Relative contributions of sex hormones, sex chromosomes, and gonads to sex differences in tissue gene regulation.

Sex differences in physiology and disease in mammals result from the effects of three classes of factors that are inherently unequal in males and females: reversible (activational) effects of gonadal hormones, permanent (organizational) effects of gonadal hormones, and cell-autonomous effects of sex chromosomes, as well as genes driven by these classes of factors. Often, these factors act together to cause sex differences in specific phenotypes, but the relative contribution of each and the interactions among them remain unclear. Here, we used the four core genotypes (FCG) mouse model with or without hormone replacement to distinguish the effects of each class of sex-biasing factors on transcriptome regulation in liver and adipose tissues. We found that the activational hormone levels have the strongest influence on gene expression, followed by the organizational gonadal sex effect, and last, sex chromosomal effect, along with interactions among the three factors. Tissue specificity was prominent, with a major impact of estradiol on adipose tissue gene regulation and of testosterone on the liver transcriptome. The networks affected by the three sex-biasing factors include development, immunity and metabolism, and tissue-specific regulators were identified for these networks. Furthermore, the genes affected by individual sex-biasing factors and interactions among factors are associated with human disease traits such as coronary artery disease, diabetes, and inflammatory bowel disease. Our study offers a tissue-specific account of the individual and interactive contributions of major sex-biasing factors to gene regulation that have broad impact on systemic metabolic, endocrine, and immune functions.

Neuroprotection in Cerebral Cortex Induced by the Pregnancy Hormone Estriol.

In multiple sclerosis (MS), demyelination occurs in the cerebral cortex, and cerebral cortex atrophy correlates with clinical disabilities. Treatments are needed in MS to induce remyelination. Pregnancy is protective in MS. Estriol is made by the fetoplacental unit, and maternal serum estriol levels temporally align with fetal myelination. Here, we determined the effect of estriol treatment on the cerebral cortex in the preclinical model of MS, experimental autoimmune encephalomyelitis (EAE). Estriol treatment initiated after disease onset decreased cerebral cortex atrophy. Neuropathology of the cerebral cortex showed increased cholesterol synthesis proteins in oligodendrocytes, more newly formed remyelinating oligodendrocytes, and increased myelin in estriol-treated EAE mice. Estriol treatment also decreased the loss of cortical layer V pyramidal neurons and their apical dendrites and preserved synapses. Together, estriol treatment after EAE onset reduced atrophy and was neuroprotective in the cerebral cortex.

Parent-of-origin differences in DNA methylation of X chromosome genes in T lymphocytes.

Many autoimmune diseases are more frequent in females than in males in humans and their mouse models, and sex differences in immune responses have been shown. Despite extensive studies of sex hormones, mechanisms underlying these sex differences remain unclear. Here, we focused on sex chromosomes using the "four core genotypes" model in C57BL/6 mice and discovered that the transcriptomes of both autoantigen and anti-CD3/CD28 stimulated CD4+ T lymphocytes showed higher expression of a cluster of 5 X genes when derived from XY as compared to XX mice. We next determined if higher expression of an X gene in XY compared to XX could be due to parent-of-origin differences in DNA methylation of the X chromosome. We found a global increase in DNA methylation on the X chromosome of paternal as compared to maternal origin. Since DNA methylation usually suppresses gene expression, this result was consistent with higher expression of X genes in XY cells because XY cells always express from the maternal X chromosome. In addition, gene expression analysis of F1 hybrid mice from CAST × FVB reciprocal crosses showed preferential gene expression from the maternal X compared to paternal X chromosome, revealing that these parent-of-origin effects are not strain-specific. SJL mice also showed a parent-of-origin effect on DNA methylation and X gene expression; however, which X genes were affected differed from those in C57BL/6. Together, this demonstrates how parent-of-origin differences in DNA methylation of the X chromosome can lead to sex differences in gene expression during immune responses.

Gene expression in oligodendrocytes during remyelination reveals cholesterol homeostasis as a therapeutic target in multiple sclerosis.

