The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually-dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in females. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation.

The male bias in autism spectrum disorder incidence is among the most extreme of all neuropsychiatric disorders, yet the origins of the sex difference remain obscure. Developmentally males are exposed to high levels of testosterone and its byproduct, estradiol. Together these steroids modify the course of brain development by altering neurogenesis, cell death, migration, differentiation, dendritic and axonal growth, synaptogenesis and synaptic pruning, all of which can be deleteriously impacted during the course of developmental neuropsychiatric disorders. Elucidating the cellular mechanisms by which steroids modulate brain development provides valuable insights into how these processes may go awry. An emerging theme is the role of inflammatory signaling molecules and the innate immune system in directing brain masculinization, the evidence for which we review here. Likewise evidence is emerging that the neuroimmune system is over-activated in individuals with autism spectrum disorder. These combined observations lead us to propose that the natural process of brain masculinization puts males at risk by moving them closer to a vulnerability threshold that could more easily be breached by inflammation during critical periods of brain development. Two brain regions are highlighted, the preoptic area and the cerebellum. Both are developmentally regulated by the inflammatory prostaglandin, PGE2, but in very different ways. Microglia, innate immune cells of the brain, and astrocytes are also critical contributors to masculinization and illustrate the importance of non-neuronal cells to the health of the developing brain.

The hormonal regulation of sexual behavior has been the topic of study for over 50 years and yet controversies persist regarding the importance of early versus late events and the identity of the critical neural and cellular substrates. We have taken a mechanistic approach toward the masculinizing actions of the gonadal steroid estradiol, as a means to understand how organization of the neuroarchitechture during a perinatal sensitive period exerts enduring influences on adult behavior. We have identified important roles for prostaglandins, FAK and paxillin, PI3 kinase and glutamate, and determined that cell-to-cell signaling is a critical component of the early organizational events. We have further determined that the mechanisms mediating different components of sexual behavior are distinct and regionally specific. The multitude of mechanisms by which the steroid estradiol, exerts divergent effects on the developing nervous system provides for a multitude of phenotypes which can vary significantly both within and between the sexes.

Prostaglandin E2 (PGE2) mediates the masculinization of adult sex behavior in rats in response to the surge in serum testosterone around birth. Measures of behavioral masculinization correlate with a two-fold increase in spinophilin protein and the density of dendritic spines in the medial preoptic area (POA). Of the four receptors for PGE2, EP2 and EP4 are required for the masculinization of behavior by PGE2. EP2 and EP4 couple to Gs-proteins, activating PKA. By using H89 and Ht31, disruptors of PKA signaling, we have determined that PKA signaling is required for the masculinization of behavior by PGE2. Glutamatergic signaling often mediates PGE2 signaling; therefore, we tested if inhibition of AMPA/kainate and metabotropic glutamate receptor signaling prevents PGE2-induced behavioral masculinization and if activation of glutamate receptors mimics PGE2. Females treated neonatally with NBQX + LY341495 combined (AMPA/kainate and mGluR inhibitors respectively) prior to PGE2 did not exhibit as many mounts or intromission-like behaviors or initiate these behaviors as quickly as animals treated with PGE2 alone. Animals neonatally treated with either kainate, ACPD (type-I-mGluR agonist), or the two combined, mounted as frequently and initiated mounting behavior as quickly as those given PGE2. Ht31 does not prevent the masculinization of behavior by ACPD + kainate cotreatment; rather the coadministration of NBQX + LY341495 prevents the forskolin-induced formation of POA dendritic spine-like processes. We conclude that PKA, AMPA/kainate and metabotropic glutamate receptor signaling are necessary for PGE2’s effects, that each receptor individually suffices to organize behavior, and that PKA is upstream of the glutamate receptors.

Gonadal steroids organize the developing brain during a perinatal sensitive period and exert enduring consequences for adult behavior. In male rodents, testicular androgens are aromatized in neurons to estrogens and initiate multiple distinct cellular processes that ultimately determine the masculine phenotype. Within specific brain regions, overall cell number and dendritic morphology are principle targets of hormonal organization. Recent advances have been made in elucidating the cellular mechanisms by which the neurological underpinnings of sexually dimorphic physiology and behavior are determined. These include estradiol-mediated prostaglandin synthesis, presynaptic release of glutamate, postsynaptic changes in glutamate receptors and changes in cell adhesion molecules. Sex differences in cell death are mediated by hormonal modulation of survival and death factors such as TNFα and Bcl-2/BAX. Sexual differentiationThe term "sexual differentiation" refers to a series of events that begins with sex determination by the SRY gene on the Y chromosome which programs the bipotential gonad to become a testis, followed by hormonally mediated cascades that direct the formation of the reproductive tract, genitalia, secondary sex characteristics and ultimately, the brain (reviewed in [1]). Production of testicular hormones during development coordinately organizes the brain and behavior in a process described by the Organizational/Activational Hypothesis of sexual differentiation [2]. This simple tenet articulates the complex notion that steroid hormones act on the developing brain to permanently organize it in a manner that directs the actions of adult hormones. Testosterone from the male testis masculinizes the brain during a perinatal sensitive period and as a result, the adult male brain is sensitive to the sex-behavior inducing effects of testosterone. Successful masculinization is evident in an adult male's mounting, intromission and ejaculatory behavior with a sexually receptive female. The opposite of masculinization is feminization, an organizational process that occurs by default when there is insufficient gonadal steroid exposure during the critical period. Successful feminization is evident in