Effect of DNA Methyltransferase Activity on Sexual Differentiation of the Rodent Brain

Abstract

Developmental exposure to gonadal steroids organizes enduring changes in brain and behavior, but the mechanisms by which early hormone exposure imparts these life-long changes are still largely unknown. The preoptic area (POA) is a brain region necessary for male reproductive behavior that is sexually differentiated by estradiol during a perinatal critical period. DNA methylation is an epigenetic process executed by DNA methyltransferase (DNMT) enzymes that impacts gene expression by altering chromatin conformation and modulating transcription factor binding. We found that females have significantly higher levels of DNMT activity and global genomic methylation in the POA compared to males during the critical period for sexual differentiation of the brain, but not thereafter. Administering a masculinizing dose of estradiol to females significantly decreases DNMT activity in the female POA, suggesting that estradiol may permanently masculinize the POA by controlling DNMT activity during the critical period. Further, pharmacological inhibition of DNMT activity in the developing female POA to mimic male DNMT activity levels results in anatomical and behavioral masculinization, and partial behavioral defeminization in adulthood. No exogenous manipulations have ever effectively masculinized the female POA if administered outside of the critical period for sexual differentiation, however we have shown that inhibiting DNMT activity following the close of the critical period masculinizes both neuronal morphology and behavior in the female rat. These observations prompted us to rethink our definition of feminization as a passive, "default" process, and led to the hypothesize that feminization of the brain requires active, life-long suppression of masculinization via epigenetic mechanisms. In addition, we hypothesized that sex differences in DNMT activity organize sex-specific gene expression in the neonatal POA. To identify novel candidate genes involved in methylation-dependent and -independent masculinization and feminization, we quantified differences in gene expression in the POA of male and female rat pups treated with a DNMT inhibitor or vehicle using RNA-Seq. We identified novel gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females.