Estradiol Signaling Research Articles

Chenodeoxycholic acid fortified diet drives ovarian steroidogenesis to improve embryo implantation through enhancing uterine receptivity via progesterone receptor signaling pathway in rats

Infertility is a worldwide reproductive health problem influenced by the embryo implantation efficiency. We previously revealed that dietary chenodeoxycholic acid (CDCA) positively influence the early embryo implantation. But how CDCA regulate embryo implantation is largely unexplored. Herein, we investigated the mechanism behind CDCA's regulation on embryo implantation in rats. Results showed that CDCA promoted uterine receptivity, leading to increased number of implantation sites. Mechanistically, CDCA reshaped maternal amino acid metabolism and enhanced serum progesterone levels. CDCA enhanced ovarian progesterone synthesis by improving steroidogenesis-related protein (StAR and CYP11A1) expression via Takeda G-protein-coupled receptor 5. Elevated progesterone exaggerated uterine progesterone but weakened the estradiol signaling in the CDCA group, contributing to better uterine receptive for embryo implantation. Additionally, elevated transcription repressor Stat5b induced the down-regulation of progesterone-metabolizing enzyme 20-hydroxysteroid dehydrogenase 20α-HSD, complementally explained uterine progesterone signaling enhancement. Overall, our data revealed that CDCA drove ovarian steroidogenesis to improve embryo implantation through enhancing uterine receptivity via progesterone receptor pathway in rats. Therefore, CDCA diet may be a potential favorable nutritional strategy for infertility and pregnancy management.

Local synthesis of estradiol in the rostral ventromedial medulla protects against widespread muscle pain in male mice.

Animal studies consistently demonstrate that testosterone is protective against pain in multiple models, including an animal model of activity-induced muscle pain. In this model, females develop widespread muscle hyperalgesia, and reducing testosterone levels in males results in widespread muscle hyperalgesia. Widespread pain is believed to be mediated by changes in the central nervous system, including the rostral ventromedial medulla (RVM). The enzyme that converts testosterone to estradiol, aromatase, is highly expressed in the RVM. Therefore, we hypothesized that testosterone is converted by aromatase to estradiol locally in the RVM to prevent development of widespread muscle hyperalgesia in male mice. This was tested through pharmacological inhibition of estrogen receptors (ER), aromatase, or ER-α in the RVM which resulted in contralateral hyperalgesia in male mice (C57BL/6J). ER inhibition in the RVM had no effect on hyperalgesia in female mice. As prior studies show modulation of estradiol signaling alters GABA receptor and transporter expression, we examined if removal of testosterone in males would decrease mRNA expression of GABA receptor subunits and vesicular GABA transporter (VGAT). However, there were no differences in mRNA expression of GABA receptor subunits of VGAT between gonadectomized and sham control males. Lastly, we used RNAscope to determine expression of ER-α in the RVM and show expression in inhibitory (VGAT+), serotonergic (tryptophan hydroxylase 2+), and μ-opioid receptor expressing (MOR+) cells. In conclusion, testosterone protects males from development of widespread hyperalgesia through aromatization to estradiol and activation of ER-α which is widely expressed in multiple cell types in the RVM.Significance Statement This study's findings reveal, for the first time, that testosterone protects males from development of widespread muscle hyperalgesia through aromatization to estradiol and activation of ER-α within the RVM. The findings also show, for the first time, ER-α expression in the RVM in male mice, and the distribution patterns of this expression among multiple cell types. Thus, together our data suggest a unique endogenous mechanism in the RVM for protection against widespread pain in males only.

Estrogen receptor beta signaling enhances extinction memory recall for heroin-conditioned cues in a sex- and region-specific manner

