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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.

Aging is a risk factor for cognitive decline and susceptibility to neurodegenerative diseases. Some aspects of aging, like the loss of sex hormones, may be preventable. Menopause is associated with cognitive deficits and brain atrophy. Since standard hormone replacement therapy (HRT) does not mitigate these brain aging outcomes, a gap in knowledge involves understanding brain region-specific, cell-specific, and receptor-specific mechanisms underlying this neurodegeneration. Here, cognitive testing and in vivo magnetic resonance imaging demonstrated that ovarian hormones in female mice at midlife protect against hippocampal-dependent cognitive impairment and dorsal hippocampal atrophy. Further, this neuroprotection in females at midlife is lost in mice with selective deletion of estrogen receptor beta (ERβ) in astrocytes, but not neurons. This preclinical evidence identifies ERβ in astrocytes as a novel therapeutic target to prevent hippocampal-dependent cognitive deficits and reduce posterior hippocampus atrophy in menopausal women, a major unmet need in half the population.

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, lastly, 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.

Multiple sclerosis (MS) is a neuroinflammatory multifocal disorder. Optic neuritis is common in MS and leads to visual disability. No current treatments repair this damage. Discerning gene expression changes within specific cell types in optic nerve (ON) may suggest new treatment targets for visual disability in MS. Astrocytes are pivotal regulators of neuroinflammation, playing either detrimental or beneficial roles. Here, we used RiboTag technology to characterize the astrocyte-specific transcriptome in ON in the experimental autoimmune encephalomyelitis (EAE) model of MS. RNA sequencing analysis showed the Complement Cascade and Cholesterol Biosynthesis Pathways as the most enriched and de-enriched pathways, respectively, in ON astrocytes in EAE. Expression of complement component 3 (C3) was confirmed to be increased in ON astrocytes at the protein level during EAE. A bigger increase in C3 expressing ON astrocytes was found in EAE females versus healthy females, as compared to that in EAE males versus healthy males. Also, there was worse retinal ganglion cell (RGC) and axonal loss in EAE females. Regression analyses showed a negative correlation between C3 expressing astrocytes and RGC density. This cell-specific and sex-specific investigation of the optic nerve provides targets for the development of therapeutic strategies tailored for optic neuritis in MS.

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.

Significance Sex differences are naturally occurring disease modifiers that, if understood, could lead to novel targets for drug development. Autoimmune diseases are more prevalent in women than in men, and sex differences in immune responses have been shown in humans and mice. Here, we discover a global parent-of-origin difference in DNA methylation on the X chromosome that affects gene expression in activated CD4+ T lymphocytes. The paternal X has more methylation than the maternal X, with higher expression of X genes in XY cells since they only express from the maternal X. Thus, parent-of-origin differences in DNA methylation of X genes can play a role in sex differences in immune responses., 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.

Multiple sclerosis (MS) is a putative T cell–mediated autoimmune disease. As with many autoimmune diseases, females are more susceptible than males. Sexual dimorphisms may be due to differences in sex hormones, sex chromosomes, or both. Regarding sex chromosome genes, a small percentage of X chromosome genes escape X inactivation and have higher expression in females (XX) compared with males (XY). Here, high-throughput gene expression analysis in CD4(+) T cells showed that the top sexually dimorphic gene was Kdm6a, a histone demethylase on the X chromosome. There was higher expression of Kdm6a in females compared with males in humans and mice, and the four core genotypes (FCG) mouse model showed higher expression in XX compared with XY. Deletion of Kdm6a in CD4(+) T cells ameliorated clinical disease and reduced neuropathology in the classic CD4(+) T cell–mediated autoimmune disease experimental autoimmune encephalomyelitis (EAE). Global transcriptome analysis in CD4(+) T cells from EAE mice with a specific deletion of Kdm6a showed upregulation of Th2 and Th1 activation pathways and downregulation of neuroinflammation signaling pathways. Together, these data demonstrate that the X escapee Kdm6a regulates multiple immune response genes, providing a mechanism for sex differences in autoimmune disease susceptibility.

