Molecular Characterisation and Reporter Gene Assay of the Three GnRH Isoforms and Two GnRH Receptors of a Clupeiform Fish, Japanese Sardine, Sardinops

The gonadotropin-releasing hormone (GnRH) is an important gene involved in fish reproduction and its action is mediated by GnRH receptor (GnRHR). Numerous GnRH-GnRHR studies have already been published in many teleost orders but information on reproduction-related genes for Clupeiformes is limited. To acquire a better understanding on the reproduction of Clupeiform fish species, GnRH isoforms and GnRHR paralogues of Japanese sardine, Sardinops sagax melanostictus (Temminck & Schlegel, 1846) were sequenced. Three GnRH isoforms (named as jsGnRH1, jsGnRH2, and jsGnRH3) and two GnRHR paralogues (named as jsGnRHR1 and jsGnRHR2) were isolated in the brain and pituitary of Japanese sardine. Based on phylogenetic analysis, jsGnRH1, jsGnRH2, and jsGnRH3 grouped into GnRH1, GnRH2, and GnRH3 forms, respectively, while jsGnRHR1 and jsGnRHR2 clustered into Type 2b and Type 1c receptors, correspondingly. The reporter gene assay showed high binding affinity of jsGnRHR1 to all three GnRH synthetic decapeptides, whereas jsGnRHR2 responded best to jsGnRH2 only. Clustering of jsGnRH1and jsGnRHR1 to other teleost GnRH1 and GnRHR2b, respectively, and activating the calcineurin/NFAT signalling pathway, suggests that these two genes are likely involved in fish reproduction.