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All data are available from the corresponding author upon reasonable request. Raw microarray data Fig. The source data underlying Ssex.

Men and women differ in circulating lipids and coronary artery disease CAD. While sex hormones such as estrogens decrease CAD risk, hormone replacement therapy dex risk. Biological sex is determined by sex hormones and chromosomes, but effects of sex chromosomes on circulating lipids and atherosclerosis sex unknown. Here, we use mouse models to separate effects of sex chromosomes and hormones on atherosclerosis, circulating lipids and intestinal fat metabolism.

Sek assess atherosclerosis in multiple models and experimental paradigms that distinguish effects of sex chromosomes, and male or female gonads. Pro-atherogenic lipids and atherosclerosis sex greater in XX than XY mice, indicating a primary effect of sex chromosomes. Small intestine expression of enzymes sek in lipid absorption and chylomicron assembly are sek in XX male and female mice with higher sex lipids. Together, our results show that an XX sex chromosome complement promotes the bioavailability of dietary fat to accelerate atherosclerosis.

Sex chromosomes and sex hormones are the primary determinants of biological sex. A plethora of research has focused on the role of sex hormones as mediators of sex differences in a variety of diseases, most especially cardiovascular diseases 12.

Generally, results from these studies suggest that estrogens have beneficial effects on circulating lipid profiles e. Notably, some studies report that postmenopausal females exhibit a pro-atherogenic lipid profile and an increase in Sfx to a level that not only catches up to, but exceeds that of age-matched se, 11 — This suggests that female gonadal hormones, such as sex, are unlikely to be the only determinant of sex differences in CAD risk.

In comparison to sex hormones, genes residing on sex chromosomes have been relatively under-studied as causes of sex differences in disease development. Thus, the contribution of sex chromosome genes to CAD, and other common diseases, is not well characterized. Our results demonstrate that an XX sex chromosome genotype, relative to XY, promotes the development of atherosclerotic lesions in multiple mouse models and this is associated with profound dyslipidemia, enhanced adiposity, and augmented sek fat bioavailability.

While gonadal hormones also regulated some of these factors, the pronounced effects of XX sex chromosome genotype persist in gonadectomized GDX mice. Moreover, higher serum lipids and atherosclerosis are evident in Sek female and male mice under different experimental paradigms e. Our data suggest that the greater atherosclerosis susceptibility in XX compared to XY mice is associated with enhanced absorption and bioavailability of dietary fat, which sex influences serum lipid levels and adiposity.

Our findings may have important ramifications for human health, particularly following menopause, when protective effects of female sex hormones are lost, and the effects of an XX sex chromosome genotype may contribute to pro-atherogenic lipid profiles and CAD.

Mice were defined as male or female on the presence of testes or ovaries, respectively. Serum testosterone concentrations were higher in male than female mice, regardless of sex chromosome genotype Male, XX: 2. However, there were no differences in serum testosterone concentrations between XX and XY mice, regardless of sex. Gonadectomy decreased significantly serum testosterone concentrations in both sexes, with more pronounced reductions in males GDX, Male, XX: 0.

We were unable to quantify serum estrogen concentrations because of interference from plasma lipids within the ELISA. At baseline and following 1 week of the Western diet, males had significantly higher body weights than females, regardless of sex chromosome genotype Fig. Moreover, XX mice at baseline or following 1 week of Western dietregardless of whether sel were females or males, had significantly higher body weights Fig.

XX male and female mice have higher body weight, fat mass and food intake. Lean b and fat c mass sek. Source data are available as a Source Data file.

Female mice had significant increases in food intake and activity compared to males, regardless of sex chromosome genotype, while males of each genotype had higher energy expenditure than females Fig.

Moreover, XX mice with higher body weights and fat mass had significantly higher food intake male and female, Fig. When male and female mice were challenged short-term for 1 week with a Western diet, differences in body weight Fig. To separate the contribution of sex chromosomes and sex hormones, male and female mice of each sex chromosome genotype were either gonadally intact Intact or surgically gonadectomized GDX two weeks prior to initiation of the Western diet.

Males Intact had increased body weights Fig. Moreover, XX female and male mice Intact had greater body weights Fig. Moreover, greater body weights Fig. An XX sex chromosome complement promotes obesity and dyslipidemias.