Author(s): MacKenzie-Graham, Allan; Brook, Jenny; Kurth, Florian; Itoh, Yuichiro; Meyer, Cassandra; Montag, Michael J; Wang, He-Jing; Elashoff, Robert; Voskuhl, Rhonda R | Abstract: IntroductionProgressive gray matter (GM) atrophy is a hallmark of multiple sclerosis (MS). Cognitive impairment has been observed in 40%-70% of MS patients and has been linked to GM atrophy. In a phase 2 trial of estriol treatment in women with relapsing-remitting MS (RRMS), higher estriol levels correlated with greater improvement on the paced auditory serial addition test (PASAT) and imaging revealed sparing of localized GM in estriol-treated compared to placebo-treated patients. To better understand the significance of this GM sparing, the current study explored the relationships between the GM sparing and traditional MRI measures and clinical outcomes.MethodsSixty-two estriol- and forty-nine placebo-treated RRMS patients underwent clinical evaluations and brain MRI. Voxel-based morphometry (VBM) was used to evaluate voxelwise GM sparing from high-resolution T1-weighted scans.ResultsA region of treatment-induced sparing (TIS) was defined as the areas where GM was spared in estriol- as compared to placebo-treated groups, localized primarily within the frontal and parietal cortices. We observed that TIS volume was directly correlated with improvement on the PASAT. Next, a longitudinal cognitive disability-specific atlas (DSA) was defined by correlating voxelwise GM volumes with PASAT scores, that is, areas where less GM correlated with less improvement in PASAT scores. Finally, overlap between the TIS and the longitudinal cognitive DSA revealed a specific region of cortical GM that was preserved in estriol-treated subjects that was associated with better performance on the PASAT.ConclusionsDiscovery of this region of overlap was biology driven, not based on an a priori structure of interest. It included the medial frontal cortex, an area previously implicated in problem solving and attention. These findings indicate that localized GM sparing during estriol treatment was associated with improvement in cognitive testing, suggesting a clinically relevant, disability-specific biomarker for clinical trials of candidate neuroprotective treatments in MS.

Significance Molecular mechanisms underlying distinct disabilities during neurological diseases may differ based on the neurological pathway involved. Multiple sclerosis (MS) is multifocal, characterized by distinct disabilities affecting walking, vision, cognition, and fatigue. Neuroprotective treatments tailored for each disability may be more effective than nonspecific treatments aiming to reduce a composite of disabilities in clinical trials. Here, we use the MS model to apply a cell-specific and region-specific gene expression approach to discover targets in distinct neuroanatomic regions. Altered cholesterol synthesis gene expression in astrocytes in spinal cord and optic nerve was identified as a potential target for walking and visual disabilities, respectively. This disability-specific discovery approach represents a strategy for finding neuroprotective treatments for multifocal neurodegenerative diseases., 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.

Klinefelter Syndrome 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 vs. XYM. Some of these genes are feminized in their expression pattern. Lastly, we also identified genes that differed only between XXYM vs. XYM and not XXM vs. XYM. Genes that are both feminized and unique to XXYM vs. 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 Klinefelter Syndrome and may aid our understanding of sex differences in brain and behavior.

Drug repurposing is an efficient approach in new treatment development since it leverages previous work from one disease to another. While multiple sclerosis (MS), Parkinson's disease (PD), and Alzheimer's disease (AD) are all neurodegenerative diseases of the central nervous system (CNS) and differ in many clinical and pathological aspects, it is possible that they may share some mechanistic features. We hypothesized that focusing on gene expression in a CNS cell type specific manner might uncover similarities between diseases that could be missed using whole tissue gene expression analyses. We found similarities and differences in gene expression in these three distinct diseases, depending upon cell type. Microglia genes were increased in all three diseases, and gene expression levels were correlated strongly among these three neurodegenerative diseases. In astrocytes and endothelia, upregulation and correlations were observed only between MS and PD, but not AD. Neuronal genes were down-regulated in all three diseases, but correlations of changes of individual genes between diseases were not strong. Oligodendrocyte showed gene expression changes that were not shared among the three diseases. Together these data suggest that treatments targeting microglia are most amenable to drug repurposing in MS, PD, and AD, while treatments targeting other CNS cells must be tailored to each disease.

