Neonatal Inhibition of DNA Methylation Alters Cell Phenotype in Sexually Dimorphic Regions of the Mouse Brain

Abstract

Many of the best-studied neural sex differences relate to differences in cell number and are due to the hormonal control of developmental cell death. However, several prominent neural sex differences persist even if cell death is eliminated. We hypothesized that these may reflect cell phenotype "decisions" that depend on epigenetic mechanisms, such as DNA methylation. To test this, we treated newborn mice with the DNA methyltransferase (DNMT) inhibitor zebularine, or vehicle, and examined two sexually dimorphic markers at weaning. As expected, control males had more cells immunoreactive for calbindin-D28k (CALB) in the medial preoptic area (mPOA) and fewer cells immunoreactive for estrogen receptor α (ERα) in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMHvl) and the mPOA than did females. Neonatal DNMT inhibition markedly increased CALB cell number in both sexes and ERα cell density in males; as a result, the sex differences in ERα in the VMHvl and mPOA were completely eliminated in zebularine-treated animals. Zebularine treatment did not affect developmental cell death or the total density of Nissl-stained cells at weaning. Thus, a neonatal disruption of DNA methylation apparently has long-term effects on the proportion of cells expressing CALB and ERα, and some of these effects are sex specific. We also found that sex differences in CALB in the mPOA and ERα in the VMHvl persist in mice with a neuron-specific depletion of either Dnmt1 or Dnmt3b, indicating that neither DNMT alone is likely to be required for the sexually dimorphic expression of these markers.

Past, present and future of epigenetics in brain sexual differentiation

“…Neonatal inhibition of DNA methylation increased ERα cell density eight‐fold in males and completely eliminated the sex difference (Figure ). A similar pattern was seen in the mPOA . This suggests that the sex differences in ERα in the VMHvl and mPOA depend on differential DNA methylation in males and females.…”

“…A sex difference in the opposite direction (male>female) is seen for calbindin‐expressing cells in the mPOA. We found that neonatally inhibiting DNA methylation significantly increased the number of calbindin‐expressing cells several weeks later, and did so in both sexes . Thus, within the same animal, neonatal DNMT inhibition feminised some cell groups (ERα in VMH and mPOA of males) and made other cell groups more male‐like (calbindin in the mPOA).…”

“…injections of the DNMT inhibitor zebularine on P0 and P1 and then examined the number of neurones expressing ERα and calbindin at weaning. The neonatal inhibition of DNA methylation led to marked, long lasting and sex‐specific increases in the number of ERα cells . For example, control females had seven‐fold more ERα cells in the VMHvl than did males.…”

Molecular mechanisms underlying sexual differentiation of the nervous system

“…Recent work by Forger’s group showed that pharmacological inhibition of DNA methyltransferase activity at birth feminized expression of Esr1, a nuclear hormone receptor for estrogen, in two hypothalamic nuclei [41•]. This suggests that DNA methylation may establish or maintain male-specific gene repression in these regions.…”

Neuroendocrine disruption of organizational and activational hormone programming in poikilothermic vertebrates

“…In mice, the organizational effects of T results in striking and delayed brain methylome and transcriptomic effects (Ghahramani et al 2014), suggesting that critical windows of exposure to NED might be even more prolonged that originally envisioned. Recently, Mosley et al (2017) reported in mice that neonatal treatment with a DNA methyltransferase (DNMT) inhibitor, zebularine, increased the number of calbindin-D28K (CALB) cell number in the medial preoptic area (mPOA) in both sexes and resulted in greater ESR1 cell density in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMHvl) and mPOA. This latter zebularine affect thus abolished sex differences in ESR1 expression observed otherwise in control individuals.…”