XX Mice Research Articles

12554 Human Sex Determination: SRY Length Regulates Its Cellular Stability And Hence The Robustness Of Testis Differentiation

Abstract Disclosure: M.A. Weiss: None. The abundance of transcription factors (TFs) mediated by the rates of degradation are subjected to be a robust to an appropriate level. This regulation via the proteasome is largely controlled by the stability of individual proteins and then could determine the direction of a gene-regulatory network. Insight is obtained through studies of bistable genetic circuits mediated by initiating transcription factors. A model is provided by SRY, a Y-encoded TF that initiates testicular differentiation. Known functions in human SRY (204 residues) majorly cluster in its high mobility group (HMG) box whereas the functions of the N- and C-terminal non-box segments are not well characterized. Here, we have used cell-based and mouse transgenic studies to measure the transcriptional threshold of SRY regulating the balance between development and dysgenesis. Our findings demonstrate a threshold length in the C-terminal domain of human SRY that determines the protein’s proteosome-enforced half-life. In a pre-Sertoli cell model, truncation of SRY resulted in the reduction of intracellular concentration and twofold attenuation of the male-specific GRN. Expression of the 1-164 fragment of human SRY in CRISPR-Cas9-edited XX mice failed to drive male differentiation whereas the 1-200 of SRY initiated male GRN development. This study provides insight into the robustness of human SRY and illustrates a powerful strategy to link biochemical properties in cultured cells and in vivo developmental outcomes. Our study reveals a checkpoint in a key TF initiating a sex-specific GRN, functioning as an experimental “control knob” in development. Our approach probes molecular determinants of cell fate and so promises to extend structure-function studies of SRY to the flanking and relatively obscure non-box domains. This result implies the balance between robustness and evolvability in metazoan is a game of numbers of initial transcription factor in the networks. Presentation: 6/1/2024

Independent contribution of gonads and sex chromosomes to sex differences in bone mass and strength in the four-core genotypes mouse model.

Vertebrate sexual dimorphism is ascribed to the presence of testes or ovaries, and, hence, to the secretion of gonad-specific hormones. However, mounting evidence indicates that sex differences in tissues and organs also stem from the presence of sex chromosomes (XX or XY). To tease out the contribution of gonads from sex chromosomes to the musculoskeletal system, we used the Four-Core Genotypes (FCG) mouse model, in which the Sry gene, which dictates testis formation, was either deleted from the Y chromosome, resulting in XY mice with ovaries (XY-SryO), or overexpressed in XX mice, resulting in XX mice with testes (XXT), together with gonadal males with XY-SryT (Sry deletion and overexpression of the Sry transgene in chromosome 3) and females with XXO. The FCG mice are generated by crossing XXO with XY-SryT mice, all of C57BL/6J background. We now show that the musculoskeletal phenotype of 2- to 4-mo-old FCG mice varies based on both gonads and sex chromosomes, depending on the age and the organ/tissue/cell analyzed. The effect of sex chromosomes on body weight, fat and lean/skeletal muscle mass, and bone mass and structure is minor in 2-/3-mo-old mice, soon after sexual maturation. The contribution of sex chromosomes (XX vs XY-Sry in mice with the same gonads and sex hormones) to several of our measurements becomes apparent in adult 4-mo-old mice. The contribution of 1X and 1Y-Sry vs 2X chromosomes varies among different measurements in gonadal males or females, and mice with XY-Sry chromosomes might have higher or lower values that XX mice. Our study shows XX vs XY-Sry chromosome contribution to the musculoskeletal phenotype, which becomes more evident as the animals reach peak bone mass, suggesting that although gonadal sex has a major role, sex chromosomes are also an unrecognized contributor to musculoskeletal mass and bone strength.

Abstract P228: Kdm6a Regulates Extracellular Matrix Genes During Arterial Stiffening and High Blood Pressure in Female Mice

