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Diseases impacting the female reproductive tract pose a critical health concern. The establishment of in vitro models to study primary endometrial cells is crucial to understanding the mechanisms that contribute to normal endometrial function and the origins of diseases. Established protocols for endometrial stromal cell culture have been in use for decades but recent advances in endometrial organoid culture have paved the way to allowing study of the roles of both epithelial and stromal endometrial cells in vitro . Due to inter-individual variability, primary cell cultures must be established from numerous persons. Generally, endometrial epithelial and stromal cells can be isolated from an endometrial biopsy, however, this is collected in a clinical setting by an invasive transcervical procedure. Our goal was to develop a non-invasive method for the isolation of paired endometrial epithelial organoids and stromal cells from menstrual fluid collected from individual women, based on recent reports describing the isolation of endometrial epithelial organoids or endometrial stromal cells from menstrual fluid. Participants recruited by the NIEHS Clinical Research Unit were provided with a menstrual cup and instructed to collect on the heaviest day of their menstrual period. Endometrial tissue fragments in the menstrual fluid samples were washed to remove blood, minced, and digested with proteinases. Following digestion, the solution was strained to separate epithelial fragments from stromal cells. Epithelial fragments were washed, resuspended in Matrigel, and plated for organoid formation. Stromal cells were separated from residual red blood cells using a Ficoll gradient and then plated in a flask. Once established, estrogen responsiveness of endometrial epithelial organoids was assessed and the decidual response of stromal cells was evaluated. Following treatments, qPCR was performed on organoids for genes induced by estradiol and on stromal cells for genes induced by decidualization. In this manner, the relative responsiveness of paired organoid and stroma cell cultures isolated from each woman could be assessed. In conclusion, we can isolate both epithelial and stromal cells from a single menstrual fluid sample, allowing us to establish organoids and cells in a paired manner. This protocol can greatly enhance our knowledge of the role of epithelial and stromal cells alone and in coordination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hewitt, Dickson, Edwards, Hampton, Garantziotis and DeMayo.)

Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion, at enhancers distal from regulated genes. Loss-of-function CTCF mutation in >20% of human endometrial tumors indicates its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcf d/d uterine tissue exhibited a marked reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcf f/f mice). Post-pubertal Ctcf d/d uteri were hypoplastic with significant reduction in both the amount of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES, and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that CTCF deletion affects a subset of progesterone-responsive genes. This finding indicates (1) Progesterone-mediated signaling remains functional following Ctcf deletion and (2) certain progesterone-regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The progesterone-responsive genes altered by CTCF ablation included Ihh, Fst, and Errfi1. CTCF-dependent progesterone-responsive uterine genes enhance critical processes including anti-tumorigenesis, which is relevant to the known effectiveness of progesterone in inhibiting progression of early-stage endometrial tumors. Overall, our findings reveal that uterine Ctcf plays a key role in progesterone-dependent expression of uterine genes underlying optimal post-pubertal uterine development., (© 2023 Federation of American Societies for Experimental Biology. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Endometrial health is affected by molecular processes that underlie estrogen responses. We assessed estrogen regulation of endometrial function by integrating the estrogen receptor α (ESR1) cistromes and transcriptomes of endometrial biopsies taken from the proliferative and mid-secretory phases of the menstrual cycle together with hormonally stimulated endometrial epithelial organoids. The cycle stage-specific ESR1 binding sites were determined by chromatin immunoprecipitation and next-generation sequencing and then integrated with changes in gene expression from RNA sequencing data to infer candidate ESR1 targets in normal endometrium. Genes with ESR1 binding in whole endometrium were enriched for chromatin modification and regulation of cell proliferation. The distribution of ESR1 binding sites in organoids was more distal from gene promoters when compared to primary endometrium and was more similar to the proliferative than the mid-secretory phase ESR1 cistrome. Inferred organoid estrogen/ESR1 candidate target genes affected formation of cellular protrusions and chromatin modification. Comparison of signaling effected by candidate ESR1 target genes in endometrium vs organoids reveals enrichment of both overlapping and distinct responses. Our analysis of the ESR1 cistromes and transcriptomes from endometrium and organoids provides important resources for understanding how estrogen affects endometrial health and function., (Published by Oxford University Press on behalf of the Endocrine Society 2022.)

Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents., (Published by Oxford University Press on behalf of the Endocrine Society 2022.)

For pregnancy to be established, uterine cells respond to the ovarian hormones, estrogen, and progesterone, via their nuclear receptors, the estrogen receptor (ESR1) and progesterone receptor (PGR). ESR1 and PGR regulate genes by binding chromatin at genes and at distal enhancer regions, which interact via dynamic 3-dimensional chromatin structures. Endometrial epithelial cells are the initial site of embryo attachment and invasion, and thus understanding the processes that yield their receptive state is important. Here, we cultured and treated organoids derived from human epithelial cells, isolated from endometrial biopsies, with estrogen and progesterone and evaluated their transcriptional profiles, their PGR cistrome, and their chromatin conformation. Progesterone attenuated estrogen-dependent gene responses but otherwise minimally impacted the organoid transcriptome. PGR ChIPseq peaks were co-localized with previously described organoid ESR1 peaks, and most PGR and ESR1 peaks were in B (inactive) compartment regions of chromatin. Significantly more ESR1 peaks were assigned to estrogen-regulated genes by considering chromatin loops identified using HiC than were identified using ESR1 peak location relative to closest genes. Overall, the organoids model allowed a definition of the chromatin regulatory components governing hormone responsiveness.

One of the endogenous estrogens, 17β-estradiol (E 2 ) is a female steroid hormone secreted from the ovary. It is well established that E 2 causes biochemical and histological changes in the uterus. However, it is not completely understood how E 2 regulates the oviductal environment in vivo. In this study, we assessed the effect of E 2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single-cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E 2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E 2 did not drastically alter the transcriptomic profile from that of endogenous E 2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E 2 -target gene in the mouse oviduct and was also expressed in human fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types, including epithelial, stromal, and muscle cells, are differentially regulated by E 2 and support gene expression changes, such as growth factors that are required for normal embryo development and transport in mouse models. Furthermore, we have identified cell-specific and region-specific gene markers for targeted studies and functional analysis in vivo., (© 2021 Federation of American Societies for Experimental Biology.)

