Frances Collins

Estrogens in Testis Biology

Abstract: Levels of estrogen within the male reproductive tract are higher than in the general circulation and the aromatase enzyme is expressed in the adult testis. Estrogens such as estradiol (E2) modify cell function by binding to high‐affinity estrogen receptors (ER). Two subtypes (ERα and ERβ) have been identified. Studies in animals have shown that over‐ or underexposure to estrogens can have an impact on testis function. For example, mice with targeted disruption of the aromatase cyp19 gene become infertile because round spermatids fail to differentiate normally. In rodents, ERα is expressed in Leydig cells; ERα mRNA and protein are not detectable in testes from humans or primates. High levels of expression of ERα occur in the efferent ductules in rodents, primates, and the human. ERβ protein has been immunolocalized to all somatic cells and to some germ cells in these same species. Messenger RNAs for splice variant isoforms of human ERβ are expressed in human testes. Homologues of the ERβ2 variant have been cloned from primates; this isoform does not exist in rodents and does not bind E2. Full‐length ERβ protein (ERβ1) and ERβ2 have differential patterns of expression in human testes. In conclusion, although estogens are synthesized in the testis and it has been suggested that E2 may function as a germ cell survival factor, the mechanisms by which estrogens influence male fertility remain uncertain and rodents may be poor models in which to examine this.

Expression of oestrogen receptors, ERα, ERβ, and ERβ variants, in endometrial cancers and evidence that prostaglandin F may play a role in regulating expression of ERα

BackgroundEndometrial cancer is the most common gynaecological malignancy; risk factors include exposure to oestrogens and high body mass index. Expression of enzymes involved in biosynthesis of oestrogens and prostaglandins (PG) is often higher in endometrial cancers when compared with levels detected in normal endometrium. Oestrogens bind one of two receptors (ERα and ERβ) encoded by separate genes. The full-length receptors function as ligand-activated transcription factors; splice variant isoforms of ERβ lacking a ligand-binding domain have also been described. PGs act in an autocrine or paracrine manner by binding to specific G-protein coupled receptors.MethodsWe compared expression of ERs, progesterone receptor (PR) and cyclooxygenase-2 (COX-2) in stage 1 endometrial adenocarcinomas graded as well (G1), moderately (G2) or poorly (G3) differentiated (n ≥ 10 each group) using qRTPCR, single and double immunohistochemistry. We used endometrial adenocarcinoma cell lines to investigate the impact of PGF2α on expression of ERs and PR.ResultsFull length ERβ (ERβ1) and two ERβ variants (ERβ2, ERβ5) were expressed in endometrial cancers regardless of grade and the proteins were immunolocalised to the nuclei of cells in both epithelial and stromal compartments. Immunoexpression of COX-2 was most intense in cells that were ERαneg/low. Expression of PR in endometrial adenocarcinoma (Ishikawa) cell lines and tissues broadly paralleled that of ERα. Treatment of adenocarcinoma cells with PGF2α reduced expression of ERα but had no impact on ERβ1. Cells incubated with PGF2α were unable to increase expression of PR mRNA when they were incubated with E2.ConclusionWe have demonstrated that ERβ5 protein is expressed in stage 1 endometrial adenocarcinomas. Expression of three ERβ variants, including the full-length protein is not grade-dependent and most cells in poorly differentiated cancers are ERβpos/ERαneg. We found evidence of a link between COX-2, its product PGF2α, and expression of ERα and PR that sheds new light on the cross talk between steroid and PG signalling pathways in this disease.

An additive interaction between the NFκB and estrogen receptor signalling pathways in human endometrial epithelial cells

BACKGROUND Human embryo implantation is regulated by estradiol (E2), progesterone and locally produced mediators including interleukin-1β (IL-1β). Interactions between the estrogen receptor (ER) and NF kappa B (NFκB) signalling pathways have been reported in other systems but have not been detailed in human endometrium. METHODS AND RESULTS Real-time PCR showed that mRNA for the p65 and p105 NFκB subunits is maximally expressed in endometrium from the putative implantation window. Both subunits are localized in the endometrial epithelium throughout the menstrual cycle. Reporter assays for estrogen response element (ERE) activity were used to examine functional interactions between ER and NFκB in telomerase immortalized endometrial epithelial cells (TERT-EEC). E2 and IL-1β treatment of TERT-EECs enhances ERE activity by a NFκB and ER dependent mechanism; this effect could be mediated by ERα or ERβ. E2 and IL-1β also positively interact to increase endogenous gene expression of prostaglandin E synthase and c-myc. This is a gene-dependent action as there is no additive effect on cyclin D1 or progesterone receptor expression. CONCLUSION In summary, we have established that NFκB signalling proteins are expressed in normal endometrium and report that IL-1β can enhance the actions of E2 in a cell line derived from healthy endometrium. This mechanism may allow IL-1β, possibly from the developing embryo, to modulate the function of the endometrial epithelium to promote successful implantation, for example by regulating prostaglandin production. Aberrations in the interaction between the ER and NFκB signalling pathways may have a negative impact on implantation contributing to pathologies such as early pregnancy loss and infertility.