Arterial stiffness measured by pulse wave velocity (PWV) increases steeply during menopause due to estradiol decline. Longitudinal studies show higher PWV in postmenopausal women compared to age-matched men. Previously, we have demonstrated increased PWV in ovariectomized (OVX) and middle-aged female mice. However, the impact of sex chromosomes on PWV is unknown in female mice. We hypothesize that a decline in estradiol unleashes the female sex chromosomes effect that promotes arterial stiffening. This study used four core genotype mice (n=6-10/group) of XX or XY sex chromosomes left intact or OVX for 8 weeks. Arterial stiffening was assessed with pulse wave Doppler and 24-hour blood pressure by radiotelemetry. Carotid artery passive myography and vascular reactivity of 2 nd order mesenteric artery were assessed by pressure myography. To induce hypertension, XX and XY mice were treated with deoxycorticosterone acetate (DOCA; 50 mg/pellet) for 21 days and 1% saline. In vitro, isolated aortic vascular smooth muscle cells (VSMC) were stretched on collagen matrix or treated with Kdm6a inhibitor GSKJ-1. PWV was significantly increased in OVX than in gonadal intact XX and XY mice (P<0.001). However, an interaction effect was indicated with higher PWV in XX than XY OVX mice (4.9±0.1 vs. 4.2±0.2 m/s; P<0.001), indicating a sex chromosome effect. Carotid artery stress-strain curves showed a leftward shift of XX than XY mice, suggesting increased structural stiffness. Masson’s trichrome staining of the aorta showed increased collagen in the adventitia of XX than XY (30% vs. 26%; P=0.005) mice. Higher systolic blood pressure (SBP) was indicated in XX than in XY mice (Baseline; 123±3 vs. 116±4 mmHg; P=0.02). Aortic RNAseq showed increased X-linked genes, including Kdm6a, in XX compared to XY mice. Stretched aortic VSMC showed increased Col1a1 and decreased Itga4 and Lama2 genes reversed by GSKJ-1. DOCA-induced hypertension blunted the PWV difference between XX and XY mice; however, SBP was higher in XX than in XY mice (Week 1: 135±13 vs.127±3 mmHg; P=0.03), but not weeks 2 and 3. We observed impaired acetylcholine-mediated relaxation in hypertensive XX than XY mice (47% vs. 24%; P=0.03). Our data suggested that OVX mice with XX sex chromosomes promote arterial stiffening and high blood pressure, and Kdm6a regulates extracellular matrix genes in XX female VSMC. DOCA-induced hypertension promotes endothelial dysfunction and blunts PWV in ovariectomized XX and XY mice.

XX Sex Chromosome Complement Promotes Arterial Stiffening and High Blood Pressure in Female Mice

Arterial stiffness measured by pulse wave velocity (PWV) increases precipitously during menopausal transition due to estradiol decline. Longitudinal studies show a steep increase in PWV of postmenopausal women compared to age-matched men. Previously, we have demonstrated increased PWV in ovariectomized and middle-aged female mice. Additionally, we showed increased oxidative stress in female than male vascular cells treated with angiotensin II in hormone-free charcoal-stripped media. We hypothesize that a decline in estradiol unleashes the female sex chromosomes effect that promotes arterial stiffening. In this study, we used the four core genotype mice (n=6-10/group) comprising females with either XX or XY sex chromosome complement that were left intact or ovariectomized for 8 weeks. Arterial stiffening was assessed with pulse wave Doppler and 24-hour blood pressure by radiotelemetry. Carotid artery passive myography and vascular reactivity of 2nd order mesenteric artery were assessed by pressure myography. To induce hypertension, ovariectomized XX and XY mice were treated with deoxycorticosterone acetate (DOCA; 50 mg/pellet) for 21 days and 1% saline as drinking water. PWV was significantly increased in ovariectomized than in gonadal intact XX and XY mice (main effect: p<0.001). However, an interaction effect was indicated with higher PWV in XX than XY ovariectomized mice (4.9±0.1 vs. 4.2±0.2 m/s; p<0.001), indicating a sex chromosome effect. Carotid artery stress-strain curves showed a leftward shift of XX than XY ovariectomized mice, suggesting increased structural stiffness. Masson’s trichrome staining of the aorta showed increased collagen in the adventitia of XX than XY ovariectomized mice (30±0.01 vs. 26±0.02 %; P=0.005). Higher systolic blood pressure (SBP) was indicated in ovariectomized XX than in XY mice (Baseline; 123±3 vs. 116±4 mmHg; P=0.02). DOCA-induced hypertension blunted the PWV difference between OVX XX and XY mice; however, SBP was higher in XX than in XY mice (Week 1: 135±13 vs.127±3 mmHg; P=0.3), (Week 2: 148±4 vs.140±1 mmHg; P=0.08), and (Week 3: 137±9 vs.127±4 mmHg; P=0.4). We observed impaired acetylcholine-mediated relaxation in mesenteric arteries of hypertensive ovariectomized XX than XY mice (47±6 % vs. 24±6 %; P=0.03). Our data suggested that ovariectomized female mice with XX sex chromosome complement promote arterial stiffening and blood pressure. However, DOCA-induced hypertension promotes endothelial dysfunction and blunts PWV in ovariectomized XX and XY mice. Future studies will identify elements in the XX sex chromosome complement contributing to arterial stiffening and high blood pressure. R00HL155841 and R25HL105400. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

