【作者】 周晓龙；

【导师】 刘红林；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 原始卵泡募集后脱离原始卵泡库开始启动发育,经过初级、次级卵泡逐渐发育成有腔卵泡,有腔卵泡对促性腺激素更加敏感开始加速生长并逐渐优势化形成优势化卵泡,优势化的卵泡命运有两个即排卵和闭锁。优势化卵泡的闭锁跟卵泡内雌激素浓度降低不能引起LH峰有很大的关系,卵泡内颗粒细胞凋亡也是卵泡闭锁的潜在机制。长期以来,人们对卵泡闭锁做了大量的相关研究工作,但调控卵泡闭锁的分子机制还不完全清楚,仍然需要进一步的研究和探索。阐明这一生理过程的分子机理对于提高动物繁殖力、治疗动物排卵障碍等生殖疾病具有十分重要的意义。本论文选取小鼠为试验动物,研究FSH调控延时排卵优势卵泡闭锁及雌激素代谢相关基因Cyplbl的分子机制。主要进行了以下几方面的研究：(1)检测FSH对延时排卵优势卵泡内颗粒细胞凋亡及凋亡相关基因的影响。(2)检测延时排卵优势卵泡内雌激素代谢相关基因的表达情况。(3)FSH对雌激素代谢关键基因Cyplbl表达调控和雌激素代谢产物四羟基雌二醇对颗粒细胞的影响。(4) Cyplbl启动子区转录因子分析。(5)FSH对Cyplbl转录因子Statl的调控作用。(6)FSH调控Statl磷酸化水平的机制研究。综合本论文的研究结果,主要得出以下结论：(1)FSH通过抑制颗粒细胞凋亡抑制延时排卵优势卵泡的闭锁颗粒细胞的凋亡是小鼠卵泡闭锁的最主要诱因之一,研究发现FSH可以抑制延时排卵优势卵泡内颗粒细胞凋亡和凋亡相关基因如Caspase-3,9, BIM等的表达。(2)FSH抑制颗粒细胞雌激素代谢相关基因Cyplbl的表达研究发现小鼠卵泡内颗粒细胞雌激素代谢相关基因中只有Cyplbl一直表达,Cyplal, Cypla2, Cyp3a在小鼠颗粒细胞中几乎不表达。FSH在体内和体外环境都可以抑制颗粒细胞内Cyplbl基因的表达,Cyplbl催化雌二醇产生的代谢产物四羟基雌二醇(兰10μM)对体外培养的颗粒细胞也有显著的促凋亡作用。(3)FSH通过调控Statl磷酸化水平调控Cyplbl表达通过网站预测和实验分析发现Statl是Cyplbl的主要转录因子之一,染色质免疫共沉淀CHIP分析发现FSH可能通过调节转录因子Statl在Cyplbl启动子区的富集度调控Cyplbl的表达。研究发现FSH对Statl蛋白水平并没有显著的调控作用且Statl酪氨酸701位点磷酸化在颗粒细胞中检测为阴性,通过信号通路分析发现FSH可以通过p38MAPK信号通路激活ser、thr磷酸酯酶PP2A的活性,致使Statl丝氨酸727位点去磷酸化,使其从基因Cyplbl启动子区脱离调控卵泡颗粒细胞雌激素代谢相关基因Cyplbl的表达。本文在FSH抑制卵泡闭锁和雌激素代谢方面做了生理生化相关的研究,确定了FSH可以通过抑制颗粒细胞凋亡来抑制延时排卵优势卵泡的闭锁。阐明了FSH调控卵泡内雌激素代谢相关基因Cyplbl的分子机制。有助于卵泡闭锁分子机制的进一步阐明。更多还原

【Abstract】 Primordial follicles raised from the primordial follicles library started development. After primary, secondary follicles gradually develops into antral follicles, antral follicles are more sensitive to gonadotropin began to accelerate growth and form of dominant follicle. The fate of the dominant follicles is ovulation or atresia. The concentration of estrogen reduce within the follicle can not cause the LH peak have a great relationship with dominant follicle atresia, and granulosa cells apoptosis is also a potential mechanism of follicular atresia. Long time, people have done a lot of research work on the follicular atresia, but the molecular mechanism of regulation of follicular atresia is not entirely clear, still need further research and exploration. Clarify the molecular mechanism of this physiological process has a very important significance for improving animal fertility, reproductive diseases, treatment of animal ovulation disorders.The papers selected mice as experimental animals, studies FSH regulation of delayed ovulation dominant follicle atresia and estrogen metabolism genes Cyplbl molecular mechanisms. Conducted a study of the following aspects:(1) Detection the function of FSH on granulosa cell apoptosis in delayed ovulation dominant follicle and related apoptosis genes.(2) Detection of the delayed ovulation dominant follicle estrogen synthesis and metabolism gene expression.(1) Detection of FSH impact on granulosa cell apoptosis and related genes of delayeded ovulation dominant follicles.(2) Detection the expression estrogen metabolism-related genes of the delayeded ovulation follicles.(3) FSH regulated estrogen metabolism key gene Cyplbl expression and estrogen metabolite4-hydroxy-estradiol effect on the granulosa cells.(4) The transcription factor analysis of Cyp1b1promoter region.(5) The role of FSH on Cyplbl transcription factor Statl.(6) FSH regulation of Statl phosphorylation level mechanism. Comprehensive results of this thesis, the following conclusions:(1) FSH suppression delayeded ovulation dominant follicle atresia by inhibiting apoptosis of granulosa cellsGranulosa cell apoptosis is one of the main cause of mouse follicular atresia, we found that FSH can inhibit granulosa cell apoptosis and apoptosis-related genes expressios, such as Caspase-3,9, BIM to rescue delayeded ovulation dominant follicle atresia.(2) FSH suppression granulosa cell estrogen metabolism genes Cyplbl expressionThe study found that the crucial gene of delayed ovulation dominant follicle granulosa cells estrogen metabolism is Cyplbl. The genes Cyplal, Cypla2, Cyp3a in granulosa cells almost no expression. FSH can inhibit Cyplbl expression in granulosa cells in vitro and in vivo, estrogen metabolite4-hydroxy-estradiol (≧10μM) by Cyplbl has a significant effect on cultured granulosa cells apoptosis.(3) FSH regulation of Statl phosphorylation levels of regulation of Cyplbl expressionSite prediction and experimental analysis found that Statl is one of the important transcription factors of Cyp1b1. Chromatin immunoprecipitation analysis showed that FSH may through the transcription factor Statl in Cyplbl promoter region play a regulatory role. Further studies showed that levels of Statl protein is not a significant regulatory by FSH. Statl tyrosine701phosphorylation is not detected in the granulosa cells, but FSH could via p38MAPK activate ser, thr phosphatase PP2A activities lead dephosphorylation of Statl on ser727, and drop from the promoter region of Cyp1b1to regulate expression of estrogen metabolism-related gene Cyplbl in follicle granulosa cells.FSH suppression delayed ovulation follicles atresia and estrogen metabolism, physiological and biochemical studies to determine FSH suppression follicular atresia by inhibiting apoptosis of granulosa cells. Elucidate the molecular mechanism of FSH regulation of follicular estrogen metabolism gene Cyplbl. Help to further elucidate the molecular mechanism of follicular atresia.更多还原