Niu Zhiwei, Zheng Liming, Wu Siyu, Mu Hailong, Ma Fanglin, Song Wencong, Zhu Haijing, Wu Jiang, He Xin, Hua Jinlian
College of Veterinary MedicineShaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China.
College of Veterinary MedicineShaanxi Centre of Stem Cells Engineering and Technology, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China
Reproduction. 2015 May;149(5):445-52. doi: 10.1530/REP-14-0506.
Spermatogonia stem cells (SSCs), also named the male germline stem cells (mGSCs), which is located at the base of the seminiferous tubules of testis, is the basis for generating sperm steadily in male animals. Currently, there are some preliminary study on the self-renewal and differentiation of SSCs, but further mechanism, especially in large animals, has not been clearly understood. Ras/ERK1/2 pathway is widely distributed in multiple cells in vivo. It plays an important role in cell proliferation, differentiation and so on. However, the study on the function for the self-renewal of dairy goats SSCs has not been investigated. In this study, the dairy goat SSCs characterization were evaluated by semi-RT-PCR, alkaline phosphatase (AP) staining, and immunofluorescence staining. Then, Ras/ERK1/2 pathway was blocked by specific MEK1/2 inhibitor PD0325901. We analyzed the proliferation by cell number, cell growth curve, Brdu incorporation assay, and cell cycle analysis. The results showed that the proliferation was significantly inhibited by PD0325901. Cell apoptosis induced by blocking the Ras/ERK1/2 pathway was analyzed by TUNEL. The expression of ETV5 and BCL6B, the downstream gene of Ras/ERK1/2 pathway, was downregulated. This study suggest that the Ras/ERK1/2 pathway plays a critical role in maintaining the self-renewal of dairy goat SSCs via regulation of ETV5 and BCL6B. This study laid a foundation for insights into the mechanism of SSCs self-renewal comprehensively.
精原干细胞（SSCs），也被称为雄性生殖系干细胞（mGSCs），位于睾丸生精小管的基部，是雄性动物稳定产生精子的基础。目前，关于SSCs自我更新和分化已有一些初步研究，但进一步的机制，尤其是在大型动物中的机制，尚未完全清楚。Ras/ERK1/2通路在体内多种细胞中广泛分布。它在细胞增殖、分化等过程中发挥重要作用。然而，关于奶山羊SSCs自我更新功能的研究尚未见报道。在本研究中，通过半定量逆转录聚合酶链反应（semi-RT-PCR）、碱性磷酸酶（AP）染色和免疫荧光染色对奶山羊SSCs的特性进行了评估。然后，用特异性MEK1/2抑制剂PD0325901阻断Ras/ERK1/2通路。我们通过细胞计数、细胞生长曲线、5-溴脱氧尿嘧啶核苷（Brdu）掺入试验和细胞周期分析来分析细胞增殖情况。结果表明，PD0325901显著抑制了细胞增殖。通过末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）分析了阻断Ras/ERK1/2通路诱导的细胞凋亡。Ras/ERK1/2通路的下游基因ETV5和BCL6B的表达下调。本研究表明，Ras/ERK1/2通路通过调节ETV5和BCL6B在维持奶山羊SSCs自我更新中起关键作用。本研究为全面深入了解SSCs自我更新机制奠定了基础。
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