In silico analysis identifies a novel role for androgens in the regulation of human endometrial apoptosis.

Context: The endometrium is a multicellular, steroid-responsive tissue that undergoes dynamic remodeling every menstrual cycle in preparation for implantation and, in absence of pregnancy, menstruation. Androgen receptors are present in the endometrium. Objective: The objective of the study was to investigate the impact of androgens on human endometrial stromal cells (hESC). Design: Bioinformatics was used to identify an androgen-regulated gene set and processes associated with their function. Regulation of target genes and impact of androgens on cell function were validated using primary hESC. Setting: The study was conducted at the University Research Institute. Patients: Endometrium was collected from women with regular menses; tissues were used for recovery of cells, total mRNA, or protein and for immunohistochemistry. Results: A new endometrial androgen target gene set (n = 15) was identified. Bioinformatics revealed 12 of these genes interacted in one pathway and identified an association with control of cell survival. Dynamic androgen-dependent changes in expression of the gene set were detected in hESC with nine significantly down-regulated at 2 and/or 8 h. Treatment of hESC with dihydrotestosterone reduced staurosporine-induced apoptosis and cell migration/proliferation. Conclusions: Rigorous in silico analysis resulted in identification of a group of androgen-regulated genes expressed in human endometrium. Pathway analysis and functional assays suggest androgen-dependent changes in gene expression may have a significant impact on stromal cell proliferation, migration, and survival. These data provide the platform for further studies on the role of circulatory or local androgens in the regulation of endometrial function and identify androgens as candidates in the pathogenesis of common endometrial disorders including polycystic ovarian syndrome, cancer, and endometriosis.

Evidence of androgen action in endometrial and ovarian cancers

Endometrial cancer (EC) and ovarian cancer are common gynaecological malignancies. The impact of androgen action in these cancers is poorly understood; however, there is emerging evidence to suggest that targeting androgen signalling may be of therapeutic benefit. Epidemiological evidence suggests that there is an increased risk of EC associated with exposure to elevated levels of androgens, and genetic variants in genes related to both androgen biosynthesis and action are associated with an increased risk of both EC and ovarian cancer. Androgen receptors (ARs) may be a potential therapeutic target in EC due to reported anti-proliferative activities of androgens. By contrast, androgens may promote growth of some ovarian cancers and anti-androgen therapy has been proposed. Introduction of new therapies targeting ARs expressed in EC or ovarian cancer will require a much greater understanding of the impacts of cell context-specific AR-dependent signalling and how ARs can crosstalk with other steroid receptors during progression of disease. This review considers the evidence that androgens may be important in the aetiology of EC and ovarian cancer with discussion of evidence for androgen action in normal and malignant endometrial and ovarian tissue.

Modulation of ERα transcriptional activity by the orphan nuclear receptor ERRβ and evidence for differential effects of long- and short-form splice variants☆

Oestrogen receptor related proteins (ERRs) affect target gene expression without binding oestradiol. We investigated the functional activity of two splice variant isoforms of ERR beta (ERR beta S [short], ERR beta L [long]) expressed in human endometrium, where they are coexpressed with the oestrogen receptor alpha (ER alpha). Over-expression of ERRbetaL enhanced ER alpha-dependent ligand-induced activation of an ERE-luciferase reporter construct, altered the induction of c-myc mRNA and increased proliferation of Ishikawa cells whereas ERR beta S was found to reduce these endpoints. Fluorescent recovery after photobleaching (FRAP) revealed that intra-nuclear mobility of YFP-ERR beta S was more rapid than YFP-ERR beta L. Fluorescence resonance energy transfer (FRET) assays revealed a close association between ERR beta L and ER alpha following addition of ligand. We speculate that ERR beta L may alter ER alpha-dependent gene activation by enhancing the recruitment of co-activators. In conclusion, variant isoforms of ERR beta have differential effects on ER alpha-dependent gene expression and this has implications for human endometrial cell function.

