Role Of Estrogen In Regulation Research Articles

Single-cell sequencing reveals the transcriptional alternations of 17β-estradiol suppressing primordial follicle formation in neonatal mouse ovaries.

Estrogen has been implicated in multiple biological processes, but the variation underlying estrogen-mediated primordial follicle (PF) formation remains unclear. Here, we show that 17β-estradiol (E2) treatment of neonatal mice led to the inhibition of PF formation and cell proliferation. Single-cell RNA sequencing (scRNA-seq) revealed that E2 treatment caused significant changes in the transcriptome of oocytes and somatic cells. E2 treatment disrupted the synchronised development of oocytes, pre-granulosa (PG) cells and stromal cells. Mechanistically, E2 treatment disrupted several signalling pathways critical to PF formation, especially down-regulating the Kitl and Smad1/3/4/5/7 expression, reducing the frequency and number of cell communication. In addition, E2 treatment influenced key gene expression, mitochondrial function of oocytes, the recruitment and maintenance of PG cells, the cell proliferation of somatic cells, as well as disordered the ovarian microenvironment. This study not only revealed insights into the regulatory role of estrogen during PF formation, but also filled in knowledge of dramatic changes in perinatal hormones, which are critical for the physiological significance of understanding hormone changes and reproductive protection.

Sex hormones and immune system: Menopausal hormone therapy in the context of COVID-19 pandemic

The fatal outcomes of COVID-19 are related to the high reactivity of the innate wing of immunity. Estrogens could exert anti-inflammatory effects during SARS-CoV-2 infection at different stages: from increasing the antiviral resistance of individual cells to counteracting the pro-inflammatory cytokine production. A complex relationship between sex hormones and immune system implies that menopausal hormone therapy (MHT) has pleiotropic effects on immunity in peri- and postmenopausal patients. The definite immunological benefits of perimenopausal MHT confirm the important role of estrogens in regulation of immune functionalities. In this review, we attempt to explore how sex hormones and MHT affect immunological parameters of the organism at different level (in vitro, in vivo) and what mechanisms are involved in their protective response to the new coronavirus infection. The correlation of sex steroid levels with severity and lethality of the disease indicates the potential of using hormone therapy to modulate the immune response and increase the resilience to adverse outcomes. The overall success of MHT is based on decades of experience in clinical trials. According to the current standards, MHT should not be discontinued in COVID-19 with the exception of critical cases.

Significance of G Protein-Coupled Estrogen Receptor in the Pathophysiology of Irritable Bowel Syndrome, Inflammatory Bowel Diseases and Colorectal Cancer.

The regulatory role of estrogens and nuclear estrogen receptors, i. e., estrogen receptor α and β has been reported in gastrointestinal diseases. However, the contribution of G protein-coupled estrogen receptor, the membrane-bound estrogen receptor, is still poorly understood. Unlike nuclear estrogen receptors, which are responsible for the genomic activity of estrogens, the G protein-coupled estrogen receptor affects the “rapid” non-genomic activity of estrogens, leading to modulation of many signaling pathways and ultimately changing gene expression. Recently, the crucial role of G protein-coupled estrogen receptor in intestinal pathogenesis has been documented. It has been shown that the G protein-coupled estrogen receptor can modulate the progression of irritable bowel syndrome, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis as well as colorectal cancer. The G protein-coupled estrogen receptor appears to be a potent factor regulating abdominal sensitivity and pain, intestinal peristalsis, colitis development, proliferation and migration potential of colorectal cancer cells and seems to be a useful target in gastrointestinal diseases. In this review, we present the current state of knowledge about the contribution of the G protein-coupled estrogen receptor to irritable bowel syndrome, inflammatory bowel diseases and colorectal cancer.

Estrogen-Receptor Expression and Function in Female Reproductive Disease.

