Estrogen receptor-beta mediates dihydrotestosterone-induced stimulation of the arginine vasopressin promoter in neuronal cells

Arginine vasopressin (AVP) is a neuropeptide involved in the regulation of fluid balance, stress, circadian rhythms, and social behaviors. In the brain, AVP is tightly regulated by gonadal steroid hormones in discrete regions with gonadectomy abolishing and testosterone replacement restoring normal AVP expression in adult males. Previous studies demonstrated that 17beta-estradiol, a primary metabolite of testosterone, is responsible for restoring most of the AVP expression in the brain after castration. However, 5alpha-dihydrotestosterone (DHT) has also been shown to play a role in the regulation of AVP expression, thus implicating the involvement of both androgen and estrogen receptors (ER). Furthermore, DHT, through its conversion to 5alpha-androstane-3beta,17beta-diol, has been shown to modulate estrogen response element-mediated promoter activity through an ER pathway. The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an estrogen or androgen receptor pathway. To that end, we overexpressed androgen receptor, ERbeta, and ERbeta splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 5alpha-androstane-3beta,17beta-diol stimulate AVP promoter activity through ERbeta in a neuronal cell line.     

Testosterone down regulates the expression of Fmr-1 gene in the cerebral cortex of gonadectomized old male mice

Sex steroid hormones are important modulators of synaptic plasticity underlying learning and memory, however, the precise molecular mechanism of their role in above is not well understood. Fragile X mental retardation protein is one of the proteins that regulate synaptic plasticity that declines during aging. Whether Fmr-1 gene expression is regulated by sex steroid hormones as a function of age is not known. To answer this, we have investigated Fmr-1 gene expression in the cerebral cortex of the gonadectomized- and testosterone-treated gonadectomized adult and old male mice. Our data reveal that Fmr-1 gene expression is significantly down regulated in adult mice brain, however, it is up regulated in old age following bilateral gonadectomy (castration). Administration of testosterone to castrated mice was found to reverse the effects of castration on Fmr-1 gene expression towards that in the Sham operated intact control old age mice whereas it has no effects as compared to that in Sham operated adult age mice. Further, to investigate the mechanism of this effect of testosterone, we studied interaction of trans-acting factors with Fmr-1 promoter by electrophoretic mobility shift assay (EMSA). The EMSA data shows formation of two complexes as a result of interaction of nuclear trans-acting factors with Fmr-1 promoter, which also exhibited age- and hormone-dependent patterns which largely corresponded to the patterns of our expression data. Our data demonstrate that testosterone regulates Fmr-1 gene expression in age-dependent manner which may throw light on the mechanisms of age- and sex steroid hormones-dependent alterations in brain function.     

Epigenetic mechanisms are involved in sexual differentiation of the brain

Sexual differentiation of the brain can be considered as a process during which effects of sex steroid hormones secreted during early development is maintained into adulthood. Epigenetic regulation is emerging as a potentially important mechanism of conveyance of long-lasting effects of the hormonal and environmental milieu in the developing brain. Evidence has accumulated to show that epigenetic regulation is involved in the control of sexual differentiation of the brain. In the preoptic area (POA), which is important for male sexual behavior, histones associated with the estrogen receptor (ER) α and aromatase (Arom) gene promoters are differentially acetylated between the sexes, and two subtypes of histone deacetylase (HDAC2 and 4) are associated with the same promoters at higher frequencies in males in the early postnatal period. Since ERα and Arom are essential genes in masculinization of the brain, these findings suggest that histone deacetylation in the early postnatal period is involved in masculinization of the brain. Indeed, inhibition of HDAC activity in males during this period abrogates brain masculinization: structural sexual dimorphism of the bed nucleus of the stria terminalis is eliminated and expression of male sexual behavior is reduced in adulthood. Previous reports have demonstrated that ERα gene expression in the POA is higher in females during the developmental and pubertal periods and in adulthood, indicating that sexually dimorphic ERα expression that appears in early postnatal development is maintained until adulthood by epigenetic programming. The ERα promoter is also more sparsely methylated in females, with an inverse correlation with ERα expression. In addition to the hormonal effect, the amount of maternal care received during postnatal development has a lasting effect on ERα expression mediated by DNA methylation of its promoter. Taken together, these results suggest that epigenetic mechanisms play a central role in the transduction and maintenance of early hormonal and social cues to organize sexually differentiated brain functions.     