Obesity is heightened during aging, and although the estrogen receptor α (ERα) has been implicated in the prevention of obesity, its molecular actions in adipocytes remain inadequately understood. Here, we show that adipose tissue ESR1/Esr1 expression inversely associated with adiposity and positively associated with genes involved in mitochondrial metabolism and markers of metabolic health in 700 Finnish men and 100 strains of inbred mice from the UCLA Hybrid Mouse Diversity Panel. To determine the anti-obesity actions of ERα in fat, we selectively deleted Esr1 from white and brown adipocytes in mice. In white adipose tissue, Esr1 controlled oxidative metabolism by restraining the targeted elimination of mitochondria via the E3 ubiquitin ligase parkin. mtDNA content was elevated, and adipose tissue mass was reduced in adipose-selective parkin knockout mice. In brown fat centrally involved in body temperature maintenance, Esr1 was requisite for both mitochondrial remodeling by dynamin-related protein 1 (Drp1) and uncoupled respiration thermogenesis by uncoupled protein 1 (Ucp1). In both white and brown fat of female mice and adipocytes in culture, mitochondrial dysfunction in the context of Esr1 deletion was paralleled by a reduction in the expression of the mtDNA polymerase γ subunit Polg1 We identified Polg1 as an ERα target gene by showing that ERα binds the Polg1 promoter to control its expression in 3T3L1 adipocytes. These findings support strategies leveraging ERα action on mitochondrial function in adipocytes to combat obesity and metabolic dysfunction., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

At birth, all female mice, including those that either lack estrogen receptor α (ERα-knockout) or that express mutated forms of ERα (AF2ERKI), have a hypoplastic uterus. However, uterine growth and development that normally accompany pubertal maturation does not occur in ERα-knockout or AF2ERKI mice, indicating ERα-mediated estrogen (E2) signaling is essential for this process. Mice that lack Cyp19 (aromatase knockout, ArKO mice), an enzyme critical for E2 synthesis, are unable to make E2 and lack pubertal uterine development. A single injection of E2 into ovariectomized adult (10 weeks old) females normally results in uterine epithelial cell proliferation; however, we observe that although ERα is present in the ArKO uterine cells, no proliferative response is seen. We assessed the impact of exposing ArKO mice to E2 during pubertal and postpubertal windows and observed that E2-exposed ArKO mice acquired growth responsiveness. Analysis of differential gene expression between unexposed ArKO samples and samples from animals exhibiting the ability to mount an E2-induced uterine growth response (wild-type [WT] or E2-exposed ArKO) revealed activation of enhancer of zeste homolog 2 (EZH2) and heart- and neural crest derivatives-expressed protein 2 (HAND2) signaling and inhibition of GLI Family Zinc Finger 1 (GLI1) responses. EZH2 and HAND2 are known to inhibit uterine growth, and GLI1 is involved in Indian hedgehog signaling, which is a positive mediator of uterine response. Finally, we show that exposure of ArKO females to dietary phytoestrogens results in their acquisition of uterine growth competence. Altogether, our findings suggest that pubertal levels of endogenous and exogenous estrogens impact biological function of uterine cells later in life via ERα-dependent mechanisms., (Published by Oxford University Press on behalf of the Endocrine Society 2020.)

Estrogen receptor α (ERα) modulates gene expression by interacting with chromatin regions that are frequently distal from the promoters of estrogen-regulated genes. Active chromatin-enriched "super-enhancer" (SE) regions, mainly observed in in vitro culture systems, often control production of key cell type-determining transcription factors. Here, we defined super-enhancers that bind to ERα in vivo within hormone-responsive uterine tissue in mice. We found that SEs are already formed prior to estrogen exposure at the onset of puberty. The genes at SEs encoded critical developmental factors, including retinoic acid receptor α (RARA) and homeobox D (HOXD). Using high-throughput chromosome conformation capture (Hi-C) along with DNA sequence analysis, we demonstrate that most SEs are located at a chromatin loop end and that most uterine genes in loop ends associated with these SEs are regulated by estrogen. Although the SEs were formed before puberty, SE-associated genes acquired optimal ERα-dependent expression after reproductive maturity, indicating that pubertal processes that occur after SE assembly and ERα binding are needed for gene responses. Genes associated with these SEs affected key estrogen-mediated uterine functions, including transforming growth factor β (TGFβ) and LIF interleukin-6 family cytokine (LIF) signaling pathways. To the best of our knowledge, this is the first identification of SE interactions that underlie hormonal regulation of genes in uterine tissue and optimal development of estrogen responses in this tissue., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Estrogen receptor α (ESR1; encoded by Esr1) is a crucial nuclear transcription factor for female reproduction and is expressed throughout the female reproductive tract. To assess the function of ESR1 in reproductive tissues without confounding effects from a potential developmental defect arising from global deletion of ESR1, we generated a mouse model in which Esr1 was specifically ablated during postnatal development. To accomplish this, a progesterone receptor Cre line (PgrCre) was bred with Esr1f/f mice to create conditional knockout of Esr1 in reproductive tissues (called PgrCreEsr1KO mice) beginning around 6 days after birth. In the PgrCreEsr1KO oviduct, ESR1 was most efficiently ablated in the isthmic region. We found that at 3.5 days post coitus (dpc), embryos were retrieved from the uterus in control littermates while all embryos were retained in the PgrCreEsr1KO oviduct. Additionally, serum progesterone (P4) levels were significantly lower in PgrCreEsr1KO compared to controls at 3.5 dpc. This finding suggests that expression of ESR1 in the isthmus and normal P4 levels allow for successful embryo transport from the oviduct to the uterus. Therefore, alterations in oviductal isthmus ESR1 signaling and circulating P4 levels could be related to female infertility conditions such as tubal pregnancy., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Insulin-like growth factor 1 (IGF1) is primarily synthesized in and secreted from the liver; however, estrogen (E2), through E2 receptor α (ERα), increases uterine Igf1 mRNA levels. Previous ChIP-seq analyses of the murine uterus have revealed a potential enhancer region distal from the Igf1 transcription start site (TSS) with multiple E2-dependent ERα-binding regions. Here, we show E2-dependent super enhancer-associated characteristics and suggest contact between the distal enhancer and the Igf1 TSS. We hypothesized that this distal super-enhancer region controls E2-responsive induction of uterine Igf1 transcripts. We deleted 430 bp, encompassing one of the ERα-binding sites, thereby disrupting interactions of the enhancer with gene-regulatory factors. As a result, E2-mediated induction of mouse uterine Igf1 mRNA is completely eliminated, whereas hepatic Igf1 expression remains unaffected. This highlights the central role of a distal enhancer in the assembly of the factors necessary for E2-dependent interaction with the Igf1 TSS and induction of uterus-specific Igf1 transcription. Of note, loss of the enhancer did not affect fertility or uterine growth responses. Deletion of uterine Igf1 in a PgrCre;Igf1f/f model decreased female fertility but did not impact the E2-induced uterine growth response. Moreover, E2-dependent activation of uterine IGF1 signaling was not impaired by disrupting the distal enhancer or by deleting the coding transcript. This indicated a role for systemic IGF1, suggested that other growth mediators drive uterine response to E2, and suggested that uterine-derived IGF1 is essential for reproductive success. Our findings elucidate the role of a super enhancer in Igf1 regulation and uterine growth.