Estrogen receptors (ER) include ER alpha, ER beta and new membrane receptor G protein-coupled receptor 30 (GPR30). Estrogen receptors are key receptors to maintain ovarian granulosa cell differentiation, follicle and oocyte growth and development, and ovulation function. The abnormal functions of estrogen, its receptors, and estradiol synthesis-related enzymes are closely related to clinical reproductive endocrine diseases, such as polycystic ovary syndrome (PCOS) and endometriosis (EMS). At present, hormone therapy is the main treatment for ovarian-related diseases, and a stable hormone environment is established by regulating ovarian function. In recent years, some estrogen-related drugs have made great progress, such as clomiphene, which is a nonsteroidal antiestrogen drug in clinical application. This article elaborates on the regulatory role of estrogen and its nuclear receptors and membrane receptors in oocyte development, especially female reproductive diseases related to the abnormal expression of estrogen and its receptors. We also highlighted the latest advances of treatment strategy for these diseases and the application of related targeted small molecule drugs in clinical research and treatment, so as to provide reference for the treatment of female reproductive diseases.

High-Fat Diet Induces Periodontitis in Mice through Lipopolysaccharides (LPS) Receptor Signaling: Protective Action of Estrogens

BackgroundA fat-enriched diet favors the development of gram negative bacteria in the intestine which is linked to the occurrence of type 2 diabetes (T2D). Interestingly, some pathogenic gram negative bacteria are commonly associated with the development of periodontitis which, like T2D, is characterized by a chronic low-grade inflammation. Moreover, estrogens have been shown to regulate glucose homeostasis via an LPS receptor dependent immune-modulation. In this study, we evaluated whether diet-induced metabolic disease would favor the development of periodontitis in mice. In addition, the regulatory role of estrogens in this process was assessed.MethodsFour-week-old C57BL6/J WT and CD14 (part of the TLR-4 machinery for LPS-recognition) knock-out female mice were ovariectomised and subcutaneously implanted with pellets releasing either placebo or 17β-estradiol (E2). Mice were then fed with either a normal chow or a high-fat diet for four weeks. The development of diabetes was monitored by an intraperitoneal glucose-tolerance test and plasma insulin concentration while periodontitis was assessed by identification of pathogens, quantification of periodontal soft tissue inflammation and alveolar bone loss.ResultsThe fat-enriched diet increased the prevalence of periodontal pathogenic microbiota like Fusobacterium nucleatum and Prevotella intermedia, gingival inflammation and alveolar bone loss. E2 treatment prevented this effect and CD14 knock-out mice resisted high-fat diet-induced periodontal defects.Conclusions/SignificanceOur data show that mice fed with a diabetogenic diet developed defects and microflora of tooth supporting-tissues typically associated with periodontitis. Moreover, our results suggest a causal link between the activation of the LPS pathway on innate immunity by periodontal microbiota and HFD-induced periodontitis, a pathophysiological mechanism that could be targeted by estrogens.

In vitro inhibition of porcine cytochrome P450 by 17β-estradiol and 17α-estradiol

Sexually mature pigs are known to possess high concentrations of testicular steroids, which have been shown to change the activities of cytochrome P450 in vitro. The aim of the present study was to evaluate the regulation of CYP1A and CYP2E1 activity by the steroids dihydrotestosterone (DHT), 3β-androstenol, 17β-estradiol and 17α-estradiol. Catalytic activities of 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD) were used as markers of CYP1A activities, while p-nitrophenol hydroxylase (PNPH) was used as a marker of CYP2E1 activities. Of the steroids tested, only 17β-estradiol and 17α-estradiol inhibited EROD and MROD activities. This inhibition was observed when a steroid concentration of 100 µM was used, while lower concentrations showed no inhibitory effect. PNPH activities were inhibited only by 100 µM of 17β-estradiol. The significance of these results in vivo is unknown because inhibition was only found when concentrations of estrogens higher than physiological levels were used. Nevertheless, the results provided further evidence on the important role of estrogens in regulation of porcine cytochrome P450 activities.