Regional specificity of testosterone regulation of proopiomelanocortin gene expression in the arcuate nucleus of the male rat brain

Endogenous opioid peptides have been implicated as mediators in the negative feedback action of gonadal steroids on GnRH secretion. We have previously demonstrated that testosterone stimulates POMC gene expression in neurons of the arcuate nucleus. However, the wide distribution and variety of actions attributed to the numerous arcuate POMC neurons suggest that these cells may be heterogeneous in their responsiveness to steroid hormones. We tested the hypothesis that testosterone modulates a select population of POMC neurons within the arcuate nucleus of the adult male rat by comparing POMC mRNA signal levels throughout the arcuate nucleus of intact, castrated, and castrated testosterone-replaced adult males. Adult male rats were castrated and implanted (sc) with a Silastic capsule (30 mm) that was either empty (n = 6) or filled with crystalline testosterone (n = 5). Control sham-operated animals (n = 6) were left intact. In each animal the arcuate nucleus was divided into four equal rostral-caudal areas within which we measured POMC mRNA content in individual cells. We report that the effects of castration and testosterone replacement are observed in POMC neurons located in the most rostral region of the arcuate nucleus. After castration, POMC mRNA content was reduced in cells of the most rostral arcuate area (intact, 152 +/- 5 grains/cell; castrate, 119 +/- 2 grains/cell; P less than 0.0005), and replacement with physiological levels of testosterone prevented the decline in POMC mRNA levels so that they remained equivalent to that of the intact animal (castrated testosterone-replaced, 153 +/- 6 grains/cell). There was no significant difference in POMC mRNA signal between intact and castrated testosterone-replaced animals in the most rostral area. POMC neurons in the more caudal aspect of the arcuate (75% of the nucleus) were unaffected by the treatments; alternatively, it is possible that a real change in POMC message content in a subpopulation of cells was obscured by larger numbers of nonresponding cells within the same tissue sections. Based on these observations we conclude that there is a heterogeneous population of POMC neurons in the arcuate nucleus and that testosterone regulates POMC gene expression in a select group of these cells located in the rostral portion of the arcuate nucleus.     

Effect of neonatal and adult testosterone treatment on the cellular composition of the adult female rat anterior pituitary

The adult female pituitary has significantly more lactotrophs than that of the male, while the later has a higher percent of somatotrophs. It is clear that GH and prolactin (PRL) gene expression and somatotroph and lactotroph proliferation are modulated by the postpubertal hormone environment; however, the role of the neonatal steroid environment in this process is not known. We have used in situ hybridization to determine the number of GH and PRL mRNA-containing cells, as well as the level of expression of these two hormones, in response to neonatal and adult testosterone treatment. Female rats exposed to testosterone during the neonatal period, adulthood or both periods, as well as normal females and males were used. Exposure to testosterone during the neonatal period significantly increased the percentage of somatotrophs (ANOVA: P<0. 005) and decreased that of lactotrophs in the adult female rat (ANOVA: P<0.001). Adult testosterone treatment had no significant effect on the percentage of somatotrophs. The percentage of lactotrophs was significantly increased by adult testosterone only in those rats also exposed to neonatal testosterone. PRL mRNA concentrations, as reflected by silver grains/cell, were reduced by neonatal testosterone and increased by adult testosterone treatment (ANOVA: P<0.0001). Overall PRL mRNA levels, measured by densitometry, were also reduced by neonatal testosterone exposure, but adult testosterone had no effect (ANOVA: P<0.001). GH mRNA levels per cell, as reflected by silver grains/cell, were increased by adult testosterone, while neonatal testosterone treatment had no effect. Overall GH mRNA levels per unit area, determined by densitometry measurements, were increased by both neonatal and adult testosterone treatment, with the combination of these two treatments resulting in adult females having levels indistinguishable from intact males (ANOVA: P<0.003). These results suggest that, in combination with postpubertal sex steroids, the neonatal gonadal steroid environment plays an important role in determining anterior pituitary hormone synthesis and cellular composition.     

