Cloning of dead end (dnd) gene in Chinese soft-shell turtle (Pelodiscus sinensis) and its expression in germ cells

Dead end (dnd) is a germ cell-specific molecular marker in most vertebrates, belonging to RNA-binding protein family. It is essential for the maintenance of the survival and migration of primordial germ cells (PGCs). In many species, dnd deficiency could block the migration of PGCs, and then they transdifferentiate into other cell types, resulting in complete loss of germ cells and sterility. Thus, dnd is essential for the survival and development of vertebrate germ cells. Dnd is originally identified in Danio rerio, proved that deletion of Dnd leads to abnormal polarization and migration of PGCs and loss of germ cells. Thereafter, dnd1 is characterized in Mus musculus and Dnd1-β is specifically expressed in adult testis. However, chicken Dnd homologue (CDH) is expressed in both gonads. In Xenopus laevis, dnd is only expressed in ovary. In teleosts, dnd has been identified as a PGCs marker and is specifically expressed in gonads that the expression level is higher in ovary than testis. Therefore, dnd shows distinct expression patterns among species. Although dnd plays a conserved role in maintaining the survival and migration of PGCs from teleosts to mammals, its expression pattern in gametogenesis varies with species. Thus, it is of great significance to identify the sequence characteristics, gene expression and distribution of dnd in turtles, to lay the foundation for studying on the origin and formation of PGCs, and gametogenesis of turtles. Pelodiscus sinensis is one of the oldest tetrapods and has unique scientific research value in study of animal morphological and reproductive evolution. Additionally, because of its high medicinal and economic values, P. sinensis occupies an important position in freshwater aquaculture in China. Here, in order to study the development and differentiation mechanism of turtles’ germ cells, the full-length cDNA of dnd was obtained by RACE form P. sinensis. The total length of P. sinensis dnd (Psdnd) cDNA was 1 251 bp (GenBank ID OL757532), including 254 bp 3' untranslated region and 1 017 bp open reading frame encoding 338 amino acids. Multiple alignment and phylogenetic tree analysis showed that Psdnd had 6 conserved domains, the same as homologous proteins of other species had, and the highly conserved RNA recognition domain was the main functional region. Besides, Psdnd had the highest sequence identity with and was the closest relative to Chelonia mydas dnd. RT-PCR showed that Psdnd was specifically expressed in gonads and the mRNA level was higher in ovary than testis. Chemical in situ hybridization showed that Psdnd mRNA was specifically expressed in germ cells. Particularly, Psdnd mRNA was mainly expressed in the cytoplasm of stage Ⅱ primary oocytes, and not expressed in oocytes stage Ⅳ onwards. Moreover, Psdnd mRNA signals were strongly detected in primary spermatocytes, followed by secondary spermatocytes and spermatogonia, and barely in spermatids. Our study indicates that dnd may play a key role in regulating the development of male and female germ cells in P. sinensis. This study lays the basis for further study of turtles’ germ cells development and differentiation mechanism.