Abstract 155: MicroRNA ‘signature’ during estrogen-mediated mammary carcinogenesis

Abstract MicroRNAs (miRNAs) play important role in a wide range of normal biological processes. Dysregulated miRNA expression has been reported with the development and progression of cancers, including breast cancer. The role of estrogens in regulation of cell proliferation and breast carcinogenesis is well known. Recent reports have associated several miRNAs with estrogen receptors in breast tumors. However, identification of miRNA ‘signature’ during estrogen-mediated carcinogenesis may be critical for understanding the effect of estrogen in human breast cancer, as well as for developing strategies for cancer chemoprevention. In this study we analyzed miRNAs at 3 weeks, 12 weeks close to the appearance of first palpable tumor and 28 weeks when tumor incidence reaches 85-90% following treatment of female ACI rats with 17ß-estradiol (E2) silastic implants. Total RNA from mammary tissues and tumors were isolated using mirVana microRNA kit and analyzed using Affymetrix GeneChip miRNAarray. This array had 351 rat specific miRNAs probes. E2 treatment resulted in significant up regulation or repression (2 fold change) of 33 miRNAs at two or more time points. A total of 9 (p<0.00128), 8 (p<0.00113) and 27 (p<0.00384) miRNAs in E2 treated normal mammary tissue, and 73 (p<0.01039) in tumor tissue were differentially expressed when compared to untreated samples from 3, 12, and 28 weeks time point, respectively, with false discovery rate (FDR) at 0.05. These miRNAs were used to predict potential target mRNAs using Partek software. Ingenuity pathway analysis was used to further predict the major functions of the target mRNAs and molecular pathways. Molecular mechanism of cancer was the top canonical pathway of putative target mRNAs at all the time points indicating influence of E2 treatment in mammary carcinogenesis. The five top-ranking miRNAs (miR-196c, miR-34c, miR-375, miR-429 and miR-206) from microarray analysis were confirmed by real-time RT-PCR using Taqman microRNA assays; 5S RNA was used as reference for normalization. The expression of these miRNAs followed the same pattern whether evaluated by microarray or qPCR. These five miRNAs were further predicted to interact with mRNAs involved in estrogen-mediated (e.g. Src, PI3K, Akt, etc.) and EGF-mediated (e.g. Sos, Ras, MEK, etc.) breast cancer pathways. Thus, our results demonstrate miRNA modulations during the estrogen-mediated mammary carcinogenesis in an in vivo model in which, histologically, the mammary tumors resemble human breast cancer. Our future studies are aimed at finding the effect of naturally-occurring anti-cancer agent(s) on these candidate miRNAs to determine their translational importance (Supported by CA-118114, CA-125152 and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 155. doi:10.1158/1538-7445.AM2011-155

Role of Estrogen in Regulation of Morphology and Synaptic Connectivity in Female Rat Subiculum

Estrogen is reported to exert neuroprotective influences on the cornu ammonis as well as the dentate gyrus, two of the three components of the hippocampal memory system. Subiculum, the third and the output component of this system, plays a vital role in retrieval of previously learnt information (recall tasks). We undertook these studies to understand estrogen's role in regulating the synaptic connectivity, neurotransmission and neural degeneration in subiculum. For this, we used brains of aged (16–18 months) post-estropausal female Wistar rats, and compared them with those of adult (4–5 months) ovary-intact, ovariectomized, ova riectomized-vehicle treated and ova riectomized-estrogen treated rats. The vehicle (sesame oil) and estrogen therapy. (17β estradiol, E2) were administered as daily subcutaneous injections (0.1 mg/kg of E2 in 0.1 ml vehicle) for 30 days. Serum estradiol assay was done to determine the levels of circulating serum estradiol levels. Brains were processed for Cresyl Violet, Golgi staining and Immunohistochemical studies [using anti-synaptophysin antibody].Our results indicate that estrogen deficiency (induced by normal ageing or by ovariectomy) results in subicular neuronal degenerative changes, decreased synaptic connectivity and reduced dendritic arborization, most of which are reasonably reversed with estrogen replenishment. These studies suggest a unique role of estrogen in preventing and even reversing the senile neurodegenerative changes in female subiculum.