Understanding why a transcription factor (TF) binds to a specific DNA element in the genome and whether that binding event affects transcriptional output remains a great challenge. In this study, we demonstrate that TF binding in the genome follows inversion symmetry (IS). In addition, the specific DNA elements where TFs bind in the genome are determined by internal IS within the DNA element. These DNA-binding rules quantitatively define how TFs select the appropriate regulatory targets from a large number of similar DNA elements in the genome to elicit specific transcriptional and cellular responses. Importantly, we also demonstrate that these DNA-binding rules extend to DNA elements that do not support transcriptional activity. That is, the DNA-binding rules are obeyed, but the retention time of the TF at these non-functional DNA elements is not long enough to initiate and/or maintain transcription. We further demonstrate that IS is universal within the genome. Thus, IS is the DNA code that TFs use to interact with the genome and dictates (in conjunction with known DNA sequence constraints) which of those interactions are functionally active., (Published by Elsevier Inc.)

Pausing of RNA polymerase II (Pol II) during early transcription, mediated by the negative elongation factor (NELF) complex, allows cells to coordinate and appropriately respond to signals by modulating the rate of transcriptional pause release. Promoter proximal enrichment of Pol II occurs at uterine genes relevant to reproductive biology; thus, we hypothesized that pausing might impact endometrial response by coordinating hormonal signals involved in establishing and maintaining pregnancy. We deleted the NELF-B subunit in the mouse uterus using PgrCre (NELF-B UtcKO). Resulting females were infertile. Uterine response to the initial decidual stimulus of NELF-B UtcKO was similar to that of control mice; however, subsequent full decidual response was not observed. Cultured NELF-B UtcKO stromal cells exhibited perturbances in extracellular matrix components and also expressed elevated levels of the decidual prolactin Prl8a2, as well as altered levels of transcripts encoding enzymes involved in prostaglandin synthesis and metabolism. Because endometrial stromal cell decidualization is also critical to human reproductive health and fertility, we used small interfering to suppress NELF-B or NELF-E subunits in cultured human endometrial stromal cells, which inhibited decidualization, as reflected by the impaired induction of decidual markers PRL and IGFBP1. Overall, our study indicates NELF-mediated pausing is essential to coordinate endometrial responses and that disruption impairs uterine decidual development during pregnancy.-Hewitt, S. C., Li, R., Adams, N., Winuthayanon, W., Hamilton, K. J., Donoghue, L. J., Lierz, S. L., Garcia, M., Lydon, J. P., DeMayo, F. J., Adelman, K., Korach, K. S. Negative elongation factor is essential for endometrial function.

Nineteen years have passed since our previous review in this journal in 1999 regarding estrogen receptors. At that time, we described the current assessments of the physiological activities of estrogen and estrogen receptors. Since that time there has been an explosion of progress in our understanding of details of estrogen receptor-mediated processes from the molecular and cellular level to the whole organism. In this review we discuss the basic understanding of estrogen signaling and then elaborate on the progress and current understanding of estrogen receptor actions that have developed using new models and continuing clinical studies.

Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and β cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of β cells in females but not in males., (Published by Elsevier Inc.)

Estrogen receptor α (ERα) action plays an important role in pancreatic β-cell function and survival; thus, it is considered a potential therapeutic target for the treatment of type 2 diabetes in women. However, the mechanisms underlying the protective effects of ERα remain unclear. Because ERα regulates mitochondrial metabolism in other cell types, we hypothesized that ERα may act to preserve insulin secretion and promote β-cell survival by regulating mitochondrial-endoplasmic reticulum (EndoRetic) function. We tested this hypothesis using pancreatic islet-specific ERα knockout (PERαKO) mice and Min6 β-cells in culture with Esr1 knockdown (KD). We found that Esr1-KD promoted reactive oxygen species production that associated with reduced fission/fusion dynamics and impaired mitophagy. Electron microscopy showed mitochondrial enlargement and a pro-fusion phenotype. Mitochondrial cristae and endoplasmic reticulum were dilated in Esr1-KD compared with ERα replete Min6 β-cells. Increased expression of Oma1 and Chop was paralleled by increased oxygen consumption and apoptosis susceptibility in ERα-KD cells. In contrast, ERα overexpression and ligand activation reduced both Chop and Oma1 expression, likely by ERα binding to consensus estrogen-response element sites in the Oma1 and Chop promoters. Together, our findings suggest that ERα promotes β-cell survival and insulin secretion through maintenance of mitochondrial fission/fusion-mitophagy dynamics and EndoRetic function, in part by Oma1 and Chop repression.