Developmental exposure to Bisphenol a impairs the uterine response to ovarian steroids in the adult

Morphoregulator genes like members of the Hox gene family regulate uterine development and are associated with endocrine-related processes such as endometrial proliferation and differentiation in the adult uterus. Exposure to neonatal endocrine disruptors could affect signaling events governed by Hox genes, altering the developmental trajectory of the uterus with lasting consequences. We investigated whether neonatal exposure to bisphenol A (BPA) alters Hoxa10 and Hoxa11 mRNA uterine expression shortly after treatment as well as in the adult. Moreover, we studied whether xenoestrogen exposure may affect the adult uterine response to hormonal stimuli. Newborn females received vehicle, 0.05 mg/kg.d BPA, 20 mg/kg*d BPA, or diethylstilbestrol (0.2 microg/kg*d) on postnatal d 1, 3, 5, and 7). At postnatal d 8, real time RT-PCR assays showed a decrease in Hoxa10 and Hoxa11 expression in all xenoestrogen-treated groups. To evaluate the long-term effects, we used adult ovariectomized rats with hormonal replacement. The subepithelial stroma in BPA- and diethylstilbestrol-treated animals showed an impaired proliferative response to steroid treatment associated with a silencing of Hoxa10 but not associated with changes in the methylation pattern of the Hoxa10 promoter. BPA animals showed that the Hoxa10 reduction was accompanied by an increased stromal expression of the silencing mediator for retinoic acid and thyroid hormone receptor. The spatial coexpression of steroid receptors Hoxa10 and silencing mediator for retinoic acid and thyroid hormone receptor was established using immunofluorescence. Our data indicate that postnatal BPA exposure affects the steroid hormone-responsiveness of uterine stroma in adulthood. Whether this impaired hormonal response is associated with effects on uterine receptivity and decidualization is currently under investigation.     

Unexpected effects of perinatal gonadal hormone manipulations on sexual differentiation of the extrahypothalamic arginine-vasopressin system in prairie voles

The sexually dimorphic extrahypothalamic arginine-vasopressin (AVP) projections from the bed nucleus of the stria terminalis to the lateral septum (LS) and lateral habenula (LHb) are denser in males than females and, in rats, require males' perinatal exposure to gonadal hormones but the absence of such exposure in females. We examined perinatal hormone effects on development of this sex difference in prairie voles (Microtus ochrogaster), which show atypical effects of hormones on sexual differentiation of some reproductive behaviors. Neonatal castration reduced the number of AVP mRNA-expressing cells in the bed nucleus of the stria terminalis and AVP immunoreactivity (ir) in the LS and LHb. Surprisingly, daily injections of 1000 microg of testosterone propionate (TP) during the first postnatal week did not maintain high levels of AVP-ir in neonatally castrated males. Furthermore, perinatal treatments with TP (75, 500, or 1000 microg), testosterone (100 microg), or dihydrotestosterone (200 microg) did not masculinize AVP-ir in the female LS or LHb. In fact, 1000 microg TP reduced it in some cases. However, 1000 microg TP lengthened anogenital distance, indicating that TP was biologically active. Neonatal estrogen receptor antagonism with tamoxifen reduced AVP-ir in the male LS, whereas treating neonatal females with the synthetic estrogen diethylstilbestrol increased septal AVP-ir. Tamoxifen and diethylstilbestrol had no effects in the LHb. Similar to rats, therefore, postnatal estrogen influences some components of the extrahypothalamic AVP system in prairie voles, but this developing system appears to be insensitive to exogenous androgens, including aromatizable androgens. Such insensitivity is atypical for a sexually dimorphic neural system in a rodent and may reflect the unusual effects of hormones on sexual differentiation of some behaviors in prairie voles.     