Three different models of MF1 strain mice were studied to measure the effects of gonadal secretions and sex chromosome type and number on body weight and composition, and on related metabolic variables such as glucose homeostasis, feeding, and activity. The 3 genetic models varied sex chromosome complement in different ways, as follows: 1) “four core genotypes” mice, comprising XX and XY gonadal males, and XX and XY gonadal females; 2) the XY* model comprising groups similar to XO, XX, XY, and XXY; and 3) a novel model comprising 6 groups having XO, XX, and XY chromosomes with either testes or ovaries. In gonadally intact mice, gonadal males were heavier than gonadal females, but sex chromosome complement also influenced weight. The male/female difference was abolished by adult gonadectomy, after which mice with 2 sex chromosomes (XX or XY) had greater body weight and percentage of body fat than mice with 1 X chromosome. A second sex chromosome of either type, X or Y, had similar effects, indicating that the 2 sex chromosomes each possess factors that influence body weight and composition in the MF1 genetic background. Sex chromosome complement also influenced metabolic variables such as food intake and glucose tolerance. The results reveal a role for the Y chromosome in metabolism independent of testes and gonadal hormones and point to a small number of X–Y gene pairs with similar coding sequences as candidates for causing these effects.

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. Developmental Dynamics 242:371–379, 2013. © 2013 Wiley Periodicals, Inc.

ImportanceMultiple sclerosis (MS) is characterized by progressive gray matter (GM) atrophy that strongly correlates with clinical disability. However, whether localized GM atrophy correlates with specific disabilities in patients with MS remains unknown.ObjectiveTo understand the association between localized GM atrophy and clinical disability in a biology-driven analysis of MS.Design, setting, and participantsIn this cross-sectional study, magnetic resonance images were acquired from 133 women with relapsing-remitting MS and analyzed using voxel-based morphometry and volumetry. A regression analysis was used to determine whether voxelwise GM atrophy was associated with specific clinical deficits. Data were collected from June 28, 2007, to January 9, 2014.Main outcomes and measuresVoxelwise correlation of GM change with clinical outcome measures (Expanded Disability Status Scale and Multiple Sclerosis Functional Composite scores).ResultsAmong the 133 female patients (mean [SD] age, 37.4 [7.5] years), worse performance on the Multiple Sclerosis Functional Composite correlated with voxelwise GM volume loss in the middle cingulate cortex (P

Historically, it was thought that the number of X chromosomes plays little role in causing sex differences in traits. Recently, selected mouse models have been used increasingly to compare mice with the same type of gonad but with one versus two copies of the X chromosome. Study of these models demonstrates that mice with one X chromosome can be strikingly different from those with two X chromosomes, when the differences are not attributable to confounding group differences in gonadal hormones. The number of X chromosomes affects adiposity and metabolic disease, cardiovascular ischaemia/reperfusion injury and behaviour. The effects of X chromosome number are likely the result of inherent differences in expression of X genes that escape inactivation, and are therefore expressed from both X chromosomes in XX mice, resulting in a higher level of expression when two X chromosomes are present. The effects of X chromosome number contribute to sex differences in disease phenotypes, and may explain some features of X chromosome aneuploidies such as in Turner and Klinefelter syndromes.

In recent years, increasing evidence suggests that sex differences in the phenotype of all tissues are influenced by the inequality of effects of sex chromosome genes in the two sexes. In birds, genes on the Z chromosome are not well dosage compensated, so that most Z genes are expressed higher in ZZ male cells than in ZW female cells. The sex difference in expression of Z and W genes is likely to cause sex differences within cells, in addition to the sex differences caused by different levels of testicular and ovarian hormones. The sexual imbalance in cell physiology has implications for aviculture and novel developments in the poultry industry.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00412-010-0308-3) contains supplementary material, which is available to authorized users.

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.

Introduction: Pulmonary hypertension (PH) is a chronic pulmonary vascular disease characterized by elevated pulmonary arterial pressures and vascular remodelling leading to right ventricular failure. It is 4 times more likely to occur in females than males. The reasons for the female predominance or male protection are still not fully elucidated. Our previous work showed that in gonadectomized (GDX) Four Core Genotypes (FCG) mice (XX and XY males, XX and XY females, C57BL6/J), XY mice having a Y-chromosome (XYM, XYF) develop less severe PH than the XX mice lacking a Y-chromosome (XXM, XXF). Hypothesis: Protection against hypoxia-induced PH in gonadectomized mice is due to the presence of Y-chromosome and not due to the number of X-chromosomes. We also examined the role of a Y-chromosome gene Ddx3y, a cellular growth suppressor expressed consistently in the lungs and heart in protection against PH. Methods: Here we used GDX XY* C57BL/6J mouse model which allows us to have male mice with XY or XXY and female mice with XX or XO genotypes (n=5/group). In addition we also used XX female mice with and without a transgenic copy of Y-chromosome gene Ddx3y (n=10/group). Mice were gonadectomized at day-75 and 4-weeks after gonadectomy, were exposed to hypoxia (10% O2) for 3-weeks. RVSP was measured through direct catheterization. Results: In the GDX XY* model, right ventricular systolic pressure (RVSP) in mice with Y-chromosome (XY=33.5±0.6, XXY=32±2mmHg) was significantly lower than that of mice without a Y-chromosome (XO=38.7±2, XX=41.5±3.8, P Conclusions: We conclude that Y-chromosome confers protection against hypoxia-induced PH in GDX mice which is not due to Ddx3y gene. Next we plan to investigate effects of the other Y-chromosome candidate genes Kdm5d, Uty, and Eif2x3y.

