Epigenetic effects of endocrine-disrupting chemicals on female reproduction: An ovarian perspective
2010 Study Abstract
The link between in utero and neonatal exposure to environmental toxicants, such as endocrine-disrupting chemicals (EDCs) and adult female reproductive disorders is well established in both epidemiological and animal studies. Recent studies examining the epigenetic mechanisms involved in mediating the effects of EDCs on female reproduction are gathering momentum. In this review, we describe the developmental processes that are susceptible to EDC exposures in female reproductive system, with a special emphasis on the ovary. We discuss studies with select EDCs that have been shown to have physiological and correlated epigenetic effects in the ovary, neuroendocrine system, and uterus. Importantly, EDCs that can directly target the ovary can alter epigenetic mechanisms in the oocyte, leading to transgenerational epigenetic effects. The potential mechanisms involved in such effects are also discussed.
DES effects on the female reproductive system
DES is a nonsteroidal synthetic estrogen that was prescribed to pregnant women at doses of 5–150 mg/day to prevent miscarriages from 1940s to 1970s). Even though early on DES was shown to be an ineffective drug, it was continued in use till the 1970s. Numerous abnormalities in the reproductive, cardiovascular, and immune systems have since been reported in both male and female offspring of women treated with DES, and validated in animal models. There are limited reports that these effects are being observed in the granddaughters of DES-treated women as well. While DES caused vaginal clear cell adenocarcinoma in only 0.1% of the female offspring, over 95 % reported reproductive tract dysfunction and poor pregnancy outcomes. Since there is evidence of multi-generational effects, epigenetic mechanisms could play an important role and were therefore investigated.
Mice injected with a single dose of 10 μg/kg DES on E15 and examined at 7 months of age had no CL and numerous atretic follicles. They were also found to have vacuolated interstitial tissue with lipid droplet inclusions. Other studies with varying doses of DES (5 μg/kg to 100 μg/kg) administered either in utero (E9-E16), or neonatally (PND1-PND5) , demonstrated that adult DES ovaries developed similar hypertrophy and vacuolation of interstitial tissue, hemorrhagic cysts and lack of corpora lutea. These animals also had high levels of testosterone. There was a dose-dependent reduction in the number of the litters as well as the number of oocytes ovulated after stimulation with exogenous gonadotropins. The oocytes derived from such treated ovaries and used in IVF showed lower levels of fertilizability, suggesting reduced oocyte quality. However, 5 µg/day DES-treated ovaries transplanted into untreated ovariectomized host mice were able to give rise to normal female offspring that in turn gave birth to normal size litters and had normal uterine morphology, suggesting that the DES treatment effects were not mediated via germ cells. However, the age at which these animals were sacrificed was 8–12 weeks, and other studies have shown that DES-treated animals do develop epithelial cancers of the uterus by 18 months of age.
DES can bind to both ERs with many fold higher affinity than estradiol. Multiple studies from Iguchi and colleagues showed that in utero (E15–18) and neonatally (PND1-5) DES-treated mice had ovaries containing excessive number of MOFs by adulthood. MOFs were also observed in ovaries that were treated in vitro at PND1-5, following their transplantation to untreated mice, suggesting a direct effect of DES in the ovary. Recent studies showed that neonatal exposure to 3 μg/kg DES induced MOFs, a process mediated by ERβ and not ERα. DES exposure was shown to reduce oocyte apoptosis (potentially suppressing oocyte nest breakdown) via ERβ signaling mechanisms. Furthermore, it was hypothesized that such alterations in the germ cell and somatic cell populations may affect the invasion of pregranulosa cells and basement membrane remodeling during primordial follicle formation. Interestingly, the incidence of MOFs has been reported with other EDC exposures as well).
The effects of DES on the sexual dimorphism of the brain have been documented. In utero and postnatal exposures increased the size of SDN-POA in females thereby defeminizing the region. It was also found that PND1-10 treatment led to a significant reduction in the levels of LH secreted although a similar effect was not found when the exposure was prenatal (E16-20), highlighting the importance of DES actions on the neuroendocrine circuits.
It is well known that DES caused T-shaped uteri and clear cell adenocarcinoma of the uterus, cervix, and vagina in women whose mothers were exposed to DES during pregnancy. There are numerous animal studies validating these human reports. For example, progeny of DES-treated mice have shown malformations of the uterus, squamous metaplasia of the luminar and glandular epithelium, endometrial hyperplasia and leiomyomas, and oviductal proliferative lesions. Ovariectomized animals when supplemented with estradiol are able to respond by a transient increase in gene expression and concomitant uterine proliferation and growth. When such a stimulus is removed, the uterus returns to its unstimulated state. However, when DES or estradiol is administered during neonatal development, expression of immediate early genes such as lactoferrin, EGF, and proto-oncogenes such as c-fos, c-jun, and c-myc is upregulated even into adulthood. Inversely, expression of genes that are necessary for uterine development, such as the Abdominal B (AbdB) Hox gene, Hoxa-10, (known to be controlled by estradiol and progesterone,), Wnt7a as well as Msx2 are repressed leading to structural abnormalities of the reproductive tract. Numerous studies have been conducted to assess the methylation patterns of promoters of several of these estrogen-responsive genes associated with uterine development.