XX Chromosome Complement Predisposes Females to Obesity-induced, Leptin-mediated Aldosterone Production and Hypertension

Despite the historical dogma that premenopausal women are protected from cardiovascular disease, clinical and experimental evidence indicate that obesity abolishes this protection, placing women at increased risk for hypertension. Previously, our laboratory demonstrated that the adipokine leptin contributes to obesity-associated hypertension in both males and females but via sex specific mechanisms: leptin-mediated sympatho-activation in males and leptin-induced aldosterone production in females. However, the origin of these sex-specific mechanisms remains unknown. Therefore, we aimed to test the contribution of both sex hormones and chromosomes. Female sex hormones were preserved (sham) or depleted via ovariectomy (OVX) in lean control and obese female agouti yellow mice. Blood pressure (BP) recording via radiotelemetry indicated that obesity increased BP (2-way ANOVA, p<0.0001) but OVX did not alter BP in agouti mice. Vascular function measured via mesenteric wire myography indicated obesity impaired (3-way ANOVA, p<0.05) endothelial-dependent relaxation but OVX did not inflict further impairment in agouti mice. OVX did not alter plasma aldosterone levels or adrenal aldosterone synthase (CYP11B2) expression in agouti mice. Leptin receptor blockade (Allo-Aca) decreased BP (2-way ANOVA, p<0.05), restored endothelial-dependent relaxation and decreased (2-way ANOVA, p<0.05) adrenal CYP11B2 expression in both intact and OVX agouti mice indicating a preservation of leptin-mediated BP elevation in the absence of female sex hormones and ruling out sex steroids as the origin of the sex specific mechanisms. Additionally, neither estrogen, progesterone, a combination of estrogen and progesterone, or dihydrotestosterone increase CYP11B2 expression in primary isolated human adrenal cells in culture further supporting that sex hormones do not mediate aldosterone production. These data suggest female sex hormones are not the source for the sex difference prompting us to utilize the four-core genotype (FCG) mouse model to assess the role of sex chromosomes. In order to mimic obesity conditions, BP was recorded at baseline and in response to leptin infusion in OVX FCG XX and XY female mice (XXF and XYF). XX females displayed higher baseline BP than XY (XXF: 109.1±2.4 vs. XYF: 100.4±1.1 mmHg, p<0.05) but leptin infusion increased BP and concurrently ablated the difference. Similarly, XXF displayed heightened plasma aldosterone (2-way ANOVA, 183.7±27 vs. 89.4±34 pg/mL, p<0.05) but leptin infusion abolished the difference. Collectively, these results indicate that XX chromosome rather than female sex steroid hormones regulates aldosterone production, and that sex chromosomes are likely the source for the sexual dimorphism in the mechanism for hypertension associated with obesity. R01HL130301, R01HL155265 (E.J.B.) F31HL168963-01 (C.T.B.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

XX sex chromosome complement modulates immune responses to heat-killed Streptococcus pneumoniae immunization in a microbiome-dependent manner.

Differences in male vs. female immune responses are well-documented and have significant clinical implications. While the immunomodulatory effects of sex hormones are well established, the contributions of sex chromosome complement (XX vs. XY) and gut microbiome diversity on immune sexual dimorphisms have only recently become appreciated. Here we investigate the individual and collaborative influences of sex chromosome complements and gut microbiota on humoral immune activation. Male and female Four Core Genotype (FCG) mice were immunized with heat-killed Streptococcus pneumoniae (HKSP). Humoral immune responses were assessed, and X-linked immune-related gene expression was evaluated to explain the identified XX-dependent phenotype. The functional role of Kdm6a, an X-linked epigenetic regulatory gene of interest, was evaluated ex vivo using mitogen stimulation of B cells. Additional influences of the gut microbiome on sex chromosome-dependent B cell activation was also evaluated by antibiotically depleting gut microbiota prior to HKSP immunization. Reconstitution of the depleted microbiome with short-chain fatty acid (SCFA)-producing bacteria tested the impact of SCFAs on XX-dependent immune activation. XX mice exhibited higher HKSP-specific IgM-secreting B cells and plasma cell frequencies than XY mice, regardless of gonadal sex. Although Kdm6a was identified as an X-linked gene overexpressed in XX B cells, inhibition of its enzymatic activity did not affect mitogen-induced plasma cell differentiation or antibody production in a sex chromosome-dependent manner ex vivo. Enhanced humoral responses in XX vs. XY immunized FCG mice were eliminated after microbiome depletion, indicating that the microbiome contributes to the identified XX-dependent immune enhancement. Reconstituting microbiota-depleted mice with select SCFA-producing bacteria enhanced fecal SCFA concentrations and increased humoral responses in XX, but not XY, FCG mice. However, exposure to the SCFA propionate alone did not enhance mitogenic B cell stimulation in ex vivo studies. FCG mice have been used to assess sex hormone and sex chromosome complement influences on various sexually dimorphic traits. The current study indicates that the gut microbiome impacts humoral responses in an XX-dependent manner, suggesting that the collaborative influence of gut bacteria and other sex-specific factors should be considered when interpreting data aimed at delineating the mechanisms that promote sexual dimorphism.