Regional differences in neurons, astrocytes, oligodendrocytes, and microglia exist in the brain during health, and regional differences in the transcriptome may occur for each cell type during neurodegeneration. Multiple sclerosis (MS) is multifocal, and regional differences in the astrocyte transcriptome occur in experimental autoimmune encephalomyelitis (EAE), an MS model. MS and EAE are characterized by inflammation, demyelination, and axonal damage, with minimal remyelination. Here, RNA-sequencing analysis of MS tissues from six brain regions suggested a focus on oligodendrocyte lineage cells (OLCs) in corpus callosum. Olig1-RiboTag mice were used to determine the translatome of OLCs in vivo in corpus callosum during the remyelination phase of a chronic cuprizone model with axonal damage. Cholesterol-synthesis gene pathways dominated as the top up-regulated pathways in OLCs during remyelination. In EAE, remyelination was induced with estrogen receptor-ß (ERß) ligand treatment, and up-regulation of cholesterol-synthesis gene expression was again observed in OLCs. ERß-ligand treatment in the cuprizone model further increased cholesterol synthesis gene expression and enhanced remyelination. Conditional KOs of ERß in OLCs demonstrated that increased cholesterol-synthesis gene expression in OLCs was mediated by direct effects in both models. To address this direct effect, ChIP assays showed binding of ERß to the putative estrogen-response element of a key cholesterol-synthesis gene (Fdps). As fetal OLCs are exposed in utero to high levels of estrogens in maternal blood, we discuss how remyelinating properties of estrogen treatment in adults during injury may recapitulate normal developmental myelination through targeting cholesterol homeostasis in OLCs.

Cell-specific and region-specific transcriptomics in the multiple sclerosis model: Focus on astrocytes.

Changes in gene expression that occur across the central nervous system (CNS) during neurological diseases do not address the heterogeneity of cell types from one CNS region to another and are complicated by alterations in cellular composition during disease. Multiple sclerosis (MS) is multifocal by definition. Here, a cell-specific and region-specific transcriptomics approach was used to determine gene expression changes in astrocytes in the most widely used MS model, experimental autoimmune encephalomyelitis (EAE). Astrocyte-specific RNAs from various neuroanatomic regions were attained using RiboTag technology. Sequencing and bioinformatics analyses showed that EAE-induced gene expression changes differed between neuroanatomic regions when comparing astrocytes from spinal cord, cerebellum, cerebral cortex, and hippocampus. The top gene pathways that were changed in astrocytes from spinal cord during chronic EAE involved decreases in expression of cholesterol synthesis genes while immune pathway gene expression in astrocytes was increased. Optic nerve from EAE and optic chiasm from MS also showed decreased cholesterol synthesis gene expression. The potential role of cholesterol synthesized by astrocytes during EAE and MS is discussed. Together, this provides proof-of-concept that a cell-specific and region-specific gene expression approach can provide potential treatment targets in distinct neuroanatomic regions during multifocal neurological diseases.

Sex chromosome contributions to sex differences in multiple sclerosis susceptibility and progression.

BACKGROUND: Why are women more susceptible to multiple sclerosis, but men have worse disability progression? Sex differences in disease may be due to sex hormones, sex chromosomes, or both. OBJECTIVE: Determine whether differences in sex chromosomes can contribute to sex differences in multiple sclerosis using experimental autoimmune encephalomyelitis. METHODS: Sex chromosome transgenic mice, which permit the study of sex chromosomes not confounded by differences in sex hormones, were used to examine an effect of sex chromosomes on autoimmunity and neurodegeneration, focusing on X chromosome genes. RESULTS: T-lymphocyte DNA methylation studies of the X chromosome gene Foxp3 suggested that maternal versus paternal imprinting of X chromosome genes may underlie sex differences in autoimmunity. Bone marrow chimeras with the same immune system but different sex chromosomes in the central nervous system suggested that differential expression of the X chromosome gene Toll-like receptor 7 in neurons may contribute to sex differences in neurodegeneration. CONCLUSION: Mapping the transcriptome and methylome in T lymphocytes and neurons in females versus males could reveal mechanisms underlying sex differences in autoimmunity and neurodegeneration.

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken-the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

X chromosome regulation of autosomal gene expression in bovine blastocysts.

Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here, we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male-to-female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient.

Feminized behavior and brain gene expression in a novel mouse model of Klinefelter Syndrome.

