Molecular identification of GnIH/GnIHR signal and its reproductive function in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides)

Gonadotropin-inhibitory hormone (GnIH) and its receptor (GnIHR) play an important role in reproduction regulation in birds, mammals and some teleost species. In protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides), the GnIH/GnIHR signaling pathway and its reproductive function have not been addressed yet. In this study, GnIH and GnIHR in orange-spotted grouper were characterized. gGnIH possessed three putative peptides (gGnIH-I, -II, -III), while gGnIHR showed the characteristics of G protein-coupled receptor and was clustered with GPR147. Functional assays demonstrated that three synthetic gGnIH peptides significantly decreased the forskolin-induced CRE promoter activity, but only gGnIH-I could significantly decrease SRE promoter activity in COS-7 cells transfected with gGnIHR. During the process of ovarian differentiation and development, gGnIH mRNA level in hypothalamus was low at the gonadal primordium stage with gonia, then increased significantly at the early differentiated gonad with primary growth oocytes, while decreased significantly at the developing gonads with cortical-alveolus and vitellogenic stage oocytes. During MT-induced sex reversal, gGnIH mRNA level in hypothalamus increased significantly when the fish completely reversed from female to male. However, gGnIHR mRNA level in pituitary decreased significantly in intersex and completely reversed male fish. Intraperitoneal injection (i.p.) of three gGnIH peptides significantly decreased GnRH1 mRNA levels in hypothalamus, and gGnIH-II significantly inhibited synthesis of LHβ in pituitary. In summary, we firstly identified the GnIH/GnIHR signal in protogynous orange-spotted grouper, which might be involved in the regulation of the reproductive function of sex differentiation, gonadal development and sex reversal via regulating the synthesis of both GnRH and GtH.