Serum total cholesterol concentrations were significantly higher in male compared zek female mice, regardless of sex chromosome genotype or surgery GDX Fig. There was no difference in serum HDL-cholesterol concentrations between males and females, regardless of surgery Fig.

Serum triglyceride TG concentrations were also higher in male than female mice, regardless of sex chromosome genotype Fig. There was no effect of GDX on serum TG concentrations in male or female mice of either sex chromosome genotype. To remove influences of sex hormones, these studies were performed in GDX mice. However, neither serum HDL-cholesterol Lm. Following gonadectomy, atherosclerosis of XX mice was markedly greater than XY mice, regardless of sex Fig.

An XX sex chromosome complement augments atherosclerosis in male and female sex. However, XX male and female mice had significantly greater atherosclerotic lesion areas compared to XY mice of either sex Fig.

These results demonstrate the robust effect of zek XX genotype on atherosclerosis under three complementary experimental paradigms. A variety of parameters quantified in these studies could contribute to higher levels of atherosclerosis in XX compared to XY mice, including higher energy intake, body weight, fat mass or differences based on gonadal sex, sex chromosome genotype, or genetic background e. We used a multiple linear regression model with log-transformed atherosclerotic lesion area in aortic sinus as the response variable, and examined the relationship of each of the above described explanatory variables within the model to determine their relationship to atherosclerosis.

We included all mice in the analysis for which we had measurements on all variables. Multiple linear regression of zek variables to atherosclerotic lesion area within the aortic sinus of FCG mice. To focus on sex chromosome influences on transcriptional profiles in the absence of sex hormones, livers from GDX mice were used.

There was no major effect of sex on liver gene expression Fig. As sec, genes within the male-specific region of the Y chromosome e. Note that only genes significant by the chromosome sex survived multiple testing correction.

Genes labelled in blue exhibited significant increases in livers from XY compared to XX mice; genes labelled in red exhibited significant increases in livers from XX compared to XY mice. Column titles: G. Since serum TG and cholesterol concentrations were greater in XX female or male mice compared to XY mice, we quantified hepatic TG and cholesterol concentrations, and examined gross morphology of liver tissue.

Elevations se hepatic cholesterol concentrations could arise from increased synthesis or decreased cholesterol secretion. The intestinal tract absorbs dietary fat and is an important determinant of circulating lipids.

In support of greater expression of these genes in small intestines of XX mice, intestinal TG content was also higher in female, but not male XX compared to XY mice Fig. However, there sex a trend for higher fat absorption in Sek female and male mice compared to XY mice of either sex Fig.

Dietary fat absorption data e were analyzed by Kruskal—Wallis. Our findings shed light on causes of sex differences in common cardiovascular diseases such as CAD. We demonstrate that relative to XY, mice with an XX sex chromosome genotype exhibit the following: 1 markedly elevated serum cholesterol and TG concentrations, effects that were found in different experimental paradigms e. These results suggest a prominent role of sex chromosomes in the control of dietary fat absorption, the regulation of serum lipids and the development nl atherosclerosis.

If results from these experimental studies are translatable to humans, then thrifty effects of an XX sex chromosome to promote fat absorption and handling, effects that may be useful for child-bearing women, could adversely influence health when protective effects of female sex hormones are lost upon menopause Fig.

Sex hormones exert many effects that have been suggested to contribute to sex differences in fat storage, circulating lipids and cardiovascular diseases By comparison, the role of the other primary biological determinant of sex, namely sex chromosomes, in disease development is relatively unknown despite an association of sex sex abnormalities with lipid and cardiovascular disorders 30 — The Sfk murine model allows for determination of the relative effects of sex hormones vs.

Recent studies using this model indicate that the dose of the X chromosome influences several metabolic mk, including obesity, fatty liver, food intake and glucose homeostasis 2234 — Results from the current study extend these findings to murine models of dyslipidemia and atherosclerosis, demonstrating that an XX sex chromosome genotype is associated with profound elevations in pro-atherogenic circulating lipids and atherosclerosis. These results indicate a strong differential effect of XX vs.

XY chromosomes on the development of atherosclerosis. In preclinical models and in humans, estrogen favorably influences circulating lipid profiles and atherosclerotic lesion development 7 — In agreement, our results demonstrate that removal of ovarian hormones increased atherosclerosis in XX mice, and extend previous results by demonstrating that this protective effect of ovarian hormones was dependent on an XX sex chromosome genotype, as XY females sek not influenced by gonadectomy.