ERβ-dependent effects on uterine endothelial cells are cell specific and mediated via Sp1

STUDY QUESTION What are the in vitro effects of estrogen receptor β (ERβ) activation on the function of endothelial cells (ECs) from different vascular beds: human endometrial ECs (HEECs; endometrium), uterine myometrial microvascular ECs (UtMVECs; myometrium) and human umbilical vein ECs (HUVECs)? SUMMARY ANSWER Studies conducted in vitro demonstrate that the ERβ agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) has EC type-specific effects on patterns of gene expression and network formation. Identification of a key role for the transcription factor Sp1 in ERβ-dependent signaling in uterine ECs offers new insights into cell-specific molecular mechanisms of estrogen action in the human uterus. WHAT IS KNOWN ALREADY Estrogens, acting via ERs (ERα and ERβ), have important, body-wide impacts on the vasculature. The human uterus is an estrogen target organ, the endometrial lining of which exhibits physiological, cyclical angiogenesis. In fixed tissue sections, human endometrial ECs are immunopositive for ERβ. STUDY DESIGN, SIZE, DURATION Cells were treated with a vehicle control or the ERβ agonist, DPN, for 2 h or 24 h (n = 5) followed by gene expression analysis. Functional assays were analyzed after a 16 h incubation with ligand (n = 5). PARTICIPANT/MATERIALS, SETTING, METHODS Analysis of DPN-treated ECs using Taqman gene array cards focused on genes involved in angiogenesis and inflammation identified cell type-specific ERβ-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ERβ signaling were investigated using bioinformatics, reporter assays, immunoprecipitation, siRNA and a specific inhibitor blocking Sp1-binding sites. The endometrium and myometrium from women with regular menses were used to validate the protein expression of candidate genes. MAIN RESULTS AND THE ROLE OF CHANCE HEECs and UtMVECs were ERβ+/ERα−. Treatment of ECs with DPN had opposite effects on network formation: a decrease in network formation in HEECs (P ≤ 0.001) but an increase in UtMVECs (P ≤ 0.05). Genomic analysis identified opposite changes in ERβ target gene expression with only three common transcripts (HEY1, ICAM1, CASP1) in all three ECs; a unique profile was observed for each. An important role for Sp1 was identified, consistent with the regulation of ERβ target genes via association with the transcription factor (‘tethered’ mechanism). LIMITATIONS, REASONS FOR CAUTION The study was mainly carried out in vitro using ECs of which one type was immortalized. Although the analysis of the protein expression of candidate genes was carried out using intact tissue samples from patients, investigations into in vivo angiogenesis were not carried out. WIDER IMPLICATIONS OF THE FINDINGS These results have implications for our understanding of the mechanisms responsible for ERβ-dependent changes in EC gene expression in hormone-dependent disorders. STUDY FUNDING/COMPETEING INTEREST(S) The study was funded by a Medical Research Council Programme Grant. E.G. is the recipient of an MRC Career Development Fellowship. The authors have nothing to disclose.

Androgens regulate scarless repair of the endometrial “wound” in a mouse model of menstruation

The human endometrium undergoes regular cycles of synchronous tissue shedding (wounding) and repair that occur during menstruation before estrogen‐dependent regeneration. Endometrial repair is normally both rapid and scarless. Androgens regulate cutaneous wound healing, but their role in endometrial repair is unknown. We used a murine model of simulated menses; mice were treated with a single dose of the nonaromatizable androgen dihydrotestosterone (DHT; 200 μg/mouse) to coincide with initiation of tissue breakdown. DHT altered the duration of vaginal bleeding and delayed restoration of the luminal epithelium. Analysis of uterine mRNAs 24 h after administration of DHT identified significant changes in metalloproteinases (Mmp3 and ‐9; P < 0.01), a snail family member (Snai3; P < 0.001), and osteopontin (Sppl; P < 0.001). Chromatin immunoprecipitation analysis identified putative androgen receptor (AR) binding sites in the proximal promoters of Mmp9, Snai3, and Spp1. Striking spatial and temporal changes in immunoexpression of matrix metalloproteinase (MMP) 3/9 and caspase 3 were detected after DHT treatment. These data represent a paradigm shift in our understanding of the role of androgens in endometrial repair and suggest that androgens may have direct impacts on endometrial tissue integrity. These studies provide evidence that the AR is a potential target for drug therapy to treat conditions associated with aberrant endometrial repair processes.—Cousins, F. L., Kirkwood, P. M., Murray, A. A., Collins, F., Gibson, D. A., Saunders, P. T. K. Androgens regulate scarless repair of the endometrial “wound” in a mouse model of menstruation. FASEB J. 30, 2802‐2811 (2016). www.fasebj.org

