Christopher L. Wright

Brain feminization requires active repression of masculinization via DNA methylation.

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 female rats. 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.

New tricks by an old dogma: Mechanisms of the Organizational / Activational Hypothesis of steroid-mediated sexual differentiation of brain and behavior

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.

Mechanisms Mediating Oestradiol Modulation of the Developing Brain

The brain has been known to be a sensitive target organ for the permanent organisational effects of gonadal steroids for close to 50 years. Recent advances have revealed a variety of unexpected cellular mechanisms by which steroids impact on the synaptic profile of hypothalamic nuclei critical to the control of reproduction. This review focuses on three in particular: 1) prostaglandins in the masculinisation of the preoptic area and control of male sexual behaviour; 2) GABA in the arcuate nucleus and potential control of the anterior pituitary; and 3) non‐genomic activation of phosphotydolinositol 3 (PI3) kinase and glutamate in the ventromedial nucleus, which is relevant to the control of female reproductive behaviour. The importance of cell‐to‐cell communication, be it between neurones or between neurones and astrocytes, is highlighted as an essential principle for expanding the impact of steroids beyond those cells that express nuclear receptors.

Prostaglandin E2-Induced Masculinization of Brain and Behavior Requires Protein Kinase A, AMPA/Kainate, and Metabotropic Glutamate Receptor Signaling

Prostaglandin E2 (PGE2) mediates the masculinization of adult sex behavior in rats in response to the surge in serum testosterone at approximately birth. Measures of behavioral masculinization correlate with a twofold 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 protein kinase A (PKA). By using H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl) 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 whether inhibition of AMPA/kainate and metabotropic glutamate receptor (mGluR) signaling prevents PGE2-induced behavioral masculinization and whether activation of glutamate receptors mimics PGE2. Females treated neonatally with NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline) plus LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] combined (AMPA/kainate and mGluR inhibitors, respectively) before 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 kainate, (±)-1-amino-1,3-cyclopentanedicarboxylic acid (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 plus kainate cotreatment; rather, the coadministration of NBQX plus 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 the effects of PGE2, that each receptor individually suffices to organize behavior, and that PKA is upstream of the glutamate receptors.

Identification of Prostaglandin E2 Receptors Mediating Perinatal Masculinization of Adult Sex Behavior and Neuroanatomical Correlates

Prostaglandin E2 (PGE2) mediates the organization of male rat sexual behavior and medial preoptic area (MPOA) neuroanatomy during a sensitive perinatal window. PGE2 is up‐regulated in response to estradiol, and initiates a two‐fold increase in dendritic spines densities on neurons. All the four receptors for PGE2 and EP1‐4 are present in developing POA, a critical region controlling male sexual behavior. Previous studies explored that EP receptors are involved in PGE2‐induction of neonatal levels of spinophilin protein, a surrogate marker for dendritic spine formation, but did not assess behavioral masculinization. Here, we used two approaches, suppression of EP receptor expression with antisense oligonucleotides and activation of EP receptors with selective agonists, to test which receptors are necessary and sufficient, respectively, for the effects of PGE2 on behavior and neuronal morphology. In female rats, neonatal treatment with antisense oligonucleotides against EP2 or EP4 but not EP1 or EP3 completely prevented the expression of adult behavior organized by PGE2 exposure. The effects of ONO‐DI‐004, ONO‐AE‐259‐01, ONO‐AE‐248, and ONO‐AE1‐329 (EP1‐4 agonists respectively) were equivalent to PGE2 treatment, which suggests activating any EP receptor neonatally suffices in masculinizing sex behavior. When given alone, not all EP agonists increased neonatal POA spinophilin levels; yet giving each agonist neonatally increased adult levels. Moreover, adult spinophilin levels significantly correlated with two measures of male sexual behavior. The body of evidence suggests that EP2 and EP4 are both necessary and sufficient for PGE2‐induced masculinization of sex behavior, whereas EP1 and EP3 provide redundant roles.

Convergence of Sex Differences and the Neuroimmune System in Autism Spectrum Disorder.

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. Evidence is also emerging that the neuroimmune system is overactivated 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 E2, but in different ways. Microglia, innate immune cells of the brain, and astrocytes are also critical contributors to masculinization and illustrate the importance of nonneuronal cells to the health of the developing brain.

Exploration of prostanoid receptor subtype regulating estradiol and prostaglandin E2 induction of spinophilin in developing preoptic area neurons

The prostaglandin E2 (PGE2) mediates estradiol-induced masculinization of sexual behavior in the rat during a perinatal sensitive period. PGE2 induces formation of dendritic spines on preoptic area (POA) neurons and this synaptic pattern change is associated with the ability to express male sexual behavior as an adult. Whether PGE2 is released from astrocytes or neurons in the developing POA is unknown. To further understanding of how PGE2 induces dendritic spine formation at the cellular level, we have explored the PGE2 receptor subtype mediating this response. There are four receptors for PGE2, EP1, EP2, EP3 and EP4, each having unique but interacting signal transduction profiles. Treatment of newborn female rats with the EP receptor agonists iloprost, butaprost and sulprostone indicated that stimulation of both the EP2 and EP3 receptors significantly increased spinophilin, a protein whose levels positively correlate to the presence of dendritic spines and masculinization of the POA. Use of antisense oligonucleotides against the mRNA for each receptor reveals that either EP2 or EP3 receptor knockdown reduces spinophilin in PGE2- or estradiol-treated females, whereas reducing EP1 or EP4 receptor levels by the same means has a smaller but also significant effect. A developmental profile of EP receptor expression indicates EP1 in particular is elevated for the first few days of life, corresponding to the critical period for masculinization, whereas mRNA levels for the other three receptors remain relatively constant.

