Shannon Whirledge

A Role for Glucocorticoids in Stress-Impaired Reproduction: Beyond the Hypothalamus and Pituitary

In addition to the well-characterized role of the sex steroid receptors in regulating fertility and reproduction, reproductive events are also mediated by the hypothalamic-pituitary-adrenal axis in response to an individuals environment. Glucocorticoid secretion in response to stress contributes to the well-characterized suppression of the hypothalamic-pituitary-gonadal axis through central actions in the hypothalamus and pituitary. However, both animal and in vitro studies indicate that other components of the reproductive system are also regulated by glucocorticoids. Furthermore, in the absence of stress, it appears that homeostatic glucocorticoid signaling plays a significant role in reproduction and fertility in all tissues comprising the hypothalamic-pituitary-gonadal axis. Indeed, as central regulators of the immune response, glucocorticoids are uniquely poised to integrate an individuals infectious, inflammatory, stress, nutritional, and metabolic status through glucocorticoid receptor signaling in target tissues. Endocrine signaling between tissues regulating the immune and stress response and those determining reproductive status provides an evolutionary advantage, facilitating the trade-off between reproductive investment and offspring fitness. This review focuses on the actions of glucocorticoids in tissues important for fertility and reproduction, highlighting recent studies that show glucocorticoid signaling plays a significant role throughout the hypothalamic-pituitary-gonadal axis and characterizing these effects as permissive or inhibitory in terms of facilitating reproductive success.

Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus

Sex hormone signaling regulates a variety of functions in the uterine endometrium essential for embryo implantation and immunity. Epithelial cells of the uterine endometrium are the target of the coordinated actions of estradiol (E(2)) and progesterone. However, little information exists regarding the interplay of estrogens with glucocorticoids in this tissue. Using the human uterine epithelial cell line ECC1, E(2) was found to antagonize induction of the glucocorticoid-induced leucine zipper (GILZ) gene expression, which is associated with several of the immune-related functions of glucocorticoids. Interestingly, E(2) antagonizes glucocorticoid regulated nascent RNA GILZ expression within 1 h of hormone treatment. Repression of glucocorticoid-induced GILZ expression requires the estrogen receptor (ER), because both treatment with the ER-antagonist ICI 182,780 and small interfering RNA knockdown of ERα block E(2)s ability to repress GILZ gene expression. Antagonism of glucocorticoid-induced GILZ expression may not be unique to ERα, as the ERβ agonist Liquiritigenin is also able to antagonize glucocorticoid signaling. Transcriptional regulation appears to be at the level of promoter binding. Both the glucocorticoid receptor and ERα are recruited to regions of the GILZ promoter containing glucocorticoid response elements and the transcriptional start site. Glucocorticoid receptor binding to these regions in the presence of dexamethasone decreases with E(2) treatment. GILZ gene expression was also found to be repressed in the whole mouse uterus treated with a combination of dexamethasone and E(2). Regulation of the antiinflammatory gene GILZ by glucocorticoids and E(2) suggests cross talk between the immune modulating functions of glucocorticoids and the reproductive actions of estradiol signaling.

Global Gene Expression Analysis in Human Uterine Epithelial Cells Defines New Targets of Glucocorticoid and Estradiol Antagonism

ABSTRACT In preparation for embryo implantation and pregnancy, the uterine epithelium undergoes a genomic and biological transition that mediates adhesion and invasion of the blastocyst. These events resemble an inflammatory response, and the immune system likely takes an active role in the establishment and maintenance of pregnancy. Although glucocorticoids are primary mediators of the immune system, the functional role of glucocorticoid signaling in the uterine epithelium is not well defined. To investigate the dynamic relationship between glucocorticoids and reproductive hormones, we performed whole-genome microarray analysis in a human uterine endometrial cancer cell line (ECC1 cells) treated with the synthetic glucocorticoid dexamethasone (Dex) alone or in combination with estradiol (E2). Over 10 000 genes were significantly regulated in the presence of Dex and/or E2. Surprisingly, unique targets of Dex and E2 together represented the largest group of regulated genes. Ingenuity pathway analysis found both overlapping and independent regulated networks for each hormone. Several hundred genes were found to be coregulated by Dex and E2, including several that were antagonistically regulated. The effects of glucocorticoids and E2 are mediated primarily through the glucocorticoid receptor (NR3C1) and estrogen receptor (ESR1), respectively. In silico promoter analysis revealed that NR3C1 and ESR1 response elements are enriched in antagonistically regulated genes, and signaling through these receptors was required for antagonism. Glucocorticoid and E2 antagonism of target genes may represent a critical junction between the immune system and female reproductive system. Moreover, identification and ontology analysis of glucocorticoid-regulated genes in a uterine epithelial-like cell line suggests that glucocorticoid signaling regulates important biological functions, including immune cell trafficking and embryonic development.