Sexually dimorphic expression of the growth hormone-releasing hormone gene is not mediated by circulating gonadal hormones in the adult rat

The sexual dimorphism characterizing GH secretion in the rat is thought to be related to differences in the hypothalamic synthesis and release of the GH-regulating peptides, GH-releasing hormone (GHRH), and somatostatin. Therefore, the influence of gender and sex steroid hormones on hypothalamic expression of the GHRH gene in adult rats were examined. GHRH messenger RNA (mRNA) levels were measured in individual rat hypothalami by Northern hybridization analysis using a 32P-labeled complementary DNA encoding rat GHRH. Destruction of hypothalamic GHRH neurons by neonatal treatment with monosodium glutamate caused similar 3-fold reductions in the levels of GHRH mRNA in adult male and female animals. In three separate experiments, hypothalamic GHRH mRNA concentrations in male rats were 2- to 3-fold greater than in randomly cycling females (four or five rats per group; P less than 0.01). In spite of the greater abundance of GHRH mRNA abundance in the male rat hypothalamus, circulating gonadal steroids lacked the ability to modulate GHRH gene expression in adult animals, since neither gonadectomy nor pharmacological sex steroid replacement changed GHRH mRNA levels in the hypothalamus of male and female adult rats. Furthermore, GHRH mRNA concentrations in female rats were similar during the proestrus, estrus, and diestrus phase of the estrous cycle. Also, GH inhibited hypothalamic GHRH gene expression in a sex-specific manner. Exposure to high levels of GH secreted by the MtTW15 tumor for 4 weeks reduced GHRH mRNA concentrations 7-fold in male rats (P less than 0.001) but only 2-fold in females (P less than 0.05). These studies demonstrate that GHRH gene expression in the rat hypothalamus is sexually dimorphic. Basal mRNA levels are greater in male rats, and expression in male hypothalami is more sensitive to feedback inhibition by GH than expression in females. There is no evidence for regulation of GHRH mRNA levels by either testosterone or estrogen in adult rats. These gender differences in GHRH gene expression likely contribute to the generation of a sex-specific pattern of GH secretion.     

Testosterone regulates pro-opiomelanocortin gene expression in the primate brain

Endogenous opioid peptides such as beta-endorphin, derived from proopiomelanocortin (POMC), have been widely implicated as serving an important role in the neuroendocrine regulation of the primate reproductive axis. In both human and nonhuman primates, POMC neurons are thought to mediate, at least in part, the negative feedback action of sex steroids on GnRH. Sex steroids, such as testosterone, are thought to inhibit GnRH secretion by enhancing the inhibitory activity of beta-endorphin; however, the cellular mechanisms by which steroid hormones regulate the activity of POMC neurons in the primate brain are unknown. In this study, we tested the hypothesis that testosterone stimulates POMC gene expression within the primate brain and that this regulation occurs within a specific subset of POMC neurons residing in the arcuate nucleus of the hypothalamus. We used in situ hybridization to compare cellular levels of POMC messenger RNA in intact (n = 4), castrated (n = 4), and castrated/testosterone-treated (n = 4) monkeys. We report that after castration of the male macaque (Macaca fascicularis), cellular POMC messenger RNA levels decline significantly (P less than 0.05) in neurons within the arcuate nucleus and that this decline is prevented by replacement with physiological doses of testosterone. Moreover, we found that this testosterone-dependent modulation of POMC gene expression is restricted to a small fraction of the numerous POMC neurons located within the most anterior region of the arcuate nucleus in the brain of this primate species. These observations provide evidence that sex steroids regulate expression of the POMC gene in the primate brain.     

Gonadotropin and steroid regulation of steroid receptor and aryl hydrocarbon receptor messenger ribonucleic acid in macaque granulosa cells during the periovulatory interval.

Although steroids play a local role(s) in ovulation and luteinization of the primate follicle, the dynamics of steroid receptor expression during the 36- to 38-h periovulatory interval has yet to be elucidated. The present study examines the regulation of messenger RNAs (mRNAs) for progesterone (PR), androgen (AR), and estrogen (ER alpha, ER beta) receptors as well as the aryl hydrocarbon receptor (AhR) in macaque granulosa cells during controlled ovarian stimulation cycles before (0 h) and after (up to 36 h) administration of the ovulatory hCG bolus with or without steroid depletion and progestin replacement. All steroid receptor mRNAs were detected in granulosa cells before the ovulatory stimulus, as determined by RT-PCR. PR mRNA increased (P < 0.05) by 12 h after hCG; 24 and 36 h after hCG, levels were intermediate between 0-12 h. PR mRNA was reduced by steroid depletion throughout the periovulatory interval (P < 0.05); however, progestin replacement returned PR mRNA to control levels at 12 h. AR mRNA increased (P < 0.05) at 24 h post-hCG and remained at this level 36 h after hCG; steroid depletion did not alter AR mRNA levels. ER alpha mRNA did not change, whereas ER beta decreased 12-36 h after the ovulatory stimulus (P < 0.05). Steroid depletion reduced ER alpha mRNA 12 h after hCG, an effect partially reversible by progestin replacement, whereas ER beta mRNA was not affected by steroids. AhR mRNA was undetectable before the administration of hCG, but increased by 12 h (P < 0.05). These data demonstrate hCG-initiated, steroid-dependent (PR, ER alpha) and -independent (AR, ER beta, AhR) expression of receptor mRNAs in primate granulosa cells during the periovulatory interval. Differences in patterns of expression may relate to diverse roles for steroid hormones and AhR ligands in periovulatory events.     

