Sex differences in epigenetic regulation of the estrogen receptor-alpha promoter within the developing preoptic area

Abstract

Sex differences in the brain are largely organized by a testicular hormone surge that occurs in males shortly after birth. Although this hormone surge is transient, sex differences in brain and behavior are lasting. Here we describe a sex difference in DNA methylation of the estrogen receptor-alpha (ERalpha) promoter region within the developing rat preoptic area, with males exhibiting more DNA methylation within the ERalpha promoter than females. More importantly, we report that simulating maternal grooming, a form of maternal interaction that is sexually dimorphic with males experiencing more than females during the neonatal period, effectively masculinizes female ERalpha promoter methylation and gene expression. This suggests natural variations in maternal care that are directed differentially at males vs. females can influence sex differences in the brain by creating sexually dimorphic DNA methylation patterns. We also find that the early estradiol exposure may contribute to sex differences in DNA methylation patterns. This suggests that early social interaction and estradiol exposure may converge at the genome to organize lasting sex differences in the brain via epigenetic differentiation.

Figure 1

ERα expression is sexually dimorphic in the rat POA on PN10. A, Male rats (n = 7) express less ERα mRNA than females (n = 6) in the POA on PN10 (*, t test P = 0.036); B, ERα immunoreactivity is also lower in the male (n = 5) compared with the female (n = 6) POA on PN10 (*, t test P = 0.029); inset, a representative immunoblot depicting ERα immunoreactivity and Ponceau S-stained bands used for loading control. Columns are means, and error bars are sem.

Figure 2

SMG masculinizes ERα exon 1b CpG methylation and ERα mRNA expression in the rat POA. A, Sequence of exon1b of the rat ERα gene. Primer annealing sites for the initial and nested PCR are underlined. Individual CpG sites are bolded and numbered for reference to the graphs in B. B, Compared with females (n = 5), the percentage of CpG methylation in exon 1b of the ERα gene within the POA is higher in males (n = 5; #, Holm-Sidak post hoc P = 0.034) and SMG-treated females (n = 5; ##, Holm-Sidak post hoc P = 0.022); by one-way ANOVA [F_(2,12) = 3.957; P < 0.05], male and SMG-treated females had similar CpG methylation (Holm-Sidak post hoc P = 0.955) (inset); *, methylation at site 1 was higher in males and SMG females compared with control females; **, methylation at site 2 was higher in males than control and SMG females. C, Control females (n = 7) express more ERα mRNA in the POA on PN10 than both males (n = 8; #, Holm-Sidak post hoc P = 0.006) and SMG females (n = 6; ##, Holm-Sidak post hoc P = 0.026); by one-way ANOVA (F = 6.719; P < 0.01), male and SMG-treated females had similar ERα mRNA expression (Holm-Sidak post hoc P = 0.364). Columns are means, and error bars are sem.

Figure 3

Neonatal EB treatment increases ERα exon 1b CpG methylation and stably decreases ERα mRNA expression in females. A, Inset, EB treatment (tx) of females (n = 4) on PN0 and PN1 increases the percentage of CpG methylation in exon 1b of the ERα gene within the POA compared with control females (n = 3; #, t test P = 0.041) (inset); *, methylation at sites 1 and 6 is greater in EB-treated compared with control females (P < 0.05). B, EB treatment of females (n = 5) on PN0 and PN1 decreases PN8 ERα mRNA in the POA compared with control female (n = 5) expression; *, t test P = 0.041. Columns are means, and error bars are sem.