Tiptanavattana Narong, Thongkittidilok Chommanart, Techakumphu Mongkol, Tharasanit Theerawat
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
J Reprod Dev. 2013;59(2):189-95. doi: 10.1262/jrd.2012-130. Epub 2013 Jan 25.
Spermatogonial stem cells (SSCs) function to regulate the balance of self-renewal and differentiation of male gametes. SSCs have been successfully isolated and cultured in vitro in several species, but not in feline. Therefore, in this study, we aimed to culture and characterize feline SSCs. In experiment 1, testes (n=5) from different pubertal domestic cats were cryosectioned and fluorescently immunolabeled to examine the expression of SSC (GFRα-1), differentiated spermatogonium (c-kit) and germ cell (DDX-4) markers. In experiments 2 and 3, testicular cells were digested and subsequently cultured in vitro. The resultant presumptive SSC colonies were then collected for SSC identification (experiment 2), or further cultured in vitro on feeder cells (experiment 3). Morphology, gene expression and immunofluorescence were used to identify the SSCs. Experiment 1 demonstrated that varying types of spermatogenic cells existed and expressed different germ cell/SSC markers. A rare population of putative SSCs located at the basement membrane of the seminiferous tubules was specifically identified by co-expression of GFRα-1 and DDX-4. Following enzymatic digestion, grape-like colonies formed by 13-15 days of culture. These colonies expressed GFRA1 and ZBTB16, but did not express KIT. Although we successfully isolated and cultured feline SSCs in vitro, the SSCs could only be maintained for 57 days. In conclusion, this study demonstrates, for the first time, that putative SSCs from testes of pubertal domestic cats can be isolated and cultured in vitro. These cells exhibited SSC morphology and expressed SSC-specific genes. However, long-term culture of these putative SSCs was compromised.
精原干细胞（SSCs）的功能是调节雄性配子自我更新和分化的平衡。在多个物种中，SSCs已成功地在体外分离和培养，但猫科动物除外。因此，在本研究中，我们旨在培养猫科动物的SSCs并对其进行特性分析。在实验1中，对来自不同青春期家猫的睾丸（n = 5）进行冷冻切片并进行荧光免疫标记，以检测SSC（GFRα-1）、分化型精原细胞（c-kit）和生殖细胞（DDX-4）标志物的表达。在实验2和实验3中，消化睾丸细胞，随后进行体外培养。然后收集所得的假定SSC集落用于SSC鉴定（实验2），或在饲养细胞上进一步进行体外培养（实验3）。通过形态学、基因表达和免疫荧光来鉴定SSCs。实验1表明存在不同类型的生精细胞，它们表达不同的生殖细胞/SSC标志物。通过GFRα-1和DDX-4的共表达，特异性鉴定出位于生精小管基底膜的稀有假定SSC群体。酶消化后，培养13 - 15天形成葡萄样集落。这些集落表达GFRA1和ZBTB16，但不表达KIT。虽然我们成功地在体外分离和培养了猫科动物的SSCs，但这些SSCs只能维持57天。总之，本研究首次证明，青春期家猫睾丸中的假定SSCs可以在体外分离和培养。这些细胞呈现出SSC形态并表达SSC特异性基因。然而，这些假定SSCs的长期培养受到了影响。
