RNA结合蛋白hnRNPU调控睾丸支持细胞的功能和作用机制研究

Testis development is a complicated biological process in which is regulated by multifactors in mammals, and contributions of Sertoli cells are a major component of the microenvironment that supports development and differentiation of germ cell in seminiferous tubule. Failure to form a functional Sertoli cell pool can lead to male infertility. However, the underlying mechanism of Sertoli cells regulate testis development is largely unknown. We previously found that RNA binding protein hnRNPU was highly expressed in mouse testis, and conditional mutation of Hnrnpu in Setoli cells caused male mice infertility. Surprisingly, we also found that the expression of hnRNPU protein was decreased in testis derived from Sertoli cell only syndrome patients. Therefore, we hypothesized that hnRNPU may have crucial roles in Sertoli cells development and testis development. In this project, we plan to utilize the Hnrnpu conditional knockout mouse model to analyze the male sterile phenotype，screen the interacting proteins of hnRNPU, and detect the abnormal expression of transcriptome upon Hnrnpu deletion in Sertoli cells by RNA-seq and RIP-seq. This research will reveal the functions and regulatory mechanism of hnRNPU in testis development, providing a new basic theory for the diagnosis and treatment of Sertoli-cell-only syndrome caused by genetic factors.

哺乳动物睾丸发育是一个受多因素精密调控的复杂过程，睾丸支持细胞在此过程中为生殖细胞的发育、分化提供稳定的生精小管内微环境。支持细胞功能异常会导致不育症的发生，但是目前对支持细胞参与调节睾丸发育的调控机理的了解还十分有限。申请人在前期工作中发现RNA结合蛋白hnRNPU在人和小鼠睾丸支持细胞中高表达。支持细胞条件性敲除Hnrnpu基因后雄性小鼠不育，并且发现唯支持细胞综合征病人睾丸中hnRNPU的表达降低。因此推测hnRNPU对睾丸发育有重要的调控作用。本课题拟利用支持细胞条件性敲除Hnrnpu的小鼠模型，首先分析敲除小鼠的不育表型，然后利用RNA-seq、RIP-seq分析hnRNPU缺失后的转录组的变化，并挖掘hnRNPU的蛋白互作网络。本项目可望揭示hnRNPU在小鼠睾丸支持细胞中的生物学功能及调控机制，为由遗传因素导致的唯支持细胞综合征的诊治提供新的理论基础。

支持细胞是分化性腺中睾丸命运的重要调节因子，然而它在调节青春期前睾丸发育中的作用和潜在的分子机制尚不清楚。尽管已经确定了支持细胞发育和功能的几个关键调节因素，但在新生睾丸发育过程中，调节生殖细胞增殖和迁移过程的外源性因素在很大程度上仍是未知的。我们的研究鉴定了一种共转录因子hnRNPU，它在小鼠和人类支持细胞中高表达，是新生支持细胞和青春期前睾丸发育所必需的。小鼠支持细胞中hnRNPU的条件敲除导致严重的睾丸萎缩、不能产生精子，表现为雄性不育，其特征是伴随新生小鼠生精小管结构异常的支持细胞和生殖细胞的快速耗竭以及青春期前睾丸发育过程中精原细胞增殖和迁移的失败。在分子水平上，我们发现hnRNPU与两个支持细胞标记物WT1和SOX9相互作用，ChIP-qPCR实验证明hnRNPU蛋白通过直接结合到它们的启动子区域来增强Sox8和Sox9两个转录因子在支持细胞中的表达。进一步的RNA-Seq和生物信息学分析揭示了支持细胞和生殖细胞命运控制所必需的关键基因，如生物粘附、增殖和迁移调控基因，在支持细胞特异性hnRNPU突变体睾丸中被异常调控。我们的发现证明了hnRNPU在支持细胞中对青春期前睾丸发育和睾丸微环境维持的重要作用，并为我们理解支持细胞功能异常相关的男性不育治疗提供了新的见解。