Department of Clinical Sciences, Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran.
Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Reprod Domest Anim. 2024 Oct;59(10):e14729. doi: 10.1111/rda.14729.
Spermatogonial stem cells (SSCs) maintain spermatogenesis through self-renewal and differentiation. The proliferation of SSCs in culture systems can provide a valuable source of germ cells. Several studies have investigated new reproductive technologies, including the production of transgenic animals and recombinant proteins secreted from milk in goats. While studies in other species exist, research on goat SSC culture remains limited. We investigated the impact of different testosterone concentrations on the survival and colonisation of cocultured goat SSCs with Sertoli cells. Cells were isolated from immature goats using two-step enzymatic digestion and enriched by differential exclusion method. DMEM/F12 culture medium containing 1% antibiotic and 5% FBS, supplemented with GDNF (20 ng/mL), EGF, bFGF and LIF (10 ng/mL), was used with different testosterone concentrations (0, 60, 120 and 240 μg/mL) and cultured for 10 days. SC subpopulations were confirmed using PGP9.5 immunocytochemistry, and the expression of germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was evaluated through RT-PCR. Alkaline phosphatase activity provided additional SSC presence. The survival rate of SSCs after isolation and the number and area of colonies on Days 4, 7 and 10 were measured using an inverted microscope. The presence of PGP 9.5 antigens and germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was confirmed by immunocytochemistry and RT-PCR, respectively. According to the results, the group with 60 μg/mL testosterone had the highest number and area of colonies. The number of colonies in the 60 μg/mL testosterone group was significantly higher than the control group (p < 0.05), but no significant difference was observed compared to other groups (p ≥ 0.05). This study suggests that a low testosterone concentration (60 μg/mL) is optimal for goat SSC colonisation and viability in coculture with Sertoli cells, potentially leading to advancements in goat reproductive technologies.
精原干细胞 (SSC) 通过自我更新和分化维持精子发生。在培养系统中 SSC 的增殖可以提供有价值的生殖细胞来源。几项研究已经探索了新的生殖技术，包括生产转基因动物和从羊奶中分泌的重组蛋白。虽然在其他物种中存在研究，但山羊 SSC 培养的研究仍然有限。我们研究了不同睾酮浓度对共培养山羊 SSC 与支持细胞存活和定植的影响。使用两步酶消化法从未成熟山羊中分离细胞，并通过差速排除法进行富集。使用含 1%抗生素和 5% FBS 的 DMEM/F12 培养基，添加 GDNF（20ng/mL）、EGF、bFGF 和 LIF（10ng/mL），并使用不同的睾酮浓度（0、60、120 和 240μg/mL）培养 10 天。使用 PGP9.5 免疫细胞化学法确认 SC 亚群，并通过 RT-PCR 评估生殖细胞标志物（ID-4、UCHL-1、THY-1、β1-整合素、BCL6B、VASA、PLZF 和 OCT-4）的表达。碱性磷酸酶活性提供了 SSC 存在的额外证据。使用倒置显微镜测量分离后 SSC 的存活率以及第 4、7 和 10 天集落的数量和面积。通过免疫细胞化学和 RT-PCR 分别证实 PGP9.5 抗原和生殖细胞标志物（ID-4、UCHL-1、THY-1、β1-整合素、BCL6B、VASA、PLZF 和 OCT-4）的存在。结果表明，睾酮浓度为 60μg/mL 的组具有最多的集落数量和面积。60μg/mL 睾酮组的集落数量明显高于对照组（p<0.05），但与其他组相比无显著差异（p≥0.05）。这项研究表明，低浓度睾酮（60μg/mL）有利于山羊 SSC 在与支持细胞共培养中的定植和存活，可能为山羊生殖技术的发展提供新的思路。