Glucocorticoids Regulate Gene Expression and Repress Cellular Proliferation in Human Uterine Leiomyoma Cells

Sex hormones and growth factors have been implicated in the pathogenesis of uterine leiomyomas. The uterus is also an abundant source of the glucocorticoid receptor but its role and function have been largely ignored. Human samples of uterine leiomyomas and matched myometrium retain expression of the glucocorticoid receptor (GR) suggesting a potential role for GR in leiomyoma function. However, hormone responsive gene expression varies between normal myometrium and leiomyoma cells. We now employ genome-wide microarray studies comparing glucocorticoid and estrogen-treated human uterine leiomyoma cells to those treated with both steroids to identify the potential role of glucocorticoids in uterine leiomyoma cells. Treatment with the synthetic glucocorticoid dexamethasone (Dex) regulated 3,128 probes. Estrogen (E2) treatment identified 2,094 probes, and in the presence of both hormones, 4,626 probes were regulated. Of the 552 probes identified, the majority of genes co-regulated by Dex, E2, and Dex + E2 exhibited co-downregulation. Interestingly, a small group of 17 genes displayed antagonistic regulation by Dex and E2, where all genes in this group, Dex reversed the E2 effect with. Ingenuity Pathway Analysis of the data identified cell growth, development, and differentiation as significant glucocorticoid regulated pathways. Flow cytometry confirmed that glucocorticoids regulated cell proliferation and significantly reduced the percentage of S-phase cells either in the presence or absence of estrogen in leiomyomas but not smooth muscle cells. Translation of our results suggest that glucocorticoids may play a significant role in regulating uterine leiomyoma gene expression and cell growth, and thus may have implications for therapeutic development of uterine leiomyoma treatment.

Uterine glucocorticoid receptors are critical for fertility in mice through control of embryo implantation and decidualization

Significance Glucocorticoids are primary stress and immune mediators of several fundamental processes, including reproduction and development. However, the direct actions of glucocorticoid signaling in the uterus are largely unknown. We have discovered that the glucocorticoid receptor (GR) in the mouse uterus is essential for normal fertility. Female mice lacking GR specifically in the uterus are subfertile due to defects in implantation related to inadequate remodeling of the endometrial stroma. Moreover, the deficiency in uterine glucocorticoid signaling leads to aberrant gene regulation during tissue remodeling events. Our findings demonstrate previously unrecognized functions of GR in both uterine physiology and fertility. In addition to the well-characterized role of the sex steroid receptors in fertility and reproduction, organs of the female reproductive tract are also regulated by the hypothalamic–pituitary–adrenal axis. These endocrine organs are sensitive to stress-mediated actions of glucocorticoids, and the mouse uterus contains high levels of the glucocorticoid receptor (GR). Although the presence of GR in the uterus is well established, uterine glucocorticoid signaling has been largely ignored in terms of its reproductive and/or immunomodulatory functions on fertility. To define the direct in vivo function of glucocorticoid signaling in adult uterine physiology, we generated a uterine-specific GR knockout (uterine GR KO) mouse using the PRcre mouse model. The uterine GR KO mice display a profound subfertile phenotype, including a significant delay to first litter and decreased pups per litter. Early defects in pregnancy are evident as reduced blastocyst implantation and subsequent defects in stromal cell decidualization, including decreased proliferation, aberrant apoptosis, and altered gene expression. The deficiency in uterine GR signaling resulted in an exaggerated inflammatory response to induced decidualization, including altered immune cell recruitment. These results demonstrate that GR is required to establish the necessary cellular context for maintaining normal uterine biology and fertility through the regulation of uterine-specific actions.

Glucocorticoids and Reproduction: Traffic Control on the Road to Reproduction

Glucocorticoids are steroid hormones that regulate diverse cellular functions and are essential to facilitate normal physiology. However, stress-induced levels of glucocorticoids result in several pathologies including profound reproductive dysfunction. Compelling new evidence indicates that glucocorticoids are crucial to the establishment and maintenance of reproductive function. The fertility-promoting or -inhibiting activity of glucocorticoids depends on timing, dose, and glucocorticoid responsiveness within a given tissue, which is mediated by the glucocorticoid receptor (GR). The GR gene and protein are subject to cellular processing, contributing to signaling diversity and providing a mechanism by which both physiological and stress-induced levels of glucocorticoids function in a cell-specific manner. Understanding how glucocorticoids regulate fertility and infertility may lead to novel approaches to the regulation of reproductive function.

