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, China.
Theriogenology. 2013 Dec;80(9):1052-60. doi: 10.1016/j.theriogenology.2013.08.003. Epub 2013 Sep 19.
In mammalian testis, male germline stem cells (mGSCs) are originated from primordial germ cells and developed into spermatocyte and spermatid. In our previous studies, we had isolated a pluripotent mGSCs from goat testes and tested their pluripotency and differentiation potential in vitro and in vivo, which revealed that the isolated and cultured dairy goat mGSCs maintained the characteristics of mGSCs. However, Thy1, a marker of mGSCs, was not examined in detail. In this study, the dairy goat mGSCs were purified by differential plating followed by magnetic-activated cell sorting (MACS) using Thy1 antibody. The quantitative reverse transcription polymerase chain reaction and immunofluorescence analyses revealed that the transcription and expression of Thy1, CD49f, Plzf, Oct4, Gfra1, and Vasa were higher in Thy1-positive cells when compared with Thy1-negative cells. The detection results of culturing dairy goat mGSCs indicated that the Thy1-positive cells maintained the characteristics of mGSCs, grew relatively faster than Thy1-negative cells, and the percentage of alkaline phosphatase-positive cells and colonies were significantly higher in Thy1-positive mGSCs than Thy1-negative cells. Collectively, these results indicate that THY1 is a marker of undifferentiated spermatogonia in goat testes, the technique of magnetic-activated cell sorting using Thy1 antibody could be an efficient method to enrich mGSCs in goat.
在哺乳动物睾丸中，雄性生殖干细胞（mGSCs）起源于原始生殖细胞，并发育为精母细胞和精子细胞。在我们之前的研究中，我们已经从山羊睾丸中分离出多能 mGSCs，并在体外和体内测试了它们的多能性和分化潜能，结果表明分离和培养的乳用山羊 mGSCs 保持了 mGSCs 的特征。然而，mGSCs 的标志物 Thy1 并没有被详细检查。在这项研究中，我们使用 Thy1 抗体通过差异平板培养和磁激活细胞分选（MACS）对乳用山羊 mGSCs 进行了纯化。定量逆转录聚合酶链反应和免疫荧光分析显示，与 Thy1 阴性细胞相比，Thy1 阳性细胞中 Thy1、CD49f、Plzf、Oct4、Gfra1 和 Vasa 的转录和表达水平更高。对培养乳用山羊 mGSCs 的检测结果表明，Thy1 阳性细胞保持了 mGSCs 的特征，比 Thy1 阴性细胞生长得相对较快，且 Thy1 阳性 mGSCs 中的碱性磷酸酶阳性细胞和集落的百分比明显高于 Thy1 阴性细胞。综上所述，这些结果表明 THY1 是山羊睾丸未分化精原细胞的标志物，使用 Thy1 抗体的磁激活细胞分选技术可能是富集山羊 mGSCs 的有效方法。
