College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling, Shaanxi 712100, PR China.
Anim Reprod Sci. 2013 Feb;137(1-2):45-52. doi: 10.1016/j.anireprosci.2012.12.005. Epub 2012 Dec 20.
Male germline stem cells (mGSC) reside in the basement of seminiferous tubules of the testis and have the capacity of self-renewal and differentiation into sperm throughout the life of animals. Reports on mice and human mGSC have demonstrated that mGSC are an unlimited resource of pluripotent stem cells for sperm production. The conditions of isolation and culture of mouse and human mGSC are well developed; however, the systematic culture conditions of dairy goat mGSC are still deficient although there have been several reports of successful cultures. With the present research, several key elements of isolation and culture of dairy goat mGSC have been determined. Details for the conditions of isolation of dairy testicular spermatogonium cells were optimized, and effects of several extracellular matrix types, ages of dairy goat, and cytokines on enrichment and culture of mGSC were compared. Biological characteristics of the cells were also evaluated by RT-PCR and immunofluorescent staining. The results indicated there is one kind of enzyme cocktail (CTHD (1mg/ml collagenase, 10μg/ml DNase, 1mg/ml hyaluronidase and 1mg/ml trypsin) combined TD (0.25% trypsin and 10mg/ml DNaseI)) that can be used to successfully isolate dairy goat testicular spermatogonium cells efficiently; and fibronectin as well as laminin were efficient extracellular matrix to enrich mGSC among the extracellular matrix types evaluated. Age of dairy goat clearly influenced the cultures of dairy goat mGSC with the efficiency of establishment of an mGSC line being greater if the age of the dairy goat is younger. Some cytokines e.g. BIO (A GSK3 inhibitor, 6-bromoindirubin-3'-oxime) and basic fibroblast growth factor (bFGF) acted positively on the maintenance of proliferation and pluripotency of mGSC. Leukemia inhibitory factor (LIF) might, however, inhibit the proliferation of dairy goat mGSC. These cultured mGSC maintained similar characteristics as mouse and human mGSC. These results provide an efficient system to isolate and culture of dairy goat mGSC.
雄性生殖干细胞（mGSC）位于睾丸曲细精管的基底，具有自我更新和分化为精子的能力，贯穿动物的一生。关于小鼠和人 mGSC 的报告表明，mGSC 是精子发生的多能干细胞的无限资源。尽管已经有几篇关于成功培养的报道，但小鼠和人 mGSC 的分离和培养条件已经得到了很好的发展；然而，奶牛山羊 mGSC 的系统培养条件仍然不足。通过本研究，已经确定了奶牛山羊 mGSC 分离和培养的几个关键要素。优化了奶牛睾丸精原细胞分离的条件，比较了几种细胞外基质类型、奶牛年龄和细胞因子对 mGSC 富集和培养的影响。还通过 RT-PCR 和免疫荧光染色评估了细胞的生物学特性。结果表明，有一种酶混合物（CTHD（1mg/ml 胶原酶、10μg/ml DNA 酶、1mg/ml 透明质酸酶和 1mg/ml 胰蛋白酶）与 TD（0.25%胰蛋白酶和 10mg/ml DNA 酶 I））可成功有效地分离奶牛睾丸精原细胞；在评估的细胞外基质类型中，纤连蛋白和层粘连蛋白是富集 mGSC 的有效细胞外基质。奶牛年龄明显影响奶牛 mGSC 的培养，奶牛年龄越小，建立 mGSC 系的效率越高。一些细胞因子，如 BIO（一种 GSK3 抑制剂，6-溴靛红-3'-肟）和碱性成纤维细胞生长因子（bFGF），对 mGSC 的增殖和多能性维持有积极作用。白血病抑制因子（LIF）可能抑制奶牛 mGSC 的增殖。这些培养的 mGSC 保持与小鼠和人 mGSC 相似的特征。这些结果提供了一种有效的分离和培养奶牛山羊 mGSC 的系统。
