Spermatogonial stem cells (SSCs) are crucial for maintaining spermatogenesis, studying germ line stem cell biology, and producing transgenic animals. Growth factors, including leukemia inhibitory factor (LIF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and glial cell line-derived neurotrophic factor (GDNF), are essential for in vitro culture of SSCs as well as their self-renewal and maintenance. In this study, we investigated the effects of these growth factors on porcine spermatogonial germ cell (pSGC) colony formation. We determined round alkaline phosphatase (AP)-positive pSGC colonies in the presence and absence of growth factors after 7 days of pSGC culture. EGF was found to be essential to support the formation of AP-positive pSGC colonies. The expression of epidermal growth factor receptor (EGFR) and Erb-B2 receptor tyrosine kinase 2 (ERBB2) was also altered in cultured pSGCs compared to that in feeder cells. We verified the effect of EGF signaling on pSGC colony formation using AG1478 as an EGFR inhibitor and AG879 as an ERBB2 inhibitor. pSGC colonies were observed in low dose AG1478-treated groups with EGF, whereas a high dose of AG1478 suppressed pSGC colony formation. AP-positive colonies were also observed in all AG879-treated groups. Taken together, EGFR and EGF signaling play a critical role in the initiation of colony formation of pSGCs. Our study provides insights into the mechanisms of EGF-mediated colony formation by SGCs derived from porcine testes, and will aid the development of transplantation techniques for the production of transgenic offspring.

精原干细胞 (SSC) 对于维持精子发生、研究生殖系干细胞生物学和生产转基因动物至关重要。生长因子，包括白血病抑制因子 (LIF)、表皮生长因子 (EGF)、碱性成纤维细胞生长因子 (bFGF) 和神经胶质细胞系衍生的神经营养因子 (GDNF)，对于体外培养至关重要SSC 的培养及其自我更新和维护。在这项研究中，我们研究了这些生长因子对猪精原生殖细胞 (pSGC) 集落形成的影响。我们在 pSGC 培养 7 天后，在存在和不存在生长因子的情况下确定了圆形碱性磷酸酶 (AP) 阳性 pSGC 集落。发现 EGF 对支持 AP 阳性 pSGC 集落的形成至关重要。与饲养细胞相比，培养的 pSGC 中表皮生长因子受体 (EGFR) 和 Erb-B2 受体酪氨酸激酶 2 (ERBB2) 的表达也发生了改变。我们使用 AG1478 作为 EGFR 抑制剂和 AG879 作为 ERBB2 抑制剂验证了 EGF 信号传导对 pSGC 集落形成的影响。在具有 EGF 的低剂量 AG1478 治疗组中观察到 pSGC 集落，而高剂量的 AG1478 抑制 pSGC 集落形成。在所有 AG879 治疗组中也观察到 AP 阳性菌落。总之，EGFR 和 EGF 信号在 pSGC 集落形成的起始中起关键作用。我们的研究提供了对源自猪睾丸的 SGC 形成 EGF 介导的集落形成机制的见解，并将有助于开发用于生产转基因后代的移植技术。