Klinefelter Syndrome (KS) is the most common sex chromosome aneuploidy in men and is characterized by the presence of an additional X chromosome (XXY). In some Klinefelter males, certain traits may be feminized or shifted from the male-typical pattern towards a more female-typical one. Among them might be partner choice, one of the most sexually dimorphic traits in the animal kingdom. We investigated the extent of feminization in XXY male mice (XXYM) in partner preference and gene expression in the bed nucleus of the stria terminalis/preoptic area and the striatum in mice from the Sex Chromosome Trisomy model. We tested for partner preference using a three-chambered apparatus in which the test mouse was free to choose between stimulus animals of either sex. We found that partner preference in XXYM was feminized. These differences were likely due to interactions of the additional X chromosome with the Y. We also discovered genes that differed in expression in XXYM versus XYM. Some of these genes are feminized in their expression pattern. Lastly, we also identified genes that differed only between XXYM versus XYM and not XXM versus XYM. Genes that are both feminized and unique to XXYM versus XYM represent strong candidates for dissecting the molecular pathways responsible for phenotypes present in KS/XXYM but not XXM. In sum, our results demonstrated that investigating behavioral and molecular feminization in XXY males can provide crucial information about the pathophysiology of KS and may aid our understanding of sex differences in brain and behavior.

Cell-autonomous sex determination outside of the gonad.

BACKGROUND: The classic model of sex determination in mammals states that the sex of the individual is determined by the type of gonad that develops, which in turn determines the gonadal hormonal milieu that creates sex differences outside of the gonads. However, XX and XY cells are intrinsically different because of the cell-autonomous sex-biasing action of X and Y genes. RESULTS: Recent studies of mice, in which sex chromosome complement is independent of gonadal sex, reveal that sex chromosome complement has strong effects contributing to sex differences in phenotypes such as metabolism. Adult mice with two X chromosomes (relative to mice with one X chromosome) show dramatically greater increases in body weight and adiposity after gonadectomy, irrespective of their gonadal sex. When fed a high-fat diet, XX mice develop striking hyperinsulinemia and fatty liver, relative to XY mice. The sex chromosome effects are modulated by the presence of gonadal hormones, indicating an interaction of the sex-biasing effects of gonadal hormones and sex chromosome genes. CONCLUSIONS: Other cell-autonomous sex chromosome effects are detected in mice in many phenotypes. Birds (relative to eutherian mammals) are expected to show more widespread cell-autonomous sex determination in non-gonadal tissues, because of ineffective sex chromosome dosage compensation mechanisms.

Sex bias and dosage compensation in the zebra finch versus chicken genomes: general and specialized patterns among birds.

We compared global patterns of gene expression between two bird species, the chicken and zebra finch, with regard to sex bias of autosomal versus Z chromosome genes, dosage compensation, and evolution of sex bias. Both species appear to lack a Z chromosome-wide mechanism of dosage compensation, because both have a similar pattern of significantly higher expression of Z genes in males relative to females. Unlike the chicken Z chromosome, which has female-specific expression of the noncoding RNA MHM (male hypermethylated) and acetylation of histone 4 lysine 16 (H4K16) near MHM, the zebra finch Z chromosome appears to lack the MHM sequence and acetylation of H4K16. The zebra finch also does not show the reduced male-to-female (M:F) ratio of gene expression near MHM similar to that found in the chicken. Although the M:F ratios of Z chromosome gene expression are similar across tissues and ages within each species, they differ between the two species. Z genes showing the greatest species difference in M:F ratio were concentrated near the MHM region of the chicken Z chromosome. This study shows that the zebra finch differs from the chicken because it lacks a specialized region of greater dosage compensation along the Z chromosome, and shows other differences in sex bias. These patterns suggest that different avian taxa may have evolved specific compensatory mechanisms.

Sex differences in the neuronal transcriptome and synaptic mitochondrial function in the cerebral cortex of a multiple sclerosis model.