Lung proinflammatory microRNA and cytokine expression in a mouse model of allergic inflammation: role of sex chromosome complement and gonadal hormones.

Epigenetic alterations such as dysregulation of miRNAs have been reported to play important roles in interactions between genetic and environmental factors. In this study, we tested the hypothesis that induction of lung inflammation by inhaled allergens triggers a sex-specific miRNA regulation that is dependent on chromosome complement and hormonal milieu. We challenged the four core genotypes (FCGs) model through intranasal sensitization with a house dust mite (HDM) solution (or PBS as a control) for 5 wk. The FCG model allows four combinations of gonads and sex chromosomes: 1) XX mice with ovaries (XXF), 2) XY mice with testes (XYM), 3) XX mice with testes (XXM), and 4) XY mice with ovaries (XYF). Following the challenge (n = 5-7/group), we assessed the expression of 84 inflammatory miRNAs in lung tissue using a PCR array and cytokine levels in bronchoalveolar lavage fluid (BAL) by a multiplex protein assay (n = 4-7 animals/group). Our results showed higher levels of the chemokine KC (an Il-8 homolog) and IL-7 in BAL from XYF mice challenged with HDM. In addition, IL-17A was significantly higher in BAL from both XXF and XYF mice. A three-way interaction among treatment, gonads, and sex chromosome revealed 60 of 64 miRNAs that differed in expression depending on genotype; XXF, XXM, XYF, and XYM mice had 45, 32, 4, and 52 differentially expressed miRNAs, respectively. Regulatory networks of miRNAs identified in this study were implicated in pathways associated with asthma. Female gonadal hormonal effects may alter miRNA expression and contribute to the higher susceptibility of females to asthma.NEW & NOTEWORTHY miRNAs play important roles in regulating gene and environmental interactions. However, their role in mediating sex differences in allergic responses and lung diseases has not been elucidated. Our study used a targeted omics approach to characterize the contributions of gonadal hormones and chromosomal components to lung responses to an allergen challenge. Our results point to the influence of sex hormones in miRNA expression and proinflammatory markers in allergic airway inflammation.

Sex hormones, sex chromosomes, and microbiota: Identification of Akkermansia muciniphila as an estrogen-responsive microbiota.

Microbiota composition is known to be linked to sex. However, separating sex hormones and sex chromosome roles in gut microbial diversity is yet to be determined. To investigate the sex chromosome role independent of sex hormones, we used the four-core genotype mouse model. In this mouse model, males with testes and females with ovaries have XX or XY sex chromosome complement. In gonadectomized four-core genotype mice, we observed a significant decrease in the levels of estradiol (P<0.001) and progesterone (P<0.03) in female and testosterone (P<0.0001) in male mice plasma samples. Independent of sex chromosome complement, microbial α diversity was increased in gonadectomized female but not male mice compared to sex-matched gonad-intact controls. β diversity analysis showed separation between male (P<0.05) but not female XX and XY mice. Importantly, Akkermansia muciniphila was less abundant in gonadectomized compared to gonadal intact female mice (P<0.0001). In the presence of β-estradiol, Akkermansia muciniphila growth exponentially increased, providing evidence for the identification of a female sex hormone-responsive bacterium (P<0.001).

Sex chromosome and gonadal hormone contributions to binge-like and aversion-resistant ethanol drinking behaviors in Four Core Genotypes mice.

