Expression pattern and functional analysis of R-spondin1 in tongue sole Cynoglossus semilaevis

Abstract

R-spondin 1 (Rspo1) is a potential female-determining gene in mammals that could regulate the Wnt/β-catenin signaling pathway. The deletion of Rspo1 causes sex reversal in females. To investigate sexual determination and differentiation, we cloned and analyzed the Rspo1 gene in Cynoglossus semilaevis. Phylogenetic and gene structure analyses revealed that Rspo1 gene exhibited high sequence conservation and contained an N-terminal signal peptide, two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat, and a C-terminal region enriched with basic charged amino acids. qRT-PCR revealed that Rspo1 expressed sexual dimorphism in gonad, with higher expression levels in the ovary than in the testis, thus, suggesting the involvement of Rspo1 in gonad differentiation. In situ hybridization results demonstrated that Rspo1 was expressed in premature germ cells, including spermatogonia and spermatocytes in the testis and stage II and stage III oocytes in the ovary. The methylation levels in two CpG sites of Rspo1 promoter significantly differed among females, males, and pseudomales. After 30days of exposure to high temperature, the expression of Rspo1 significantly decreased in female individuals, some of which were prone to males. However, no difference of Rspo1 gene expression was observed between the control group and high-temperature group in males. These preliminary findings suggested that Rspo1 played a crucial role in sex determination and development. This study laid the groundwork for further sex control breeding techniques in C. semilaevis.