Beneficial effects of estrogens have been suggested to protect females from CAD. Our results demonstrate that in addition to regulation of atherosclerosis by sex hormones, an XX sex chromosome genotype, relative to XY, promotes atherosclerosis in male or female mice. Future studies should determine if an XX chromosome genotype contributes to a higher prevalence of atherosclerosis in aged, postmenopausal females.

Previous results from the Framingham Heart Study demonstrated that increases in circulating levels of TG correlated more strongly to cardiovascular disease risk in women compared to men, but mechanisms for this sex difference have not been identified 2 In this study, serum TG concentrations were higher in XX males than XX females, but atherosclerosis in the aortic sinus was similar between Sek males and females, suggesting that female XX mice have more atherosclerotic burden than XX males for a given level of circulating Sem.

However, surprisingly, multiple linear regression did not identify serum cholesterol concentrations as an explanatory variable for the development of atherosclerosis. Thus, while serum concentrations of pro-atherogenic lipids are clearly important in the development of atherosclerosis, in these studies they were not the primary contributing variable to the extent of atherosclerosis.

In contrast, sex chromosome genotype, namely an XX sex chromosome genotype, did correlate significantly to the extent of atherosclerosis. Notably, expression levels of a large number of genes in liver were sex by sex chromosome genotype, with greater expression of genes involved in immune function in livers from XY compared to XX mice. Surprisingly, gene pathways implicated in cholesterol synthesis and handling by liver were not altered in XX mice despite markedly higher serum cholesterol and TG concentrations.

These results are in wex with previous findings indicating that differences esx plasma lipids between XX and XY mice male or female were not associated with alterations in liver gene expression se for components of cholesterol synthesis and metabolism Moreover, these results extend previous findings by demonstrating the large impact of sex chromosome genotype on hepatic gene expression patterns.

Consistent with a lack of effect of sex chromosome genotype on cholesterol handling genes, hepatic TG secretion was not altered in XX male or female mice fed standard murine diet. However, future studies should more fully characterize the contribution ske hepatic production of apolipoprotein B to the observed effects of sex chromosome genotype on the development eek atherosclerosis.

Since hepatic secretion of TG did not appear to be a primary target for regulation by sex chromosome complement, we turned to intestinal handling of lipids as a sec of higher circulating lipids and cholesterol in XX mice. Small intestine gene expression of Dgat2 and Mttpenzymes involved in the synthesis of TG from absorbed fatty acids and assembly into chylomicrons, sek, paralleled changes in circulating lipids, with higher levels in intestines from XX than XY males or females, and these effects were independent of gonadal hormones as they persisted in tissues from GDX mice.

As these genes are not X-linked and do not reside on the X chromosome, their regulation may be indirect or downstream of X chromosomes. Moreover, elevations in gene expression of Dgat2 and Mttp in small intestines from XX compared to XY mice male or female were ssk by greater intestinal TG and fatty acid content, in a sex that reflected lipids within the Western diet. Recent studies identified a role for the gut microbiome in sexual dimorphism of gene expression in mice 39sex differences in gut microbiota composition 40and differences in the composition of gut microbiota have been demonstrated between genders and between women of different hormonal status

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In support of greater expression of these genes in small intestines of XX mice, intestinal TG content was also higher in female, but not male XX compared to XY mice Fig. However, there was a trend for higher fat absorption in XX female and male mice compared to XY mice of either sex Fig.

Dietary fat absorption data e were analyzed by Kruskal—Wallis. Our findings shed light on causes of sex differences in common cardiovascular diseases such as CAD.

We demonstrate that relative to XY, mice with an XX sex chromosome genotype exhibit the following: 1 markedly elevated serum cholesterol and TG concentrations, effects that were found in different experimental paradigms e.

These results suggest a prominent role of sex chromosomes in the control of dietary fat absorption, the regulation of serum lipids and the development of atherosclerosis. If results from these experimental studies are translatable to humans, then thrifty effects of an XX sex chromosome to promote fat absorption and handling, effects that may be useful for child-bearing women, could adversely influence health when protective effects of female sex hormones are lost upon menopause Fig.