Background The majority of preclinical biomedical research involves studies of males rather than females. It is thought that researchers have avoided females based on the idea that female traits are more variable than those of males because of cyclic variation in effects of ovarian hormones. Methods To test the assumption of inherently greater female variability, we analyzed 293 microarray datasets measuring gene expression in various tissues of mice and humans, comprising analysis of more than 5 million probes. Results Meta-analysis showed that on average, male gene expression is slightly more variable than that of females although the difference is small. We also tested if the X chromosome of humans shows greater variability in gene expression in males than in females, as might be expected because of hemizygous exposure of polymorphic X alleles but again found little sex difference. Conclusion Our analysis supports and extends previous studies reporting no overall greater phenotypic variability in females. Electronic supplementary material The online version of this article (doi:10.1186/s13293-015-0036-8) contains supplementary material, which is available to authorized users.

Introduction: Sex differences in susceptibility to ischemia/reperfusion (I/R) injury have been mostly attributed to sex hormones. Recently we examined the role of sex chromosomes in sex differences in myocardial I/R injury. We discovered that gonadectomized mice with two X chromosomes (XX or XXY) have ~50% larger infarct size after I/R injury, compared to mice with one X chromosome (XY or XO). Only few X genes escape X inactivation and are expressed higher in XX than XY individuals. Here we examined the role of “X escapee” histone demethylase Kdm6a which is important in cardiac development. Methods: Female mice with a heterozygous global knockout of Kdm6a (Kdm6a+/-) and with 2 copies of Kdm6a (Kdm6a+/+, regular WT) were used. Isolated mouse hearts were subjected to 30 min global normothermic ischemia followed by 60 min reperfusion. RNA-Seq analysis was performed by comparing gene expression in hearts of Kdm6a+/+ vs. Kdm6a+/- females at baseline before ischemia. We calculated an unbiased composite score of relevance in which the level of significance of the Kdm6a effect on expression (p value) was integrated with the size of the KDM6A effect on expression (fold change), and with the amount of H3K27me3 mark found on the genes in the heart based on online ChIP-Seq data. Two way ANOVA was used for statistical analysis. P Results: Kdm6a+/+ female mice had significantly lower heart functional recovery compared to their littermate Kdm6a+/- (LVDP: 46.7±9.8% vs. 79.8±3.5%; RPP: 44.1±10.5% vs. 76.2±8.5%, n=6-8 mice/group, p Conclusion: Histone demethylase KDM6A contributes to X chromosome dependent I/R injury via epigenetic regulation

Background The “four core genotypes” (FCG) mouse model has emerged as a major model testing if sex differences in phenotypes are caused by sex chromosome complement (XX vs. XY) or gonadal hormones or both. The model involves deletion of the testis-determining gene Sry from the Y chromosome and insertion of an Sry transgene onto an autosome. It produces XX and XY mice with testes, and XX and XY mice with ovaries, so that XX and XY mice with the same type of gonad can be compared to assess phenotypic effects of sex chromosome complement in cells and tissues. Findings We used PCR to amplify the Sry transgene and adjacent genomic sequences, to resolve the location of the Sry transgene to chromosome 3 and confirmed this location by fluorescence in situ hybridization (FISH) of the Sry construct to metaphase chromosomes. Using quantitative PCR, we estimate that 12–14 copies of the transgene were inserted. The anogenital distance (AGD) of FCG pups at 27–29 days after birth was not different in XX vs. XY males, or XX vs. XY females, suggesting that differences between XX and XY mice with the same type of gonad are not caused by difference in prenatal androgen levels. Conclusion The Sry transgene in FCG mice is present in multiple copies at one locus on chromosome 3, which does not interrupt known genes. XX and XY mice with the same type of gonad do not show evidence of different androgen levels prenatally.