Gene regulatory programs are encoded in the sequence of the DNA. Since the completion of the Human Genome Project, millions of gene regulatory elements have been identified in the human genome. Understanding how each of those sites functionally contributes to gene regulation, however, remains a challenge for nearly every field of biology. Transcription factors influence cell function by interpreting information contained within cis-regulatory elements in chromatin. Whereas chromatin immunoprecipitation-sequencing has been used to identify and map transcription factor-DNA interactions, it has been difficult to assign functionality to the binding sites identified. Thus, in this study, we probed the transcriptional activity, DNA-binding competence, and functional activity of select nuclear receptor mutants in cellular and animal model systems and used this information to define the sequence constraints of functional steroid nuclear receptor cis-regulatory elements. Analysis of the architecture within sNR chromatin interacting sites revealed that only a small fraction of all sNR chromatin-interacting events is associated with transcriptional output and that this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides. These findings define the transcriptional grammar necessary to predict functionality from regulatory sequences, with a multitude of future implications., (Copyright © 2017 Endocrine Society.)

Aberrant regulation of uterine cell growth can lead to endometrial cancer and infertility. To understand the molecular mechanisms of estrogen-induced uterine cell growth, we removed the estrogen receptor α (Esr1) from mouse uterine stromal cells, where the embryo is implanted during pregnancy. Without ESR1 in neighboring stroma cells, epithelial cells that line the inside of the uterus are unable to grow due to a lack of growth factors secreted from adjacent stromal cells. Moreover, loss of stromal ESR1 caused mice to deliver fewer pups due in part due to inability of some embryos to implant in the uterus, indicating that stromal ESR1 is crucial for uterine cell growth and pregnancy.

Estrogen (E2) signaling through its nuclear receptor, E2 receptor α (ERα) increases insulinlike growth factor 1 (IGF1) in the rodent uterus, which then initiates further signals via the IGF1 receptor. Directly administering IGF1 results in similar biological and transcriptional uterine responses. Our studies using global ERα-null mice demonstrated a loss of uterine biological responses of the uterus to E2 or IGF1 treatment, while maintaining transcriptional responses to IGF1. To address this discrepancy in the need for uterine ERα in mediating the IGF1 transcriptional vs growth responses, we assessed the IGF1 transcriptional responses in PgrCre+Esr1f/f (called ERαUtcKO) mice, which selectively lack ERα in progesterone receptor (PGR) expressing cells, including all uterine cells, while maintaining ERα expression in other tissues and cells that do not express Pgr. Additionally, we profiled IGF1-induced ERα binding sites in uterine chromatin using chromatin immunoprecipitation sequencing. Herein, we explore the transcriptional and molecular signaling that underlies our findings to refine our understanding of uterine IGF1 signaling and identify ERα-mediated and ERα-independent uterine transcriptional responses. Defining these mechanisms in vivo in whole tissue and animal contexts provides details of nuclear receptor mediated mechanisms that impact biological systems and have potential applicability to reproductive processes of humans, livestock and wildlife.

Background: Treatment with estrogen in early menopausal women protects against development of hepatic steatosis and nonalcoholic fatty liver disease but estrogen has undesirable side effects, which negate its beneficial effects in premenopausal and postmenopausal women. Targeted therapies require better understanding of the target sites and mechanisms by which estrogen signaling exerts its protective effects in women. Estrogen receptor α (ERα) is thought to be the primary mediator for estrogen signaling to protect against hepatic steatosis. ERα has several mechanisms for signal transduction: (1) inducing gene transcription by direct binding to specific DNA sequences, (2) inducing tethered transcription with other DNA-binding factors, and (3) stimulating nongenomic action through membrane-associated ERα. However, it is still unclear which mechanisms mediate ERα-dependent protection against hepatic steatosis., Methods: To understand the mechanisms of estrogen signaling for protection against hepatic steatosis in females, we analyzed the global ERα knockout mouse (αERKO), ERα DNA-binding domain mutant mouse (KIKO) and liver-specific ERα knockout mouse (LERKO) fed high-fat diets (HFD). The KIKO mouse disrupts the direct DNA-binding transcription activity but retains tethered transcription regulation and nongenomic action. Hepatic steatosis was evaluated by scoring the macrovesicular and microvesicular steatosis as well as serum alanine aminotransferase (ALT) levels. We analyzed serum testosterone to assess its correlation with hepatic steatosis., Results: Liver fat accumulation was far greater in HFD-fed αERKO and KIKO females than in HFD-fed wild-type (WT) controls. Conversely, HFD-fed LERKO females did not accumulate excess liver fat. HFD-fed αERKO and KIKO females showed higher microvesicular steatosis and ALT levels than WT controls that correlated with increased serum testosterone levels., Conclusions: ERα-mediated direct transcription in non-hepatic tissues is essential for estrogen-mediated protection against hepatic steatosis in HFD-fed females. The balance between non-hepatic estrogen signaling and hepatic or non-hepatic testosterone action may control hepatic steatosis.