Prostaglandin E2 Regulates AMPA Receptor Phosphorylation and Promotes Membrane Insertion in Preoptic Area Neurons and Glia during Sexual Differentiation

Sexual differentiation of the rodent brain is dependent upon the organizing actions of the steroid hormone, estradiol. In the preoptic area, a brain region critical for the expression of adult reproductive behavior, there are twice as many dendritic spine synapses per unit length on newborn male neurons compared to female neurons and this sex difference correlates with the expression of adult male copulatory behavior. The sex difference in the POA is achieved via estradiols upregulation of the membrane-derived lipid signaling molecule prostaglandin E2 (PGE2); PGE2 is necessary and sufficient to masculinize both dendritic spine density and adult sexual behavior in rats. We have previously shown that PGE2 activates EP2 and EP4 receptors which increases protein kinase A (PKA) activity and that masculinized dendritic spine density and sex behavior are both dependent upon PKA as well as activation of AMPA type glutamate receptors. In the current experiments, we build upon this signaling cascade by determining that PGE2 induces phosphorylation of the AMPA receptor subunit, GluR1, which leads to increased AMPA receptor insertion at the membrane. Treating female pups on the day of birth with PGE2 induced the phosphorylation of GluR1 at the PKA-sensitive site within 2 hours of treatment, an effect that was blocked by co-administration of the PKA/AKAP inhibitor, HT31 with PGE2. Brief treatment of mixed neuronal/glial POA cultures with PGE2 or the cAMP/PKA stimulator, forskolin, increased membrane associated GluR1 in both neurons and glia. We speculate that PGE2 induced increases in AMPA receptor associated with the membrane underlies our previously observed increase in dendritic spine density and is a critical component in the masculinization of rodent sex behavior.

Prostaglandin E2 Stimulates Estradiol Synthesis in the Cerebellum Postnatally with Associated Effects on Purkinje Neuron Dendritic Arbor and Electrophysiological Properties

Prostaglandins (PGs) are ubiquitous membrane-derived, lipid-signaling molecules with wide ranging effects throughout the body. In the brain, PGE(2) is the key regulator of fever after inflammation but is also implicated in neural development and synaptic plasticity. The steroid hormone estradiol is also a key regulator of neural development and synaptic plasticity. Recently, we showed that administering cyclooxygenase (COX) inhibitors to block PGE(2) production increased the total length of Purkinje cell dendrites, the number of dendritic spines, and the level of spinophilin protein, which is enriched in dendritic spines. Correspondingly, PGE(2) administration into the cerebellum decreased spinophilin protein content. We now report that PGE(2) stimulates estradiol synthesis in the immature rat cerebellum via enhanced activity of the aromatase enzyme. Treatment with cyclooxygenase inhibitors reduced cerebellar aromatase activity and estradiol content whereas PGE(2) administration increased both. Treatment with either PGE(2) or estradiol stunted Purkinje neuron dendritic length and complexity and produced a corresponding reduction in spinophilin content. Treatment with formestane to inhibit aromatase activity led to excessive sprouting of the dendritic tree, whereas elevated estradiol had the opposite effect. Electrophysiological measurements from Purkinje neurons revealed novel sex differences in input resistance and membrane capacitance that were abolished by estradiol exposure, whereas a sex difference in the amplitude of the afterhyperpolarization after an action potential was not. Correlated changes in action potential threshold suggest that prolonged alterations in neuronal firing activity could be a consequence of increased estradiol content during the second week of life. These findings reveal a previously unappreciated role for PG-stimulated steroidogenesis in the developing brain and a new potential route for inflammation-mediated disruption of neuronal maturation.

Masculinization induced by neonatal exposure to PGE2 or estradiol alters c-fos induction by estrous odors in adult rats

Processing of relevant olfactory and pheromonal cues has long been known as an important process necessary for social and sexual behavior in rodents. Several nuclei that receive input from the vomeronasal projection pathway are involved in sexual behavior and show changes in immediate early gene expression after stimulation with a variety of sex-related stimuli. The nuclei in this pathway are sexually dimorphic due to the early patterning events induced by estradiol derived from testicular androgens, which developmentally defeminize and masculinize the brain and adult sexual behavior. Masculinization can be induced independently of estradiol via prostaglandin-E(2) (PGE(2)), and therefore assessed separately from defeminization. Here we examined the effects of brain defeminization and masculinization on neuronal response to sex-related odors using Fos, the protein product of the immediate early gene c-fos, as an indicator of activity. Female rat pups treated with a cyclooxygenase-2 inhibitor, to reduce PGE(2), plus estradiol, estradiol alone, and PGE(2) alone were exposed to estrous female odor as adults and the resulting Fos expression was examined in the medial amygdala, preoptic area, and ventromedial nucleus of the hypothalamus. Defeminized and/or masculinized females all showed patterns of Fos activity similar to control males and significantly different from control females. These results suggest that early exposure to estradiol and PGE(2) do not affect olfaction in females, but switch the activity pattern of sex-related nuclei in females to resemble that of males following exposure to sexually-relevant cues.