Identification of a promoter element mediating kisspeptin-induced increases in GnRH gene expression in sheep.

Gonadotropin-releasing hormone (GnRH) plays an important role in regulating the activities of other components downstream of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining the normal reproductive cycle of animals. However, the molecular mechanisms by which GnRH synthesis and secretion are regulated in sheep remains unclear. In this study, a series of eight recombinant vectors with deletion fragments were constructed and cotransfected with pGL3-Basic and pRL-SV40 into sheep hypothalamic neuronal cells. After treatment with 1 nM kisspeptin, the core promoter of the sheep GnRH gene was identified to be in the region of -1912 bp to -1461 bp by dual-luciferase reporter assay. Bioinformatics analysis showed that there was a binding site for the transcription factor Otx-2 in the core promoter region (-1786 to -1770 bp) that was highly conserved among different species. The expression patterns of Kiss-1, Otx-2 and GnRH in the sheep hypothalamus were the same, and the expression of Kiss-1, Otx-2 and GnRH was significantly higher in the breeding season than in nonbreeding season (P < 0.01). In addition, when hypothalamic neurons were cultured in vitro with kisspeptin, kisspeptin induced the expression of GnRH and Otx-2. In conclusion, these results provide evidence that the core promoter region (-1786 to -1770 bp) of the GnRH gene is involved in the regulation of hypothalamic activity by kisspeptin and that binding of the transcription factor Otx-2 mediates this activation.