Liu Xiaoli, Liu Fang, Xu Haoyang, Yang Yanping, Wang Yakun, Hong Xiaoyou, Li Wei, Yu Lingyun, Chen Chen, Xu Hongyan, Zhu Xinping
Key Laboratory of Tropical and Subtropical Fishery Resources Application and Cultivation, Ministry of Agriculture and Rural Affairs, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China.
Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), College of Fisheries, Southwest University, Chongqing 402460, China.
Biology (Basel). 2022 Sep 26;11(10):1404. doi: 10.3390/biology11101404.
Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes' functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells' culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we established a new ovarian cell line from the young Asian yellow pond turtle (), which exhibited a normal diploid karyotype with high alkaline phosphatase activity. The cell line, designated as YTO2, was then characterized through the analysis of gene expression profiles. The transcriptome analysis and the reverse transcription polymerase chain reaction (RT-PCR) showed that the cells expressed germline genes such as , , , , and . Moreover, the immunostaining showed that the germ cell markers, Tdrd7 and Rad51 proteins, were detected predominant in cytoplasm of perinuclear region, while proliferation marker, PCNA, was dominantly observed in the nuclei of cultured cells. Intriguingly, the cells could respond to the retinoic acid induction with significantly increasing the expression level of some meiosis genes, including , , , and . Furthermore, YTO2 cells could be efficiently transfected with the pHBAd-BHG-EGFP adenovirus and properly expressed the exogenous genes. To sum up, an ovarian cell line of the Asian yellow pond turtle had been established and could be stably propagated under in vitro culture condition, as well as being capable of efficiently expressing the exogenous gene . This cell line would provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle, even in reptiles.
性腺细胞系具有自我更新和分化的能力，被用作分析参与性别分化和配子发生的基因功能的有效工具。尽管在包括硬骨鱼和哺乳动物在内的多个门类的性腺细胞培养或操作方面已经取得了一些重大成就，但关于龟类性腺细胞操作的研究却很有限。在本研究中，我们从幼年亚洲黄喉拟水龟建立了一种新的卵巢细胞系，其显示出具有高碱性磷酸酶活性的正常二倍体核型。然后通过基因表达谱分析对该命名为YTO2的细胞系进行了表征。转录组分析和逆转录聚合酶链反应（RT-PCR）表明，这些细胞表达了如、、、、和等生殖系基因。此外，免疫染色显示，生殖细胞标记物Tdrd7和Rad51蛋白主要在核周区域的细胞质中被检测到，而增殖标记物PCNA则主要在培养细胞的细胞核中被观察到。有趣的是，这些细胞可以对视黄酸诱导作出反应，显著提高包括、、、和在内的一些减数分裂基因的表达水平。此外，YTO2细胞可以用pHBAd-BHG-EGFP腺病毒有效转染并正确表达外源基因。综上所述，已经建立了亚洲黄喉拟水龟的卵巢细胞系，其能够在体外培养条件下稳定增殖，并且能够有效表达外源基因。该细胞系将为阐明龟类甚至爬行动物生殖细胞发育、分化和卵子发生背后的分子机制提供有价值的工具。