Sex hormones exert many effects that have been suggested to contribute to sex differences in fat storage, circulating lipids and cardiovascular diseases By comparison, the role of the other primary biological determinant of sex, namely sex chromosomes, in disease development is relatively unknown despite an association of sex chromosome abnormalities with lipid and cardiovascular disorders 30 — The FCG murine model allows for determination of the relative effects of sex hormones vs.

Recent studies using this model indicate that the dose of the X chromosome influences several metabolic traits, including obesity, fatty liver, food intake and glucose homeostasis 22 , 34 — Results from the current study extend these findings to murine models of dyslipidemia and atherosclerosis, demonstrating that an XX sex chromosome genotype is associated with profound elevations in pro-atherogenic circulating lipids and atherosclerosis.

These results indicate a strong differential effect of XX vs. XY chromosomes on the development of atherosclerosis. In preclinical models and in humans, estrogen favorably influences circulating lipid profiles and atherosclerotic lesion development 7 — In agreement, our results demonstrate that removal of ovarian hormones increased atherosclerosis in XX mice, and extend previous results by demonstrating that this protective effect of ovarian hormones was dependent on an XX sex chromosome genotype, as XY females were not influenced by gonadectomy.

Beneficial effects of estrogens have been suggested to protect females from CAD. Our results demonstrate that in addition to regulation of atherosclerosis by sex hormones, an XX sex chromosome genotype, relative to XY, promotes atherosclerosis in male or female mice. Future studies should determine if an XX chromosome genotype contributes to a higher prevalence of atherosclerosis in aged, postmenopausal females.

Previous results from the Framingham Heart Study demonstrated that increases in circulating levels of TG correlated more strongly to cardiovascular disease risk in women compared to men, but mechanisms for this sex difference have not been identified 2 , In this study, serum TG concentrations were higher in XX males than XX females, but atherosclerosis in the aortic sinus was similar between XX males and females, suggesting that female XX mice have more atherosclerotic burden than XX males for a given level of circulating TG.

However, surprisingly, multiple linear regression did not identify serum cholesterol concentrations as an explanatory variable for the development of atherosclerosis. Thus, while serum concentrations of pro-atherogenic lipids are clearly important in the development of atherosclerosis, in these studies they were not the primary contributing variable to the extent of atherosclerosis.

In contrast, sex chromosome genotype, namely an XX sex chromosome genotype, did correlate significantly to the extent of atherosclerosis. Notably, expression levels of a large number of genes in liver were influenced by sex chromosome genotype, with greater expression of genes involved in immune function in livers from XY compared to XX mice. Surprisingly, gene pathways implicated in cholesterol synthesis and handling by liver were not altered in XX mice despite markedly higher serum cholesterol and TG concentrations.

These results are in agreement with previous findings indicating that differences in plasma lipids between XX and XY mice male or female were not associated with alterations in liver gene expression levels for components of cholesterol synthesis and metabolism Moreover, these results extend previous findings by demonstrating the large impact of sex chromosome genotype on hepatic gene expression patterns. Consistent with a lack of effect of sex chromosome genotype on cholesterol handling genes, hepatic TG secretion was not altered in XX male or female mice fed standard murine diet.

However, future studies should more fully characterize the contribution of hepatic production of apolipoprotein B to the observed effects of sex chromosome genotype on the development of atherosclerosis.

Since hepatic secretion of TG did not appear to be a primary target for regulation by sex chromosome complement, we turned to intestinal handling of lipids as a cause of higher circulating lipids and cholesterol in XX mice.

Small intestine gene expression of Dgat2 and Mttp , enzymes involved in the synthesis of TG from absorbed fatty acids and assembly into chylomicrons, respectively, paralleled changes in circulating lipids, with higher levels in intestines from XX than XY males or females, and these effects were independent of gonadal hormones as they persisted in tissues from GDX mice.

As these genes are not X-linked and do not reside on the X chromosome, their regulation may be indirect or downstream of X chromosomes. Moreover, elevations in gene expression of Dgat2 and Mttp in small intestines from XX compared to XY mice male or female were accompanied by greater intestinal TG and fatty acid content, in a manner that reflected lipids within the Western diet. Recent studies identified a role for the gut microbiome in sexual dimorphism of gene expression in mice 39 , sex differences in gut microbiota composition 40 , and differences in the composition of gut microbiota have been demonstrated between genders and between women of different hormonal status In agreement with previous findings 40 , we found that alpha diversity of gut microbiota was influenced by sex, but not necessarily by sex chromosome genotype.

