Nr5a1b promotes and Nr5a2 inhibits transcription of lhb in the orange-spotted grouper, Epinephelus coioides†

Abstract

Abstract Nr5a1 (Sf-1) up-regulates lhb expression across vertebrates; however, its regulatory roles on fshb remain to be defined. Moreover, the involvement of Nr5a2 in the regulation of gonadotropin expression is not clear either. In the present study, the involvement of Nr5a1b (a homologue of Nr5a1) and Nr5a2 in the regulation of lhb and fshb expression in the orange-spotted grouper was examined. Dual fluorescent immunohistochemistry using homologous antisera showed that in the pituitary of orange-spotted groupers, Lh cells contain both immunoreactive Nr5a1b and Nr5a2 signals, whereas Fsh cells contain neither of them. In LβT2 cells, Nr5a1b up-regulated basal activities of lhb and fshb promoters possibly via Nr5a sites, and synergistically (on lhb promoter) or additively (on fshb promoter) with forskolin. Surprisingly, Nr5a2 inhibited basal activities of lhb promoter possibly via Nr5a sites and attenuated the stimulatory effects of both forskolin and Nr5a1b. In contrast, Nr5a2 had no effects on fshb promoter. Chromatin immunoprecipitation analysis showed that both Nr5a1b and Nr5a2 bound to lhb promoter, but not fshb promoter in the pituitary of the orange-spotted grouper. The abundance of Nr5a1b bound to lhb promoter was significantly higher at the vitellogenic stage than the pre-vitellogenic stage, whereas that of Nr5a2 exhibited an opposite trend. Taken together, data of the present study demonstrated antagonistic effects of Nr5a1b and Nr5a2 on lhb transcription in the orange-spotted grouper and revealed novel regulatory mechanisms of differential expression of lhb and fshb genes through Nr5a homologues in vertebrates.