While substantial research has focused on the contribution of sex hormones to driving elevated levels of alcohol drinking in female rodents, fewer studies have investigated how genetic influences may underlie sex differences in this behavior. We used the Four Core Genotypes (FCG) mouse model to explore the contribution of sex chromosome complement (XX/XY) and gonad type [ovaries (Sry-)/testes (Sry+)] to ethanol (EtOH) consumption and quinine-resistant drinking across two voluntary self-administration tasks: limited access consumption in the home cage and an operant response task. For limited access drinking in the dark, XY/Sry + (vs. XX/Sry +) mice consumed more 15% EtOH across sessions while preference for 15% EtOH vs. water was higher in XY vs. XX mice regardless of gonad type. XY chromosomes promoted quinine-resistant drinking in mice with ovaries (Sry-) and the estrous cycle did not affect the results. In the operant response task, responding for EtOH was concentration dependent in all genotypes except XX/Sry + mice, which maintained consistent response levels across all concentrations (5-20%) of EtOH. When increasing concentrations of quinine (100-500 μM) were added to the solution, FCG mice were insensitive to quinine-punished EtOH responding, regardless of sex chromosome complement. Sry + mice were further found to be insensitive to quinine when presented in water. Importantly, these effects were not influenced by sensitivity to EtOH's sedative effect, as no differences were observed in the time to lose the righting reflex or the time to regain the righting reflex between genotypes. Additionally, no differences in EtOH concentration in the blood were observed between any of the genotypes once the righting reflex was regained. These results provide evidence that sex chromosome complement regulates EtOH consumption, preference, and aversion resistance and add to a growing body of literature suggesting that chromosomal sex may be an important contributor to alcohol drinking behaviors. Examination of sex-specific genetic differences may uncover promising new therapeutic targets for high-risk drinking.

Somatic FGFR2 is Required for Germ Cell Maintenance in the Mouse Ovary.

During sex determination in the mouse, fibroblast growth factor 9 signals through the fibroblast growth factor receptor 2c isoform (FGFR2c) to trigger Sertoli cell and testis development from 11.5 days post coitum (dpc). In the XX gonad, the FOXL2 and WNT4/RSPO1 pathways drive granulosa cell and ovarian development. The function of FGFR2 in the developing ovary, and whether FGFR2 is required in the testis after sex determination, is not clear. In fetal mouse gonads from 12.5 dpc, FGFR2 shows sexually dimorphic expression. In XX gonads, FGFR2c is coexpressed with FOXL2 in pregranulosa cells, whereas XY gonads show FGFR2b expression in germ cells. Deletion of Fgfr2c in XX mice led to a marked decrease/absence of germ cells by 13.5 dpc in the ovary. This indicates that FGFR2c in the somatic pregranulosa cells is required for the maintenance of germ cells. Surprisingly, on the Fgfr2c-/- background, the germ cell phenotype could be rescued by ablation of Foxl2, suggesting a novel mechanism whereby FGFR2 and FOXL2 act antagonistically during germ cell development. Consistent with low/absent FGFR2 expression in the Sertoli cells of 12.5 and 13.5 dpc XY gonads, XY AMH:Cre; Fgfr2flox/flox mice showed normal testis morphology and structures during fetal development and in adulthood. Thus, FGFR2 is not essential for maintaining Sertoli cell fate after sex determination. Combined, these data show that FGFR2 is not necessary for Sertoli cell function after sex determination but does play an important role in the ovary.

Genetic variation in the Y chromosome and sex-biased DNA methylation in somatic cells in the mouse

Several lines of evidence suggest that the presence of the Y chromosome influences DNA methylation of autosomal loci. To better understand the impact of the Y chromosome on autosomal DNA methylation patterns and its contribution to sex bias in methylation, we identified Y chromosome dependent differentially methylated regions (yDMRs) using whole-genome bisulfite sequencing methylation data from livers of mice with different combinations of sex-chromosome complement and gonadal sex. Nearly 90% of the autosomal yDMRs mapped to transposable elements (TEs) and most of them had lower methylation in XY compared to XX or XO mice. Follow-up analyses of four reporter autosomal yDMRs showed that Y-dependent methylation levels were consistent across most somatic tissues but varied in strains with different origins of the Y chromosome, suggesting that genetic variation in the Y chromosome influenced methylation levels of autosomal regions. Mice lacking the q-arm of the Y chromosome (B6.NPYq-2) as well as mice with a loss-of-function mutation in Kdm5d showed no differences in methylation levels compared to wild type mice. In conclusion, the Y-linked modifier of TE methylation is likely to reside on the short arm of Y chromosome and further studies are required to identify this gene.

Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Chromosomal and gonadal sex drive sex differences in lipids and hepatic gene expression in response to hypercholesterolemia and statin treatment

BackgroundBiological sex impacts susceptibility and presentation of cardiovascular disease, which remains the leading cause of death for both sexes. To reduce cardiovascular disease risk, statin drugs are commonly prescribed to reduce circulating cholesterol levels through inhibition of cholesterol synthesis. The effectiveness of statin therapy differs between individuals with a sex bias in the frequency of adverse effects. Limited information is available regarding the mechanisms driving sex-specific responses to hypercholesterolemia or statin treatment.MethodsFour Core Genotypes mice (XX and XY mice with ovaries and XX and XY mice with testes) on a hypercholesteremic Apoe–/– background were fed a chow diet without or with simvastatin for 8 weeks. Plasma lipid levels were quantified and hepatic differential gene expression was evaluated with RNA-sequencing to identify the independent effects of gonadal and chromosomal sex.ResultsIn a hypercholesterolemic state, gonadal sex influenced the expression levels of more than 3000 genes, and chromosomal sex impacted expression of nearly 1400 genes, which were distributed across all autosomes as well as the sex chromosomes. Gonadal sex uniquely influenced the expression of ER stress response genes, whereas chromosomal and gonadal sex influenced fatty acid metabolism gene expression in hypercholesterolemic mice. Sex-specific effects on gene regulation in response to statin treatment included a compensatory upregulation of cholesterol biosynthetic gene expression in mice with XY chromosome complement, regardless of presence of ovaries or testes.ConclusionGonadal and chromosomal sex have independent effects on the hepatic transcriptome to influence different cellular pathways in a hypercholesterolemic environment. Furthermore, chromosomal sex in particular impacted the cellular response to statin treatment. An improved understanding of how gonadal and chromosomal sex influence cellular response to disease conditions and in response to drug treatment is critical to optimize disease management for all individuals.

Gonadal hormones and sex chromosome complement differentially contribute to ethanol intake, preference, and relapse-like behaviour in four core genotypes mice.

Alcohol use and high-risk alcohol drinking behaviours among women are rapidly rising. In rodent models, females typically consume more ethanol (EtOH) than males. Here, we used the four core genotypes (FCG) mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviours. FCG mice were given access to escalating concentrations of EtOH in a two-bottle, 24-h continuous access drinking paradigm to assess consumption and preference. Relapse-like behaviour was measured by assessing escalated intake following repeated cycles of deprivation and re-exposure. Twenty-four-hour EtOH consumption was greater in mice with ovaries (Sry-), relative to those with testes, and in mice with the XX chromosome complement, relative to those with XY sex chromosomes. EtOH preference was higher in XX versus XY mice. For both consumption and preference, the influences of the Sry gene and sex chromosomes were concentration dependent. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). Mice with ovaries (Sry- FCG mice and C57BL/6J females) were also found to consume more water than mice with testes. These results demonstrate that aspects of EtOH drinking behaviour may be independently regulated by sex hormones and chromosomes and inform our understanding of the neurobiological mechanisms which contribute to EtOH dependence in male and female mice. Future investigation of the contribution of sex chromosomes to EtOH drinking behaviours is warranted. We used the FCG mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviours, including the alcohol deprivation effect. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). These results demonstrate that aspects of EtOH drinking behaviour may be independently regulated by sex hormones and chromosomes.

X, but not Y, Chromosomal Complement Contributes to Stroke Sensitivity in Aged Animals.

Post-menopausal women become vulnerable to stroke and have poorer outcomes and higher mortality than age-matched men, and previous studies suggested that sex chromosomes play a vital role in mediating stroke sensitivity in the aged. It is unknown if this is due to effects of the X or Y chromosome. The present study used the XY* mouse model (with four genotypes: XX and XO gonadal females and XY and XXY gonadal males) to compare the effect of the X vs. Y chromosome compliment in stroke. Aged (18-20months) and gonadectomized young (8-12weeks) mice were subjected to a 60-min middle cerebral artery occlusion. Infarct volume and behavioral deficits were quantified 3days after stroke. Microglial activation and infiltration of peripheral leukocytes in the aged ischemic brain were assessed by flow cytometry. Plasma inflammatory cytokine levels by ELISA, and brain expression of two X chromosome-linked genes, KDM6A and KDM5C by immunochemistry, were also examined. Both aged and young XX and XXY mice had worse stroke outcomes compared to XO and XY mice, respectively; however, the difference between XX vs. XXY and XO vs. XY aged mice was minimal. Mice with two copies of the X chromosome showed more robust microglial activation, higher brain-infiltrating leukocytes, elevated plasma cytokine levels, and enhanced co-localization of KDM6A and KDM5C with Iba1+ cells after stroke than mice with one X chromosome. The number of X chromosomes mediates stroke sensitivity in aged mice, which might be processed through the X chromosome-linked genes and the inflammatory responses.