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα + luminal cells and not the mammary stem cells (MaSCs) that are ERα neg . Since ERα + luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα + subset of EpCAM + /CD24 + /CD49f hi MaSCs. We show that the MaSC population has a distinct SCA-1 + population that is abundant in pre-pubertal mammary glands. The SCA-1 + MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1 neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1 + MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα + , estrogen-sensitive subpopulation within the CD24 + /CD49f hi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The hormone estrogen is involved in both female and male reproduction, as well as numerous other biological systems including the neuroendocrine, vascular, skeletal, and immune systems. Therefore, it is also implicated in many different diseases and conditions such as infertility, obesity, osteoporosis, endometriosis, and a variety of cancers. Estrogen works through its two distinct nuclear receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Various transcriptional regulation mechanisms have been identified as the mode of action for estrogen, mainly the classical mechanism with direct DNA binding but also a nongenomic mode of action and one using tethered or indirect binding. The expression profiles of ERα and ERβ are unique with the primary sites of ERα expression being the uterus and pituitary gland and the main site of ERβ expression being the granulosa cells of the ovary. Mouse models with knockout or mutation of Esr1 and Esr2 have furthered our understanding of the role of each individual receptor plays in physiology. From these studies, it is known that the primary roles for ERα are in the uterus and neuroendocrine system, as female mice lacking ERα are infertile due to impaired ovarian and uterine function, whereas female mice lacking ERβ are subfertile due to ovarian defects. The development of effective therapies for estrogen-related diseases has relied on an understanding of the physiological roles and mechanistic functionalities of ERα and ERβ in human health and disease., (© 2017 Elsevier Inc. All rights reserved.)

Impaired estrogen receptor α (ERα) action promotes obesity and metabolic dysfunction in humans and mice; however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle is a primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERα expression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test this relationship, we generated muscle-specific ERα knockout (MERKO) mice. Impaired glucose homeostasis and increased adiposity were paralleled by diminished muscle oxidative metabolism and bioactive lipid accumulation in MERKO mice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment in basal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A-regulator of calcineurin 1-calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolism in MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERα deficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findings indicate the retention of dysfunctional mitochondria in MERKO muscle and implicate ERα in the preservation of mitochondrial health and insulin sensitivity as a defense against metabolic disease in women., (Copyright © 2016, American Association for the Advancement of Science.)

While the function of progesterone receptor (PR) has been studied in the mouse vagina and uterus, its regulation and function in the cervix has not been described. We selectively deleted epithelial PR in the female reproductive tracts using the Cre/LoxP recombination system. We found that epithelial PR was required for induction of apoptosis and suppression of cell proliferation by progesterone (P4) in the cervical and vaginal epithelium. We also found that epithelial PR was dispensable for P4 to suppress apoptosis and proliferation in the uterine epithelium. PR is encoded by the Pgr gene, which is regulated by estrogen receptor α (ERα) in the female reproductive tracts. Using knock-in mouse models expressing ERα mutants, we determined that the DNA-binding domain (DBD) and AF2 domain of ERα were required for upregulation of Pgr in the cervix and vagina as well as the uterine stroma. The ERα AF1 domain was required for upregulation of Pgr in the vaginal stroma and epithelium and cervical epithelium, but not in the uterine and cervical stroma. ERα DBD, AF1, and AF2 were required for suppression of Pgr in the uterine epithelium, which was mediated by stromal ERα. Epithelial ERα was responsible for upregulation of epithelial Pgr in the cervix and vagina. Our results indicate that regulation and functions of epithelial PR are different in the cervix, vagina, and uterus.

Estrogen receptor alpha (ERα) is a critical player in development and function of the female reproductive system. Perturbations in ERα response can affect wide-ranging aspects of health in humans as well as in livestock and wildlife. Because of its long-known and broad impact, ERα mechanisms of action continue to be the focus on cutting-edge research efforts. Consequently, novel insights have greatly advanced understanding of every aspect of estrogen signaling. In this review, we attempt to briefly outline the current understanding of ERα mediated mechanisms in the context of the female reproductive system., (© 2016 Society for Endocrinology.)

Development of uterine endometrial receptivity for implantation is orchestrated by cyclic steroid hormone-mediated signals. It is unknown if these signals are necessary for oviduct function in supporting fertilization and preimplantation development. Here we show that conditional knockout (cKO) mice lacking estrogen receptor α (ERα) in oviduct and uterine epithelial cells have impaired fertilization due to a dramatic reduction in sperm migration. In addition, all successfully fertilized eggs die before the 2-cell stage due to persistence of secreted innate immune mediators including proteases. Elevated protease activity in cKO oviducts causes premature degradation of the zona pellucida and embryo lysis, and wild-type embryos transferred into cKO oviducts fail to develop normally unless rescued by concomitant transfer of protease inhibitors. Thus, suppression of oviductal protease activity mediated by estrogen-epithelial ERα signaling is required for fertilization and preimplantation embryo development. These findings have implications for human infertility and post-coital contraception.

Background: Concerns regarding potential endocrine-disrupting chemicals (EDCs) have led to a need for methods to evaluate candidate estrogenic chemicals. Our previous evaluations of two such EDCs revealed a response similar to that of estradiol (E2) at 2 hr, but a less robust response at 24 hr, similar to the short-acting estrogen estriol (E3)., Objectives: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective. Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses., Methods: We used biological end points in uterine tissue and a signature pattern-recognizing tool that identified coexpressed transcripts to develop and test a panel of transcripts in order to classify potentially estrogenic compounds using an in vivo system. The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species., Results: We evaluated biological and transcriptional end points with proven short- and long-acting estrogens and verified the use of our approach using a phytoestrogen. With our model, we were able to classify the diarylheptanoid D3 as a short-acting estrogen., Conclusions: We have developed a panel of transcripts as biomarkers which, together with biological end points, might be used to screen and evaluate potentially estrogenic chemicals and infer mode of activity.

Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The estrogens are female sex hormones that are involved in a variety of physiological processes, including reproductive development and function, wound healing, and bone growth. They are mainly known for their roles in reproductive tissues--specifically, 17β-estradiol (E2), the primary estrogen, which is secreted by the ovaries and induces cellular proliferation and growth of the uterus and mammary glands. In addition to the role of estrogens in promoting tissue growth and development during normal physiological states, they have a well-established role in determining susceptibility to disease, particularly cancer, in reproductive tissues. The responsiveness of various tissues to estrogen is genetically controlled, with marked quantitative variation observed across multiple species, including humans. This variation presents both researchers and clinicians with a veritable physiological puzzle, the pieces of which--many of them unknown--are complex and difficult to fit together. Although genetics is known to play a major role in determining sensitivity to estrogens, there are other factors, including parent of origin and the maternal environment, that are intimately linked to heritable phenotypes but do not represent genotype, per se. The objectives of this review article were to summarize the current knowledge of the role of genotype, and uterine and neonatal environments, in phenotypic variation in the response to estrogens; to discuss recent findings and the potential mechanisms involved; and to highlight exciting research opportunities for the future., (© FASEB.)