Introduction: Multiple sclerosis (MS) affects the cerebral cortex, inducing cortical atrophy and neuronal and synaptic pathology. Despite the fact that women are more susceptible to getting MS, men with MS have worse disability progression. Here, sex differences in neurodegenerative mechanisms are determined in the cerebral cortex using the MS model, chronic experimental autoimmune encephalomyelitis (EAE). Methods: Neurons from cerebral cortex tissues of chronic EAE, as well as age-matched healthy control, male and female mice underwent RNA sequencing and gene expression analyses using RiboTag technology. The morphology of mitochondria in neurons of cerebral cortex was assessed using Thy1-CFP-MitoS mice. Oxygen consumption rates were determined using mitochondrial respirometry assays from intact as well as permeabilized synaptosomes. Results: RNA sequencing of neurons in cerebral cortex during chronic EAE in C57BL/6 mice showed robust differential gene expression in male EAE compared to male healthy controls. In contrast, there were few differences in female EAE compared to female healthy controls. The most enriched differential gene expression pathways in male mice during EAE were mitochondrial dysfunction and oxidative phosphorylation. Mitochondrial morphology in neurons showed significant abnormalities in the cerebral cortex of EAE males, but not EAE females. Regarding function, synaptosomes isolated from cerebral cortex of male, but not female, EAE mice demonstrated significantly decreased oxygen consumption rates during respirometry assays. Discussion: Cortical neuronal transcriptomics, mitochondrial morphology, and functional respirometry assays in synaptosomes revealed worse neurodegeneration in male EAE mice. This is consistent with worse neurodegeneration in MS men and reveals a model and a target to develop treatments to prevent cortical neurodegeneration and mitigate disability progression in MS men.

Estrogen receptor beta in astrocytes modulates cognitive function in mid-age female mice.

Menopause is associated with cognitive deficits and brain atrophy, but the brain region and cell-specific mechanisms are not fully understood. Here, we identify a sex hormone by age interaction whereby loss of ovarian hormones in female mice at midlife, but not young age, induced hippocampal-dependent cognitive impairment, dorsal hippocampal atrophy, and astrocyte and microglia activation with synaptic loss. Selective deletion of estrogen receptor beta (ERß) in astrocytes, but not neurons, in gonadally intact female mice induced the same brain effects. RNA sequencing and pathway analyses of gene expression in hippocampal astrocytes from midlife female astrocyte-ERß conditional knock out (cKO) mice revealed Gluconeogenesis I and Glycolysis I as the most differentially expressed pathways. Enolase 1 gene expression was increased in hippocampi from both astrocyte-ERß cKO female mice at midlife and from postmenopausal women. Gain of function studies showed that ERß ligand treatment of midlife female mice reversed dorsal hippocampal neuropathology.

Possible differences in the two Z chromosomes in male chickens and evolution of MHM sequences in Galliformes.

The male hypermethylated (MHM) region of the chicken Z chromosome encodes a non-coding RNA that is expressed only in females. The MHM sequence is found only in galliform birds, and Z genes near this region show an unusual degree of dosage compensation between males and females despite the overall low level of dosage compensation in Z chromosome gene expression in birds. Here we report that the MHM locus shows a dramatic sex difference in the configuration of chromatin, open in females and condensed in males, based on DNA fluorescent in situ hybridization of an MHM probe in interphase nuclei. The demethylating agent 5-aza-cytidine causes an asymmetric effect on the two Z chromosomes of males, altering the chromatin configuration, MHM RNA expression, and H4K16Ac modification, suggesting an inequality in the methylation status and chromatin of the two Z chromosomes. We identified numerous MHM-related genomic and RNA sequences that possess a short conserved sequence common to the majority of clones, suggesting the functional importance of the MHM region. Some of the RNA sequences, which like MHM are expressed in females but not in males, are likely to be polyadenylated and have genomic intron/exon structure. The turkey, another galliform bird, has repetitive sequences in the predicted turkey MHM region, raising the question of regional dosage compensation in the turkey as in the chicken.

Karyotypic polymorphism of the zebra finch Z chromosome.

We describe a karyotypic polymorphism on the zebra finch Z chromosome. This polymorphism was discovered because of a difference in the position of the centromere and because it occurs at varying frequencies in domesticated colonies in the USA and Germany and among two zebra finch subspecies. Using DNA fluorescent in situ hybridization to map specific Z genes and measurements of DNA replication, we show that this polymorphism is the result of a large pericentric inversion involving the majority of the chromosome. We sequenced a likely breakpoint for the inversion and found many repetitive sequences. Around the breakpoint, there are numerous repetitive sequences and several copies of PAK3 (p21-activated kinase 3)-related sequences (PAK3Z) which showed testes-specific expression by RT-PCR. Our findings further suggest that the sequenced genome of the zebra finch may be derived from a male heterozygote for the Z chromosome polymorphism. This finding, in combination with regional differences in the frequency of the polymorphism, has important consequences for future studies using zebra finches.

