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BACKGROUND: Diethylstilbestrol (DES) is a synthetic estrogen associated with adverse effects on reproductive organs. DES-induced toxicity of the mouse seminal vesicle (SV) is mediated by estrogen receptor α (ERα), which alters expression of seminal vesicle secretory protein IV (Svs4) and lactoferrin (Ltf) genes. OBJECTIVES: We examined a role for nuclear receptor activity in association with DNA methylation and altered gene expression. METHODS: We used the neonatal DES exposure mouse model to examine DNA methylation patterns via bisulfite conversion sequencing in SVs of wild-type (WT) and ERα-knockout (αERKO) mice. RESULTS: The DNA methylation status at four specific CpGs (-160, -237, -306, and -367) in the Svs4 gene promoter changed during mouse development from methylated to unmethylated, and DES prevented this change at 10 weeks of age in WT SV. At two specific CpGs (-449 and -459) of the Ltf gene promoter, DES altered the methylation status from methylated to unmethylated. Alterations in DNA methylation of Svs4 and Ltf were not observed in αERKO SVs, suggesting that changes of methylation status at these CpGs are ERα dependent. The methylation status was associated with the level of gene expression. In addition, gene expression of three epigenetic modifiers--DNMT3A, MBD2, and HDAC2--increased in the SV of DES-exposed WT mice. CONCLUSION: DES-induced hormonal toxicity resulted from altered gene expression of Svs4 and Ltf associated with changes in DNA methylation that were mediated by ERα. Alterations in gene expression of DNMT3A, MBD2, and HDAC2 in DES-exposed male mice may be involved in mediating the changes in methylation status in the SV. 10.1289/ehp.1307351, Introduction Endocrine-disrupting chemicals (EDCs) are substances in the environment, food sources, and manufactured products that can interfere with the normal functioning of the body's endocrine system (Diamanti-Kandarakis et al. 2009). [...]

The ever-increasing body of knowledge in the field of estrogen biology, especially as applied to the female reproductive processes is reviewed. It is noted that the way of perturbing homeostasis could reveal mechanisms of many components important in reproduction, both directly related to estrogen signaling and secondary to homeostatic disruptions.

Estrogen receptors (ERs) are thought to play a crucial role in development, reproduction and normal physiology. Using gene targeting techniques, we have produced lines of transgenic mice homozygous for the disrupted ERa gene ([Alpha]ERKO) and ER[Beta] genes ([Beta]ERKO). Western blot analysis showed no detectable ER protein in AERKO uteri. Ribonuclease protection assay analysis has detected comparable levels of ER[Beta] mRNA in tissues of [Alpha]ERKO mice, suggesting that ER[Beta] expression is not dependent on ER[Alpha]. [Alpha]ERKO mice were totally unresponsive to uterotropic assays with estradiol, hydroxytamoxifen and diethylstilbestrol. Further support came from the failure of treatment with estrogen or epidermal growth factor (EGF) to induce DNA synthesis in aERKO uteri, even though EGF signaling was shown to be intact by stimulation of c-fos, a gene regulatory response. Progesterone receptor mRNA was detected in [Alpha]ERKO mice but not stimulated by estrogen in the uterus, mammary gland and ovary, indicating an estrogen-dependent and estrogen-independent gene regulation. [Alpha]ERKO females are infertile and have hypoplastic uteri and hyperemic ovaries with no corpora lutea. The [Alpha]ERKO ovarian phenotype occurs developmentally by age 30 d and continues to become more overt. Ovarian gonadotropin receptor levels, serum estrogen and luteinizing hormone are elevated compared with wild type (WT) females. Analysis of the mammary glands of adult [Alpha]ERKO females showed a primitive ductal rudiment rather than the fully developed ductal tree seen in WT or [Beta]ERKO mice. [Alpha]ERKO males are also infertile, with atrophy of the testes and seminiferous tubule dysmorphogenesis resulting in decreased spermatogenesis and inactive sperm. Sperm transplantation studies of [Alpha]ERKO males rescues the infertility phenotype. Males have reduced bone density and some alterations in cardiovascular function. Phenotypic differences were seen in sex behavior and aggressive behavior in both [Alpha]ERKO males and females compared with the patterns in WT mice. Initial findings in [Beta]ERKO females assessed by continuous breeding studies and superovulation are arrested folliculogenesis and subfertility. In contrast to the [Alpha]ERKO males, the [Beta]ERKO males are fertile and have normal sexual behavior. Recent development of a viable double ER [Alpha]/[Beta] knockout shows a unique ovarian phenotype of transdifferentiation of granulosa to Sertoli cells. Further characterization of the mice and comparison of the individual and double ER gene knockout phenotypes will be required to more fully understand the physiological consequences of ER-mediated actions and the specific roles of the two different forms of ER in estrogen hormone responsiveness.