Potential role of estrogen in regulation of the Insulin-like growth factor2–H19 locus in the rat testis

The selective estrogen receptor modulator, tamoxifen, has been shown to reduce DNA methylation at Insulin-like growth factor 2/H19 differentially methylated region ( Igf2/H19 DMR) in the spermatozoa of the Holtzman rats. Since imprint at this locus is acquired during spermatogenesis in the male germ-line, we hypothesized role for estrogen signaling in the methylation dynamics in the testis. The present study was designed to identify putative estrogen response elements (ERE) at Igf2/H19 DMR and their interaction with DNA methylation pathway. Here, we demonstrate presence of functional ERE at 2637/2655 base pair on Igf2/H19 DMR in testicular germ cells, which was found to bind to estrogen receptor β (ERβ) in the chromatin immunoprecipitation assay. Tamoxifen attenuated ERβ-ERE association thereby acting as an estrogen antagonist at this locus. Further mechanistic study involving colocalization and immunoprecipitation assay revealed interaction of ERβ and Dnmt1 in the testis. The study provides evidence for the role for estrogen in acquisition of imprint at Igf2/H19 DMR in testis and help in understanding molecular mechanism of environmental estrogens impacting male fertility.

The Regulation of MS-KIF18A Expression and Cross Talk with Estrogen Receptor

This study provides a novel view on the interactions between the MS-KIF18A, a kinesin protein, and estrogen receptor alpha (ERα) which were studied in vivo and in vitro. Additionally, the regulation of MS-KIF18A expression by estrogen was investigated at the gene and protein levels. An association between recombinant proteins; ERα and MS-KIF18A was demonstrated in vitro in a pull down assay. Such interactions were proven also for endogenous proteins in MBA-15 cells were detected prominently in the cytoplasm and are up-regulated by estrogen. Additionally, an association between these proteins and the transcription factor NF-κB was identified. MS-KIF18A mRNA expression was measured in vivo in relation to age and estrogen level in mice and rats models. A decrease in MS-KIF18A mRNA level was measured in old and in OVX-estrogen depleted rats as compared to young animals. The low MS-KIF18A mRNA expression in OVX rats was restored by estrogen treatment. We studied the regulation of MS-KIF18A transcription by estrogen using the luciferase reporter gene and chromatin immuno-percipitation (ChIP) assays. The luciferase reporter gene assay demonstrated an increase in MS-KIF18A promoter activity in response to 10−8 M estrogen and 10−7M ICI-182,780. Complimentary, the ChIP assay quantified the binding of ERα and pcJun to the MS-KIF18A promoter that was enhanced in cells treated by estrogen and ICI-182,780. In addition, cells treated by estrogen expressed higher levels of MS-KIF18A mRNA and protein and the protein turnover in MBA-15 cells was accelerated. Presented data demonstrated that ERα is a defined cargo of MS-KIF18A and added novel insight on the role of estrogen in regulation of MS-KIF18A expression both in vivo and in vitro.

Molecular Interaction of BMP-4, TGF-β, and Estrogens in Lactotrophs: Impact on the PRL Promoter

The regulatory role of estrogen, bone morphogenetic protein-4 (BMP-4), and TGF-beta has a strong impact on hormone secretion, gene transcription, and cellular growth of prolactin (PRL)-producing cells. In contrast to TGF-beta, BMP-4 induces the secretion of PRL in GH3 cells. Therefore, we studied the mechanism of their transcriptional regulation. Both BMP-4 and TGF-beta inhibited the transcriptional activity of the estrogen receptor (ER). Estrogens had no effect on TGF-beta-specific Smad protein transcriptional activity but presented a stimulatory action on the transcriptional activity of the BMP-4-specific Smads. BMP-4/estrogen cross talk was observed both on PRL hormone secretion and on the PRL promoter. This cross talk was abolished by the expression of a dominant-negative form for Smad-1 and treatment with ICI 182780 but not by point mutagenesis of the estrogen response element site within the promoter, suggesting that Smad/ER interaction might be dependent on the ER and a Smad binding element. By serial deletions of the PRL promoter, we observed that indeed a region responsive to BMP-4 is located between -2000 and -1500 bp upstream of the transcriptional start site. Chromatin immunoprecipitation confirmed Smad-4 binding to this region, and by specific mutation and gel shift assay, a Smad binding element responsible site was characterized. These results demonstrate that the different transcriptional factors involved in the Smad/ER complexes regulate their transcriptional activity in differential ways and may account for the different regulatory roles of BMP-4, TGF-beta, and estrogens in PRL-producing cells.

