Sheila Macpherson

Differential Expression of Estrogen Receptor-α and -β and Androgen Receptor in the Ovaries of Marmosets and Humans

Abstract Estrogens and androgens are essential for the maturation of the ovarian follicle and normal fertility in the female. We have used antibodies specific for both forms of estrogen receptor (alpha [ERα] and beta [ERβ]) and androgen receptor (AR) to investigate the pattern of receptor expression in ovaries obtained from women and from a New World primate, the Common marmoset (Callthrix jacchus). On Western blots, three antibodies directed against different peptides within human ERβ all recognized recombinant human (h) ERβ but did not bind to recombinant hERα. The ERβ protein was extracted from human ovary and prostate and marmoset ovary. In marmoset and human ovaries, ERβ protein was detected in the nuclei of granulosa cells in all sizes of follicle (both before and after formation of the antrum), and it was also detected in thecal cells, corpora lutea, surface epithelium, and stroma. In contrast, ERα protein was not detected in the nuclei of granulosa cells in preantral follicles, was low/absent from stromal and thecal cells, but was expressed in granulosa cells of antral follicles and in the surface epithelium. The pattern of expression of AR protein more closely resembled that of ERβ than ERα. In conclusion, three independent antibodies have demonstrated convincingly that ERβ is expressed in a wide range of cells in the primate ovary. Granulosa cells in preantral follicles could contain ERβ:β dimers. In antral follicles, however, ERα is also expressed, and the formation of homo- or heterodimers containing ERα may influence the pattern of gene activation within these cells.

Human oestrogen receptors: differential expression of ERalpha and beta and the identification of ERbeta variants

Two structurally related subtypes of oestrogen receptor (ER), known as alpha (ER alpha, NR3A1) and beta (ER beta, NR3A2) have been identified. ER beta mRNA and protein have been detected in a wide range of tissues including the vasculature, bone, and gonads in both males and females, as well as in cancers of the breast and prostate. In many tissues the pattern of expression of ER beta is distinct from that of ER alpha. A number of variant isoforms of the wild type beta receptor (ER beta 1), have been identified. In the human these include: (1). use of alternative start sites within the mRNA leading to translation of either a long (530 amino acids, hER beta 1L) or a truncated form (487aa hER beta 1s); (2). deletion of exons by alternative splicing; (3). formation of several isoforms (ER beta 2-beta 5) due to alternative splicing of exons encoding the carboxy terminus (F domain). We have raised monoclonal antibodies specific for hER beta1 as well as to three of the C terminal isoforms (beta2, beta 4 and beta 5). Using these antibodies we have found that ER beta 2, beta 4 and beta 5 proteins are expressed in nuclei of human tissues including the ovary, placenta, testis and vas deferens. In conclusion, in addition to the differential expression of full length ER alpha and ER beta a number of ER variant isoforms have been identified. The impact of the expression of these isoforms on cell responsiveness to oestrogens may add additional complexity to the ways in which oestrogenic ligands influence cell function.

Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells

Significance Men are defined by androgens (testosterone), which drive fetal masculinization (male development) and puberty and maintain masculinity in adulthood, including sex drive, erectile function, and fertility. Moreover, Western cardiometabolic diseases are all associated with lowered testosterone levels in men. Therefore, influences on testosterone levels in adulthood have pervasive importance for masculinity and health. Our study shows, for the first time, to our knowledge, that testosterone levels during fetal masculinization can (re)program adult testosterone levels through effects on stem cells, which develop into adult Leydig cells (the source of testosterone) after puberty. These stem cells are present in fetal testes of humans and animals, and using the latter, we show how these cells are reprogrammed to affect adult testosterone levels. Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.

