Sex differences in neural development are established via a number of cellular processes (i.e., migration, death and survival).One critical factor identified is the neonatal rise in testosterone (T) which activates gene transcription via androgen (AR) and, after aromatization to estradiol, estrogen receptors (ERα and β). Recent evidence shows that AR and ERs interact with histone modifying enzymes. Post-translational modifications of histones, including acetylation and methylation, are involved in transcriptional regulation during normal development. Therefore, we hypothesized that acetylation and/or methylation of histone H3 may underlie sexual differentiation, at least in some regions of the brain. We measured levels of acetylated (H3K9/14Ac) and trimethylated (H3K9Me3) H3 in whole neonatal mouse brains and in three regions: preoptic area + hypothalamus, amygdala and cortex + hippocampus (CTX/HIP). Sex differences in H3K9/14Ac and H3K9Me3 (males > females) were noted in the CTX/HIP on embryonic day 18, the day of birth, and six days later. To determine if T mediates these changes in H3 modifications, pregnant dams received vehicle or T for the final four days of gestation; pup brains were collected at birth. Methylation of H3 was sexually dimorphic despite hormone treatment. In contrast, H3 acetylation in the CTX/HIP of females from T-treated dams rose to levels equivalent to males. Thus, H3 modifications are sexually dimorphic in the developing mouse CTX/HIP and acetylation, but not methylation, is masculinized in females by T in utero. This is the first demonstration that histone modification is associated with neural sexual differentiation.

The cerebral cortex and hippocampus are important for the control of cognitive functions and social behaviors, many of which are sexually dimorphic and tightly regulated by gonadal steroid hormones via activation of their respective nuclear receptors. As different levels of sex steroid hormones are present between the sexes during early development and their receptors act as transcription factors to regulate gene expression, we hypothesize that sexually dimorphic gene expression in the developing mouse cortex and hippocampus might result in sex differences in brain structures and neural circuits governing distinct behaviors between the sexes as adults. To test our hypothesis, we used gene expression microarrays to identify 90 candidate genes differentially expressed in the neonatal cortex/hippocampus between male and female mice, including 55 male-biased and 35 female-biased genes. Among these genes, sexually dimorphic expression of eight sex chromosome genes was confirmed by reverse transcription with quantitative PCR (RT-qPCR), including three located on the X chromosome (Xist, Eif2s3x, and Kdm6a), three on the Y chromosome (Ddx3y, Eif2s3y, and Kdm5d), and two in the pseudoautosomal region of the X and Y chromosomes (Erdr1 and Mid1). In addition, five autosomal genes (Cd151, Dab2, Klk8, Meg3, and Prkdc) were also validated for their sexually dimorphic expression in the neonatal mouse cortex/hippocampus. Gene Ontology annotation analysis suggests that many of these sexually dimorphic genes are involved in histone modifications, cell proliferation/death, androgen/estrogen signaling pathways, and synaptic organization, and these biological processes have been implicated in differential neural development, cognitive function, and neurological diseases between the sexes.

Sexual behavior is variable between individuals, ranging from celibacy to sexual addictions. Within normal populations of individual men, ranging from young to middle aged, testosterone levels do not correlate with libido. To study the genetic mechanisms that contribute to individual differences in male sexual behavior, we used hybrid B6D2F1 male mice, which are a cross between two common inbred strains (C57BL/6J and DBA/2J). Unlike most laboratory rodent species in which male sexual behavior is highly dependent upon gonadal steroids, sexual behavior in a large proportion of these hybrid male mice after castration is independent of gonadal steroid hormones and their receptors; thus, we have the ability to discover novel genes involved in this behavior. Gene expression arrays, validation of gene candidates, and transgenic mice that overexpress one of the genes of interest were used to reveal genes involved in maintenance of male sexual behavior. Several genes related to neuroprotection and neurodegeneration were differentially expressed in the hypothalamus of males that continued to mate after castration. Male mice overexpressing the human form of one of these candidate genes, amyloid ␤ precursor protein (APP), displayed enhanced sexual behavior before castration and maintained sexual activity for a longer duration after castration compared with controls. Our results reveal a novel and unexpected relationship between APP and male sexual behavior. We speculate that declining APP during normal aging in males may contribute to the loss of sexual function.

17beta-Estradiol (E2)-stimulated estrogen receptor (ERalpha) transcription is accompanied by protein degradation via the 26S-proteasome pathway. Inhibition of proteasome activity stabilizes ERalpha protein and abolishes E2-activated transcription, suggesting functional linkages between transcription and degradation. It is not known whether ligand-independent ERalpha activation is coupled to proteolysis. In pituitary cells, forskolin (FSK) stimulates ERalpha transcription through the protein kinase A (PKA) pathway. This study examined interactions between E2-dependent and PKA-stimulated pathways in GH(3) cells by measuring transcription of a transfected reporter gene and endogenous ERalpha levels. E2 stimulated estrogen response element-mediated transcription 2- to 3-fold and decreased ERalpha protein levels to 40%. In contrast, FSK stimulated ERalpha transcription without decreasing ERalpha protein. Treatment with FSK plus E2 resulted in synergistic ERalpha transactivation, and FSK specifically prevented E2-induced ERalpha degradation. PKA is required for protection and was prevented by H89 (a PKA inhibitor), but not PD98059 (a MAPK kinase inhibitor). Propyl-pyrazole-triol and R,R-diethyl-tetrahydrochrysene, selective ERalpha agonists, reduced ERalpha protein by 50% while stimulating ERalpha transcriptional activity 4- to 8-fold. The antagonist ICI 182,780 similarly decreased ERalpha levels, but prevented ER activation. FSK prevented all ligand-induced ERalpha degradation. Lactacystin, a proteasome inhibitor, abolished E2-stimulated, but not FSK-stimulated, ERalpha transcription. Thus, stimulation of ERalpha transcription by the PKA-dependent pathway is dissociated from receptor degradation and proteasome activity. These data suggest a mechanism of ERalpha transcriptional activation by PKA that is distinct from E2 activation and that may contribute to the synergistic transcriptional activation of ERalpha by ligand-dependent and PKA-dependent pathways.