Return to use, or relapse, is a major challenge in the treatment of opioid use disorder (OUD). Relapse can be precipitated by several factors, including exposure to drug-conditioned cues. Identifying successful treatments to mitigate cue-induced relapse has been challenging, perhaps due to extinction memory recall (EMR) deficits. Previously, inhibition of estradiol (E2) signaling in the basolateral amygdala (BLA) impaired heroin-cue EMR. This effect was recapitulated by antagonism of BLA estrogen receptors (ER) in a sex-specific manner such that blocking ERα in males, but ERβ in females, impaired EMR. However, it is unclear whether increased E2 signaling, in the BLA or systemically, enhances heroin-cue EMR. We hypothesized that ERβ agonism would enhance heroin-cue EMR in a sex- and region-specific manner. To determine the capacity of E2 signaling to improve EMR, we pharmacologically manipulated ERβ across several translationally designed experiments. First, male and female rats acquired heroin or sucrose self-administration. Next, during a cued extinction session, we administered diarylpropionitrile (DPN, an ERβ agonist) and tested anxiety-like behavior on an open field. Subsequently, we assessed EMR in a cue-induced reinstatement test and, finally, measured ERβ expression in several brain regions. Across all experiments, females took more heroin and sucrose than males and had greater responses during heroin-cued extinction. Administration of DPN in the BLA enhanced EMR in females only, driven by ERβ’s impacts on memory consolidation. Interestingly, however, systemic DPN administration improved EMR for heroin cues in both sexes across several different tests, but did not impact sucrose-cue EMR. Immunohistochemical analysis of ERβ expression across several different brain regions showed that females only had greater expression of ERβ in the basal nucleus of the BLA. Here, in several preclinical experiments, we demonstrated that ERβ agonism enhances heroin-cue EMR and has potential utility in combatting cue-induced relapse.

FOXL2 regulates RhoA expression to change actin cytoskeleton rearrangement in granulosa cells of chicken pre-ovulatory follicles†.

Forkhead box L2 (FOXL2) is an indispensable key regulator of female follicular development, and it plays important roles in the morphogenesis, proliferation, and differentiation of follicle granulosa cells, such as establishing normal estradiol signaling and regulating steroid hormone synthesis. Nevertheless, the effects of FOXL2 on granulosa cell morphology and the underlying mechanism remain unknown. Using FOXL2 ChIP-seq analysis, we found that FOXL2 target genes were significantly enriched in the actin cytoskeleton-related pathways. We confirmed that FOXL2 inhibited the expression of RhoA, a key gene for actin cytoskeleton rearrangement, by binding to TCATCCATCTCT in RhoA promoter region. In addition, FOXL2 overexpression in granulosa cells induced the depolymerization of F-actin and disordered the actin filaments, resulting in a slowdown in the expansion of granulosa cells, while FOXL2 silencing inhibited F-actin depolymerization and stabilized the actin filaments, thereby accelerating granulosa cell expansion. RhoA/ROCK pathway inhibitor Y-27632 exhibited similar effects to FOXL2 overexpression, even reversed the actin polymerization in FOXL2 silencing granulosa cells. This study revealed for the first time that FOXL2 regulated granulosa cell actin cytoskeleton by RhoA/ROCK pathway, thus affecting granulosa cell expansion. Our findings provide new insights for constructing the regulatory network of FOXL2 and propose a potential mechanism for facilitating rapid follicle expansion, thereby laying a foundation for further understanding follicular development.

Systemic metabolic benefits of 17α-estradiol are not exclusively mediated by ERα in glutamatergic or GABAergic neurons.

17α-Estradiol (17αE2), a less-feminising enantiomer of 17β-estradiol, has been shown to prolong lifespan and improve metabolic health in a sex-specific manner in male, but not in female mice. Recent studies have demonstrated the pivotal role of estrogen receptor α (ERα) in mediating the effects of 17αE2 on metabolic health. However, the specific tissues and/or neuronal signalling pathways that 17αE2 acts through remain to be elucidated. ERα expression in glutamatergic and GABAergic neurons (principal excitatory and inhibitory neurons respectively) in the hypothalamus is essential for estradiol signalling. Therefore, we hypothesised that knocking out ERα from one of these neuronal populations would attenuate the established beneficial metabolic effects of 17αE2 in male mice exposed to a high fat diet. To test this hypothesis we used two established brain specific ERα KO models, targeting either glutamatergic or GABAergic neurons (Vglut2/Vgat-ERαKO). We show that both of these ERα KO models exhibit a strong reduction in ERα expression in the arcuate nucleus of the hypothalamus, a control centre for metabolic regulation. Deletion of ERα from GABAergic neurons significantly diminished the effect of 17αE2 on body weight relative to controls, although these animals still show metabolic benefits with 17αE2 treatment. The response to 17αE2 was unaffected by ERα deletion in glutamatergic neurons. Our results support a benefit of 17αE2 treatment in protection against metabolic dysfunction, but these effects do not depend on exclusive ERα expression in glutamatergic and GABAergic neurons and persist when ERα expression is strongly reduced in the arcuate nucleus of the hypothalamus.

Estradiol mediates greater germinal center responses to influenza vaccination in female than male mice.