Comparative contents of mRNAs of sex steroid receptors and enzymes of their metabolism in arterial walls of men

The potential role of estrogens in regulation of metabolism in arteries of men was studied. Contents of mRNAs of sex hormone receptors, of some enzymes of their metabolism, and of some potential markers of the hormone effects were determined by real-time polymerase chain reaction in fragments of 18-54-year-old men's large arteries with and without atherosclerotic lesions. Contents of estrogen receptor alpha (ERalpha) and transferrin receptor mRNAs were significantly different in undamaged fragments of the aorta and of the carotid and coronary arteries. Contents of some mRNAs in the carotid artery and aorta were found to correlate, which suggested a similarly directed regulation of their expressions. The levels of ERalpha and aromatase mRNAs negatively correlated with the blood plasma concentration of estradiol. Levels of steroid sulfatase and aromatase mRNAs were lower and the level of estrogen sulfotransferase mRNA was higher in blood vessel fragments with atherosclerotic lesions than in undamaged fragments. It is suggested that large arteries should be different in sensitivity to estrogens and that atherosclerotic lesions could lead to local suppression of the effect of estrogen on the cells of arteries.

Reducing endogenous estrogen during development alters hormone production by porcine Leydig cells and seminiferous tubules

High levels of estrogen produced by boar testes and the presence of estrogen receptors in both interstitial and tubular compartments are consistent with a direct role for estrogen in regulation of testicular cell function. This study investigated the importance of estrogen on hormone production by Leydig cells and seminiferous tubules in the developing boar. Thirty-six 1-week-old littermate pairs of boars were treated weekly with vehicle or 0.1 mg/kg BW Letrozole, an aromatase inhibitor, until castration at 2, 3, 4, 5, 6, 7, or 8 months. Tissue was collected and Leydig cells and seminiferous tubules were isolated. In a separate study, five untreated boars (ages 1.5–4 months) were castrated and Letrozole was added in vitro to Leydig cell and seminiferous tubule cultures. Leydig cells were cultured for 24 h with and without porcine LH. Media were assayed for estradiol (E 2) and testosterone (T) concentrations by RIA. Seminiferous tubules were cultured for 4 h with and without porcine FSH; media were assayed for E 2 and immunoreactive inhibin (INH). In vivo aromatase inhibition decreased basal E 2 and increased basal T production by cultured Leydig cells. Basal seminiferous tubule production of E 2 but not INH was reduced. Decreasing estrogen synthesis in vivo did not alter LH-induced Leydig cell E 2 production or FSH-induced seminiferous tubule INH production. INH production decreased with advancing age regardless of treatment. In conclusion, in vivo aromatase inhibition altered baseline steroid production by cultured Leydig cells and seminiferous tubules but had little effect on response to gonadotropins.

Role of estrogen in regulation of cellular differentiation: A study using human placental and rat Leydig cells

Estrogen classically is recognized as a growth-promoting hormone. Recent evidence suggests that estrogens are also involved in a wide variety of cellular and physiological functions involving the central nervous system, immune system, cardiovascular system and bone homeostasis. Our studies in cytotrophoblasts and BeWo cells, demonstrated that 17β-estradiol induces terminal differentiation of placental trophoblasts directly and this differentiation is coupled with an increased production of TGFβ1, which, in turn, affects telomerase activity and telomerase associated components at the level of hTERT. Furthermore, using rats treated in vivo with either EDS or estradiol and in vitro Leydig cell cultures, we proposed that 17β-estradiol mediated down-regulation of collagen IV α4 expression could be one of the possible mechanisms for the inhibition of progenitor Leydig cell proliferation. In this review, we summarize the results from both the model systems, the human placental cytotrophoblast and rat Leydig cells to conclude that 17β-estradiol has a unique stage-specific role in differentiation.

