The presence of robust gender differences in the frequency and intensity of mental health and neurological disorders underscores a need to identify the fundamental mechanisms mediating the organization of sex differences in the brain. That developmental exposure to gonadal steroids organizes enduring changes in brain and behavior is one of the central tenets of neuroendocrinology, but the mechanisms by which early hormone exposure imparts these life-long changes are still largely unknown. The preoptic area (POA) of the laboratory rat is sexually differentiated by estradiol during a perinatal critical period and thereby provides an ideal model system to explore these unknown mechanisms. DNA methylation is the epigenetic process by which a DNA methyltransferase (DNMT) enzyme covalently modifies cytosine dinucleotides within a gene's promoter region via addition of a methyl group, resulting in changes in chromatin conformation and gene silencing. Our preliminary data indicate that newborn males and estradiol-treated females have lower levels of DNMT activity and decreased methylation of genes associated with masculinization of the POA compared to females. Based on these findings, we hypothesize that estradiol-induced sex differences in DNMT activity organize enduring sex differences in neural morphology and behavior. Our first goal (Specific Aim 1) is to determine the role of sex differences in DNMT activity in maintaining sex-specific dendritic morphology. The density of dendritic spine synapses within the POA is 2-3 times higher in males and hormonally masculinized females compared to control females, and this pattern is maintained from birth through adulthood. With the use of live cell imaging of POA neuron/glia cultures, we will record dynamic changes in synaptic patterning following DNMT inhibition. We predict that low levels of DNMT activity (typical of the neonatal male) will result in masculinized dendritic spine density and patterning in cultured POA neurons. Our second goal (Specific Aim 2) is to associate sex differences in neonatal DNMT activity with sex-specific behavior in adulthood. Based on a preliminary finding that lowering neonatal DNMT activity defeminizes sexual behavior in adulthood, we predict that DNMT enzyme inhibition during the perinatal critical period will result in masculinized sex behavior in testosterone-primed adult female rats. Finally, under Specific Aim 3, our goal is to identify differentially regulated genetic targets of sex-specific DNMT activity. We will use a genome-wide microarray analysis of mRNA from male and female animals treated with DNMT inhibitors or vehicle to categorize differential gene expression. Completion of these studies will provide novel insight into the mechanisms maintaining sex differences in the brain and will address for the first time how hormonal changes established neonatally endure across the life-span. These studies have significance for understanding gender biases in the etiology of mental health and neurological disorders, as well as to understanding fundamental mechanisms of brain development.

Public Health Relevance

The pervasiveness of strong gender biases in the prevalence and intensity of mental health and neurological disorders highlights the importance of understanding the biological basis of sex differences in the brain. Studying epigenetic control of sex-specific gene expression patterns during neonatal development, and its impact on the organization of sex-specific neural morphology and behavior, is a necessary step towards understanding brain development as a whole in addition to understanding the mechanisms controlling life-long establishment of synaptic patterning.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-F02A-J (20))
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Gnadt, James W
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University of Maryland Baltimore
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McCarthy, Margaret M; Nugent, Bridget M (2015) At the frontier of epigenetics of brain sex differences. Front Behav Neurosci 9:221
Nugent, Bridget M; Wright, Christopher L; Shetty, Amol C et al. (2015) Brain feminization requires active repression of masculinization via DNA methylation. Nat Neurosci 18:690-7
McCarthy, M M; Nugent, B M (2013) Epigenetic contributions to hormonally-mediated sexual differentiation of the brain. J Neuroendocrinol 25:1133-40
Nugent, Bridget M; Valenzuela, Carla V; Simons, Timothy J et al. (2012) Kinases SPAK and OSR1 are upregulated by estradiol and activate NKCC1 in the developing hypothalamus. J Neurosci 32:593-8
Nugent, B M; Tobet, S A; Lara, H E et al. (2012) Hormonal programming across the lifespan. Horm Metab Res 44:577-86
Nugent, Bridget M; McCarthy, Margaret M (2011) Epigenetic underpinnings of developmental sex differences in the brain. Neuroendocrinology 93:150-8