Sexual dimorphism in body weight, fat distribution, and metabolic disease has been attributed largely to differential effects of male and female gonadal hormones. Here, we report that the number of X chromosomes within cells also contributes to these sex differences. We employed a unique mouse model, known as the “four core genotypes,” to distinguish between effects of gonadal sex (testes or ovaries) and sex chromosomes (XX or XY). With this model, we produced gonadal male and female mice carrying XX or XY sex chromosome complements. Mice were gonadectomized to remove the acute effects of gonadal hormones and to uncover effects of sex chromosome complement on obesity. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had up to 2-fold increased adiposity and greater food intake during daylight hours, when mice are normally inactive. Mice with two X chromosomes also had accelerated weight gain on a high fat diet and developed fatty liver and elevated lipid and insulin levels. Further genetic studies with mice carrying XO and XXY chromosome complements revealed that the differences between XX and XY mice are attributable to dosage of the X chromosome, rather than effects of the Y chromosome. A subset of genes that escape X chromosome inactivation exhibited higher expression levels in adipose tissue and liver of XX compared to XY mice, and may contribute to the sex differences in obesity. Overall, our study is the first to identify sex chromosome complement, a factor distinguishing all male and female cells, as a cause of sex differences in obesity and metabolism.

Author Summary

Differences exist between men and women in the development of obesity and related metabolic diseases such as type 2 diabetes and cardiovascular disease. Previous studies have focused on the sex-biasing role of hormones produced by male and female gonads, but these cannot account fully for the sex differences in metabolism. We discovered that removal of the gonads uncovers an important genetic determinant of sex differences in obesity—the presence of XX or XY sex chromosomes. We used a novel mouse model to tease apart the effects of male and female gonads from the effects of XX or XY chromosomes. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had increased body fat and ate more food during the sleep period. Mice with two X chromosomes also had accelerated weight gain, fatty liver, and hyperinsulinemia on a high fat diet. The higher expression levels of a subset of genes on the X chromosome that escape inactivation may influence adiposity and metabolic disease. The effect of X chromosome genes is present throughout life, but may become particularly significant with increases in longevity and extension of the period spent with reduced gonadal hormone levels.

The “four core genotypes” (FCG) model comprises mice in which sex chromosome complement (XX vs. XY) is unrelated to the animal's gonadal sex. The four genotypes are XX gonadal males or females, and XY gonadal males or females. The model allows one to measure (1) the differences in phenotypes caused by sex chromosome complement (XX vs. XY), (2) the differential effects of ovarian and testicular secretions, and (3) the interactive effects of (1) and (2). Thus, the FCG model provides new information regarding the origins of sex differences in phenotype that has not been available from studies that manipulate gonadal hormone levels in normal XY males and XX females. Studies of the FCG model have uncovered XX vs. XY differences in behaviors (aggression, parenting, habit formation, nociception, social interactions), gene expression (septal vasopressin), and susceptibility to disease (neural tube closure and autoimmune disease) not mediated by gonadal hormones. Some sex chromosome effects are mediated by sex differences in dose of X genes or their parental imprint. Future studies will identify the genes involved and their mechanisms of action.

In animal studies of nociception, females are often more sensitive to painful stimuli, whereas males are often more sensitive to analgesia induced by μ agonists. Sex differences are found even at birth, and in adulthood are likely caused, at least in part, by differences in levels of gonadal hormones. Here we investigate nociception and analgesia in neonatal mice, and assess the contribution of the direct action of sex chromosome genes in hotplate and tail withdrawal tests. We used the four core genotypes mouse model, in which gonadal sex is independent of the complement of sex chromosomes (XX vs. XY). Mice were tested at baseline and then injected with μ-opioid agonist morphine (10mg/kg), or with the κ-opioid agonist U50,488H (U50, 12.5mg/Kg) with or without the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.1mg/kg). On the day of birth, XX mice showed faster baseline latencies than XY in tail withdrawal, irrespective of their gonadal type. Gonadal males showed greater effects of morphine than gonadal females in the hotplate test, irrespective of their sex chromosome complement. U50 and morphine were both effective analgesics in both tests, but MK-801 did not block the U50 effect. The results suggest that sex chromosome complement and gonadal secretions both contribute to sex differences in nociception and analgesia by the day of birth.

Perspective: Sex differences in pain may stem not only from the action of gonadal hormones on pain circuits, but from the sex-specific action of X and Y genes. Identification of sex chromosome genes causing sex differences could contribute to better pain therapy in females and males.

We tested the role of sex chromosome complement and gonadal hormones in sex differences in several different paradigms measuring nociception and opioid analgesia using “four core genotypes” C57BL/6J mice. The genotypes include XX and XY gonadal males, and XX and XY gonadal females. Adult mice were gonadectomized and tested 3–4 weeks later, so that differences between sexes (mice with testes vs. ovaries) were attributable mainly to organizational effects of gonadal hormones, whereas differences between XX and XY mice were attributable to their complement of sex chromosomes. In experiment 1 (hotplate test of acute morphine analgesia), XX mice of both gonadal sexes had significantly shorter hotplate baseline latencies prior to morphine than XY mice. In experiment 2, (test of development of tolerance to morphine), mice were injected twice daily with 10mg/kg morphine or saline for 6 days. Saline or the competitive NMDA antagonist CPP [3-]2-carboxypiperazin-4yl)propyl-1-phospionic acid] (10mg/kg) was co-injected. On day 7, mice were tested for hotplate latencies before and after administration of a challenge dose of morphine (10mg/kg). XX mice showed shorter hotplate latencies than XY mice at baseline, and the XX-XY difference was greater following morphine. In experiment 3, mice were injected with morphine (10mg/Kg) or saline,15 minutes before intraplantar injection of formalin (5%/25µl). XX mice licked their hindpaw more than XY mice within 5 minutes of formalin injection. The results indicate that X- or Y-linked genes have direct effects, not mediated by gonadal secretions, on sex differences in two different types of acute nociception.

Hundreds of genes show aberrant DNA hypermethylation in cancer, yet little is known about the causes of this hypermethylation. We identified RIL as a frequent methylation target in cancer. In search for factors that influence RIL hypermethylation, we found a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Pyrosequencing of homozygous tumors revealed a 2.1-fold higher methylation for the short alleles (P<0.001). Bisulfite sequencing of cancers heterozygous for RIL showed that the short alleles are 3.1-fold more methylated than the long (P<0.001). The comparison of expression levels between unmethylated long and short EBV-transformed cell lines showed no difference in expression in vivo. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on. However, stable transfection of methylation-seeded constructs showed gradually decreasing transcription levels from the short allele with eventual spreading of de novo methylation. In contrast, the long allele showed stable levels of expression over time as measured by luciferase and ∼2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that, in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.

Author Summary

The factors that guide DNA hypermethylation in cancer are poorly understood. We identified the candidate tumor-suppressor gene, RIL, as a frequent methylation target in cancer. Here, we report on a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Methylation analysis showed that, in aging colon, colon cancer, and leukemias, the short allele had 2.1–3.1-fold higher methylation than the long allele (P<0.001). Short and long alleles had similar expression levels in EBV-transformed cell lines. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors. Transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on, but methylation-seeded constructs showed gradually decreasing transcription from the short allele with eventual spreading of de novo methylation. By contrast, the long allele showed stable expression over time as measured by luciferase, and ∼2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.