Molecular cloning and expression of R-spondin1 gene in Carassius auratus

In order to study the roles of R-spondin1(Rspo1) gene in the sex determination and differentiation of the Qihe crucian carp (Carassius auratus), a natural gynogenesis triploid fish, we cloned its cDNA by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA 5′ and 3′ ends (RACE). We also examined Rspo1 spatio-temporal expression profiles by real-time PCR, at the same time, expression level of Rspo1 in the testis induced by Letrozole or high temperature treatment was also examined. The results showed that the cDNA of Rspo1 was 1243 bp including a 318 bp 5′-untranslated region (UTR), a 127 bp 3′UTR, and a 798 bp open reading frame (ORF) encoding a polypeptide of 265 amino acids. Sequence analysis revealed that Rspo1 from Qihe crucian carp showed the highest similarity with Cyprinidae fish, while had low similarity with other vertebrates including mammalians and reptiles. Tissue distribution by real-time PCR revealed that Rspo1 was abundantly expressed in the muscle, and then in the ovary, gill, intestine, and was barely detectable in other tissues. The expression of Rspo1 was gradually downregulated from fertilization stage, and then upregulated from the tailbud stage, so Rspo1 might be a maternal factor. Ontogenic analysis demonstrated that Rspo1 was expressed abundantly from 20 days after hatching (dah) in the gonad. However, Rspo1 was upregulated in the gonad of sex-reversed males by Letrozole or high temperature treatment, indicating that Rspo1 exhibited a sexual dimophic expression pattern. These results suggested that, as a maternal factor, Rspo1 might be involved in both the ovarian differentiation and spermatogenesis in the testis of C. auratus.