Adult females of reproductive age develop greater antibody responses to inactivated influenza vaccines (IIV) than males. How sex, age, and sex steroid concentrations impact B cells and durability of IIV-induced immunity and protection over 4 months post-vaccination (mpv) was analyzed. Vaccinated adult females had greater germinal center B cell and plasmablast frequencies in lymphoid tissues, higher neutralizing antibody responses 1-4 mpv, and better protection against live H1N1 challenge than adult males. Aged mice, regardless of sex, had reduced B cell frequencies, less durable antibody responses, and inferior protection after challenge than adult mice, which correlated with diminished estradiol among aged females. To confirm that greater IIV-induced immunity was caused by sex hormones, four core genotype (FCG) mice were used, in which the testes-determining gene, Sry, was deleted from chromosome Y (ChrY) and transferred to Chr3 to separate gonadal sex (i.e., ovaries or testes) from sex chromosome complement (i.e., XX or XY complement). Vaccinated, gonadal female FCG mice (XXF and XYF) had greater numbers of B cells, higher antiviral antibody titers, and reduced pulmonary virus titers following live H1N1 challenge than gonadal FCG males (XYM and XXM). To establish that lower estradiol concentrations cause diminished immunity, adult and aged females received either a placebo or estradiol replacement therapy prior to IIV. Estradiol replacement significantly increased IIV-induced antibody responses and reduced morbidity after the H1N1 challenge among aged females. These data highlight that estradiol is a targetable mechanism mediating greater humoral immunity following vaccination among adult females.IMPORTANCEFemales of reproductive ages develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection against influenza were mediated by estradiol signaling in B cells. Using diverse mouse models ranging from advanced-age mice to transgenic mice that separate sex steroids from sex chromosome complement, those mice with greater concentrations of estradiol consistently had greater numbers of antibody-producing B cells in lymphoid tissue, higher antiviral antibody titers, and greater protection against live influenza virus challenge. Treatment of aged female mice with estradiol enhanced vaccine-induced immunity and protection against disease, suggesting that estradiol signaling in B cells is critical for improved vaccine outcomes in females.

Deciphering the Roles & Regulation of Estradiol Signaling during Female Mini-Puberty: Insights from Mouse Models.

Mini-puberty of infancy is a short developmental phase occurring in humans and other mammals after birth. In females, it corresponds to transient and robust activation of the hypothalamo-pituitary-ovarian (HPO) axis revealed by high levels of gonadotropin hormones, follicular growth, and increased estradiol production by the ovary. The roles of estradiol signaling during this intriguing developmental phase are not yet well known, but accumulating data support the idea that it aids in the implementation of reproductive function. This review aims to provide in-depth information on HPO activity during this particular developmental phase in several mammal species, including humans, and to propose emerging hypotheses on the putative effect of estradiol signaling on the development and function of organs involved in female reproduction.

Proteomic Analysis of Human Breast Cancer MCF-7 Cells to Identify Cellular Targets of the Anticancer Pigment OR3 from Streptomyces coelicolor JUACT03.

Search for ideal compounds with known pathways of anticancer mechanism is still a priority research focus for cancer, as it continues to be a major health challenge across the globe. Hence, in the present study, anticancer potential of a yellow pigment fraction, OR3, isolated from Streptomyces coelicolor JUACT03 was assessed on the breast cancer cell line MCF-7. TLC-fractionated OR3 pigment was subjected to HPLC and GC-MS analysis for characterization and identification of the bioactive component. MCF-7 cells were treated with IC50 concentration of OR3 and the molecular alterations were analyzed using mass spectrometry-based quantitative proteomic analysis. Bioinformatics tools such as STRING analysis and Ingenuity Pathway Analysis were performed to analyze proteomics data and to identify dysregulated signaling pathways. As per our obtained data, OR3 treatment decreased cell proliferation and induced apoptotic cell death due to significant dysregulation of protein expressions in MCF-7 cells. Altered expression included the ribosomal, mRNA processing and vesicle-mediated transport proteins as a result of OR3 treatment. Downregulation of MAPK proteins, NFkB, and estradiol signaling was identified in OR3-treated MCF-7 cells. Mainly eIF2, mTOR, and eIF4 signaling pathways were altered in OR3-treated cells. GC-MS data indicated the presence of novel compounds in OR3 fraction. It can be concluded that OR3 exhibits potent anticancer activity on the breast cancer cells mainly through altering the expression and affecting the signaling proteins which are involved in different cell proliferation/apoptotic pathways thereby causing inhibition of cancer cell proliferation, survival and metastasis.