A possible regulatory role of 17β-estradiol and tamoxifen on glyoxalase I and glyoxalase II genes expression in MCF7 and BT20 human breast cancer cells

The glutathione-dependent glyoxalases system, composed of glyoxalase I (GloI) and glyoxalase II (GloII) enzymes, is involved in the detoxification of methylglyoxal, a by-product of cell metabolism. Aberrations in the expression of glyoxalase genes in several human cancers have been reported. Sometimes, these aberrations seem to differ depending on the organs and on the sensitivity of the tumours to estrogens, as we previously detected in the hormone-responsive breast cancer compared to the hormone-independent bladder cancer. To investigate a possible regulatory role of estrogens, as well as antiestrogens, on glyoxalases system, estrogen receptor (ER)-positive MCF7 and ER-negative BT20 human breast cancer cells were cultured in the presence of 17beta-estradiol (E2) and tamoxifen (TAM) performing two independent experiments. After a 24 h or 4 days treatment, we evaluated GloI and GloII mRNA levels, by Ribonuclease Protection Assay (RPA), enzymatic activities spectrophotometrically and cell proliferation by [3H]thymidine incorporation. We found that both steroid molecules affected glyoxalases gene expression and proliferation in a different manner in the cell lines. The modifications in mRNA levels were accompanied by parallel changes in the enzymatic activities. The possibility that modulation of glyoxalase genes by E2 and TAM are due to the presence of estrogen response elements (ERE) or cross-talk mechanisms by proteins of the estrogen signal transduction pathways are discussed. Knowledge regarding the regulation of glyoxalases by E2 and TAM may provide insights into the importance of this enzymes in human breast carcinomas in vivo.

Developmental maturation of primate placental trophoblast: placental cytosolic and secretory phospholipase A 2 expression after estrogen suppression of baboons

We have demonstrated that the baboon placenta expressed the mRNAs and proteins for secretory and cytosolic phospholipase A 2 (PLA 2) enzymes and that cPLA 2 expression increased with advancing gestation in association with the increase in placental estrogen production. To determine whether estrogen regulates placental PLA 2 expression, as it does other aspects of syncytiotrophoblast functional differentiation, we compared sPLA 2 and cPLA 2 mRNA levels in placentas obtained on day 165 of gestation (term = day 184) from baboons that were untreated or treated during the second half of gestation with the aromatase inhibitor CGS 20267 or CGS 20267 and estradiol. Maternal saphenous and uterine vein estradiol levels were reduced ( P < 0.05) by approximately 95% by treatment with CGS 20267 and restored by concomitant administration of CGS 20267 and estrogen. However, sPLA 2 and cPLA 2 mRNA levels expressed as a ratio of β-actin were similar in whole villous placenta from baboons that were untreated or treated with CGS 20267 or CGS 20267 plus estrogen. PLA 2 expression in an enriched fraction of nontrophoblast cells of the baboon placenta was also not altered by CGS 20267 treatment. Collectively these findings indicate that placental cPLA 2 and sPLA 2 expression is not estrogen-dependent. Because estrogen has been shown to regulate other aspects of placental steroidogenesis, we suggest that the regulatory role of estrogen on syncytiotrophoblast functional maturation is specific.

