Tiptanavattana Narong, Radtanakatikanon Araya, Hyttel Poul, Holm Hanne, Buranapraditkun Supranee, Setthawong Piyathip, Techakumphu Mongkol, Tharasanit Theerawat
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
J Reprod Dev. 2015;61(6):581-8. doi: 10.1262/jrd.2015-094. Epub 2015 Sep 27.
The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4-6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron microscopy and immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3). Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1(+) cells (14.89 ± 5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed mRNA for GFRA1, ZBTB16, RET and POU5F1. Our study found that the G/SSC transition occurs at 4-6 months of age. This period is useful for isolation and improves the establishment efficiency of cat SSCs in vitro.
猫生殖细胞的发育尚未得到完整记录，尤其是从生殖母细胞向精原干细胞（G/SSC）的过渡阶段。本研究的目的是检查睾丸发育情况并确定G/SSC过渡，以便在体外分离和培养精原干细胞。根据供体年龄将睾丸分为3组（I组，小于4个月；II组，4 - 6个月；III组，大于6个月）。在实验1中，我们通过组织学、透射电子显微镜和免疫组织化学研究睾丸发育。在实验2中，我们测定了GFRα - 1、DDX - 4和c - kit的表达并进行了流式细胞术检测。从II组和III组分离出的精原干细胞通过逆转录聚合酶链反应（RT - PCR）和透射电子显微镜（TEM）进行鉴定（实验3）。结果表明了G/SSC过渡中的时间变化。精原干细胞的大小、形态和超微结构与生殖母细胞不同。结果显示，与其他组相比，II组每生精索/小管中精原干细胞数量最多（17.66 ± 2.20%），GFRα - 1(+)细胞数量也最多（14.89 ± 5.66%）。这些发现与II组相比III组精原干细胞衍生效率的提高相一致（74.33 ± 2.64%对23.33 ± 2.23%）。这些集落表达GFRA1、ZBTB16、RET和POU5F1的信使核糖核酸（mRNA）。我们的研究发现G/SSC过渡发生在4 - 6个月龄。这个时期有利于精原干细胞的分离，并提高了猫精原干细胞体外建立效率。