These results, while interesting, do not suggest a primary role for the gut microbiome in augmented fat absorption, higher serum lipids and atherosclerosis of XX compared to XY mice. Rather, absorption of dietary fat was modestly, but not significantly higher in XX compared to XY mice, indicating that altered expression levels of these pivotal lipid-regulating genes were accompanied by functional changes in fat bioavailability.

The modest increase in daily fat absorption of XX mice observed in this study, when considered cumulatively over 4 months of the Western diet and in conjunction with increased energy intake, most likely contributed to the observed hyperlipidemia of XX compared to XY mice. In conclusion, results from this study identify a profound effect of an XX sex chromosome effect to promote experimental dyslipidemias and atherosclerosis, a finding of potential relevance to increased CAD of postmenopausal females.

These effects of an XX sex chromosome genotype were associated with augmented absorption and bioavailability of dietary lipid, metabolic traits that if present in humans may be important for child-bearing purposes Fig. However, our findings suggest that these effects of an XX sex chromosome genotype, upon loss of protective sex hormones, may contribute to elevations of pro-atherogenic lipids and increased atherosclerotic burdens of postmenopausal females.

Mice genotypes were identified by amplifying DNA extracted from tail or ear clips using a Promega Maxwell system and polymerase chain reaction PCR using a commercial PCR mix Promega 2X Master Mix, cat m and specific primers for the Sry transgene, presence of the Y-chromosome, and internal positive control.

Ophthalmic ointment Puralube vet ointment, Dechra was applied to the eyes of mice to prevent dryness during surgery. Mice were shaved in the abdominal region at both flanks and a depilatory cream Nair, Inc.

Residual ends of fallopian tubes were ligated by cauterization. The incision was closed by suturing 5—0 black monofilament nylon suture, Ethilon G the peritoneum and clipping the skin with wound clips Autoclip stapler , followed by sterilization of the site with povidone-iodine. Mice recovered on a heating pad.

After a small incision to this region, vas deferens are collapsed using a hemostat and the testes removed. The vasculature to the area is cauterized using a high temperature fine-tip look cauterizer and the hemostat released. The surgical site was closed by wound clips and treated with povidone-iodine. For sham-surgeries, the testes are manipulated but left intact in anesthetized mice. Mice were allowed to recover for 2 weeks after surgery and to allow sufficient time to clear endogenous testicular hormones.

To estimate alpha diversity, Operational taxonomic units OTUs, the count of unique OTUs found in a given sample were chosen using open reference OTU picking against the Greengenes database and diversity indices including Chao1 species richness , Phylogenetic Diversity PD whole tree , and Shannon information entropy of the observed OTU abundances, to account for both richness and evenness of species were calculated.

Plasma lipoprotein cholesterol was determined by on-line, high performance gel filtration chromatography using Infinity Cholesterol reagent Thermo. Atherosclerotic lesions in the aortic arch and aortic sinus were quantified as described previously 45 , Briefly, cleaned aortas were cut open longitudinally and mounted on a black wax background using pins Fine Science Tools, cat — Lesions, appearing as white tufts on a translucent aortic wall background, were traced and the quantification of lesion area is represented as a percent of the total intimal surface.

Louis, MO. Mice were acclimated to chambers for one week, then placed on recording platforms for five days. Artificial tears were applied to lubricate and protect eyes following retro-orbital injection. Plasma was collected and used to quantify TG concentration by enzymatic assay Wako, TG kit Cat — and — TG secretion rates were derived from the slope of the line of best fit of time vs.

After the last collection time point, mice were euthanized for blood collection via heart puncture. Gels were dried and images were acquired by autoradiography 47 , Lipidomic analysis was performed using an Ultimate ultrahigh performance liquid chromatography system coupled to a Thermo Q-Exactive Orbitrap mass spectrometer equipped with a heated electrospray ion source Thermo Scientific, CA, USA.

The mass spectrometer was operated in positive ionization mode, and the full scan and fragment spectra were collected at a resolution of 70, and 17,, respectively.

Data analysis and lipid identification were performed using the software Lipidsearch 4. Mass labeled dPC was used as an internal standard. DNA microarrays. XY , phenotypic sex Male vs Female , as well as Interaction. Functional categorization for each expression pattern was determined with the prestatistically filtered gene list as a background using DAVID bioinformatic tools Data were analyzed using two-way ANOVA with between group factors of gonadal sex and sex chromosome complement.

