State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, China.
Int J Mol Sci. 2024 Aug 25;25(17):9215. doi: 10.3390/ijms25179215.
Spermatogonial stem cells (SSCs) possess the characteristics of self-renewal and differentiation, as well as the ability to generate functional sperm. Their unique stemness has broad applications in male infertility treatment and species preservation. In rodents, research on SSCs has been widely reported, but progress is slow in large livestock such as cattle and pigs due to long growth cycles, difficult proliferation in vitro, and significant species differences. Previously, we showed that histone 3 (H3) lysine 9 (K9) trimethylation (H3K9me3) is associated with the proliferation of bovine SSCs. Here, we isolated and purified SSCs from calf testicular tissues and investigated the impact of different H3K9me3 levels on the in vitro proliferation of bovine SSCs. The enriched SSCs eventually formed classical stem cell clones in vitro in our feeder-free culture system. These clones expressed glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1, specific marker for SSCs), NANOG (pluripotency protein), C-KIT (germ cell marker), and strong alkaline phosphatase (AKP) positivity. qRT-PCR analysis further showed that these clones expressed the pluripotency genes and and the SSC-specific marker gene . To investigate the dynamic relationship between H3K9me3 levels and SSC proliferation, H3K9me3 levels in bovine SSCs were first downregulated using the methyltransferase inhibitor, chaetocin, or transfection with the siRNA of H3K9 methyltransferase suppressor of variegation 3-9 homologue 1 (SUV39H1). The EDU (5-Ethynyl-2'-deoxyuridine) assay revealed that SSC proliferation was inhibited. Conversely, when H3K9me3 levels in bovine SSCs were upregulated by transfecting lysine demethylase 4D (KDM4D) siRNA, the EDU assay showed a promotion of cell proliferation. In summary, this study established a feeder-free culture system to obtain bovine SSCs and explored its effects on the proliferation of bovine SSCs by regulating H3K9me3 levels, laying the foundation for elucidating the regulatory mechanism underlying histone methylation modification in the proliferation of bovine SSCs.
精原干细胞（SSCs）具有自我更新和分化的特性，以及产生功能性精子的能力。它们独特的干细胞特性在男性不育症治疗和物种保护方面有广泛的应用。在啮齿动物中，SSCs 的研究已经得到了广泛的报道，但在牛和猪等大牲畜中进展缓慢，这是由于生长周期长、体外增殖困难以及物种差异显著等原因。此前，我们已经表明组蛋白 3（H3）赖氨酸 9（K9）三甲基化（H3K9me3）与牛 SSCs 的增殖有关。在这里，我们从小牛睾丸组织中分离和纯化了 SSCs，并研究了不同 H3K9me3 水平对牛 SSCs 体外增殖的影响。在我们无饲养层培养系统中，富集的 SSCs 最终形成了经典的干细胞克隆。这些克隆在体外表达神经胶质细胞衍生的神经营养因子家族受体α-1（GFRα1，SSCs 的特异性标记物）、NANOG（多能蛋白）、C-KIT（生殖细胞标记物）和强碱性磷酸酶（AKP）阳性。qRT-PCR 分析进一步表明，这些克隆表达了多能基因 和 以及 SSC 特异性标记基因 。为了研究 H3K9me3 水平与 SSC 增殖之间的动态关系，我们首先使用甲基转移酶抑制剂 chaetocin 下调牛 SSCs 中的 H3K9me3 水平，或转染 H3K9 甲基转移酶 SUV39H1 抑制子的 siRNA。EDU（5-乙炔基-2'-脱氧尿苷）检测表明 SSC 增殖受到抑制。相反，当通过转染赖氨酸去甲基酶 4D（KDM4D）siRNA 上调牛 SSCs 中的 H3K9me3 水平时，EDU 检测显示细胞增殖得到促进。总之，本研究建立了一种无饲养层培养系统来获得牛 SSCs，并通过调节 H3K9me3 水平探索其对牛 SSCs 增殖的影响，为阐明组蛋白甲基化修饰在牛 SSCs 增殖中的调控机制奠定了基础。
