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

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.

Genome-wide identification, evolution and expression analysis of bone morphogenetic protein (BMP) gene family in chinese soft-shell turtle (Pelodiscus sinensis)

“…RA is critical for the production of oocytes and sperm in mammals ( Endo et al, 2019 ). In Mauremys mutica , the expression levels of some meiosis genes in ovarian cells were significantly increased by RA treatment ( Liu et al, 2022 ). Exogenous RA treatment activates Dmrt1a and Amh , thereby inhibiting germ cell differentiation, and genetic knockout of the RA degrading enzyme Cyp26a promoted meiosis and oogenesis in medaka ( Adolfi et al, 2020 ).…”

A chromosome-level genome assembly of the Asian giant softshell turtle Pelochelys cantorii

“…In order to further determine the chromosome number of the Asian giant softshell turtle, about 1 mL blood was extracted from the neck blood vessels of four 1 to 3 years-old young individuals from the turtle domestication base of the Pearl River Fisheries Research Institute. After 72 h of culture, cells were collected for the preparation of chromosomes analysis as previously described 14 . A total of 43 metaphase mitotic cells with a higher number of chromosomes were obtained (Figure S2 , Figure S3 ).…”