Effects of estrogen on the condylar cartilage of the rat mandible in organ culture

Purpose: The effects of estrogen on bone have been well documented. However, very little is known about the regulatory role of estrogen on cartilage and, in particular, the secondary cartilage of the mandibular condyle. The aims of this study were to determine whether estrogen receptors are present in the condylar cartilage of the rat mandible and to assess the effect of varying 17β-estradiol (E 2) concentrations on the proteoglycan content of this tissue. Materials and Methods: Mandibular condyles of 16 female Sprague-Dawley rats were resected. Eighteen of these condyles were divided into three groups and the condylar cartilage was removed and placed in organ culture for 4 days with media containing different concentrations of estrogen: 10 −11 mol/L, 10 −8 mol/L, and 10 −6 mol/L. The cartilage then was analyzed for proteoglycan content along with six specimens not passed through the organ culture. Six intact mandibular condyles also were resected and placed in organ culture with the same varying E 2 concentrations, and the condylar cartilage was analyzed for estrogen receptors along with two condyles not passed through the culture system. Results: Estrogen receptors were evenly distributed within the chondroblastic and hypertrophic zones in the control group and the group with 10 −11 mol/L E 2. With E 2 concentrations of 10 −8 mol/L and 10 −6 mol/L, there was a qualitative decrease in hypertrophic chondroblasts, thickness of the condylar cartilage, and a significant decrease in proteoglycan content. Conclusions: This study shows the presence of estrogen receptors in the secondary cartilage of the rat mandibular condyle. Estrogen has the potential to cause a decrease in extracellular matrix and thickness of this cartilage.

High affinity binding of the estrogen receptor to a DNA response element does not require homodimer formation or estrogen

We have developed an antibody-based DNA binding assay to study the effects of the absence or presence of an estrogen receptor agonist and two antagonists on the thermodynamic binding parameters for estrogen receptor binding to a consensus estrogen response element in vitro. We first demonstrate that antibodies raised against a region of the estrogen receptor directly adjacent to the DNA-binding domain do not interfere with the receptor's ability to preferentially bind to the vitellogenin estrogen response element in plasmid DNA. Exploiting this property to develop a quantitative DNA binding assay, we demonstrate that estrogen is not required for high affinity binding of the receptor for an oligonucleotide containing this element, nor do antiestrogens alter this interaction. In addition, we find that 1 mol of estrogen receptor is complexed with high affinity to 1 mol of vitellogenin estrogen response element, instead of two estrogen receptors/response element as would be predicted if estrogen receptor homodimer formation was required for high affinity DNA binding. Our data best fit a model in which the active estrogen receptor form is a monomer or heterodimer, but not a homodimer, and the regulatory role of estrogen on the estrogen receptor is the induction of protein-protein, but not protein-DNA, interactions.

Role of estrogens in regulation of the bile acid composition of the enterohepatic system of rabbits and monkeys with extrahepatic cholestasis

Role of estrogens in regulation of the bile acid composition of the enterohepatic system of rabbits and monkeys with extrahepatic cholestasis

Presence of Immunoreactive Vasoactive Intestinal Polypeptide in Anterior Pituitary of Normal Male and Long Term Estrogen-Treated Female Rats: A Light Microscopic Immunohistochemical Study*

The presence and synthesis of vasoactive intestinal polypeptide (VIP) has been demonstrated in the rat anterior pituitary. It was recently confirmed that immunoreactive VIP is present in the anterior pituitary, and in a thyroid-deficient state, VIP could be detected by light microscopic immunohistochemistry. It has also been suggested that VIP plays a stimulatory role in PRL secretion. To gain more detailed information on the localization of VIP and the conditions that alter the synthesis of VIP, we examined VIP immunoreactivity using immunohistochemistry in pituitaries of normal male and cycling female rats and in those states in which PRL secretion was enhanced (pregnancy, lactation, long term estrogen treatment, and pituitary implanted under kidney capsule of normal or estrogen-treated female rats). In situ, implanted and cultured pituitary cells were stained for VIP immunoreactivity using the peroxidase-antiperoxidase method. VIP immunoreactivity was observed in about 45% of the male rats, in all estrogen-treated female rats, in the implanted pituitaries under the kidney capsule (three of five from estrogen-treated and one of five from intact females, respectively), and in the pituitary cell cultures derived from estrogen-treated rats. Using a double labeling procedure we have also observed PRL immunoreactivity in a small population of the VIP-positive cells. These results suggest a positive regulatory role of estrogen in expression of the VIP gene. The physiological and pathophysiological significance of VIP in PRL secretion, however, remains to be clarified.