Measurement of the mRNAs encoding kisspeptin and its receptor GPR54 in the MBH demonstrated that Kiss1 mRNA abundance increases in this brain region between EJ and LJ, and that inhibition of DNA methylation prevented this change (Fig. 5a). In contrast Gpr54 mRNA levels remained unaltered in LJ animals as compared with EJ rats, but were significantly increased by the inhibition of DNA methylation (Fig. 5b). These results suggest that the hyper-responsiveness of the GnRH neuronal network to kisspeptin seen in Aza-treated rats may be related, at least in part, to a reduced endogenous production of kisspeptin in the presence of upregulated levels of its GPR54 receptor. Methylation of the Kiss1 promoter remained unchanged in the MBH of LJ animals as compared to EJ rats (Fig. 5c), indicating that the increase in Kiss1 mRNA abundance observed at the end of juvenile development is not caused by alterations in promoter methylation. Although Aza decreased Kiss1 promoter methylation (Fig. 5c), it obliterated the pubertal increase in Kiss1 mRNA levels, suggesting that inhibition of DNA methylation prevents the pubertal increase in Kiss1 expression by mechanisms other than changes in Kiss1 promoter methylation.
