睾丸支持细胞Uhrf1基因缺失致雄性不育的分子基础及机制研究

Structural and functional abnormalities of Sertoli cells (SCs) are closely related to male infertility. It has been suggested that the dysregulation of epigenetic modifications, in particular the DNA methylation, are involved in the development of male infertility. However, the underlying epigenetic mechanisms need to be further investigated. The epigenetic regulator UHRF1 has well-established roles in the maintenance of DNA methylation, and therefore contributes to several physiological and pathological processes including cell cycle, differentiation, embryonic development and tumorigenesis. Of note, the role of UHRF1 in testis development and spermatogenesis has not been reported yet. We previously discovered the unique temporal and spatial expression pattern of UHRF1 in SCs during testis development in mice. We successfully generated a conditional knockout mouse model with SCs-specific deletion of Uhrf1 gene. Interestingly, we observed that the ablation of Uhrf1 in SCs caused damage to the morphological structure of SCs, induced DNA hypomethylation in SCs, and led to infertility in male mice. This project intends to use knockout mice and their primary SCs, using high-throughput sequencing and other molecular technologies to reveal the effects of the ablation of UHRF1 in SCs on genomic DNA methylation and proteomics, to identify key proteins interacting with UHRF1. This research will uncover the biological functions and molecular mechanisms of UHRF1 underlying the cross-talk between SCs and germs cells during spermatogenesis. Our overall goal of this project is to provide novel insights to enhance our understanding of the epigenetic mechanisms underlying the regulation of spermatogenesis by SCs, thus putting forward a new theoretical basis for the targeted therapy for idiopathic azoospermia.

睾丸支持细胞(SCs)结构和功能异常与男性不育的发生密切相关，以DNA甲基化为代表的表观遗传改变参与以上病理过程，但确切机制尚不清楚。DNA甲基化调控因子UHRF1参与调控细胞周期、分化、胚胎发育及肿瘤发生等生理病理过程，但UHRF1与睾丸发育和精子发生的关系尚未见报道。申请人前期研究发现UHRF1在小鼠睾丸组织SCs中呈时空特异性表达；条件性敲除SCs中Uhrf1基因导致SCs形态结构异常、DNA低甲基化和小鼠不育，由此推测UHRF1在SCs分化发育中发挥重要作用。本项目拟采用基因敲除小鼠及其原代SCs，应用高通量测序等技术检测Uhrf1基因敲除对SCs基因组DNA甲基化和蛋白组的影响，验证与UHRF1互作的关键蛋白质，阐释UHRF1在SCs和生精细胞“交互对话”过程中的生物学功能及分子机制，为SCs调控精子发生的表观遗传修饰机制和特发性无精子症的靶向治疗提供新的理论依据。

睾丸支持细胞 (Sertol cells, SCs) 结构和功能异常与男性不育的发生密切相关，以DNA甲基化为代表的表观遗传改变参与以上病理过程，但确切机制尚不清楚。DNA甲基化调控因子UHRF1参与调控细胞周期、分化、胚胎发育及肿瘤发生等生理病理过程，但UHRF1与睾丸发育和精子发生的关系尚未见报道。本项目研究小鼠SCs中UHRF1在精子发生中的作用，同时，探讨SCs中Uhrf1对SCs结构和功能的影响及其构筑的生精微环境调控生精细胞发生的具体分子机制。我们发现UHRF1在人和小鼠睾丸部分不成熟SCs中表达，而在成熟SCs中不表达，体外促增殖环境可诱导成熟原代SCs中UHRF1重新表达。SCs中Uhrf1敲除导致雄性小鼠睾丸体积明显变小，生殖细胞凋亡、数量减少、大量不成熟生殖细胞从生精小管上皮脱落，从而造成雄性不育，但SCs中UHRF1缺失对早期生精细胞增殖分化不产生影响。SCs中Uhrf1敲除后，SCs的增殖能力在P3至P5时期降低，之后增殖能力无明显改变，后期分化成熟相对有所延迟；Uhrf1敲除的SCs极性消失，F-actin分布紊乱，在圆形精子周围大量异常聚集，尤其是在精子发生的第I-VIII期；同时Uhrf1 缺失导致SCs内脂滴异常堆积，脂代谢异常，睾丸血-睾丸屏障受损，SCs与生殖细胞粘附与沟通遭破坏；Uhrf1缺失的SCs中与细胞外基质构成和细胞黏附相关基因大部分表达显著上调，同时伴随部分转座子的异常激活，尤其是IAP转座子；过表达细胞外基质和细胞黏附相关基因同样会破坏SCs骨架结构及SCs-生殖细胞间连接，导致大量不成熟SCs从生精小管上皮脱落。Uhrf1作为表观遗传调控因子主要调控SCs的DNA甲基化，控制SCs细胞外基质和细胞间黏附相关基因的甲基化水平，调节SCs和生殖细胞间连接和沟通，对精子发生是必需的。SCs中UHRF1功能一旦缺失，SCs细胞微观结构、细胞极性及脂质代谢受损，进而诱发SCs-生殖细胞连接沟通出现障碍，最终导致生殖细胞大量脱落，精子发生异常和雄性不育。虽然我们在本项目中揭示了UHRF1同样在人不成熟SCs中表达，但其具体的作用机制还有待在临床研究中进一步去验证，从而更好地为临床男性不育的诊断及治疗提供理论性指导。