Abstract 82: The Second X Chromosome Contributes to Stroke Sensitivity in the Absence of Activational Hormonal Effects

Introduction: Stroke is a sexually dimorphic disease. Stroke sensitivity in the elderly is altered by the decline of estrogen levels in females. Our previous studies suggested a X chromosomal effect on stroke sensitivity in aged mice; however, these mice were gonadal intact and the activational effect of hormones cannot be ruled out. In this study, we utilized the XY* mice model in which mice of four genotypes were produced (XX, XO, XY, and XXY) to dissociate the effect of X chromosome from that of Y. To rule out the activational effect of hormones, we gonadectomized all mice at 3 weeks old, and let them survive for 6-8 weeks to achieve “surgical post-menopause”. We hypothesized that the second X chromosome mediates the stroke sensitivity in the absence of activational sex hormones. Two X escapee genes ( Kdm6A and Kdm5C ) that escape from the X chromosome inactivation and involved in microglial activation, were also examined to explore the potential mechanisms. Methods: “Surgical postmenopausal” XY* mice were subjected to a 60-min middle cerebral artery occlusion (MCAO) (n=6-8/gp for stroke and = 4-5/gp for sham). Infarct volumes and behavior deficits were quantified 3 days after MCAO. Plasma inflammatory cytokine (TNFα, IL-1β, IL-4, iNOS, TGF-β1 and IL-10) levels were determined by ELISA. KDM6A and KDM5C expression in microglia was examined by IHC. Results: XX mice had significantly larger infarct size than XO mice in striatum (55.02±12.38% vs. 36.06±9.25%, P=0.0387) and the ipsilateral hemisphere (45.33±10.77% vs. 26.55±6.92%, p=0.0413); the same pattern was seen in XXY vs. XY group in striatum (58.47±14.26% vs. 34.22±15.24%, P=0.0027). Neurological deficit scores were higher in XX vs. XO mice. Mice with two copies of the X chromosome had significantly higher plasma levels of pro-inflammatory cytokines (TNFα, IL-1β) than the mice with one X. Significantly higher levels of KDM6A or KDM5C in Iba-1 positive microglial cells were also found in mice with two vs. one copy of X. Conclusion: The second X chromosome contributes to stroke sensitivity in the absence of activational sex hormones. Our future goal is to use Kdm6A and Kdm5C microglial conditional knockout mice to explore the molecular mechanisms underlying chromosomal factors in stroke sensitivity.

Generation and mutational analysis of a transgenic mouse model of human SRY.

SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage the pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive testis determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRY's three domains (N-terminal, HMG box, and C-terminal) and performed a detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRYand present a versatile and powerful system in which to functionally analyze variations of SRY including known and novel pathogenic variants found in DSD.

Abstract MP62: Sars-cov-2 Spike Protein Regulation Of Angiotensin Converting Enzyme 2 And Tissue Renin-angiotensin Systems: Influence Of Biologic Sex

Angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor and an enzyme of the renin-angiotensin system (RAS), is on the X chromosome and stimulated by estrogen. Male sex is a risk factor for SARS-CoV-2 severity. Previous investigators demonstrated that the SARs-CoV-2 Spike (S) protein decreases tissue ACE2 by protein internalization or shedding. This study defined sex differences in tissue ACE2 expression and their impact on SARS-CoV-2 S protein regulation of ACE2 activity and AngII levels. Male and female intact or gonadectomized (GDX) low density lipoprotein receptor deficient ( Ldlr -/- ) mice, and Four Core Genotype (FCG) male (XY or XX) or female (XX or XY) mice were fed a Western diet for 4 months. In lung, ACE2 mRNA abundance was similar in male and female mice and reduced by GDX (Male XY intact: 1.04 ± 0.15; Female XX intact: 1.13 ± 0.13; Male XY GDX: 0.11 ± 0.03; Female XX GDX: 0.18 ± 0.04 ΔΔCt; P&lt;0.05). Lungs from XX mice had higher ACE2 mRNA abundance than XY mice regardless of gonadal sex (P&lt;0.05), and GDX reduced ACE2 mRNA abundance in lungs of XX, but not XY females (XX Female GDX: 0.18 ± 0.04; XY Female GDX: 0.38 ± 0.09; P&lt;0.05). In adipose, XX females had higher ACE2 mRNA abundance than XY males (XX female: 5.4 ± 0.7; XY male: 1.0 ± 0.1; P&lt;0.05), regardless of gonadal sex (XY females: 3.3 ± 0.7; XX males: 1.5 ± 0.3; P&lt;0.05). Male XY and female XX Ldlr -/- mice were administered vehicle or SARS-CoV-2 S protein (2 nmol/kg, ip, 3 doses) with tissue harvest six hours later. In lung, AngII levels were increased by S protein in male, but not female mice (Male, vehicle: 12.3 ± 2.3; Male, S protein: 33.6 ± 7.1; Female, vehicle: 16.1 ± 2.0; Female, S protein: 20.2 ± 1.3 pg/μg protein; P&lt;0.05). In adipose, ACE2 activity was reduced by S protein in male, but not female mice (Male, vehicle: 63.6 ± 13.9; Male, S protein: 26.1 ± 1.9; Female, vehicle: 32.5 ± 1.9; Female, S protein: 25.1 ± 1.3 RFU/hr/mg tissue; P&lt;0.05). SARS-CoV-2 S protein (35 nM) decreased ACE2 activity in type II lung alveolar cells (Vehicle: 2.0 x 10 4 ; S protein: 1.2 x 10 4 RFU/10 6 cells) and 3T3-L1 adipocytes (Vehicle: 2.1 x 10 4 ± 0.3 x 10 4 ; S protein: 1.1 x 10 4 ± 0.8 x 10 3 RFU/10 5 cells; P&lt;0.05). Biologic sex regulation of ACE2 may protect females from SARS-CoV-2 S protein-mediated ACE2 reductions and activation of the local RAS.

Effect of sex chromosomes versus hormones in neonatal lung injury.

The main mechanisms underlying sexually dimorphic outcomes in neonatal lung injury are unknown. We tested the hypothesis that hormone- or sex chromosome–mediated mechanisms interact with hyperoxia exposure to impact injury and repair in the neonatal lung. To distinguish sex differences caused by gonadal hormones versus sex chromosome complement (XX versus XY), we used the Four Core Genotypes (FCG) mice and exposed them to hyperoxia (95% FiO2, P1–P4: saccular stage) or room air. This model generates XX and XY mice that each have either testes (with Sry, XXM, or XYM) or ovaries (without Sry, XXF, or XYF). Lung alveolarization and vascular development were more severely impacted in XYM and XYF compared with XXF and XXM mice. Cell cycle–related pathways were enriched in the gonadal or chromosomal females, while muscle-related pathways were enriched in the gonadal males, and immune-response–related pathways were enriched in chromosomal males. Female gene signatures showed a negative correlation with human patients who developed bronchopulmonary dysplasia (BPD) or needed oxygen therapy at 28 days. These results demonstrate that chromosomal sex — and not gonadal sex — impacted the response to neonatal hyperoxia exposure. The female sex chromosomal complement was protective and could mediate sex-specific differences in the neonatal lung injury.

Sex chromosome complement affects multiple aspects of reversal-learning task performance in mice.

Determining the mechanisms by which the sex-chromosome complement (SCC) affects learning, attention, and impulsivity has implications for observed sex differences in prevalence, severity, and prognosis of psychiatric/neurodevelopmental disorders and syndromes associated with sex-chromosome aneuploidy. Here, Four Core Genotypes (FCG) mice were evaluated in order to assess the separable and/or interacting effects of gonads (testes vs. ovaries) and their secretions and/or SCC (XX vs. XY) acting via non-gonadal mechanisms on behavior. We tested FCG mice on a reversal-learning task that enables the quantification of aspects of learning, attention and impulsivity. Across testing phases (involving the initial acquisition of a spatial discrimination and subsequent reversal learning), overall error rate was larger in XY compared with XX mice. Although XX and XY groups did not differ in the total number of trials required in order to reach a preset performance criterion, analyses of reversal error types showed more perseverative errors in XY than XX mice, with no difference in regressive errors. Additionally, prepotent-response latencies during the reversal phase were shorter in XY males, as compared with both XX gonadal males and females of either SCC, and failures to sustain the observing response were more frequent in XY mice than XX mice during the acquisition phase. These results indicate that SCC affects the characteristic pattern of response selection during acquisition and reversal performance without affecting the overall learning rate. More broadly, these results show direct effects of the SCC on cognitive processes that are relevant to psychiatric/neurodevelopmental disorders and syndromes associated with sex-chromosome aneuploidies.