mTORC1信号通路介导精氨酸调控公猪精原干细胞增殖的作用机制研究

Decreasing of semen volume and total sperm count is the universal problem of boar. In the previous study, the project applicant has found that arginine supplementation in the boar diet promoted the generation of sperm, improve semen production. The quantity and quality of boar spermatogonial stem cells is the premise and basis of sperm production. Research about the expression of target of rapamycin (mTOR) in different ages boar, suggested a critical role of mTOR in testicular development. However, whether the proliferation of boar spermatogonial stem cells and increasing sperm production by arginine is mediated by mTORC1 signaling pathway, has not been reported previously. The project will comparatively analyze the different genome expression of germ cell, and screening the candidate target genes of mTORC1 signaling pathway by using the transcriptomics technology and immunohistochemical method after boar fed with dietary arginine. Further, the cell model will be used for exploring the mechanism of mTORC1 signaling pathway in promoting cell proliferation induced by arginine by using flow cytometry, gene and protein expression detection technology. The study will play an important role in elucidating how mTORC1 signaling pathway mediate the effect of spermatogonial stem cells proliferation by arginine, understanding the mechanism of arginine in increasing the sperm count in-depth. This project is of great practical significance to improve boar fertility.

精子数量减少和精液产量下降是当前公猪普遍存在的问题。项目申请人前期研究发现，饲粮中添加精氨酸可促进公猪精子生成，提高精液产量。睾丸中精原干细胞的数量和质量是公猪精子生成的前提和基础，对公猪睾丸雷帕霉素靶蛋白（mTOR）时空表达规律的研究揭示了mTOR在睾丸发育中的关键作用。然而有关mTORC1信号通路如何介导精氨酸调控公猪精原干细胞增殖，进而影响公猪精子生成尚未见报道。本项目通过在后备公猪饲粮中添加精氨酸，利用RNA-seq高通量测序技术分析精原干细胞差异表达基因，筛选mTORC1信号通路的候选靶基因；结合精原干细胞体外培养模型，运用流式细胞仪、基因和蛋白表达检测技术，解析mTORC1信号通路介导精氨酸调控精原干细胞增殖的作用机制。该研究对阐明mTORC1信号通路介导精氨酸调节精原干细胞增殖和精子生成的机制具有重要的理论意义，对提高公猪繁殖力将具有重大的实践意义。

睾丸中精原干细胞的数量和质量公猪精子生成的前提和基础，对不同日龄公猪睾丸雷帕霉素靶蛋白（mTOR）时空表达规律的研究，揭示了mTOR在睾丸生殖细胞中的关键作用。本项目通过在后备公猪饲粮中添加精氨酸，结合蛋白组学、RT-qPCR、Western Blot等技术，发现饲粮添加0.8%精氨酸使后备公猪睾丸精原细胞数量增加16%，睾丸总重显著增加；精氨酸的补充显著提高了血清中FSH、LH和NO水平，增加睾丸中精氨酸和腐胺的浓度；蛋白组学154个差异表达蛋白质，包括76个上调蛋白质，78个下调蛋白质，其中有18个差异蛋白与雄性生殖能力相关，通过差异蛋白分析进一步揭示了精氨酸调控睾丸发育的关键通路是mTOR和WNT信号通路，随后通过WB技术验证了mTOR信号通路关键蛋白在精氨酸调控睾丸发育中的作用。在此基础上，采用精原干细胞体外培养模型，结合流式细胞仪、RT-qPCR以及抑制剂等方式，揭示了0.8 mM精氨酸处理24 h时细胞增殖率最佳，该条件下促进了细胞周期进程，显著提高了增殖相关基因Oct4和Plzf基因的表达量；其促进增殖的主要机理是精氨酸介导mTOR/S6K1信号通路调控Plzf和Nanos2的表达。通过体内与体外相结合的模型，系统的研究了精氨酸对后备公猪睾丸发育的作用机理，解析了mTORC1信号通路介导精氨酸促进细胞精原干细胞增殖的作用机制。该研究对阐明mTORC1信号通路如何介导精氨酸调节精原干细胞增殖的机理，深入认识精氨酸增加精子数量的机制具有重要意义，同时对提高我国优秀后备公猪的培育以及繁殖力将具有重大的实践意义。

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