The zebra finch (Taeniopygia guttata) is often studied because of its interesting behaviour and neurobiology. Genetic information on this species has been lacking, making analysis of informative mutants difficult. Here we report on an improved cytological method for preparation of metaphase chromosomes suitable for fluorescent in situ hybridization of adult birds. We found that individual chicken chromosome paints usually hybridized to single zebra finch chromosomes, indicating only minor chromosomal rearrangements since the evolutionary divergence of these two species, and suggesting that the genomic location of chicken genes will predict the location of zebra finch orthologues. Chicken chromosome 1 appears to have split into two macrochromosomes in zebra finches, and chicken chromosome 4 paint hybridizes to a zebra finch macrochromosome and a microchromosome. This pattern was confirmed by mapping the androgen receptor (AR), which is located on chicken chromosome 4 but on a zebra finch microchromosome. We detected a telocentric/submetacentric polymorphism of chromosome 6 in our colony of zebra finches, and found that the polymorphism was inherited in a Mendelian pattern.

The brains of males and females differ, not only in regions specialized for reproduction, but also in other regions (controlling cognition, for example) where sex differences are not necessarily expected. Moreover, males and females are differentially susceptible to neurological and psychiatric disease. What are the origins of these sex differences? Two major sources of sexually dimorphic information could lead to sex differences in brain function. Male and female brain cells carry a different complement of sex chromosome genes and are influenced throughout life by a different mix of gonadal hormones. Until recently all sex differences in the brain have been attributed to the differential action of gonadal hormones. Recent findings, however, suggest that brain cells that differ in their genetic sex are not equivalent, and that difference may contribute to sex differences in brain function. Here we discuss evidence for sex chromosome effects on both neural and nonneural systems, which together provide support for the idea that XX and XY cells differentiate even before they are influenced by gonadal hormones, and even if they are exposed to similar levels of gonadal steroids. Fortunately, new model systems for studying sex chromosome effects have recently been developed, and they should help in testing further the role of sex chromosome genes.

A genomic clone, pWS44, isolated from the chicken W chromosome-specific genomic library contained a partial (226-bp) sequence of a novel SspI-family repetitive sequence. A genomic clone, pWPRS09, containing a 508-bp SspI fragment (a repeating unit of the family) was subsequently obtained and sequenced. This 0.5-kb unit is tandemly repeated about 11 300 times. FISH to mitotic and lampbrush W chromosomes indicates that the SspI-family is located on the chromomere 6 between heterochromatic and distal non-heterochromatic regions on the short arm. The SspI-family sequence was proved to be a good positional marker in FISH mapping of active genes in the non-heterochromatic region on the lampbrush W chromosome. The presence of SspI-family repetitive sequence is limited to the genus Gallus (chickens and jungle fowls). The 0.5-kb repeating unit contains a 120-bp stretch of polypurine/polypyrimidine sequence (GGAGA repeats), shows no DNA curvature, and rapid electrophoretic mobility in 4% polyacrylamide gel at 4°C. The SspI-family forms a relatively diffused chromatin structure in nuclei. These features are distinctly different from those of XhoI- and EcoRI-family sequences on the W chromosome. The total amount of non-repetitive DNA in the chicken W chromosome is estimated to be about 10 Mb.

Objectives: The objective of this study is to demonstrate the effect on blood lipids in humans, of a supplement which contained peptides and amino acids originating from oyster meat. Methods: Proteins in oyster meat were hydrolyzed by enzymes, followed by preparing oyster meat extract powder containing the peptides and free amino acids as a supplement. We performed the following 2 clinical trials in which male and female adults with dyslipidemia received the oyster meat supplement for specified periods; in the first open label trial, the oyster meat supplement was given to 14 subjects (average age 48.7 years old) for 8 weeks, while in the second crossover controlled trial, the oyster meat supplement and its placebo were taken by 19 subjects (average age 52.4 years old) for 4 weeks, respectively. In these trials, the effect of the supplement on blood lipids was evaluated by conducting serum chemistry and body composition measurement before and after taking the supplement. Results: In the open label trial, at the end of Week 4 from the start of taking the supplement, a decrease of very low-density lipoprotein (VLDL) and an increase of high-density lipoproteins (HDL) in blood lipoproteins were observed with statistically significant difference (P

