NLR家族基因在生殖细胞分化及精子发生中的功能和调控机制研究

Male germ cells development that starts from primordial germ cells (PGCs) to mature sperms is a long and rigorous regulatory process. Although the expression of many genes is differentially regulated in this process, most of their functions are elusive. Recent progress in the induced differentiation of PGC-like cells from embryonic stem cells (ESCs) in vitro has enabled the systematic study of genes and their regulatory mechanisms during PGC development. From our preliminary results, a novel gene, Nlrp14, was found to have important roles in PGC differentiation and spermatogenesis. Nlrp14 is one of the most differentially regulated genes during PGC differentiation, knockdown or knockout of which strongly inhibited PGC differentiation from mESCs. In vivo study revealed that Nlrp14 deficiency caused male infertility by reducing sperm counts, motility issues and increased percentage of abnormal sperms. Most of germs cells were trapped in the spermatogonia and pre-meiotic stage in knockout mice. In this proposal, we would like to comprehensively explore the molecular mechanism of mouse Nlrp14 in germ cell differentiation and spermatogenesis through a combination of knockout and transgenic mouse models, cell biological and biochemical approaches. Our study will not only provide a full capture of the Nlrp14 molecular mechanism in male germ cells development in mice but also provide a potential new target for diagnosis and intervention therapeutic in human infertility treatment.

小鼠的生殖细胞发育是一个复杂精密的分子调控过程，仍存在大量基因的功能和调控机制不明确。胚胎干细胞（ESCs）向原始生殖细胞样细胞（PGCLCs）体外分化体系的建立为开展系统性基因功能发掘和机制解析带来新途径。在前期研究中，经转录组数据分析结合基因编辑，我们发现Nlrp14缺失显著抑制PGCLCs分化；Nlrp14敲除雄鼠亦呈现精子数量少、活力低、畸形精子比例增加，且大部分细胞停滞在精原细胞期和减数分裂前期，提示Nlrp14在生殖干细胞发育与分化中具有重要功能。基于此，本项目拟通过Nlrp14敲除小鼠和转基因小鼠模型，结合细胞与生化技术手段，深入探究Nlrp14在生殖细胞分化和精子发生过程中的作用和调控机制，其不仅有利于解析雄性生殖细胞发育中新基因的新功能，也可为进一步开展疾病诊断和干预提供新靶点、新思路。

小鼠的生殖细胞发育是一个复杂精密的分子调控过程，目前仍存在大量基因的功能和调控机制不明确。胚胎干细胞（ESCs）向原始生殖细胞样细胞（PGCLCs）体外分化体系的建立为开展系统性基因功能发掘和机制解析带来新途径。基于此，本项目从已发表的转录组数据出发，经分析筛选获得10个候选基因，并基于shRNA敲降系统分别建立10个候选基因敲降的小鼠胚胎干细胞（mESCs），通过PGCLCs体外分化系统最终鉴定出Nlrp14在PGCLCs的体外分化过程中具有重要作用。随后的Nlrp14敲除小鼠的构建，从在体水平发现Nlrp14的缺失显著降低雌雄小鼠的生殖能力，导致雄性小鼠精子数量减少，活力降低，畸形精子数量增加，并使大部分雄性生殖细胞停滞在精原干细胞期及减数分裂前期。机制方面，在小鼠精原干细胞系C18-4中过表达Nlrp14后，经免疫共沉淀（co-IP）及质谱分析发现，NLRP14与HSPA2具有互作关系，而HSPA2已被报道参与雄性生殖细胞的分化及精子发生。进一步实验研究显示，NLRP14通过招募BAG2与HSPA2互作保护HSPA2免于泛素化介导的蛋白酶体降解过程，进而促使HSPA2的核转移。而以往研究显示HSPA2与精子DNA的包装相关。值得注意的是，人源NLRP14-HSPA2-BAG2复合物的形成也可以保护HSPA2免于泛素化介导的蛋白酶体降解过程，而该复合物很可能与男性生殖相关。综上所述，该研究首次揭示了Nlrp14与HSPA2及BAG2形成的复合物抑制HSPA2的泛素化并促使其核转移，进而参与原始生殖细胞样细胞（PGCLCs）与精子发生过程。该发现揭示了一种由蛋白酶体调控的，非经典的在PGCLCs和精子发生过程中Nlp14的功能，为性腺特异性NLRs家族成员在生殖系统发育过程中的分子机制提供全新的视野，也可为进一步开展疾病诊断和干预提供新靶点、新思路。