Gonadal Development and Sex Determination Related Genes Expression in Metamorphic Korean Fire-Bellied Toad (Bombina orientalis).

In amphibians, although sex chromosomes are responsible for sex determination with either male heterogamety (XY/XX) or female heterogamety (ZW/ZZ), the environmental factors and hormones can override the genotypic mechanism of sex determination and induce sex reversal. However, the fire-bellied toad (Bombina orientalis) which belongs to male heterogamety group is refractory to sex hormone in external sex determination. In amphibians, testis or ovary develops during metamorphosis from bipotential gonad of intermediate mesoderm origin. Sex-determining gene (Sry) has central role in testis development and autosomal Sry-related HMG box gene 9 (Sox9) also directly participates in testis determination. Transcription factors including Sox3, SF1, WT1, Wnt4, Dmrt1, Dax1, Fgf9 and GATA4 also involved in gonad differentiation. In an effort to elucidate the genetic regulation of sex differentiation in B. orientalis, cDNA sequences for Sox9, Sox3, SF1, WT1, Wnt4, Dmrt1, Dax1, Fgf9, and GATA4 were determined. Furthermore, the expression of above genes during metamorphosis was analyzed together with morphological analysis of gonad differentiation. Sex differentiation started in B. orientalis at stage 32-33 and thereafter female gonad was divided into medulla and cortex regions. Realtime PCR revealed that SF1, GATA4, WT1+KTS, and WT1-KTS mRNA levels were high during pre-metamorphic stage. In particular, SF1, GATA4, and WT1+KTS were highly expressed in male gonad but WT1-KTS was highly expressed in developing ovary, suggesting WT1-KTS functions in ovary development in B. orientalis. Sox9 mRNA was largely found in male gonad and reach to the highest levels in metamorphosis complete stage tadpoles. Fgf9 mRNA were appeared in male gonad before sex determination. Sox3 mRNA was found in both sexes before sex determination and the highest in ovary at post-metamorphic stage. Dax1 mRNA was appeared in male gonad at mid-metamorphic stage. Dmrt1 mRNA was detected in ovary at post-metamorpic stage. This expression patterns of Dmrt1 and Dax1 mRNA was not coincided with their formerly reported functions, suggesting that they are the Dmrt1 and Dax1 isotypes having unique role in gonad development in B. orientalis. This is the first report on the characteristics of metamorphic and gonadal development stages and genes involved in the development of gonad in Korean amphibian species. Together with the morphological feature of gonad development, molecular information obtained from B. orientalis will be helpful for the understanding the sex-reversal by environmental factors including endocrine disruptors as well as molecular mechanism for gonadal development in this Korean amphibian species. (poster)