Immunoprofiling of human uterine mast cells identifies three phenotypes and expression of ERβ and glucocorticoid receptor

Background: Human mast cells (MCs) are long-lived tissue-resident immune cells characterised by granules containing the proteases chymase and/or tryptase. Their phenotype is modulated by their tissue microenvironment. The human uterus has an outer muscular layer (the myometrium) surrounding the endometrium, both of which play an important role in supporting a pregnancy. The endometrium is a sex steroid target tissue consisting of epithelial cells (luminal, glandular) surrounded by a multicellular stroma, with the latter containing an extensive vascular compartment as well as fluctuating populations of immune cells that play an important role in regulating tissue function. The role of MCs in the human uterus is poorly understood with little known about their regulation or the impact of steroids on their differentiation status. The current study had two aims: 1) To investigate the spatial and temporal location of uterine MCs and determine their phenotype; 2) To determine whether MCs express receptors for steroids implicated in uterine function, including oestrogen (ERα, ERβ), progesterone (PR) and glucocorticoids (GR). Methods: Tissue samples from women (n=46) were used for RNA extraction or fixed for immunohistochemistry. Results: Messenger RNAs encoded by TPSAB1 (tryptase) and CMA1 (chymase) were detected in endometrial tissue homogenates. Immunohistochemistry revealed the relative abundance of tryptase MCs was myometrium>basal endometrium>functional endometrium. We show for the first time that uterine MCs are predominantly of the classical MC subtypes: (positive, +; negative, -) tryptase+/chymase- and tryptase+/chymase+, but a third subtype was also identified (tryptase-/chymase+). Tryptase+ MCs were of an ERβ+/ERα-/PR-/GR+ phenotype mirroring other uterine immune cell populations, including natural killer cells. Conclusions: Endometrial tissue resident immune MCs have three protease-specific phenotypes. Expression of both ERβ and GR in MCs mirrors that of other immune cells in the endometrium and suggests that MC function may be altered by the local steroid microenvironment.

The impact of 27-hydroxycholesterol on endometrial cancer proliferation

Endometrial cancer (EC) is the most common gynaecological malignancy. Obesity is a major risk factor for EC and is associated with elevated cholesterol. 27-hydroxycholesterol (27HC) is a cholesterol metabolite that functions as an endogenous agonist for Liver X receptor (LXR) and a selective oestrogen receptor modulator (SERM). Exposure to oestrogenic ligands increases risk of developing EC; however, the impact of 27HC on EC is unknown. Samples of stage 1 EC (n = 126) were collected from postmenopausal women undergoing hysterectomy. Expression of LXRs (NR1H3, LXRα; NR1H2, LXRβ) and enzymes required for the synthesis (CYP27A1) or breakdown (CYP7B1) of 27HC were detected in all grades of EC. Cell lines originating from well-, moderate- and poorly-differentiated ECs (Ishikawa, RL95, MFE 280 respectively) were used to assess the impact of 27HC or the LXR agonist GW3965 on proliferation or expression of a luciferase reporter gene under the control of LXR- or ER-dependent promoters (LXRE, ERE). Incubation with 27HC or GW3965 increased transcription via LXRE in Ishikawa, RL95 and MFE 280 cells (P < 0.01). 27HC selectively activated ER-dependent transcription (P < 0.001) in Ishikawa cells and promoted proliferation of both Ishikawa and RL95 cells (P < 0.001). In MFE 280 cells, 27HC did not alter proliferation but selective targeting of LXR with GW3965 significantly reduced cell proliferation (P < 0.0001). These novel results suggest that 27HC can contribute to risk of EC by promoting proliferation of endometrial cancer epithelial cells and highlight LXR as a potential therapeutic target in the treatment of advanced disease.