Ghrelin Partially Protects Against Cisplatin-Induced Male Murine Gonadal Toxicity in a GHSR-1a-Dependent Manner

ABSTRACT The chemotherapeutic drug cisplatin causes a number of dose-dependent side effects, including cachexia and testicular damage. Patients receiving a high cumulative dose of cisplatin may develop permanent azoospermia and subsequent infertility. Thus, the development of chemotherapeutic regimens with the optimal postsurvival quality of life (fertility) is of high importance. This study tested the hypothesis that ghrelin administration can prevent or minimize cisplatin-induced testicular damage and cachexia. Ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR-1a), are expressed and function in the testis. Targeted deletion of ghrelin, or its receptor, significantly increases the rate of cell death in the testis, suggesting a protective role. Intraperitoneal administration of vehicle, ghrelin, or cisplatin alone or in combination with ghrelin, in cycles of 9 or 18 days, to adult male C57Bl/6 mice was performed. Body weight was measured daily and testicular and epididymal weight, sperm density and motility, testicular histology, and testicular cell death were analyzed at the time of euthanization. Ghrelin coadministration decreased the severity of cisplatin-induced cachexia and gonadal toxicity. Body, testicular, and epididymal weights significantly increased as testicular cell death decreased with ghrelin coadministration. The widespread damage to the seminiferous epithelium induced by cisplatin administration was less severe in mice simultaneously treated with ghrelin. Furthermore, ghrelin diminished the deleterious effects of cisplatin on testis and body weight homeostasis in wild-type but not Ghsr−/− mice, showing that ghrelins actions are mediated via GHSR. Ghrelin or more stable GHSR agonists potentially offer a novel therapeutic approach to minimize the testicular damage that occurs after gonadotoxin exposure.

Glucocorticoid Signaling in Health and Disease: Insights From Tissue-Specific GR Knockout Mice

Glucocorticoids are adrenally produced hormones critically involved in development, general physiology, and control of inflammation. Since their discovery, glucocorticoids have been widely used to treat a variety of inflammatory conditions. However, high doses or prolonged use leads to a number of side effects throughout the body, which preclude their clinical utility. The primary actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a transcription factor that regulates many complex signaling pathways. Although GR is nearly ubiquitous throughout the body, glucocorticoids exhibit cell- and tissue-specific effects. For example, glucocorticoids stimulate glucose production in the liver, reduce glucose uptake in the skeletal muscle, and decrease insulin secretion from the pancreatic β-cells. Mouse models represent an important approach to understanding the dynamic functions of GR signaling in normal physiology, disease, and resistance. In the absence of a viable GR null model, gene-targeting techniques utilizing promoter-driven recombination have provided an opportunity to characterize the tissue-specific actions of GR. The aim of the present review is to describe the organ systems in which GR has been conditionally deleted and summarize the functions ascribed to glucocorticoid action in those tissues.

Genistein disrupts glucocorticoid receptor signaling in human uterine endometrial Ishikawa cells.

Background: The link between environmental estrogen exposure and defects in the female reproductive tract is well established. The phytoestrogen genistein is able to modulate uterine estrogen receptor (ER) activity, and dietary exposure is associated with uterine pathologies. Regulation of stress and immune functions by the glucocorticoid receptor (GR) is also an integral part of maintaining reproductive tract function; disruption of GR signaling by genistein may also have a role in the adverse effects of genistein. Objective: We evaluated the transcriptional response to genistein in Ishikawa cells and investigated the effects of genistein on GR-mediated target genes. Methods: We used Ishikawa cells as a model system to identify novel targets of genistein and the synthetic glucocorticoid dexamethasone through whole genome microarray analysis. Common gene targets were defined and response patterns verified by quantitative real-time reverse-transcription polymerase chain reaction. The mechanism of transcriptional antagonism was determined for select genes. Results: Genistein regulated numerous genes in Ishikawa cells independently of estradiol, and the response to coadministration of genistein and dexamethasone was unique compared with the response to either estradiol or dexamethasone alone. Furthermore, genistein altered glucocorticoid regulation of GR target genes. In a select set of genes, co-regulation by dexamethasone and genistein was found to require both GR and ERα signaling, respectively. Conclusions: Using Ishikawa cells, we observed that exposure to genistein resulted in distinct changes in gene expression and unique differences in the GR transcriptome. Citation: Whirledge S, Senbanjo LT, Cidlowski JA. 2015. Genistein disrupts glucocorticoid receptor signaling in human uterine endometrial Ishikawa cells. Environ Health Perspect 123:80–87; http://dx.doi.org/10.1289/ehp.1408437

Stress and the HPA Axis: Balancing Homeostasis and Fertility

An organism’s reproductive fitness is sensitive to the environment, integrating cues of resource availability, ecological factors, and hazards within its habitat. Events that challenge the environment of an organism activate the central stress response system, which is primarily mediated by the hypothalamic–pituitary–adrenal (HPA) axis. The regulatory functions of the HPA axis govern the cardiovascular and metabolic system, immune functions, behavior, and reproduction. Activation of the HPA axis by various stressors primarily inhibits reproductive function and is able to alter fetal development, imparting a biological record of stress experienced in utero. Clinical studies and experimental data indicate that stress signaling can mediate these effects through direct actions in the brain, gonads, and embryonic tissues. This review focuses on the mechanisms by which stress activation of the HPA axis impacts fertility and fetal development.