Two populations of kisspeptin neurons, located in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV), are considered to be involved in generating GnRH pulse and surge respectively. The present study aimed to determine the region-specific enhancer for ARC Kiss1 gene expression by in vivo reporter assay using transgenic (Tg) mice. Three GFP reporter constructs (long-, middle-, and short-length) were generated by insertion of GFP cDNA in the translational start site of Kiss1 gene in the mouse BAC clone. The DNA constructs were microinjected into the pronucleus of fertilized one-cell stage embryos of BDF1 mice. GFP expressions in the hypothalamus were validated in ovariectomized (OVX) Tg female mice in the presence or absence of estradiol-17β. Tg lines bearing long- and 5′-truncated middle-length constructs showed GFP-immunoreactivities in both AVPV and ARC in female mice. On the other hand, Tg mice bearing 5′-truncated short-length construct showed few GFP signals in the ARC kisspeptin neurons, while the Tg line showed GFP-immunoreactivities in the AVPV kisspeptin neurons. Chromosome conformation capture assay showed an interaction between the promoter and 5′ region of Kiss1 locus in the ARC, but not in the AVPV. Taken together, these results suggest that the upstream region of Kiss1 locus is essential for ARC Kiss1 gene expression in mice via formation of chromatin loop with the proximal promoter of Kiss1 gene. This work was supported in part by the Research Program on Innovative Technologies for Animal Breeding, Reproduction, and Vaccine Development.
02 Sep 2014 - 04 Sep 2014