FSH效应因子SF-1调控精原干细胞增殖机制及其激动剂促进模型小鼠生精研究

Male factor infertility is a common disease mainly with spermatogenesis abnormalities including oligozoospermia, asthenospermia and azoospermia. Follicle stimulating hormone (FSH) treatment improved the recovery of spermatogenic function among some patients. The targets as well as regulatory pathways in male germ cells regulated by FSH are still unknown. Our previously reported results indicated that FSH promoted the proliferation of spermatogonial stem cells (SSCs) co-clutured with Sertoli cells and that FSH responsive genes,SF-1 directly binded to the promoter of GDNF which is a key gowth factor for SSCs self-renewal. Therefore, we suppose that FSH is the main hormone in regulation of the proliferation of SSCs in vivo and affects the GDNF expression by SF-1 within the Sertoli cells to improve the proliferation of male spermatogonial stem cell in paracrine manner. In the present study, many methods including co-immunoprecipitation, electrophoresis mobility shift assays, site-directed mutagenesis and gene knockout animal models will be used to analyze the regulation mechanisms of SF-1 on the proliferation of SSCs in vivo and the interactions between SSCs and Sertoli cells. It will clarify the molecular mechanisms of SSCs development in vivo to find out the pathological causes and provide new therapeutic targets for male infertility.

男性因素引起的不孕不育症是临床常见疾病，主要表现为少、弱或无精子症。卵泡刺激素（FSH）治疗，部分患者症状改善，恢复生精功能。但是FSH调控雄性生殖细胞靶点不明，调控路径未知。我们前期的研究结果表明，在精原干细胞-支持细胞共培养体系中添加FSH，可以促进精原干细胞增殖；FSH的效应基因SF-1直接与精原干细胞增殖的关键细胞因子GDNF的启动子结合，促进GDNF转录。因此，我们认为：FSH是调控体内精原干细胞增殖的主要激素，其作用方式是通过支持细胞内SF-1调控GDNF表达，以旁分泌的方式影响雄性精原干细胞的增殖。本研究中，我们将通过凝胶迁移实验、基因定点突变、基因敲除动物模型等技术，分析SF-1调控体内精原干细胞增殖的机制、支持细胞与精原干细胞之间的相互作用，阐明精原干细胞体内发育的分子机制，为临床药物干预无精子症提供方案与依据。

本研究使用免疫组织化学染色、Western blot、RT-qPCR、ELISA、流式细胞术、ChIP、荧光素酶报告基因等实验方法，条件性敲除小鼠模型等，探究了（1）EHD3在小鼠睾丸组织中的表达规律及其生物学功能；（2）EHD3对睾酮分泌的影响及其作用机制；（3）在条件性敲除小鼠模型中验证EHD3对睾酮的分泌及其作用机制。结果如下：.1．EHD3在小鼠睾丸间质细胞中呈先增加后降低的表达趋势，并且在出生28及42天的睾丸间质细胞中有特异性的高表达。.2．EHD3具有促进细胞增殖，抑制细胞凋亡的生物学功能。.3．EHD3过表达后促进cAMP水平及其合成酶的表达，上调类固醇合成酶StAR、3β-HSD、Cyp11a1及Cyp17a1的表达，显著增加睾酮的分泌。EHD3敲降后，细胞中cAMP水平显著降低，cAMP合成酶PKAβ、PKAα、Ar2α、Lhcgr、AC及类固醇合成酶的表达均显著下调，睾丸间质细胞睾酮分泌受到明显抑制。更重要的是，干扰EHD3后，睾丸间质细胞摄取外泌体的内吞转运功能受到显著抑制。.4．转录因子NR5A1(SF-1) 结合在EHD3启动子区的NBRE元件上，通过促进EHD3启动子活性，正向调控EHD3的转录。.5．NR5A1过表达促进细胞内cAMP水平及其合成酶PKAβ、PKAα、Ar2α、Lhcgr、AC的表达，上调类固醇合成酶StAR、3β-HSD、Cyp11a1及Cyp17a1的表达，促进睾酮分泌。相反，NR5A1干扰后，细胞中cAMP水平显著降低，cAMP合成酶及类固醇合成酶的表达显著下调，睾酮浓度显著降低。与EHD3结果一致的是，干扰NR5A1后，间质细胞摄取外泌体的内吞转运功能受到抑制。在NR5A1过表达的原代睾丸间质细胞中转染EHD3干扰RNA，睾酮浓度显著降低。.6．NR5A1敲除后显著抑制EHD3的表达，类固醇合成酶StAR及3β-HSD的表达也受到显著抑制。NR5A1敲除后，睾丸组织中cAMP水平及血清中睾酮浓度显著降低。NR5A1敲除后显著抑制内吞作用依赖的关键蛋白Clathrin的表达。.以上结果揭示了EHD3及NR5A1(SF -1)在小鼠睾丸组织中的时空表达规律，发现EHD3受上游转录因子NR5A1的正向调控，通过介导细胞内吞作用影响睾酮分泌。为进一步探索激素分泌机制提供新的研究方向，也为解决睾酮缺乏导致的临床疾病提供新