Key Laboratory of Aquatic Genomics, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China.
Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China.
J Cell Biochem. 2018 Sep;119(9):7667-7677. doi: 10.1002/jcb.27114. Epub 2018 Jun 20.
Gonadal cell lines provide valuable tools for studying gametogenesis, sex differentiation, and manipulating germ cells in reproductive biology. Female germline stem cells have been characterized and isolated from ovaries of mammalian species, including mice and human, but there has been very few studies on female germline stem cells in reptiles. Here, we described an ovarian stem cell-like line isolated and cultured from the Chinese soft-shell turtle (Pelodiscus sinensis), designated as PSO1. The cells showed high alkaline phosphatase activity with a normal diploid karyotype. As shown by reverse transcription-polymerase chain reaction, the cells were positive for the expression of germ cell-specific genes, vasa and dazl, as well as a stem cell marker, nanog, but negative for the expression of the folliculogenesis-specific gene, figla. Likewise, through fluorescent immunostaining analyses, both the Dazl and Vasa proteins were detected abundantly in the cytoplasm of perinuclear region, whereas Nanog and PCNA were dominantly observed in the nuclei in PSO1 cells. Moreover, PSO1 cells transfected with pCS2:h2b-egfp could properly express the fusion protein in the nuclei. Taken together, the findings suggested that the germline stem cells exist in the ovary of juvenile Chinese soft-shell turtle and these cells can be isolated for a long-term in vitro culture under experimental conditions. This study has provided a valuable basis for further investigations on the molecular mechanisms whereby the germline stem cells develop and differentiate into gametes in turtles. Also, it has paved the way for studies on oogenesis in turtles, even in the other reptiles.
性腺细胞系为研究配子发生、性别分化和在生殖生物学中操纵生殖细胞提供了有价值的工具。雌性生殖干细胞已从包括小鼠和人类在内的哺乳动物的卵巢中得到鉴定和分离，但关于爬行动物的雌性生殖干细胞的研究却很少。在这里，我们描述了从中华鳖（Pelodiscus sinensis）的卵巢中分离和培养的一种卵巢干细胞样系，命名为 PSO1。这些细胞具有高碱性磷酸酶活性，并且具有正常的二倍体核型。如逆转录聚合酶链反应所示，这些细胞表达生殖细胞特异性基因 vasa 和 dazl，以及干细胞标记物 nanog，但不表达卵泡发生特异性基因 figla。同样，通过荧光免疫染色分析，在核周区细胞质中大量检测到 Dazl 和 Vasa 蛋白，而 Nanog 和 PCNA 则主要观察到在 PSO1 细胞的核中。此外，用 pCS2:h2b-egfp 转染的 PSO1 细胞可以在核内正确表达融合蛋白。总之，这些发现表明，幼中华鳖的卵巢中存在生殖干细胞，并且这些细胞可以在实验条件下进行长期体外培养。这项研究为进一步研究生殖干细胞在海龟中发育和分化为配子的分子机制提供了有价值的基础。此外，它为海龟甚至其他爬行动物的卵子发生研究铺平了道路。