Aim Sex differences in coronary heart disease have been attributed to sex hormones, whereas the potential role of the sex chromosomes has been ignored so far. Here, we investigated the role of the sex chromosomes in causing sex differences in myocardial ischaemia/reperfusion (I/R) injury. Methods and results We used two unique mouse models, the ‘four core genotypes’ [XX mice with ovaries (XXF) or testes (XXM) and XY mice with ovaries (XYF) or testes (XYM)] and XY* (gonadal male or female mice with one or two X chromosomes). All mice were gonadectomized (GDX). In vivo or isolated Langendorff-perfused hearts were subjected to I/R injury. The in vivo infarct size in XY mice was significantly smaller than XX mice regardless of their gonadal type (24.5 ± 4.1% in XYF and 21.8 ± 3.3% in XYM vs. 37.0 ± 3.2% in XXF and 35.5 ± 2.1% in XXM, P < 0.01). Consistent with the results in vivo , the infarct size was markedly smaller and cardiac functional recovery was significantly better in XY mice compared with XX ex vivo . The mitochondrial calcium retention capacity was significantly higher in XY compared with XX mice (nmol/mg protein: XXF = 126 ± 9 and XXM = 192 ± 45 vs. XYF = 250 ± 56 and XYM = 286 ± 51 , P < 0.05). In XY* mice, mice with 2X chromosomes had larger infarct size (2X females = 41.4 ± 8.9% and 2X males = 46.3 ± 9.5% vs. 1X females = 23.7 ± 3.9% and 1X males = 26.6 ± 6.9%, P < 0.05) and lower heart functional recovery, compared with those with 1X chromosome. Several X genes that escape X inactivation ( Eif2s3x , Kdm6a , and Kdm5c ) showed higher expression in XX than in XY hearts. Conclusion XX mice have higher vulnerability to I/R injury compared with XY mice, which is due to the number of X chromosomes rather than the absence of the Y chromosome.

Two W chromosome-linked cDNA clones, p5fm2 and p5fm3, were obtained from a subtracted (female minus male) cDNA library prepared from a mixture of undifferentiated gonads and mesonephroi of male or female 5-d (stages 26-28) chicken embryos. These two clones were demonstrated to be derived from the mRNA encoding an altered form of PKC inhibitor/interacting protein (PKCI), and its gene was named Wpkci. The Wpkci gene reiterated approximately 40 times tandemly and located at the nonheterochromatic end of the chicken W chromosome. The W linkage and the moderate reiteration of Wpkci were conserved widely in Carinatae birds. The chicken PKCI gene, chPKCI, was shown to be a single-copy gene located near the centromere on the long arm of the Z chromosome. Deduced amino acid sequences of Wpkci and chPKCI showed approximately 65% identity. In the deduced sequence of Wpkci, the HIT motif, which is essential for PKCI function, was absent, but the alpha-helix region, which was conserved among the PKCI family, and a unique Leu- and Arg-rich region, were present. Transcripts from both Wpkci and chPKCI genes were present at significantly higher levels in 3- to 6-d (stages 20-29) embryos. These transcripts were detected in several embryonic tissues, including undifferentiated left and right gonads. When the green fluorescent protein-fused form of Wpkci was expressed in male chicken embryonic fibroblast, it was located almost exclusively in the nucleus. A model is presented suggesting that Wpkci may be involved in triggering the differentiation of ovary by interfering with PKCI function or by exhibiting its unique function in the nuclei of early female embryos.

A small amount of DNA extracted from a single plucked contour feather from four captive young Oriental White Storks Ciconia boyciana, a sexually monomorphic bird, was used for gender determination by the method of polymerase chain reaction (PCR) based on its sex chromosome-specific DNA sequences, the 0.6-kb Xba I -HindIII fragment(XH0.6) on the W chromosome and the region homologous to the XH0.6sequence (XH0.6-RSM) on the Z chromosome. After the agarose gel electrophoresis of the PCR products, two bands which correspond to the W and Z chromosome-specific fragments were observed from the female DNA. By contrast only one Z chromosome-specific fragment was observed from the male DNA. These results were consistent with those obtained by Southern blot hybridization using the DNA from red blood cells and the discriminant analysis of body measurements. This PCR method using a single feather is easy, fast, accurate, and also relatively stress-less to the birds which are not used to being handled for sampling. It will become one of useful tools in the captive propagation program and reintroduction plan for the C. boyciana, a special natural monument of Japan.

