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Abstract
Kisspeptin, and its G-protein-coupled receptor, Kiss1r, participate in the regulation of gonadotropin-releasing hormone (GnRH) secretion, which regulates feedback pathways that control reproductive hormones. Neuronal Kisspeptin signaling is sensitive to varying estrogen levels during the estrous cycle, but it is not known whether GnRH neuron sensitivity to Kisspeptin is affected by estrogen. To address this, we determined the promoter region of Kiss1r by analyzing the nucleotide sequence upstream of the gene. First, Rapid Amplification of 5' cDNA Ends (RACE) was performed to locate the Kiss1r mRNA 5' Transcription Start Site (TSS). We found that, contrary to the TSS's indicated in the UCSC Genome Browser for other tissues, the mouse Kiss1r TSS is either approximately 199 base pairs (bp) or 248 bp upstream from the Kiss1r translation start site in mouse brain. Next, to further characterize the nucleotide sequences upstream of the ATG site, potential Kiss1r promoter regions were cloned into pGL3 reporter vectors. GT1-7 and HeLa cell lines were co-transfected with these constructs along with ERα expression vector and a TKβgal control for subsequent estrogen treatments. Luciferase and β-galactosidase assays were performed to determine whether estrogen treatment induced an effect on the expression of our potential Kiss1r promoters. We concluded that estrogen induces an increase in activity within the region -4971 to -1681 bp upstream of Kiss1r. Overall, this approach will help determine whether estrogen regulation of Kiss1r has a role in GnRH neuronal signaling and the feedback pathways that regulate reproductive function.
