Within the hypothalamic-pituitary-ovarian axis, inhibition of DNA methylation did not affect the capacity of the ovary to respond to gonadotropin stimulation with estrogen release, and failed to alter the pituitary gonadotropin response to GnRH, suggesting a central site of action. Direct assessment of the GnRH response to kisspeptin, a major GnRH secretagogue 24, revealed that GnRH neurons of Aza-treated animals are hyper-responsive, instead of unresponsive, to kisspeptin. Although 5-Aza, like other DNMT inhibitors, may also act via mechanisms other than DNA methylation 45, 46, our results are consistent with the interpretation that pharmacological inhibition of DNA methylation prevents a methylation event scheduled to occur at the onset of puberty. Without ruling out GnRH neurons as direct targets of epigenetic control 47, our results suggest that: a) the pubertal delay caused by inhibition of DNA methylation involves cellular subsets functionally connected to the GnRH neuronal network, and b) the deficit may result from the activation of repressive genes whose expression would normally decrease at puberty. By inference, these repressors would be expected to negatively control the expression of downstream genes which need to be activated for puberty to occur.