Estrogens exert their activity through estrogen receptor alpha (ERalpha) to stimulate hypertrophy and hyperplasia in the uterus. A uterine epithelial ERalpha conditional knockout mouse model (Wnt7a(Cre+);Esr1(f/f) or cKO) demonstrated that ERalpha in the epithelial cells was dispensable for an initial uterine proliferative response to 17beta-estradiol (E2) but required for subsequent uterine biological responses. This study aimed to characterize the differential gene expression patterns induced by E2 in the presence or absence of epithelial ERalpha. RNA microarray analysis revealed that approximately 20% of the genes differentially expressed at 2 h were epithelial ERalpha independent, as they were preserved in the cKO uteri. This indicates that early uterine transcripts mediated by stromal ERalpha are sufficient to promote initial proliferative responses. However, more than 90% of the differentially expressed transcripts at 24 h were not regulated in the cKO, indicating that the majority of later transcriptional regulation required epithelial ERalpha, especially those involved in mitosis. This shows that loss of regulation of these later transcripts results in blunted subsequent uterine growth after 3 days of E2 treatment. Additionally, progesterone's ability to inhibit E2-induced epithelial cell proliferation was impaired, consistent with a uterine receptivity defect that contributes to cKO infertility. These transcriptional profiles correlate with our previously observed biological responses, in which the initial proliferative response is independent of epithelial ERalpha and thus dependent on stromal ERalpha, yet epithelial ERalpha is essential for subsequent tissue responsiveness., (© 2014 by the Society for the Study of Reproduction, Inc.)

The uterotropic response of the uterus to 17β-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous genetic studies from our laboratory using inbred mice that are high (C57BL6/J; B6) or low (C3H/HeJ; C3H) responders to E2 led to the identification of quantitative trait loci (QTL) associated with phenotypic variation in uterine growth and leukocyte infiltration. Like the uterus, phenotypic variation in the responsiveness of the mammary gland to E2 during both normal and pathologic conditions has been reported. In the current experiment, we utilized an E2-specific model of mammary ductal growth combined with a microarray approach to determine the degree to which genotype influences the responsiveness of the mammary gland to E2, including the associated transcriptional programs, in B6 and C3H mice. Our results reveal that E2-induced mammary ductal growth and ductal morphology are genetically controlled. In addition, we observed a paradoxical effect of mammary ductal growth in response to E2 compared with what has been reported for the uterus; B6 is a high responder for the uterus and was a low responder for mammary ductal growth, whereas the reverse was observed for C3H. In contrast, B6 was a high responder for mammary ductal side branching. The B6 phenotype was associated with increased mammary epithelial cell proliferation and apoptosis, and a distinct E2-induced transcriptional program. These findings lay the groundwork for future experiments designed to investigate the genes and mechanisms underlying phenotypic variation in tissue-specific sensitivity to systemic and environmental estrogens during various physiological and disease states.

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

The uterotropic response of the uterus to 17β-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous genetic studies from our laboratory using inbred mice that are high [C57BL/6J (B6)] or low [C3H/HeJ (C3H)] responders to E2 led to the identification of quantitative trait (QT) loci associated with phenotypic variation in uterine growth and leukocyte infiltration. The mechanisms underlying differential responsiveness to E2, and the genes involved, are unknown. Therefore, we used a microarray approach to show association of distinct E2-regulated transcriptional signatures with genetically controlled high and low responses to E2 and their segregation in (C57BL/6J×C3H/HeJ) F1 hybrids. Among the 6664 E2-regulated transcripts, analysis of cellular functions of those that were strain specific indicated C3H-selective enrichment of apoptosis, consistent with a 7-fold increase in the apoptosis indicator CASP3, and a 2.4-fold decrease in the apoptosis inhibitor Naip1 (Birc1a) in C3H vs. B6 following treatment with E2. In addition, several differentially expressed transcripts reside within our previously identified QT loci, including the ERα-tethering factor Runx1, demonstrated to enhance E2-mediated transcript regulation. The level of RUNX1 in uterine epithelial cells was shown to be 3.5-fold greater in B6 compared to C3H. Our novel insights into the mechanisms underlying the genetic control of tissue sensitivity to estrogen have great potential to advance understanding of individualized effects in physiological and disease states.

Background: Diarylheptanoid (D3) isolated from the medicinal plant, Curcuma comosa, has estrogenic activity., Objective: We aimed to elucidate the mechanism(s) of D3 action and compare it with that of 17β-estradiol (E2) using both in vitro and in vivo uterine models., Methods: We used human uterine (Ishikawa) cells to determine the estrogenic action of D3 on the activation and nuclear translocation of estrogen receptor α (ERα). In addition, we further characterized the uterine response to D3 treatment in vivo., Results: D3 activated an estrogen responsive element (ERE) luciferase reporter through ERα, and molecular modeling suggested that D3 could be accommodated in the ERα binding pocket. Using modified ERα to assay ligand-dependent nuclear translocation, we observed D3-dependent ERα interaction and translocation. In mouse uteri, early- and late-phase estrogen-regulated gene responses were increased in D3-treated ovariectomized wild-type animals, in a manner similar to that of E2; no response was seen in ERα knockout animals. We observed a divergence in estrogen responses after D3 treatment: D3 induced robust DNA synthesis in uterine epithelial cells, linked to an increase in cell-cycle-related genes; however, no increase in uterine weight was observed 24 hr after treatment. D3 also affected uterine progesterone receptor expression patterns similar to E2. When D3 and E2 were administered together, we observed no additive or antagonistic effects of D3 on E2. Our findings suggest that D3 is a weak estrogenic agonist compound., Conclusion: D3 is a weakly acting phytoestrogen that mimics the mitogenic responses produced by E2 in an ERα-dependent manner, but it is unable to increase uterine weight or enhance or antagonize the effects of estrogen.