The suitable fabrication condition for the two types of reaction synthesis processes of FeAl were investigated. In the reactive sintering of pre-mixed elemental powders, homogination and densification above 99% of the relative density were achieved by vacuum hot pressing at 1273–1500 K with 20 MPa of applied pressure. The isothermal holding at the temperatures above 1273 K is necessary to remove pores generated during exothermic reaction at 933 K in this hot pressing condition. The another process studied in this work is the squeeze casting of aluminum melt into porous iron sintered bodies (SQ method). The homogeneous aluminides were not formed by in-situ reaction in the SQ method, however, dense and uniform FeAl was obtain by the combination of SQ method and post hot pressing (pseudo-isostatic pressure) treatment.

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. 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. 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.

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 zygote. These factors, and downstream factors such as gonadal hormones, act directly on tissues to produce sex differences, and to antagonize each other to reduce sex differences. Recent study of mouse models such as the Four Core Genotypes has begun to distinguish between 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 more information is needed about sex differences in the epigenome.

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.

The zebra finch (Taeniopygia guttata) has been intensively studied in many research fields including neuroscience, behavioral neurobiology, and evolution of the genome. Although numerous molecular and genomic resources are available for this model species, immortalized cell lines have been lacking. We have established two zebra finch cell lines derived from spontaneous tumors. ZFTMA is a tetraploid female cell line and G266 as a diploid male cell line. These first zebra finch cell lines should facilitate development of research on this model species.

Background Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E2) is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4), an enzyme that converts E2 to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ) and females (ZW) because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E2 in their brains than females. Results Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain. Conclusions Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E2 metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms.

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.

The zebra finch (Taeniopygia guttata) germline-restricted chromosome (GRC) is the largest chromosome and has a unique system of transmission in germ cells. In the male, the GRC exists as a single heterochromatic chromosome in the germline and is eliminated from nuclei in late spermatogenesis. In the female, the GRC is bivalent and euchromatic and experiences recombination. These characteristics suggest a female-specific or female-beneficial function of the GRC. To shed light on the function of GRC, we cloned a portion of the GRC using random amplified polymorphic DNA-polymerase chain reaction and analyzed it using molecular genetic and cytogenetic methods. The GRC clone hybridized strongly to testis but not blood DNA in genomic Southern blots. In fluorescent in situ hybridization analysis on meiotic chromosomes from synaptonemal complex spreads, the probe showed hybridization across a large area of the GRC, suggesting that it contains repetitive sequences. We isolated a sequence homologous to the GRC from zebra finch chromosome 3 and a region of chicken chromosome 1 that is homologous to zebra finch chromosome 3; the phylogenetic analysis of these three sequences suggested that the GRC sequence and the zebra finch chromosome 3 sequence are most closely related. Thus, the GRC sequences likely originated from autosomal DNA and have evolved after the galliform-passeriform split. The present study provides a foundation for further study of the intriguing GRC. Fil: Itoh, Yuichiro. University of California at Los Angeles; Estados Unidos Fil: Kampf, Kathy. University of California at Los Angeles; Estados Unidos Fil: Pigozzi, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Arnold, Arthur P.. University of California at Los Angeles; Estados Unidos

Intensive study of a few genetically tractable species with XX/XY sex chromosomes has produced generalizations about the process of sex chromosome dosage compensation that do not fare well when applied to ZZ/ZW sex chromosome systems, such as those in birds. The inherent sexual imbalance in dose of sex chromosome genes has led to the evolution of sex-chromosome-wide mechanisms for balancing gene dosage between the sexes and relative to autosomal genes. Recent advances in our knowledge of avian genomes have led to a reexamination of sex-specific dosage compensation (SSDC) in birds, which is less effective than in known XX/XY systems. Insights about the mechanisms of SSDC in birds also suggest similarities to and differences from those in XX/XY species. Birds are thus offering new opportunities for studying dosage compensation in a ZZ/ZW system, which should shed light on the evolution of SSDC more broadly.