Nanos2维持水牛精原干细胞干性特征机制的研究

Nanos2 is an important RNA-binding protein, which plays a pivotal role in the sexual differentiation of male germ cells and is required for maintaining homeostasis of SSCs. However, the function and the underlying mechanism of Nanos2 in male germ cell of domestic animals are poorly understood, especially in buffalo. This project will focus on the following contents with the buffalo as research object: We firstly investigate co-localization status of NANOS2 and candidate SSCs membrane surface proteins by immunofluorescence staining to screen the specific membrane surface protein of buffalo SSCs. And then SSCs are sorted with the selected biomarker. We establish buffalo SSCs cell lines of Nanos2-overexpresison, Nanos-RNAi and Nanos2-mutation to investigated the changes of stem cell characteristics and epigenetics status in these genetic modified SSCs cell lines. Meanwhile, we jointly analyze the mass spectrometry data collected by high resolution mass spectrometry with TMT labeling and high throughput transcriptase sequence data of these genetic modified SSCs, to further excavate the potential important signaling pathways that participate the function of Nanos2 maintaining the buffalo SSCs characteristic. The expected results of this project will help to improve the efficiency of the buffalo SSCs sorting system and expand our understanding of the molecular mechanisms by which Nanos2 regulates SSC characteristics，laying a theoretical foundation for promoting the related research on the culture and differentiation of domestic animals SSCs in vitro.

Nanos2是调控小鼠雄性生殖细胞发育的重要RNA结合蛋白，能促进生殖细胞向雄性分化并维持精原干细胞（spermatogonial stem cells, SSCs）动态平衡，但大家畜Nanos2相关研究缺乏且机制不清楚。本项目拟以水牛为研究对象，主要开展以下研究：①采用Nanos2与候选SSCs膜表面蛋白免疫荧光共染色方法，筛选出水牛SSCs膜特异表面蛋白；②以特异表面蛋白作为标记分选SSCs，构建Nanos2过表达、RNAi和移码突变SSCs细胞系；③观察这三种细胞系的干性特征及表观遗传学变化；④联合分析这三种细胞系TMT外标定量质谱分析数据和转录组测序数据，挖掘潜在参与Nanos2维持SSCs干性特征的重要信号通路。本项目预期结果有助于进一步提高水牛SSCs分选效率，拓展我们对Nanos2维持SSCs干性特征相关分子机制的认知，为推动大家畜SSCs体外培养及分化相关研究奠定理论基础。

Nanos2是调控小鼠雄性生殖细胞发育的重要RNA结合蛋白，能促进生殖细胞向雄性分化并维持精原干细胞（spermatogonial stem cells, SSCs）动态平衡，但大家畜Nanos2相关研究缺乏且机制不清楚。我们通过探讨生物活性物质（Nanos2、LIF、GDNF、FGF2/FGF5）、小分子化合物（Melatonin、CHIR99021）及培养方式（SSCs与Sertoli细胞接触或非接触共培养）对水牛SSCs体外增殖的影响，优化了水牛SSCs体外培养系统，实现了水牛SSCs体外长久培养（＞3个月）。在此基础上，我们构建了水牛Nanos2过表达和Nanos2敲除的SSCs，并通过转录学解析了Nanos2过表达后SSCs代谢通路特征。研究结果显示，培养液中添加水牛源LIF和GDNF与商品化小鼠LIF和GDNF相比，能显著提高水牛SSCs体外增殖能力，显著提高Nanos2的表达水平；培养液中添加CHIR99021有利于水牛SSCs体外增殖和干性维持，显著提高Nanos2的表达水平；培养液添加FGF2和FGF5皆能促进水牛SSCs的体外增殖，但FGF2效果更佳；尤其是水牛SSCs与Sertoli细胞非接触培养方式能很好地支持SSCs体外增殖和干性维持，能有效维持水牛SSCs体外长久培养。差异转录组学分析结果表明，水牛SSCs中Nanos2的过表达，与对照组相比，具有显著性差异基因2659个，这些显著差异表达基因多数参与了与SSC自我更新和增值相关信号通路。显著上调基因多数参与了与SSC自我更新、增值及干性维持相关信号通路，显著影响了甲基化和rRNA甲基化等与表观修饰相关信号通路；而显著下调基因多数参与了细胞凋亡相关通路。在Nanos2过表达后，促进SSC分化的基因BMP4和SOX4表达量显著下调，而与SSC增殖及干性维持相关的基因FZD9、SOX17和WNT5B表达量显著上调。总之，通过本项目研究，建立起了水牛SSCs体外长久培养系统，初步阐明了Nanos2维持水牛SSCs干性特征的分子机制，为水牛SSCs体外诱导和基因编辑等研究奠定了基础。