Endometriosis results from ectopic invasion of endometrial tissue within the peritoneal cavity. Aberrant levels of the estrogen receptor (ER), ERα and ERβ, and higher incidence of autoimmune disorders are observed in women with endometriosis. An immunocompetent mouse model of endometriosis was used in which minced uterine tissue from a donor was dispersed into the peritoneal cavity of a recipient. Wild-type (WT), ERα-knockout (αERKO), and βERKO mice were donors or recipients to investigate the roles of ERα, ERβ, and estradiol-mediated signaling on endometriosis-like disease. Mice were treated with vehicle or estradiol, and resulting location, number, and size of endometriosis-like lesions were assessed. In comparison with WT lesions in WT hosts, αERKO lesions in WT hosts were smaller and fewer in number. The effect of ER status and estradiol treatment on nuclear receptor status, proliferation, organization, and inflammation within lesions were examined. αERKO lesions in WT hosts did not form distal to the incision site, respond to estradiol, or proliferate but did have increased inflammation. WT lesions in αERKO hosts did respond to estradiol, proliferate, and show decreased inflammation with treatment, but surprisingly, progesterone receptor expression and localization remained unchanged. Only minor differences were observed between WT lesions in βERKO hosts and βERKO lesions in WT hosts, demonstrating the estradiol-mediated signaling responses are predominately through ERα. In sum, these results suggest ER in both endometriosis-like lesions and their environment influence lesion characteristics, and understanding these interactions may play a critical role in elucidating this enigmatic disease.

To advance understanding of mechanisms leading to biological and transcriptional endpoints related to estrogen action in the mouse uterus, we have mapped ERα and RNA polymerase II (PolII) binding sites using chromatin immunoprecipitation followed by sequencing of enriched chromatin fragments. In the absence of hormone, 5184 ERα-binding sites were apparent in the vehicle-treated ovariectomized uterine chromatin, whereas 17,240 were seen 1 h after estradiol (E₂) treatment, indicating that some sites are occupied by unliganded ERα, and that ERα binding is increased by E₂. Approximately 15% of the uterine ERα-binding sites were adjacent to (<10 kb) annotated transcription start sites, and many sites are found within genes or are found more than 100 kb distal from mapped genes; however, the density (sites per base pair) of ERα-binding sites is significantly greater adjacent to promoters. An increase in quantity of sites but no significant positional differences were seen between vehicle and E₂-treated samples in the overall locations of ERα-binding sites either distal from, adjacent to, or within genes. Analysis of the PolII data revealed the presence of poised promoter-proximal PolII on some highly up-regulated genes. Additionally, corecruitment of PolII and ERα to some distal enhancer regions was observed. A de novo motif analysis of sequences in the ERα-bound chromatin confirmed that estrogen response elements were significantly enriched. Interestingly, in areas of ERα binding without predicted estrogen response element motifs, homeodomain transcription factor-binding motifs were significantly enriched. The integration of the ERα- and PolII-binding sites from our uterine sequencing of enriched chromatin fragments data with transcriptional responses revealed in our uterine microarrays has the potential to greatly enhance our understanding of mechanisms governing estrogen response in uterine and other estrogen target tissues.

Uterine gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition. Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition.

ERα is expressed in macrophages and other immune cells known to exert dramatic effects on glucose homeostasis. We investigated the impact of ERα expression on macrophage function to determine whether hematopoietic or myeloid-specific ERα deletion manifests obesity-induced insulin resistance in mice. Indeed, altered plasma adipokine and cytokine levels, glucose intolerance, insulin resistance, and increased adipose tissue mass were observed in animals harboring a hematopoietic or myeloid-specific deletion of ERα. A similar obese phenotype and increased atherosclerotic lesion area was displayed in LDL receptor-KO mice transplanted with ERα(-/-) bone marrow. In isolated macrophages, ERα was necessary for repression of inflammation, maintenance of oxidative metabolism, IL-4-mediated induction of alternative activation, full phagocytic capacity in response to LPS, and oxidized LDL-induced expression of ApoE and Abca1. Furthermore, we identified ERα as a direct regulator of macrophage transglutaminase 2 expression, a multifunctional atheroprotective enzyme. Our findings suggest that diminished ERα expression in hematopoietic/myeloid cells promotes aspects of the metabolic syndrome and accelerates atherosclerosis in female mice.

Background: Interest and concern regarding potentially estrogenic substances have resulted in development of model systems to evaluate mechanisms of such chemicals. Microarray studies have indicated that estradiol (E2)-stimulated uterine responses can be divided into early and late phases. Comparison of E2 uterine transcript profiles and those of other estrogenic chemicals of interest in vivo indicates mechanisms and activities of test compounds., Objectives: We compared transcript responses and mechanisms of response using mouse reproductive tracts after treatment with E2, estriol (E3), bisphenol A (BPA), and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE)., Methods: Uterine RNA from ovariectomized wild-type mice, estrogen receptor α (ERα) knockout (αERKO) mice, and mice expressing a DNA-binding-deficient ERα (KIKO) treated with E2, E3, BPA, or HPTE for 2 or 24 hr was analyzed by microarray. Resulting regulated transcripts were compared by hierarchical clustering and correlation analysis, and response patterns were verified by reverse-transcription real-time polymerase chain reaction (RT-PCR)., Results: Both xenoestrogens, BPA and HPTE, showed profiles highly correlated to that of E2 in the early response phase (2 hr), but the correlation diminished in the later response phase (24 hr), similar to the known weak estrogen E3. Both xenoestrogens also mimicked E2 in samples from KIKO mice, indicating that they are able to utilize the indirect tethering mode of ERα signaling. No response was detected in ERα-null uteri, indicating that ERα mediates the responses., Conclusion: Our study forms a basis on which patterns of response and molecular mechanisms of potentially estrogenic chemicals can be assessed.

