R-spondin1 in loach (Misgurnus anguillicaudatus): Identification, characterization, and analysis of its expression patterns and DNA methylation in response to high-temperature stress.

With a well-understood function in mammals, R-spondin1 (Rspo1) is an important regulator of ovarian development via the Wnt/β-catenin pathway. Rspo1 deficiency causes retardation of ovarian development in XX fish, and increases Rspo1 function induces femininity and sex reversal in XY fish. In this study, Rspo1 was successfully cloned from loach (Misgurnus anguillicaudatus), and its expression profile was analyzed. The full-length cDNA of Misgurnus anguillicaudatus Rspo1 (MaRspo1) comprised 1322 bp and included an open reading frame (ORF) of 795 bp, which encoded a predicted polypeptide measuring 264 amino acids in length. Phylogenetic and gene structure analyses showed a highly conserved sequence of MaRspo1 (identical to the Rspo1 genes of other species), consisting of an N-terminal signal peptide (SP), two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat (TSP1) and a C-terminal region. Real-time PCR revealed the female-biased expression profile of MaRspo1, with the highest expression level among tested tissues detected in ovary. Investigation of MaRspo1 expression levels throughout the early development stage (10-60 days post hatching) under three temperature treatments (25 °C, 28 °C, and 31 °C) revealed significantly differential expression of MaRspo1 among the three temperature groups, with decreased MaRspo1 expression in the high-temperature (31 °C) group. The results of DNA methylation analysis indicated that exposure to high temperature during early development can increase the average promoter methylation level of MaRspo1 in both females and males. Taken together, the results of this study provide the basis for the further investigation of the molecular mechanism of Rspo1 in response to temperature.