Male mice with a knockout of the estrogen receptor (ER)-[Alpha] gene, a ligand-activated transcription factor, showed reduced levels of intromissions and no ejaculations whereas simple mounting behavior was not affected. In contrast, all components of sexual behaviors were intact in male mice lacking the novel ER-[Beta] gene. Here we measure the extent of phenotype in mice that lack both ER-[Alpha] and ER-[Beta] genes ([Alpha]ERKO). [Alpha]ERKO male mice did not show any components of sexual behaviors, including simple mounting behavior. Nor did they show ultrasonic vocalizations during behavioral tests with receptive female mice. On the other hand, reduced aggressive behaviors of [Alpha][Beta]ERKO mice mimicked those of single knockout mice of ER-[Alpha]gene ([Alpha]ERKO). They showed reduced levels of lunge and bite aggression, but rarely showed offensive attacks. Thus, either one of the ERs is sufficient for the expression of simple mounting in male mice, indicating a redundancy in function. Offensive attacks, on the other hand, depend specifically on the ER-[Alpha] gene. Different patterns of natural behaviors require different patterns of functions by ER genes. testosterone | mounts | intromissions | ejaculation | aggression

The protective effects of estrogen in the cardiovascular system result from both systemic effects and direct actions of the hormone on the vasculature. Two estrogen receptors have been identified, ER[Alpha] and ER [Beta]. We demonstrated previously that estrogen inhibits the response to vascular injury in both wild-type and ER[Alpha]-deficient mice, and that ER [Beta] is expressed in the blood vessels of each, suggesting a role for ER [Beta] in the vascular protective effects of estrogen. In the present study, we examined the effect of estrogen administration on mouse carotid arterial injury in ER [Beta]-deficient mice. Surprisingly, in ovariectomized female wild-type and ER [Beta] knockout mice, 17 [Beta]-estradiol markedly and equally inhibited the increase in vascular medial area and the proliferation of vascular smooth muscle cells after vascular injury. These data demonstrate that ER [Beta] is not required for estrogenmediated inhibition of the response to vascular injury, and suggest that either of the two known estrogen receptors is sufficient to protect against vascular injury, or that another unidentified estrogen receptor mediates the vascular protective effects of estrogen.

Previously, it was shown that the lack of a functional estrogen receptor (ER) [Alpha] gene (ER[Alpha]) greatly affects reproduction-related behaviors in both female and male mice. However, widespread expression of a novel second ER gene, ER[Beta], demanded that we examine the possible participation of ER[Beta] in regulation of these behaviors. In dramatic contrast to our results with ER[Alpha] knockout ([Alpha]ERKO) males, [Beta]ERKO males performed at least as well as wild-type controls in sexual behavior tests. Moreover, not only did [Beta]ERKO males exhibit normal male-typical aggressive behavior, including offensive attacks, but they also showed higher levels of aggression than wild-type mice under certain conditions of social experience. These data revealed a significant interaction between genotype and social experience with respect to aggressive behavior. Finally, females lacking a functional [Beta] isoform of the ER gene showed normal lordosis and courtship behaviors, extending in some cases beyond the day of behavioral estrus. These results highlight the importance of ER[Alpha] for the normal expression of natural reproductive behaviors in both sexes and also provide a background for future studies evaluating ER [Beta] gene contributions to other, nonreproductive behaviors. testosterone | progesterone | lordosis | sexual behavior | aggression