To address issues resulting from α estrogen receptor-knockout (αERKO) residual N-terminal truncated estrogen receptor α, and to allow tissue-selective deletion of ERα, we generated loxP-flanked exon 3 mice. Initial characterization of global sox2 cre-derived exon 3-deleted Ex3αERKO mice indicated no ERα protein in uterine tissue and recapitulation of previously described female phenotypes, confirming successful ablation of ERα. Body weights of Ex3αERKO female mice were 1.4-fold higher than wild-tupe (WT) females and comparable to WT males. Microarray indicated the Ex3αERKO uterus is free of residual estrogen responses. RT-PCR showed Nr4a1 is increased 41-fold by estrogen in WT and 7.4-fold in αERKO, and not increased in Ex3αERKO. Nr4a1, Cdkn1a, and c-fos transcripts were evaluated in WT and Ex3αERKO mice following estrogen, IGF1, or EGF injections. All 3 were increased by all treatments in WT. None were increased by estrogen in Ex3αERKO. Nr4a1 increased 24.5- and 14.7-fold, Cdkn1a increased 14.2- and 12.3-fold, and c-fos increased 20.9-fold and 16.2-fold after IGF1 and EGF treatments, respectively, in the Ex3αERKO mice, confirming that growth factor regulation is independent of ERα. Our Ex3α ERα model will be useful in studies of complete or selective ablation of ERα in target tissues.

Disruption of the Esr1 gene encoding estrogen receptor α (ERα) by insertion of a neomycin resistance gene (neo) into exon 2 (αERKO mice) was shown previously to cause infertility in male mice. While full-length ERα protein was not expressed in αERKO mice, alternative splicing resulted in the low-level expression of a truncated form lacking the N-terminus A/B domain and containing the DNA- and ligand-binding domains. Thus, it was unclear whether the reproductive phenotype in αERKO males was only due to the lack of full-length ERα or was affected by the presence of the variant ERα isoform. The present study examined male mice with deletion of exon 3 of Esr1 gene, lacking the DNA-binding domain, and null for ERα (Ex3αERKO). Dilation of some seminiferous tubules was apparent in male Ex3αERKO mice as early as postnatal day 10 and was pronounced in all tubules from day 20 onward. At 6 weeks of age, sperm numbers and sperm motility were lower in Ex3αERKO mice than in wild-type (WT) mice, and the rete testis and efferent ductules were dilated. Mating studies determined that adult Ex3αERKO males were infertile and failed to produce copulatory plugs. Serum testosterone levels and Hsd17b3 and Cyp17a1 transcript levels were significantly higher, but serum estradiol, progesterone, LH, and FSH levels and Cyp19a1 transcript levels were not significantly different from those in WT mice. These results confirm and extend those seen in other studies on male mice with deletion of exon 3 of Esr1 gene. In addition, the reproductive phenotype of male Ex3αERKO mice recapitulated the phenotype of αERKO mice, strongly suggesting that the αERKO male infertility was not due to the presence of the DNA-binding domain in the truncated form of ERα and that full-length ERα is essential for maintenance of male fertility.

Female fertility requires estrogen to specifically stimulate estrogen receptor α (ERα)-dependent growth of the uterine epithelium in adult mice, while immature females show proliferation in both stroma and epithelium. To address the relative roles of ERα in mediating estrogen action in uterine epithelium versus stroma, a uterine epithelial-specific ERα knockout (UtEpiαERKO) mouse line was generated by crossing Esr mice with Wnt7a-Cre mice. Expression of Wnt7a directed Cre activity generated selective deletion of ERα in uterine epithelium, and female UtEpiαERKO are infertile. Herein, we demonstrate that 17β-estradiol (E(2))-induced uterine epithelial proliferation was independent of uterine epithelial ERα because DNA synthesis and up-regulation of mitogenic mediators were sustained in UtEpiαERKO uteri after E(2) treatment. IGF-1 treatment resulted in ligand-independent ER activation in both wild-type (WT) and UtEpiαERKO and mimicked the E(2) stimulatory effect on DNA synthesis in uterine epithelium. Uterine epithelial ERα was necessary to induce lactoferrin, an E(2)-regulated secretory protein selectively synthesized in the uterine epithelium. However, loss of uterine epithelial ERα did not alter the E(2)-dependent progesterone receptor (PR) down-regulation in epithelium. Strikingly, the uterine epithelium of UtEpiαERKO had robust evidence of apoptosis after 3 d of E(2) treatment. Therefore, we surmise that estrogen induced uterine hyperplasia involves a dispensable role for uterine epithelial ERα in the proliferative response, but ERα is required subsequent to proliferation to prevent uterine epithelial apoptosis assuring the full uterine epithelial response, illustrating the differential cellular roles for ERα in uterine tissue and its contribution during pregnancy.

Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ERalpha also enhances insulin synthesis in cultured islets. Whether ERalpha stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERalpha knockout mouse (PERalpha KO(-/-)) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and beta cells. Whereas 17beta-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERalpha lox/lox mice, these E2 effects are abolished in PERalpha KO(-/-) mice. We find that E2-activated ERalpha increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ERalpha eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ERalpha interacts with the tyrosine kinase Src, which activates extracellular signal-regulated kinases(1/2), to increase nuclear localization and binding to the insulin promoter of the transcription factor NeuroD1. This study supports the importance of ERalpha in beta cells as a regulator of insulin synthesis in vivo.

FOXA1, estrogen receptor alpha (ERalpha) and GATA3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERalpha and GATA3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERalpha expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike ERalpha and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERalpha expression, but not GATA3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERalpha-positive breast cancers, at least in part, through its control of ERalpha expression.