In this study three potential Otx binding
In this study, three potential Otx-2 ApoBrdU DNA Fragmentation Assay Kit were found in the promoter region of the sheep GnRH gene: −1786 bp to −1770 bp, −825 bp to −819 bp, and − 529 bp to −523 bp. Combined with the results of the double-luciferase assay, it could be inferred that the binding site of Otx-2 in the core promoter region from −1786 bp to −1770 bp may promote the transcription of the GnRH gene. The RNA-Seq dataset from GenBank included two hypothalamus samples, for which GnRH expression was not detected. Analysis of the expression of the Kiss-1 and Otx-2 genes, the finding suggested that the expression patterns of Kiss-1 and Otx-2 are highly correlated and that these genes play an important biological role in the hypothalamus. There are many inconsistencies between Fig. 4A and Fig. 4B because of the different species and physiological stages. Through analysis of the expression patterns of GnRH and Otx-2 in sheep in different seasons, it was found that the expression patterns of GnRH and Otx-2 were highly associated. The expression of GnRH and Otx-2 was higher in the breeding season than in nonbreeding season (P < 0.01). These results suggest that the Kiss-1 gene participates in the regulation of sheep seasonal reproduction and GnRH transcription by inducing Otx-2 in sheep hypothalamic neurons. Kisspeptin is a major regulatory factor of gonadotropin secretion. Kisspeptin is crucial for pubertal development, steroid-mediated feedback regulation and fertility control (Skorupskaite et al., 2014). Studies have shown that endogenous kisspeptin could be used as a key factor in GnRH activation or inhibition (Verma et al., 2014). Studies by Colledge and Kirilov have shown that kisspeptin binds to GPR54 in GnRH neurons to activate the HPG axis in mammals. GnRH neurons are the key reproductive sites related to kisspeptin/GPR54 signal transduction (Colledge, 2009; Kirilov et al., 2013). In addition, the effects of reproduction-related gene mutations have been reported. The results of associative analyses have shown that these mutations are significantly related to the number of lambs produced (Cui et al., 2018; Wang et al., 2018). Other studies have shown that kisspeptin neurons in female mice may be the earliest neurons involved in aging and may be involved in initiating early reproductive decline (Zhang et al., 2014). In this study, the expression of GnRH and Otx-2 in hypothalamic neurons was significantly upregulated by kisspeptin at a concentration of 1 nM, similar to previous findings of the concentration-dependent activation of GnRH neurons by kisspeptin (Verma et al., 2014). Through analysis of potential functional elements in the GnRH promoter region, 33 potential transcription factor binding sites, including sites for Otx-2, GATA-1 and Oct-1, were identified. This study found that only the Otx-2 binding site was in the core region of the GnRH promoter. Otx-2 is a member of the Otx transcription factor family, a conserved family in vertebrates. Otx-2 is homologous to the Drosophila Otd gene and plays an important role in the development of photoreceptors and the medulla oblongata (Koike et al., 2007; Spatazza et al., 2013; Samuel et al., 2014). As confirmed by in vitro experiments on mouse neurons, there are Otx binding sites in the promoter region of GnRH that regulate the expression of GnRH in mouse hypothalamic neurons. Otx-2 is a homologous domain protein necessary for the formation of the forebrain that is overexpressed in GnRH neurons, activating GnRH promoter activity in neuronal cell lines. The deletion of Otx-2 in GnRH neurons results in significant reductions in GnRH neurons in the hypothalamus, delayed puberty and abnormal estrous cycles. After kisspeptin treatment, the mRNA and protein levels of the GnRH promoter transcription factor Otx-2 are increased. Otx-2 knock-out results in severe embryonic dysplasia in mice, especially brain dysplasia, indicating that Otx-2 plays an important regulatory role in brain development (Diaczok et al., 2011). Our study found that the expression of the GnRH and Otx-2 genes changed markedly with the same trend after treatment with 1 nM kisspeptin, indicating that the kisspeptin-mediated transcription factor Otx-2 acts on the upstream core promoter of GnRH to regulate GnRH transcription in sheep.