Regulation of estrogen receptor alpha and beta expression by testosterone in the rat prostate gland

Although ER beta is known to be expressed at high levels in the rat prostate gland, its regulation is not well understood. Here we examined ER mRNA expression and the effects of testosterone administration in male rats at 1, 4 and 9 weeks of age who were castrated and/or treated with testosterone for a week, and then sacrificed. ER alpha was the major type of ER expressed in 2 week-old animals while dominant expression of ER beta mRNA was apparent in older age groups. Interestingly while ER beta expression was diminished and ER alpha mRNA increased in the castrated group, testosterone administration reversed this effect. A time-course study indicated that induction of ER beta mRNA increased within 9 hr and ER alpha decreased in 2 days after an injection (i.p.) of testosterone. Our results suggested that 1) testosterone up-regulates ER beta mRNA expression while ER alpha is down-regulated; and that 2) great changes in ER alpha and beta expression in the prostate gland during development from the newborn to adult may be due to the influence of testosterone.     

Androgen receptor gene expression in leucocytes is hormonally regulated: implications for gender differences in disease pathogenesis

OBJECTIVE: There is evidence that male sex hormones influence the rate of progression of inflammatory and cardiovascular diseases. We have previously shown that human leucocytes and arterial cells isolated from male donors express more androgen receptor (AR) than those from female cells, with potentially pro-atherogenic effects. We now investigate whether the gender difference in AR expression is due to genetic or hormonal regulation. DESIGN AND PATIENTS: The influence of hormones on AR expression were studied in hpg mice (a mouse model of androgen deficiency) treated with testosterone, oestradiol or dihydrotestosterone (DHT). Blood samples were obtained for leucocyte AR expression and hormone levels from 53 subjects, grouped into: 12 male [six young adult (27-45 years), six elderly (71-79 years)] and six female (young adult 25-45 years) healthy controls; six male-to-female transsexuals (M2F; 20-50 years) receiving stable pharmacological oral oestrogen treatment; six female-to-male transsexuals (F2M; 31-51 years) receiving stable androgen replacement therapy; five younger men (18-56 years) who had been receiving long-term androgen replacement therapy for hypogonadal disease; six elderly men (72-88 years) who had undergone medical castration for prostate cancer treatment; and 12 male bone marrow transplant recipients (BMT; 23-65 years) from either male or female donors. MEASUREMENTS: Serum testosterone and oestradiol concentrations were measured by established immunoflurometric assays from unextracted human serum. AR mRNA levels were measured by RT-PCR and AR protein levels by western blot (cell culture) or immunohistochemistry (mouse arteries). RESULTS: We found that AR mRNA levels were significantly down-regulated in the leucocytes of hpg mice that were treated with exogenous testosterone, oestradiol or DHT. AR protein levels were also lower in aortic tissue from the same mice. In humans, we found AR expression was significantly down-regulated by exogenous treatment with testosterone in F2M (31 +/- 13%, compared with control) or oestradiol in M2F (22 +/- 5%) but was significantly up-regulated by endogenous testosterone in BMT (128 +/- 17%). Low androgen levels measured in castrated older men were associated with markedly increased AR expression (207 +/- 26%, P < 0.05) compared with age-matched older male controls (100 +/- 2%). CONCLUSIONS: Our results indicate a regulated ability of vascular cells to respond to sex hormones, with the effects of exogenous therapies differing markedly from those due to endogenous sex hormones. We conclude that the gender difference in AR expression in vascular cells is hormonally, rather than genetically, controlled.     