Estrogen receptor α (ERα) is a major transducer of estrogenmediated physiological signals. ERα is a member of the nuclear receptor superfamily, which encompasses ligand-dependent transcription factors. The C terminus of nuclear receptors, termed the F domain, is the least homologous region among the members of this family. The ERα F domain possesses 45 amino acids; however, its function remains unclear. We noticed that the homology of the F domains between mouse and human ERαs is remarkably lower (75.6% similarity) than that between the entire proteins (94.7% similarity). To assess the functionality of the ERα F domains, here we generated chimeric ERα expression constructs with mouse-human-exchanged F domains. Using cell-based in vitro assays, we analyzed the transcriptional coactivator interaction and ligand-binding domain dimerization activities of these mouse-human F domain-swapped ERαs. We found that the transcriptional activity of the mouse WT ERα is more potent than that of the human WT ERα in the human hepatoma cell line HepG2. 4-Hydroxytamoxifen (4OHT)-mediated transcriptional activity of mouse-human F domain-swapped ERαs was the inverse of the WT ERα activities but not estradiol-mediated transcriptional activities. Further experiments with constructs containing deletion or point mutations of a predicted β-strand region within the F domain suggested that this region governs the species-specific 4OHTmediated transcriptional activity of ERα. We conclude that the ERα F domain has a species-specific function in 4OHTmediated receptor transactivation and that mouse-human F domain-swapped ERα mutants enable key insights into ERα F domain structure and function. [ABSTRACT FROM AUTHOR]

Early transient developmental exposure to an endocrine active compound, diethylstilbestrol (DES), a synthetic estrogen, causes late-stage effects in the reproductive tract of adult mice. Estrogen receptor alpha (ERa) plays a role in mediating these developmental effects. However, the developmental mechanism is not well known in male tissues. Here, we present genome-wide transcriptome and DNA methylation profiling of the seminal vesicles (SVs) during normal development and after DES exposure. ERa mediates aberrations of the mRNA transcriptome in SVs of adult mice following neonatal DES exposure. This developmental exposure impacts differential diseases between male (SVs) and female (uterus) tissues when mice reach adulthood due to most DES-altered genes that appear to be tissue specific during mouse development. Certain estrogen-responsive gene changes in SVs are cell-type specific. DNA methylation dynamically changes during development in the SVs of wild-type (WT) and ERa-knockout (aERKO) mice, which increases both the loss and gain of differentially methylated regions (DMRs). There are more gains of DMRs in aERKO compared with WT. Interestingly, the methylation changes between the two genotypes are in different genomic loci. Additionally, the expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of endocrine-disrupting chemicals (EDCs), such as DES, during development in the male mouse tissues. This unique evidence contributes to our understanding of developmental actions of EDCs in human health. [ABSTRACT FROM AUTHOR]

BACKGROUND: Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) that might be harmful to human health. Recently, there has been widespread usage of bisphenol chemicals (BPs), such as bisphenol AF (BPAF) and bisphenol S (BPS), as replacements for BPA. However, the potential biological actions, toxicity, and the molecular mechanism of these compounds are still poorly understood. OBJECTIVES: Our objective was to examine the estrogenic effects of BPA, BPAF, and BPS and the molecular mechanisms of action in the estrogen receptor alpha (ERa) complex. METHODS: In vitro cell models were used to compare the estrogenic effects of BPA, BPAF, and BPS to estrogen. Microarray Assay for Real-Time Coregulator-Nuclear receptor Interaction (MARCoNI) analysis was used to identify coregulators of BPA, BPAF, and BPS, and molecular dynamic (MD) simulations were used to determine the compounds binding in the ERα complex. RESULTS: We demonstrated that BPA and BPAF have agonistic activity for both ERα and ERb, but BPS has ERα-selective specificity. We concluded that coregulators were differentially recruited in the presence of BPA, BPAF, or BPS. Interestingly, BPS recruited more corepressors when compared to BPA and BPAF. From a series of MD analysis, we concluded that BPA, BPAF, and BPS can bind to the ER-ligand-binding domain with differing energetics and conformations. In addition, the binding surface of coregulator interactions on ERα was characterized for the BPA, BPAF, and BPS complexes. CONCLUSION: These findings further our understanding of the molecular mechanisms of EDCs, such as BPs, in ER-mediated transcriptional activation, biological activity, and their effects on physiological functions in human health. [ABSTRACT FROM AUTHOR]