BACKGROUND:In mammals, R-spondin (Rspo), an activator of the Wnt/β-catenin signaling pathway, has been shown to be involved in ovarian differentiation. However, the role of the Rspo/Wnt/β-catenin signaling pathway in fish gonads is still unknown.RESULTS:In the present study, full-length cDNAs of Rspo1, 2 and 3 were cloned from the gonads of medaka (Oryzias latipes). The deduced amino acid sequences of mRspo1-3 were shown to have a similar structural organization. Phylogenetic analysis showed that Rspo1, 2 and 3 were specifically clustered into three distinct clads. Tissue distribution revealed that three Rspo genes were abundantly expressed in the brain and ovary. Real-time PCR analysis around hatching (S33-5dah) demonstrated that three Rspo genes were specifically enhanced in female gonads from S38. In situ hybridization (ISH) analysis demonstrated that three Rspo genes were expressed in the germ cell in ovary, but not in testis. Fluorescence multi-color ISH showed that Rspo1 was expressed in both somatic cells and germ cells at 10dah. Exposure to ethinylestradiol (EE2) in XY individuals for one week dramatically enhanced the expression of three Rspo genes both at 0dah and in adulthood.CONCLUSIONS:These results suggest that the Rspo-activating signaling pathway is involved in the ovarian differentiation and maintenance in medaka.