Sertoli细胞分泌因子FGF5调控精原干细胞增殖与自我更新的分子机制

The proliferation and self-renewal of spermatogonial stem cell (SSC) highly depend on the strict regulation provided by the Niche, which is mainly composed of growth factors secreted by Sertoli cells. However, human SSC cannot be cultured and differentiated to mature sperm in vitro using growth factors known. Our previous studies have revealed that the expression of FGF5 in Sertoli cells from Sertoli cell-only syndrome patients is significantly lower than that from men with normal spermatogenesis. Further studies demonstrate that FGF5 promotes the proliferation of SSC in vitro via activating ERK1/2 and PI3K/AKT pathway, indicating that FGF5 plays crucial roles in the fate determination of SSC. However, the underlying molecular mechanisms remain unknown. Therefore, we will conduct this important project with the following aims: i) to validate the expressions characteristics of FGF5 and its receptors in human testes; ii) to explore the effects of FGF5 exerted on the proliferation and self-renewal of SSC using gene overexpression and silencing techniques via in vitro Sertoli cell and SSC co-culture system; iii) to reveal the biological effects of FGF5 in vivo using seminiferous tubule transplantation and ectopic seminiferous tubule regeneration models. Eventually, the molecular mechanisms involved in SSC proliferation and self-renewal could be elucidated. The present study is of particular significance, because it could optimize the human SSC culture system, underlie a novel pathological mechanism of spermatogenic dysfunction and, further offer new targets for the repair of spermatogenic damage and the management of azoospermia.

精原干细胞（SSC）的增殖和自我更新，高度依赖Sertoli细胞分泌因子等微环境的严密调控。然而应用已知细胞因子，无法实现人SSC长期培养扩增，更无法获得SSC来源的成熟精子。课题组前期研究发现FGF5在唯支持细胞综合征患者Sertoli细胞表达异常降低，并证实FGF5能激活ERK1/2和PI3K/AKT通路，促进SSC体外增殖，这提示FGF5在SSC命运调控中发挥关键作用。然而其分子机制尚未阐明。本课题拟：1）明确FGF5及其受体在人睾丸组织的定位与表达特征；2）在Sertoli细胞与SSC共培养体系中，采用基因过表达或沉默技术，探讨FGF5如何调控SSC增殖和自我更新；3）应用生精小管注射和生精小管异位重建技术，揭示FGF5在体内对SSC的影响。本课题将揭示FGF5调控SSC命运的分子机制，有助于优化SSC的培养体系，阐释精子发生障碍的机理，提供生精功能损伤修复及无精子症诊治的新靶点。

精子发生是精原干细胞（Spermatogonial stem cell, SSC）自我更新和分化为成熟精子的过程。Sertoli细胞是生精小管内唯一的体细胞，能够分泌大量的细胞因子，为SSC提供适宜微环境（Niche），使其不断自我更新和增殖，保证精子发生持续稳定地进行。Sertoli细胞分泌功能异常将引起Niche稳态失调，造成SSC自我更新和分化障碍，生精功能损伤，导致无精子症。然而Sertoli细胞参与Niche和精子发生调控的具体分子机制尚未阐明。申请者通过RNA-Seq数据分析提示FGF5在生精功能正常男性Sertoli细胞中表达丰度高，而在生精功能异常的唯支持细胞综合征（SCOS）患者Sertoli细胞中表达异常降低，提示FGF5是Sertoli细胞分泌的调控Niche与SSS增殖和自我更新的关键因子。基于此，课题组围绕FGF5开展了本研究，主要内容包括FGF5及其受体在人睾丸组织的定位与表达特征；FGF5信号通路调控SSC增殖和自我更新的作用和机制。通过本研究，课题组发现FGF5在人类睾丸组织中仅表达于Sertoli细胞，在SSC并无表达。同时我们发现FGF5受体FGFR1和FGFR2基因在SSC均有较高表达。申请者通过向SSC细胞培养液中添加FGF5因子，进一步研究发现FGF5能提高细胞增殖活性，该作用具有时间和剂量依赖性。我们采用信号通路芯片研究方法，筛选出FGF5所激活的下游信号通路；进一步通过Western-blot验证显示，FGF5能激活ERK1/2和AKT，上调PCNA、Cyclin A2 和Cyclin E1。睾丸组织体外培养研究发现FGF5对睾丸组织存活和维持生精上皮完整性有一定作用，动物在体实验研究发现FGF5也影响异体移植人类睾丸组织存活。这些结果表明FGF5作为Sertoli细胞的重要分泌因子，在SSC的增殖和自我更新过程发挥十分关键的作用。本课题揭示了FGF5调控SSC命运的分子机制，有助于优化SSC的培养体系，阐释精子发生障碍的机理， FGF5可能是培养SSC和治疗男性不育的新靶点。