There is sufficient evidence to indicate that kisspeptin pla

There is sufficient evidence to indicate that kisspeptin plays a role in regulating sheep reproduction. However, the molecular mechanism by which kisspeptin regulates GnRH K-Ras(G12C) inhibitor 12 and reproduction has remained unclear. Therefore, in this study on GnRH in sheep, the promoter region of GnRH was cloned, and reporter vectors with fragments of the promoter region deleted were constructed. The core promoter region of GnRH was identified by dual-fluorescence reporter assay and bioinformatics method, and the transcription factors mediating GnRH gene expression were identified. From a cellular perspective, the results elucidated the molecular mechanism by which kisspeptin regulates the expression of the GnRH gene via Otx-2. The study results provide a theoretical basis for demonstrating the molecular mechanism by which ewe kisspeptin mediates GnRH gene expression and have theoretical importance for further elucidating the foundations of reproductive regulation in animals and understanding animal reproductive behaviors.
Materials and methods
Results
Discussion Seasonal reproduction is a common adaptive method that allows for breeding at times when it is most advantageous for the growth of offspring. In recent years, the neural and neuroendocrine circuitry responsible for seasonal sheep reproduction has been extensively studied; kisspeptin regulates GnRH neurons and changes in the activity of relevant neurons in the hypothalamus (Clarke and Cummins, 1982; Titi et al., 2010; Javed et al., 2015). However, the molecular mechanism by which kisspeptin regulates GnRH has remained unclear in sheep and thus required further elucidation. Therefore, we looked into the key region of the GnRH gene promoter that controls the expression of GnRH in sheep—animals with seasonal reproduction—and also evaluated the transcription factors that could bind with these key segments and control GnRH expression in sheep. We thus investigated the role of kisspeptin in modulating GnRH transcription/translation activity. Our results provide evidence that the core promoter region from −1912 bp to −1461 bp of the GnRH gene maybe involve in the regulation of hypothalamic GnRH by kisspeptin and that binding of the transcription factor Otx-2 potentially mediates this activation. Analysis of Otx-2 binding sites in different species, the results suggested that in mammals, Otx-2 can bind to the GnRH promoter region, regulating GnRH expression. Promoters consist of a core promoter region and a regulatory region. The former region is for gene transcription, while the latter region properly regulates gene expression (Bucher, 1990; Alfred et al., 2009). Many eukaryotic promoters feature 80–220 bp sequences upstream of the transcriptional initiation site that can markedly increase low basal levels of transcription (Schumacher et al., 2003). The experimental results showed that the recombinant plasmids with the GnRH gene promoter had obvious transcriptional activity compared with the empty pGL3-Basic vector, indicating that the cloned promoter region of the GnRH gene had transcriptional activity. In addition, the transcriptional activity of the recombinant plasmid pGL3-8 was not quite as high but was not significantly different from that of pGL3-3, pGL3-4, pGL3-5, pGL3-6 or pGL3-7, which indicated that the TATA signal sequence in the −80 bp to +174 bp region was one of the major transcriptional elements of GnRH, though it was not regulated by kisspeptin. Additionally, the activity of pGL3-2 was significantly higher than that of the other seven recombinant plasmids (P < 0.05), indicating that there were important transcription factor binding sites in the pGL3-2 and pGL3-3 differential sequence from −1912 bp to −1461 bp. In contrast, the activity of pGL3-1 was significantly lower than that of pGL3-2, suggesting that negative transcriptional regulators or silencers may exist in the region from −2251 bp to −1913 bp upstream of the transcriptional initiation site. In addition to transcriptional regulation of the GnRH gene, recent studies have shown that increased GnRH mRNA expression at the onset of puberty in small-tailed Han sheep may be associated with decreased methylation levels at specific sites in the promoter region of the hypothalamic GnRH gene (Ding et al., 2018); however, no methylation islands were found in the promoter region of sheep GnRH in this study.