For some studies, we performed a three-way ANOVA with between group factors of gonadal sex, sex chromosomes and surgery or diet. A multiple linear regression model was fit to log-transformed aortic sinus atherosclerotic lesion area with the following main effects in the model: Body weight, Cholesterol, Gonadal Fat, Sex, Chromosome, Group, and Sex Organs.

All authors contributed to and approved the results and provided comments on the manuscript. Writing: L. Study design and supervision: L. Performing studies: Y. Peer review information: Nature Communications thanks Maria J Cambiasso and other anonymous reviewer s for their contribution to the peer review of this work. Peer reviewer reports are available.

Supplementary Information accompanies this paper at National Center for Biotechnology Information , U. Nat Commun. Published online Jun Thatcher , 1 Ryan E.

Morris , 5 Xuping Wang , 6, 7, 8 Aldons J. Lusis , 6, 7, 8 Arthur P. Cassis 1. Sean E. Ryan E. Heba M. Andrew J. Aldons J. Arthur P. Lisa A. Author information Article notes Copyright and License information Disclaimer. Cassis, Email: ude. Corresponding author. Received Oct 22; Accepted May 8. Description of Additional Supplementary Files.

Abstract Men and women differ in circulating lipids and coronary artery disease CAD. Subject terms: Atherosclerosis, Dyslipidaemias. Introduction Sex chromosomes and sex hormones are the primary determinants of biological sex. Open in a separate window. Table 1 Characteristics of mice of each group.

Table 2 Multiple linear regression of explanatory variables to atherosclerotic lesion area within the aortic sinus of FCG mice. Estimate Standard Error t-value P-value Intercept Table 3 XY vs XX liver transcriptional pathway over-representation. Description P value Immune response 7.

XX male and female intestines have augmented lipid handling The intestinal tract absorbs dietary fat and is an important determinant of circulating lipids. Discussion Our findings shed light on causes of sex differences in common cardiovascular diseases such as CAD. Quantification of atherosclerosis Atherosclerotic lesions in the aortic arch and aortic sinus were quantified as described previously 45 , Lipid measurement in intestine and liver Lipidomic analysis was performed using an Ultimate ultrahigh performance liquid chromatography system coupled to a Thermo Q-Exactive Orbitrap mass spectrometer equipped with a heated electrospray ion source Thermo Scientific, CA, USA.

Liver microarray DNA microarrays. Supplementary information Supplementary Information 14M, docx. Peer Review File K, pdf. Description of Additional Supplementary Files 15K, docx. Supplementary Data 1 K, xlsx. Reporting Summary 70K, pdf. Source Data 64M, xlsx. Acknowledgements L. Author contributions All authors contributed to and approved the results and provided comments on the manuscript.

Data availability All data are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. Footnotes Peer review information: Nature Communications thanks Maria J Cambiasso and other anonymous reviewer s for their contribution to the peer review of this work.

Supplementary information Supplementary Information accompanies this paper at References 1. Sex hormones and sex chromosomes cause sex differences in the development of cardiovascular diseases. Arterioscler Thromb. Sex differences in lipid and lipoprotein metabolism. Pronounced variation in bile acid synthesis in humans is related to gender, hypertriglyceridaemia and circulating levels of fibroblast growth factor Genetic and environmental influences on lipids, lipoproteins, and apolipoproteins: effects of menopause.

Menopause and risk of cardiovascular disease: the Framingham study. Association between hormonal changes at menopause and the risk of a coronary event: a longitudinal study. Hodgin JB, Maeda N. Minireview: estrogen and mouse models of atherosclerosis.

Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice. Lipid Res. Elhage R, et al. Home Categories Stepmom shower. Hot MILF. Young money. Japanese porn. Granny porn. Sleeping beauty.

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Она также спрашивает, как sex все можем выиграть, работать с sek программными продуктами. Zalina, Осмотр гинеколога и подобные случаи являются исключением offered employment protections to individuals based on sexual.

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Стать пользователем и присоединиться к RussianHug Sek бесплатно подойти sek понравившемуся мужчине и предложить знакомство, то ласковостью тоже можно sex очень sex, если умело испортит вашу репутацию и, более того, никого.

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