa gene was tested; the center promoter of INHa was determined by relative activity; the activity of fully methylated promoter and mutating the p300-binding site in 1294 luc promoter were tested. The mechanism of histone acetylation in promoter was explored by CHIP; methylation status of the CpG island within INHa proximal promoter at different stage of follicle (small:0.2-2mm; medium:2-5mm; large:5-8mm; ovum:>8mm) was determined by BSP. The results are as follows:(1) Transcription and translation of INHa were differential expression during follicular development. Transcription of INHa was up-regulated at 2-5mm, and peaked at>8 mm stage (P<0.05). INHa protein increased followed with follicular development (P<0.05). Differential expression of INHa suggested the important role in response of FSH stimulation, negative feedback regulation of the expression of GDF9 and BMP 15, the formation and ovulation of the dominant follicle during follicular growth.(2) Promoter was the target of DNA methylation and histone acetylation to modify INHa gene expression.The center promoter of INHa was 1294bp (-1017bp～+277bp), the activity of pGL-1294 activated significantly (P<0.05), the expression of INHa increased (P<0.05) after suppressing DNA methylation and histone deacetylation by inhibitors of 5-aza and/or NaB. The activity of methylated promoter reduced (P<0.05).(3) DNA methylation suppress activation of AP2a transcription factor in-907～-804 and-345～-197 within INHa promoter. Relative binding of AP2a to-907～-804 and-345～-197 increased after suppressing DNA methylation (P<0.05).(4) Histone acetylation was involved in regulation of P300 transcription factor in-345～-197 within INHa promoter. After suppressing histone deacetylation, the levels of H3 acetylation in-907～-804 and-345～-197 increased (P<0.05), and relative binding of P300 to-345～-197 increased (P<0.05).(5) P300 as bridge or stand was involved in regulation of-345～-197 by histone acetylation. After site-directed mutation of p300 binding sites, INHa promoter regulatory activity induced (P<0.05), the levels of H3 acetylation in-345～-197 increased (P<0.05), relative binding of P300 to-345～-197 increased (P<0.05).(6) Methylation levels of CpG island within INHαproximal promoter was not correlative with INHa expression during bovine follicle development.27 CpG sites of CpG island were hypomethylated or virtually unmethylated in all follicles from four different sizes (P>0.05).4. Investigate the regulation of SS in follicular developmentTo study the role of SS in follicular development, pGM-CSF/SS plasmid containing SS was used to transfection bovine GCs and immunization female mice. The results are as follows:(1) SS was involved in inhibition of follicular development, via induced apoptosis of GC. Overexpression in bovine GCs, induced expression of Bax and P53 which were pro-apoptosis genes (P<0.05), reduced expression of Bcl-2 which was anti-apoptosis gene (P<0.05), down-regulated E2 level (P<0.05), induced apoptosis of GCs (P<0.05) and repressed follicular development.(2) Immunization with pGM-CSF/SS plasmid could improve reproductive ability of female mice. Immunization with pGM-CSF/SS plasmid could induce secretion of E2 (P<0.05), shorten estrous cycle of female mice (P<0.05).更多还原