Intrauterine position affects estrogen receptor α expression in the ventromedial nucleus of the hypothalamus via promoter DNA methylation

There is well-established evidence in many mammalian species for effects of the intrauterine position (IUP) (the sex-specific positioning of the embryo) on postnatal brain function and behavior. We found that the IUP affects estrogen receptor (ER)α expression in adult female rats in the ventrolateral region of the hypothalamic ventromedial nucleus (vlVMH), which is associated with sexual behavior. The ERα expression level in the vlVMH was higher in females that developed in utero between two male siblings (2M females) than in those that developed between female siblings (2F females). We also found that the cytosine methylation status across the ERα promoter in the vlVMH was affected by the IUP, with greater methylation in 2F females. These findings showed a negative correlation between ERα expression levels in the vlVMH and methylation frequency in the ERα promoter. This suggests that genomic methylation sustains the effect of the fetal IUP on ERα expression in the vlVMH.     

Developmental exposure to vasopressin increases aggression in adult prairie voles

Although the biological roots of aggression have been the source of intense debate, the precise physiological mechanisms responsible for aggression remain poorly understood. In most species, aggression is more common in males than females; thus, gonadal hormones have been a focal point for research in this field. Although gonadal hormones have been shown to influence the expression of aggression, in many cases aggression can continue after castration, indicating that testicular steroids are not completely essential for the expression of aggression. Recently, the mammalian neuropeptide arginine vasopressin (AVP) has been implicated in aggression. AVP plays a particularly important role in social behavior in monogamous mammals, such as prairie voles (Microtus ochrogaster). In turn, the effects of social experiences may be mediated by neuropeptides, including AVP. For example, sexually na?ve prairie voles are rarely aggressive. However, 24 h after the onset of mating, males of this species become significantly aggressive toward strangers. Likewise, in adult male prairie voles, central (intracerebroventricular) injections of AVP can significantly increase intermale aggression, suggesting a role for AVP in the expression of postcopulatory aggression in adult male prairie voles. In this paper, we demonstrate that early postnatal exposure to AVP can have long-lasting effects on the tendency to show aggression, producing levels of aggression in sexually na?ve, adult male prairie voles that are comparable to those levels observed after mating. Females showed less aggression and were less responsive to exogenous AVP, but the capacity of an AVP V(1a) receptor antagonist to block female aggression also implicates AVP in the development of female aggression.     

Age- and sex-related analysis of methylation of 5′-upstream sequences of <Emphasis Type="Italic">Fmr-1</Emphasis> gene in mouse brain and modulation by sex steroid hormones

Fmr-1 gene is implicated in synaptic plasticity and thereby learning, memory and cognition, and methylation of Fmr-1 gene is necessary for memory development that is an age-dependent phenomenon. Aging in general has been reported to affect methylation of gene, however, nothing is known on the age dependent variation in methylation of Fmr-1 gene. Using the brain tissues from male and female mice of various age groups and sex steroid hormones (testosterone or 17beta-estradiol) as modulators, restriction enzymes Hpa II and Msp I and Southern blotting technique, we studied methylation of 5'-upstream sequences of Fmr-1 gene. Our data reveal that the methylation of the 5'-upstream sequences that include CpG islands in promoter and 5'-untraslated region (5'-UTR) gradually increases due to advancing age in both the sexes. 17beta-estradiol lowers the methylation significantly in the brain of mouse of both male and female mouse in age-dependent manner where as testosterone does not affect it appreciably. The alteration in the methylation may be attributed to altered DNA methyl transferase (DNMT) activity as the age increases from young to old, and the 17beta-estradiol may down regulate the DNMT activity in both the age and sex groups whereas the testosterone may not have similar effect on DNMT. Down regulation of methylation of Fmr-1 CpG island and/or 5'-UTR by 17beta-estradiol might lead to derepression of Fmr-1 gene especially in old age. This finding on Fmr-1 methylation is novel and it might have implications in understanding fragile X related disorders and age-dependent alteration in LTP and LTD.     

Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis.

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.