Characterization and Localization of Calb2 in Both the Testis and Ovary of the Japanese Flounder (Paralichthys olivaceus)

SIMPLE SUMMARY: Calretinin (CALB2), which is a Ca(2+)-binding protein, plays a known pivotal role in the neural system in vertebrates. The role of CALB2 in mammalian gonads has been gradually recognized; however, little information on the function of CALB2 in fish gonads has been reported. Therefore, we firstly identified the calb2 gene in Paralichthys olivaceus (P. olivaceus), and then investigated its tissue distribution and localization in the gonads by real-time PCR, western blotting, and immunohistochemistry. The P. olivaceus calb2 mRNA was relatively highly expressed in both the testis and ovary. The CALB2 protein is located in Leydig cells of the testis and ovarian germ epithelial cells in P. olivaceus. This study provides a basis for further explorations on the function and regulation mechanism of calb2 in fish gonads. ABSTRACT: Although its function in mammalian gonads has been gradually recognized, the expression and function of calretinin (CALB2)—a Ca(2+)-binding protein—in the testis and ovary of fish are still unclear. Here, we identified the cDNA sequences of calb2 in Paralichthys olivaceus (P. olivaceus); analyzed its gene structure and phylogenetic and syntenic relationship by bioinformatics; and investigated its tissue distribution and localization in the gonads by real-time PCR, western blotting, and immunohistochemistry. The P. olivaceus calb2 gene has 11 exons and 10 introns, and the full-length cDNA is 1457 bp, including an open reading frame (ORF) of 816 bp encoding 271 amino acids. The CALB2 of P. olivaceus has a higher homology with Lates calcarifer (99%) compared with other species. The conserved synteny of calb2 neighboring gene loci was also detected in fish. Real-time PCR showed that the expression of calb2 mRNA is abundant not only in the brain, but also in the gonads, and exhibits a higher expression in the testis than in the ovary. Western blotting indicated that the CALB2 protein has a higher expression in the testis compared with the ovary. Immunohistochemistry demonstrated that the CALB2 protein appears in Leydig cells and the ovarian germ epithelium. These results reveal that calb2 plays an important role in the gonads of P. olivaceus.