H3K9me3对牛精原干细胞干性的调控作用及其分子机制

Spermatogonial stem cells (SSCs) are natural adult stem cells capable of continuous self-renewal and gene transfer to offspring. They have great potential application prospects in basic research and livestock husbandry. Currently, there are still several essential issues regarding researches of bovine SSCs, such as the difficulty in isolation and identification, instability of the culture system as well as the insufficient maintenance of their stemness, etc. Previously, we carried out the cryopreservation of bovine testicular tissues and conducted the isolation and enrichment, verification of the surface marker, optimization of the culture system, and regulation of the epigenetic modifications, in bovine SSCs. We found that histone H3 lysine 9 trimethylation (H3K9me3) is closely related to the stemness of bovine SSCs. However, its regulatory function and the mechanism are still unclear. Using immunohistochemistry, RNA interference (RNAi), gene overexpression, RNA-Seq technology, etc. this project was proposed to characterize new surface markers and establish a long-term culture system of bovine SSCs, assess the effects of this system on the stemness maintenance and H3K9me3 level of bovine SSCs, determine the key histone methyltransferases and demethylases of H3K9me3 modification, and investigate the regulatory effects of down- and up-regulation of H3K9me3 level on the stemness of bovine SSCs. Together, this project aims to reveal the regulatory function and molecular mechanism of H3K9me3 modification on the stemness maintenance of bovine SSCs, thereby provide reference for the applications of SSCs-related theory and techniques in the protection of germplasm resources of endangered animals, genetic breeding of livestock, and generation of transgenic animals, etc.

精原干细胞（SSCs）是体内能持续自我更新并将基因传至子代的天然成体干细胞，在基础研究和畜牧业生产等领域有广阔应用前景。目前有关牛SSCs研究还存在分离鉴定难、培养体系不稳定、干性难维持等诸多问题。我们前期开展了牛睾丸组织冻存及SSCs分离纯化、表面标记确证、培养体系优化、表观修饰调控等研究，发现组蛋白甲基化修饰H3K9me3与牛SSCs干性的维持有密切关系，但其调控作用和机制尚不清楚。本项目拟寻找牛SSCs新的标记分子，建立其长期培养体系，采用免疫组化、RNAi、基因过表达、转录组学等技术，评价该体系对其干性维持和H3K9me3水平的影响，确定H3k9me3建立和去除的关键催化酶，探明上调和下调H3K9me3水平对牛SSCs干性的调节作用,揭示H3k9me3修饰调控牛SSCs干性的分子机制，为SSCs相关理论和技术在濒危动物种质资源保护、家畜遗传繁育、转基因动物生产等领域的应用提供依据。

精原干细胞（SSCs）是体内能持续自我更新并将基因传至子代的天然成体干细胞，在基础研究和畜牧业生产等领域有广阔应用前景。目前有关牛SSCs研究还存在分离鉴定难、培养体系不稳定、干性难以维持等诸多问题。针对以上问题，在前期基础上我们采用免疫组化、荧光定量PCR、RNAi、基因过表达、转录组学、细胞重编程、生物信息学等技术，开展了牛睾丸组织冻存及SSCs分离纯化、表面标记确证、培养体系优化、表观修饰调控、支持细胞重编程及睾丸炎预防和治疗等研究，取得了以下结果：1）确证了GFRα-1在牛SSCs及其前体细胞中的表达，可作为其分子标记；PLD6在牛生精细胞中表达，在特定阶段可作为牛生精细胞的分子标记；2）2i培养条件有促进牛SSCs及其前体细胞增殖和保持其干性的作用，可能是通过Mek1/2和Gsk3β信号通路抑制了甲基转移酶Suv39h1/2介导的H3K9me3而发生的；3）通过甲基转移酶抑制剂Chaetocin处理牛SSCs及用siRNA干扰SSCs的甲基转移酶SUV39H1，降低牛SSCs中的H3K9me3水平，发现SSCs增殖被抑制；而用去甲基化酶赖氨酸脱甲基酶4D（KDM4D）的siRNA上调SSCs中H3K9me3的水平，则促进SSCs增殖；4）用经典OSKM 4因子（Oct4, Sox2, Klf4, c-Myc）诱导法，将分离纯化的犊牛睾丸支持细胞诱导为iPSCs并分析了其转录组学特征，进一步将其重编程为原始态样（naïve-like）iPSCs，这些细胞核型正常，有体内外发育能力，可反复冻存后长期传代培养；5）建立了牛睾丸炎体内外模型，证实了抗菌肽MPX对牛睾丸炎有良好的预防和治疗效果。以上研究成果为阐明牛等大家畜精子发生机理及后续牛生精细胞体外发育机制研究奠定了基础，也具有在优质高产抗病家畜、特种经济价值动物及珍稀濒危动物繁育、种质资源保存等方面应用的潜在广阔前景。