Expression of oestrogen receptor beta (ERβ1) protein in human breast cancer biopsies

Oestrogen action is mediated via specific receptors that act as ligand-activated transcription factors. A monoclonal antibody specific to the C-terminus of human oestrogen receptor beta has been characterized and the prevalence of expression of oestrogen receptor beta protein investigated in a well defined set of breast cancers. Reverse transcription-polymerase chain reaction analysis of RNA from tissue biopsies detected oestrogen receptor beta in all samples examined. The anti-oestrogen receptor beta antibody cross reacted specifically with both long (∼59 Kd) and short (∼53 Kd) forms of recombinant oestrogen receptor beta. Western blot analysis of breast tumours contained both forms of oestrogen receptor beta protein although in some samples lower molecular weight species (32–45 Kd) were identified. Fifty-one breast cancer biopsies were examined using immunohistochemistry; 41 (80%) were immunopositive for oestrogen receptor alpha, 48 (94%) were immunopositive for oestrogen receptor beta and 38 (74.5%) co-expressed both receptors. Expression of oestrogen receptor beta was exclusively nuclear and occurred in multiple cell types. There was no quantitative relationship between staining for the two ERs although in tumours in which both receptors were present immunoexpression of oestrogen receptor alpha was invariably more intense. The significance of oestrogen receptor beta protein expression in breast cancers to therapy remains to be determined but the availability of a well characterized antibody capable of detecting oestrogen receptor beta in archive material will facilitate the process.

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.

Modulation of gene expression by androgen and oestrogens in the testis and prostate of the adult rat following androgen withdrawal

Androgens are important for the structural and functional integrity of the testis and the prostate and this may in part be mediated by the aromatisation of testosterone to oestradiol. The aim of the present study was to establish an in vivo model that would allow the identification of genes, the expression of which was regulated acutely by androgen and/or oestrogen in the male reproductive system. In rats in which the Leydig cells were ablated by administration of ethane dimethane sulfonate (EDS) 6 days earlier, testosterone esters (T) were administered from day 0 (To), and additional animals were administered either T, 17beta-oestradiol benzoate (EB) or diethylstilbestrol (DES) for 1 or 4 h on day 6 after EDS-treatment. Nuclear immunoexpression of the androgen receptor (AR) was reduced or absent from the testis but unaffected in the ventral prostate following these treatments. ERbeta immunoexpression in these tissues was unchanged. Northern blot analysis showed that EB and DES as well as T administration 4 h earlier could modulate mRNA expression of two androgen-responsive genes, C3 and SGP-2, in the prostate. The co-administration of T or EB with the AR antagonist, flutamide, or with the ER antagonist, ICI 182,780 (ICI), did not block the suppression of SGP-2 mRNA expression by T or EB. In contrast, the upregulation of C3 mRNA expression by T was successfully antagonised by both flutamide and by ICI. A preliminary evaluation of the expression of three Sertoli cell and five germ cell mRNAs revealed that their expression was not steroid regulated. Our results support the hypothesis that the action of testosterone in the male reproductive system may in part be mediated by its conversion to oestradiol. This in vivo model should prove of value in future studies to identify androgen and oestrogen regulated genes in the male reproductive system.

Estrogen receptor related beta is expressed in human endometrium throughout the normal menstrual cycle

BACKGROUND Estrogen receptor related beta (ERRβ, ESRRB/NR3B2) is an orphan receptor that shares significant sequence homology with estrogen receptors ERα and ERβ. ERR family members are reported to exhibit constitutive transcriptional activity; however, little is known about the biological function of ERRβ. In an attempt to delineate its role, we examined expression of ERRβ in normal human endometrium, a tissue that undergoes cyclic remodelling under the influence of estrogen and progesterone. METHODS Well-characterized endometrial tissue (n = 31), including full-thickness biopsies, was obtained from women with regular menstrual cycles. RT–PCR was used to measure mRNA encoding ERRβ, the peroxisome proliferator activated receptor gamma coactivators (PGC)-1α and β and to determine whether ERRβ splice variant mRNAs were expressed. ERRβ was immunolocalized using both single and double antibody immunohistochemistry. RESULTS Total ERRβ mRNA appeared higher in proliferative phase samples but results did not reach significance. Transcripts corresponding to the long- and short-splice variants of ERRβ as well as PGC1α and β were detected but ERRβΔ10 was absent. ERRβ protein was localized to cell nuclei within multiple endometrial cell types including the glands, stroma, endothelium and immune cells, including uterine natural killer (uNK) cells and macrophages. Fluorescent immunohistochemistry revealed that some cells co-expressed ERRβ and ERα or ERβ, for example, endothelial and uNK cells were ERRβ+/ERβ+. CONCLUSIONS ERRβ mRNA and protein are expressed in healthy human endometrium. Further studies are warranted to characterize the functional impact of ERRβ on endometrial biology.

