DNA methylation and demethylation underlie the sex difference in estrogen receptor alpha in the arcuate nucleus

Introduction Neurons expressing estrogen receptor (ER) α in the arcuate nucleus (ARC) of the hypothalamus sex-specifically control energy homeostasis and bone density. Females have more of these neurons than do males, but how this sex difference develops is unknown. Objective We tested the hypothesis that DNA methylation and/or demethylation control the development of a sex difference in ERα in the ARC. Methods ERα immunoreactive neurons were quantified at birth and at weaning in male, female and testosterone-treated female mice that received neonatal, intracerebroventricular injections of vehicle or zebularine, a DNA methyltransferase inhibitor. Methylation status of Esr1 was determined in the ARC and ventromedial hypothalamus (VMH) using bisulfite conversion of DNA followed by pyrosequencing. Small interfering RNAs against ten-eleven translocases were used to examine effects of demethylation on ERα cell number. Results A sex difference in ERα cell number in the ARC, favoring females, developed between birth and weaning and was due to programming effects of testosterone. Zebularine treatment eliminated the sex difference in ERα in the ARC at weaning by decreasing ERα in females to male-like levels. Previously, the same treatment increased ERα in males in the VMH. A promoter region of Esr1 exhibited sex differences in opposite directions in percent of total methylation in the ARC (females > males) and VMH (males > females). Moreover, neonatal inhibition of demethylation increased ERα in the ARC of males. Conclusion DNA methylation and demethylation regulate ERα cell number in the ARC, and methyl marks may paradoxically activate Esr1 in this region.