Isolation and in vitro culture of ovarian stem cells in Chinese soft‐shell turtle (<i>Pelodiscus sinensis</i>)

Abstract

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.