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.

Comparative Effects of Di(n-Butyl) Phthalate Exposure on Fetal Germ Cell Development in the Rat and in Human Fetal Testis Xenografts

Background Phthalate exposure induces germ cell effects in the fetal rat testis. Although experimental models have shown that the human fetal testis is insensitive to the steroidogenic effects of phthalates, the effects on germ cells have been less explored. Objectives We sought to identify the effects of phthalate exposure on human fetal germ cells in a dynamic model and to establish whether the rat is an appropriate model for investigating such effects. Methods We used immunohistochemistry, immunofluorescence, and quantitative real-time polymerase chain reaction to examine Sertoli and germ cell markers on rat testes and human fetal testis xenografts after exposure to vehicle or di(n-butyl) phthalate (DBP). Our study included analysis of germ cell differentiation markers, proliferation markers, and cell adhesion proteins. Results In both rat and human fetal testes, DBP exposure induced similar germ cell effects, namely, germ cell loss (predominantly undifferentiated), induction of multinucleated gonocytes (MNGs), and aggregation of differentiated germ cells, although the latter occurred rarely in the human testes. The mechanism for germ cell aggregation and MNG induction appears to be loss of Sertoli cell–germ cell membrane adhesion, probably due to Sertoli cell microfilament redistribution. Conclusions Our findings provide the first comparison of DBP effects on germ cell number, differentiation, and aggregation in human testis xenografts and in vivo in rats. We observed comparable effects on germ cells in both species, but the effects in the human were muted compared with those in the rat. Nevertheless, phthalate effects on germ cells have potential implications for the next generation, which merits further study. Our results indicate that the rat is a human-relevant model in which to explore the mechanisms for germ cell effects. Citation van den Driesche S, McKinnell C, Calarrão A, Kennedy L, Hutchison GR, Hrabalkova L, Jobling MS, Macpherson S, Anderson RA, Sharpe RM, Mitchell RT. 2015. Comparative effects of di(n-butyl) phthalate exposure on fetal germ cell development in the rat and in human fetal testis xenografts. Environ Health Perspect 123:223–230; http://dx.doi.org/10.1289/ehp.1408248

Diethylstilboestrol exposure does not reduce testosterone production in human fetal testis xenografts.

In rodents, in utero exposure to exogenous estrogens including diethylstilboestrol (DES) results in major suppression of steroidogenesis in fetal testes. Whether similar effects occur in the human fetal testis is equivocal. Based on the results of the rodent studies, we hypothesised that exposure of human fetal testes to DES would result in a reduction in testosterone production. We show, using a xenograft approach, that testosterone production is not reduced in human fetal testis following DES exposure. Human fetal testes (15–19 weeks’ gestation, n = 6) were xenografted into castrate male nude mice which were then treated for 35 days with vehicle or 100 µg/kg DES three times a week. For comparison, similar treatment was applied to pregnant rats from e13.5–e20.5 and effects on fetal testes evaluated at e21.5. Xenograft testosterone production was assessed by measuring host seminal vesicle (SV) weights as an indirect measure over the entire grafting period, and single measurement of serum testosterone at termination. Human fetal testis xenografts showed similar survival in DES and vehicle-exposed hosts. SV weight (44.3 v 26.6 mg, p = 0.01) was significantly increased in DES compared to vehicle-exposed hosts, respectively, indicating an overall increase in xenograft testosterone production over the grafting period, whilst serum testosterone at termination was unchanged. In contrast intra-testicular testosterone levels were reduced by 89%, in fetal rats exposed to DES. In rats, DES effects are mediated via Estrogen Receptor α (ESR1). We determined ESR1 protein and mRNA expression in human and rat fetal testis. ESR1 was expressed in rat, but not in human, fetal Leydig cells. We conclude that human fetal testis exposure to DES does not impair testosterone production as it does in rats, probably because ESR1 is not expressed in human fetal Leydig cells. This indicates that DES exposure is likely to pose minimal risk to masculinization of the human fetus.