Genetic Mechanisms Mediating Kisspeptin Regulation Of GnRH Gene Expression In Sheep

Genetics study shows, KISS-1 and Gn RH genes are the key genes to control the seasonal oestrus of seasonal breeding animals, including sheep. Although we have known that kisspeptin regulated expression of Gn RH in sheep, the specific molecular mechanism is unclear. Therefore we looked into the key region of Gn RH gene promoter that control expression of Gn RH in sheep, and transcription factors that could bind with these key segments and control Gn RH expression by using Altay sheep--the seasonal breeding animals as experimental animal, and combined techniques such as dual luciferase report system, the hypothalamus primary cell culture and bioinformatics analysis as approaches.The main results are as follows. First, we successfully developed the system for culturing of the hypothalatic nerve cells of sheep. The fetal sheep hypothalamic cells retained proper morphological and functional characteristics of hypothalamic neurons in the in vitro system. Then we constructed a serial of recombinants that fused different length of Gn RH gene promoter region from-2313 bp to +173bp with luciferase reporter gene. Through the dual fluorescence detection and bioinformatics analysis found that, compared with the empty plasmid p GL3-Basic, the plasmids of p GL3-1, p GL3-2, p GL3-3, p GL3-4, p GL3-5, p GL3-6, p GL3-7 and p GL3-8 have obvious activity(P<0.05), among them, p GL3-1, p GL3-3, p GL3-4, p GL3-5, p GL3-6, p GL3-7 and p GL3-8 plasmids expression of the activity is relatively similar(P>0.05). The plasmid of p GL3-2 activity was significantly higher than p GL3-Basic and other plasmids(P<0.05).We used RT-PCR and real-time fluorescent quantitative PCR techniques to research the tissue expression profile of KISS-1, Otx-2, Gn RH and MT1 genes. From the cell and individual levels, we proved that kisspeptin may regulate the expression of Gn RH gene by Otx-2.The results suggested that the core promoter of sheep Gn RH gene located at-1843 bp ~-1507 bp in about regions of 300 bp, and some negative transcriptional regulatory elements in the regions of-2313bp~-1843 bp. Otx-2 transcription factors can be combined in-1508bp~-1503 bp on the promoter, and kisspeptin may regulate the expression of Gn RH gene by Otx-2 in sheep.