Characterization of the In Vitro Cultured Ovarian Cells in the Asian Yellow Pond Turtle (Mauremys mutica)

SIMPLE SUMMARY: 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 isolated an ovarian cell line (fGSCs), from the juvenile Asian yellow pond turtle in vitro. The cultured fGSCs were characterized with germline stem cell specific markers’ expression using transcriptome sequencing on Illumina platform, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence (IF). Our study aims to provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle even in reptiles, and also, the established cell line has potential applications to enrich the conservation strategy of aquatic animal germplasm resources. ABSTRACT: 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 (Mauremys mutica), 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 tdrd7, nanos1, klf5, igtb1, hsd17b4 and rad51. 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 vasa, dazl, figla, and dmc1. 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 tdrd7. 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.