The genetics and epigenetics of sex differences in the brain.

The major drivers underlying sexually dimorphic brain development are gonadal hormones, namely testosterone (T). During the perinatal sensitive period, a time when the embryonic brain is maximally sensitive to changes in the levels of gonadal hormones, exposure to T has permanent organizing effects on the brain, the molecular basis of which is not known. One potential mechanism for the long term permanence may be DNA methylation. To examine the contribution of epigenetic mechanisms to both the establishment and maintenance of sex differences, I compared the methylomes of male, female, and female mice treated with testosterone. Methylation maps were generated for sexually dimorphic brain regions such as the striatum at postnatal day 4 (PN4) during the sensitive period and PN60 during adulthood using reduced representation bisulfite sequencing. I found that testosterone altered the methylation of a few genes during the sensitive period but a much greater number in adulthood. I next investigated whether administration of a single dose of testosterone to females on the day of birth could induce a shift in DNA methylation from a female-typical to a more male-typical pattern. The results demonstrated that the masculinizing effect of testosterone was mostly evident at PN60 but not at PN4. This observation provided a new perspective on the mechanisms underlying organizational effects of testosterone because contrary to the expectation that testosterone leaves a strong, stable imprint shortly after exposure, testosterone effects on DNA methylation were not immediately evident but emerged later. Based on these data, I concluded that sex differences in methylation are not the result of the immediate early actions of testosterone on the brain. Rather, the neural molecular patterns found in adults are conditioned by early hormonal exposures, the effects of which might emerge over a period of time. Gene Ontology analysis on the set of genes whose methylation was altered by testosterone revealed a significant enrichment of genes belonging to signaling components associated with dopamine modulation as well as movement disorders that display a male-bias. These data are consistent with striatum's role in regulation of movement.In addition to assessing the contribution of hormones to brain sexual differentiation, I also investigated the impact of sex chromosomes on sex differences in brain and behavior. To test for sex chromosome effects, I used the four core genotypes mouse model and found sex differences in expression of a subset of striatal genes caused by XX vs. XY differences in mice with the same gonadal type. Moreover, comparison of animals with different numbers of sex chromosomes in a novel mouse model of Klinefelter Syndrome (KS), the Sex Chromosome Trisomy Model, indicated that presence of an additional X chromosome and/or its interaction with the Y in XXY male mice can contribute to some of the behavioral and molecular phenotypes observed in KS. Interestingly, analysis of striatal transcriptome in KS mice revealed a feminized molecular signature in the brain of KS male mice. Such information is crucial knowledge in elucidating not only the pathophysiology of KS, but also the origin of sex differences in brain and behavior. Altogether, my work demonstrates the significance of genetics and epigenetics in the process of brain development as it relates to sex. The results presented in this dissertation suggest that (1) the sex chromosomes carry genes that could influence brain function and behavior; and (2) the long lasting effects of steroid hormones on the brain could be mediated by epigenetic mechanisms such as DNA methylation.