What a difference an X or Y makes: sex chromosomes, gene dose, and epigenetics in sexual differentiation.

A modern general theory of sex determination and sexual differentiation identifies the factors that cause sexual bias in gene networks, leading to sex differences in physiology and disease. The primary sex-biasing factors are those encoded on the sex chromosomes that are inherently different in the male and female zygotes. These factors, and downstream factors such as gonadal hormones, act directly on tissues to produce sex differences and antagonize each other to reduce sex differences. Recent studies of mouse models such as the four core genotypes have begun to distinguish between the direct effects of sex chromosome complement (XX vs. XY) and hormonal effects. Several lines of evidence implicate epigenetic processes in the control of sex differences, although a great deal of information is needed about sex differences in the epigenome.

The X factor in neurodegeneration.

Given the aging population, it is important to better understand neurodegeneration in aging healthy people and to address the increasing incidence of neurodegenerative diseases. It is imperative to apply novel strategies to identify neuroprotective therapeutics. The study of sex differences in neurodegeneration can reveal new candidate treatment targets tailored for women and men. Sex chromosome effects on neurodegeneration remain understudied and represent a promising frontier for discovery. Here, we will review sex differences in neurodegeneration, focusing on the study of sex chromosome effects in the context of declining levels of sex hormones during aging.

Sex Differences in the Immune System Become Evident in the Perinatal Period in the Four Core Genotypes Mouse.

We have used the four core genotypes (FCG) mouse model, which allows a distinction between effects of gonadal secretions and chromosomal complement, to determine when sex differences in the immune system first appear and what influences their development. Using splenic T cell number as a measure that could be applied to neonates with as yet immature immune responses, we found no differences among the four genotypes at postnatal day 1, but by day 7, clear sex differences were observed. These sex differences were unexpectedly independent of chromosomal complement and similar in degree to gonadectomized FCG adults: both neonatal and gonadectomized adult females (XX and XY) showed 2-fold the number of CD4+ and 7-fold the number of CD8+ T cells versus their male (XX and XY) counterparts. Appearance of this long-lived sex difference between days 1 and 7 suggested a role for the male-specific perinatal surge of testicular testosterone. Interference with the testosterone surge significantly de-masculinized the male CD4+, but not CD8+ splenic profile. Treatment of neonates demonstrated elevated testosterone limited mature cell egress from the thymus, whereas estradiol reduced splenic T cell seeding in females. Neonatal male splenic epithelium/stroma expressed aromatase mRNA, suggesting capacity for splenic conversion of perinatal testosterone into estradiol in males, which, similar to administration of estradiol in females, would result in reduced splenic T cell seeding. These sex steroid effects affected both CD4+ and CD8+ cells and yet interference with the testosterone surge only significantly de-masculinized the splenic content of CD4+ cells. For CD8+ cells, male cells in the thymus were also found to express one third the density of sphingosine-1-phosphate thymic egress receptors per cell compared to female, a male characteristic most likely an indirect result of Sry expression. Interestingly, the data also support a previously unrecognized role for non-gonadal estradiol in the promotion of intra-thymic cell proliferation in neonates of both sexes. Microarray analysis suggested the thymic epithelium/stroma as the source of this hormone. We conclude that some immune sex differences appear long before puberty and more than one mechanism contributes to differential numbers and distribution of T cells.

Disruption of FEM1C-W gene in zebra finch: evolutionary insights on avian ZW genes.

Sex chromosome genes control sex determination and differentiation, but the mechanisms of sex determination in birds are unknown. In this study, we analyzed the gene FEM1C which is highly conserved from Caenorhabditis elegans to higher vertebrates and interacts with the sex determining pathway in C. elegans. We found that FEM1C is located on the Z and W chromosome of zebra finches and probably other Passerine birds, but shows only Z linkage in other avian orders. In the zebra finch, FEM1C-W is degraded because of a point mutation and possibly because of loss of the first exon containing the start methionine. Thus, FEM1C-W appears to have degenerated or been lost from most bird species. FEM1C-Z is expressed in a cytoplasmic location in zebra finch fibroblast cells, as in C. elegans. FEM1C represents an interesting example of evolutionary degradation of a W chromosome gene.