Geng Qingqing, Hu Cai, Zhao Ziduo, Wang Zhe, Cheng Fufu, Chen Jing, Zuo Qisheng, Zhang Yani
Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China; College of Veterinary Medicine, Yangzhou University, Yangzhou, PR China.
Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China.
Poult Sci. 2025 Jan;104(1):104583. doi: 10.1016/j.psj.2024.104583. Epub 2024 Nov 25.
Spermatogonial stem cells (SSCs) have vast application prospects in livestock and poultry production, genetic engineering, and medical research. However, the scarcity of SSCs and the complexity of their development limit the elucidation and verification of the mechanism of SSCs in vitro. Although miRNAs have been identified as critical players in germ cell development, upstream regulatory mechanisms by which miRNAs regulate SSCs formation are rarely reported. In this study, miR-1458, which was differentially expressed during SSCs formation, was selected by transcriptomic sequencing. We found that miR-1458, inhibited in an in vitro SSCs induction model, significantly upregulated the expression of germline marker genes (Cvh and integrin β1). Further analysis using Immunofluorescence and Flow Cytometry confirmed that miR-1458 inhibition promotes the formation of spermatogonial stem-like cells (SSCLCs). Immunohistochemical significantly increased the number of SSCs in the testis in vivo. However, significant upregulation of miR-1458 showed opposite results. High-throughput sequencing results showed that miR-1458 interacted with TBX6, one of the target genes of miR-1458, involved in affecting cell differentiation, and dual-luciferase reporter vectors confirmed the targeting relationship between the two. TBX6 overexpression and knockdown in vitro and in vivo have validated its function in SSCs formation. We found that overexpression of TBX6 promoted SSCs formation. Additionally, we identified Vitamin B6, a key metabolite affecting SSCs formation, as an upstream regulator of miR-1458 expression. The results showed that low concentrations of Vitamin B6 led to low expression of miR-1458 by decreasing histone demethylation levels. Overall, our findings suggest that miR-1458 is involved in SSCs formation, which is inhibited by low concentrations of Vitamin B6 and subsequently regulates the formation of SSCs by targeting TBX6, an essential gene involved in embryonic stem cell differentiation. Our study demonstrates the critical role of the Vitamin B6-miR-1458-TBX6 regulatory axis in spermatogonial stem cell formation in Rugao Yellow Chicken, providing new insights into the regulatory mechanisms by which miRNAs affect SSCs formation. It should be noted that most of the germline findings related to miRNAs were obtained by in vitro studies, and in vivo studies are needed to validate our results for clinical applications.
精原干细胞（SSCs）在畜禽生产、基因工程和医学研究中具有广阔的应用前景。然而，SSCs的稀缺性及其发育的复杂性限制了体外对SSCs机制的阐明和验证。尽管miRNAs已被确定为生殖细胞发育的关键参与者，但miRNAs调控SSCs形成的上游调控机制鲜有报道。在本研究中，通过转录组测序筛选出在SSCs形成过程中差异表达的miR-1458。我们发现，在体外SSCs诱导模型中受到抑制的miR-1458显著上调了生殖系标记基因（Cvh和整合素β1）的表达。使用免疫荧光和流式细胞术的进一步分析证实，抑制miR-1458可促进精原干细胞样细胞（SSCLCs）的形成。免疫组化显著增加了体内睾丸中SSCs的数量。然而，miR-1458的显著上调显示出相反的结果。高通量测序结果表明，miR-1458与miR-1458的靶基因之一TBX6相互作用，TBX6参与影响细胞分化，双荧光素酶报告载体证实了两者之间的靶向关系。在体外和体内对TBX6进行过表达和敲低验证了其在SSCs形成中的功能。我们发现，过表达TBX6可促进SSCs的形成。此外，我们鉴定出影响SSCs形成的关键代谢物维生素B6是miR-1458表达的上游调节因子。结果表明，低浓度的维生素B6通过降低组蛋白去甲基化水平导致miR-1458表达降低。总体而言，我们的研究结果表明，miR-1458参与SSCs的形成，低浓度的维生素B6对其产生抑制作用，随后通过靶向TBX6（一个参与胚胎干细胞分化的关键基因）来调节SSCs的形成。我们的研究证明了维生素B6-miR-1458-TBX6调控轴在如皋黄鸡精原干细胞形成中的关键作用，为miRNAs影响SSCs形成的调控机制提供了新的见解。需要注意的是，大多数与miRNAs相关的生殖系研究结果是通过体外研究获得的，需要进行体内研究以验证我们的结果用于临床应用。