Neonatal DES exposure in mice caused nearly 90% incidence of epithelial cancers of the uterus by 18 months of age. In mice similarly treated with DES, the promoter region of the lactoferrin gene was found to be hypomethylated in the adult uterus. However, if the animals were exposed for the same length of time during adulthood, no such methylation or expression defects were observed. Subsequently, it was also found that exon 4 of the c-fos gene was extensively hypomethylated while the promoter region and intron 1 was unaffected, thereby potentially allowing for the upregulation of c-fos expression. QPCR studies performed by Sato and colleagues examining the expression of Dnmts in neonatally DES exposed C57BL/6 mice, revealed that expression of Dnmt1 and Dnmt3b was decreased at PND5 in DES-treated mice, and the pattern continued until PND14. Interestingly, it was found that human leiomyoma samples had alterations in the levels of Dnmts as well, with concomitant global hypomethylation.
As mentioned above, DES down-regulates Hoxa gene expression. These effects are akin to those associated with uterine abnormalities found in Hoxa KO mice. The predominant phenotype is the loss of boundary between the oviduct and uterus. It has been shown that the anterior to posterior specific pattern of Hoxa-9 is essential for the normal development and function of the uterus and that DES causes a posterior shift of Hoxa-9 and Hoxa-10 expression and homeotic anterior transformations. A recent report by Bromer and colleagues has shown that after in utero (E9-16) exposure to 10 μg/kg DES, there is hyper-methylation in the promoter and intron 1 regions of Hoxa-10 gene, in the caudal part of the uterus with a concomitant increase in the Hoxa-10 expression in the same region. While these data are interesting from the point of epigenetic regulation of the regionalization of the uterus, the authors suggest that the apparently conflicting data i.e., increased methylation vs increased gene expression might be due to differential binding to transcriptional repressors. Since previous studies have shown that no epigenetic changes were detected in the promoters of Hoxa-10 and Hoxa-11 genes, continuation of these studies is warranted.
Alworth and colleagues showed that in utero exposure (E12-18) of CD-1 mice to DES doses of 0.1–100 μg/kg followed by estradiol administration at 7–8 months of age caused opposite responses between low and high doses. The lower dose enhanced the response to exogenous estrogens, resulting in an increase in uterine weight, while the high dose dampened the response resulting in lighter uteri. A global methylation assay was conducted employing DMH, which was suitable to detect hypermethylation events. Over 300 CpG island loci were examined and five candidates were identified in 18S rDNA and 45S pre-rDNA methylation, suggesting a role for ribosomal assembly and protein synthesis in the mediation of DES effects.
Couse and colleagues have shown that ERα is essential for the mediation of DES effects in the uterus: αERKO female mice exhibited a complete resistance to the effects of DES while βERKO mice did not. Additionally, as mentioned earlier, ERα induction is necessary for activation of estrogen responsive gene expression including that of the lactoferrin and c-fos genes. Since these genes are all downstream of ERα signaling, it is imperative to thoroughly examine the potential role of epigenetic mechanisms in the regulation of ERα expression after EDC exposure. Interesting new studies by Bredfeldt and colleagues have now provided a link between ER signaling and regulation of histone modifications. It was found that rapid PI3K/Akt signaling downstream of membrane-associated ER, in response to estradiol as well as DES, caused reduction in trimethylation of H3K27. More interestingly, activation of this non-genomic signaling caused reprogramming of the uterine gene expression profile. Whether such altered epigenetic mechanisms are transgenerational would be of great interest to the field.
Tang and colleagues recently investigated whether neonatal DES/genistein exposure could cause epigenetic changes and alter gene expression in adult uteri and whether there are interactions between adult ovarian hormones and such epigenetic reprogramming. CD-1 mice were exposed to DES (1 μg and 1000 μg/kg) or genistein (50 mg/kg) from PND1-5. Subsequently, some animals were sacrificed at PND19 while others were aged to 6 and 18 months with or without ovariectomies. Genome-wide methylation analysis was conducted with MSRF and candidate genes were identified. Of interest was the nucleosomal binding protein 1 (Nsbp1), which was shown to be hypomethylated at PND19 and hyper-methylated by puberty, in the control. Low-dose DES- and genistein- treated vs high-dose DES-treated animals had opposing methylation patterns. Furthermore, it was shown that in the aged animals, both DES and genistein caused hyper-methylation in the ovariectomized animals but remained hypomethylated in non-ovariectomized animals. These data suggest that Nsbp1 is hyper-methylated in intact mice with age and that DES and genistein have opposing effects on the methylation patterns in intact vs ovariectomized aging animals (hypomethylation vs hyper-methylation), respectively. These studies highlighted the age-dependent aspect of epigenetic reprogramming and also its interaction with steroid hormones.
Transgenerational epigenetic effects associated with DES exposures
Documentation of the transgenerational epigenetic effects of EDCs has been accumulating such as the case of transgenerational effects of DES exposure reported in humans. Exposure to DES while in utero was shown to affect the female F2 generation but not males in mice. Later studies with DES exposure showed that it leads to reproductive tract tumors in both F2 generation males and female mice.
- Full text (free access) : Epigenetic effects of endocrine-disrupting chemicals on female reproduction: An ovarian perspective, Frontiers in neuroendocrinology, NCBI PubMed, PMC3009556, 2010 Jul 4.
- Featured image credit Luis Sarabia.