Cloning and expression analysis of tsn in medaka (Oryzias latipes)

WANG Siyu;GUO Haiyan;LI Xi;LIAO Min;LI Yayuan;GUAN Guijun;Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University;International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University;

gsdf;;tsn;;germ cell;;sex differentiation;;ovary;;medaka

Although mechanisms of sex determination in sexual reproduction are diverse, there is a common feature of germ cells to undergo mitotic division and proliferation, following the entery of meiosis to develop oocyte or sperm. It is unclear whether germ cells are sexually dimorphic during the mitotic proliferation prior to the onset of meiosis, or how spermatogenesis or oogenesis is initiated. Tsn(Translin) protein products were significantly higher in gsdf deficient(gsdf ~(-/-)) XY ovaries than that in normal(gsdf~(+/+)) XX ovaries, revealed by the proteomic expression profiles among normal(gsdf~(+/+))XX ovaries, normal(gsdf~(+/+))XY testises and gsdf(gonadal soma-derived factor) deficient(gsdf ~(-/-)) ovaries in medaka(Oryzias latipes), suggesting that Tsn protein expression might be regulated by dmy(DM domain on Y chromosome) and downstream Gsdf male signals in gonad development. The full-length Open Reading Frame(ORF) fragment of tsn was cloned from gonadal cDNA libraries in medaka. Phylogenetic tree and alignment of amino acid sequence analysis shows that Tsn is well conserved in vertebrates with the high homology of amino acid sequence sharing between medaka and zebrafish(Danio rerio). Although the tsn transcription was detectable in multiple tissues by reverse transcription PCR and real-time quantitative PCR, the testicular level of tsn mRNA was significantly higher than that of ovaries in medaka. Immunofluorescence revealed that cystic germ cells positive to anti-medaka Tsn antibody were significantly increased in gsdf ~(-/-) XY ovaries than that in gsdf~(+/+) XX ovaries as a control. This is consistent with the previous report of high Tsn protein products in gsdf ~(-/-) XY ovaries detected by the high-throughput proteomics analysis. A conclusion has been drawn that Tsn-mediated germ cell proliferation was promoted in male pathway under the regulation of dmy(DM-domain on Y chromosome) but inhibited by Gsdf signaling. Lack of Gsdf released the inhibition of Tsn expressing germ cell proliferation during medaka testicular differentiation. The results may provide clues for defining sexual difference between male and female germ cells in the process of vertebrate mitosis and proliferation.