Epigenetic Mechanisms of Brain Sexual Differentiation.

Across vertebrate species, gonadal hormones coordinate physiology with behavior to facilitate social interactions essential for reproduction and survival. In adulthood, these hormones activate neural circuits that regulate behaviors presenting differently in females and males, such as parenting and territorial aggression. Yet long before sex-typical behaviors emerge at puberty, transient hormone production during sensitive periods of neurodevelopment establish the circuits upon which adult hormones act. How transitory waves of early-life hormone signaling exert lasting effects on the brain remains a central question. Here we discuss how perinatal estradiol signaling organizes cellular and molecular sex differences in the rodent brain. We review classic anatomic studies revealing sex differences in cell number, volume, and neuronal projections, and consider how single-cell sequencing methods enable distinction between sex-biased cell-type abundance and gene expression. Finally, we highlight the recent discovery of a gene regulatory program activated by estrogen receptor α (ERα) following the perinatal hormone surge. A subset of this program displays sustained sex-biased gene expression and chromatin accessibility throughout the postnatal sensitive period, demonstrating a bona fide epigenetic mechanism. We propose that ERα-expressing neurons throughout the social behavior network use similar gene regulatory programs to coordinate brain sexual differentiation.

A Causal Link Between Disrupted AKT Signaling and Hyperproliferative Endometrial Diseases.

Most endometrial diseases are hyperproliferative and caused by excessive estradiol (E2) signaling, diminished progesterone (P4) signaling, or both. By example, endometriosis, adenomyosis, and type I endometrial cancer are all driven by excessive E2 exposure or aberrant estrogen receptor (ESR1) signaling. Endometriosis is also commonly associated with P4 resistance in which ectopic endometriotic lesions and eutopic endometrium have abnormally low expression of progesterone receptor (PGR). While it is well established that genomic events coordinate many of the most profound and well-studied sex steroid-initiated actions in the endometrium, activation of intracellular phosphorylation cascades, production of rapid calcium transients, changes to luminal epithelial cell height, and edema occur much too rapidly to be caused by the classical pathway, which involves transcription and translation. Work from several laboratories over the past 2 decades has revealed the importance of rapid acting steroid hormone signaling collectively called non-classical signaling. By example, P4 signals rapidly through PGR that is tethered to the plasma membrane by palmitoylate lipid moieties and this activates several phosphorylation cascades (1). Some non-classical E2 signaling events are thought to be mediated by membrane bound ESR1 (mESR1) and mESR2 (1). It is now clear that membrane bound forms of sex steroid receptors (ie, ESR1/2, PGR, androgen receptor) potentiate the actions of their soluble cytosolic/nuclear receptor equivalents through activation of phosphorylation cascades (1, 2). Such phosphorylation cascades result in selective phosphorylation of cytosolic/nuclear isoforms, and this in turn enhances/reduces protein-protein interactions with transcriptional co-activators/repressors to fine-tune genomic responses.

Estradiol Signaling at the Heart of Folliculogenesis: Its Potential Deregulation in Human Ovarian Pathologies.

Estradiol (E2) is a major hormone controlling women fertility, in particular folliculogenesis. This steroid, which is locally produced by granulosa cells (GC) within ovarian follicles, controls the development and selection of dominant preovulatory follicles. E2 effects rely on a complex set of nuclear and extra-nuclear signal transduction pathways principally triggered by its nuclear receptors, ERα and ERβ. These transcription factors are differentially expressed within follicles, with ERβ being the predominant ER in GC. Several ERβ splice isoforms have been identified and display specific structural features, which greatly complicates the nature of ERβ-mediated E2 signaling. This review aims at providing a concise overview of the main actions of E2 during follicular growth, maturation, and selection in human. It also describes the current understanding of the various roles of ERβ splice isoforms, especially their influence on cell fate. We finally discuss how E2 signaling deregulation could participate in two ovarian pathogeneses characterized by either a follicular arrest, as in polycystic ovary syndrome, or an excess of GC survival and proliferation, leading to granulosa cell tumors. This review emphasizes the need for further research to better understand the molecular basis of E2 signaling throughout folliculogenesis and to improve the efficiency of ovarian-related disease therapies.

Spatiotemporal AMPK\u03b12 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only

BackgroundThe AMP-activated protein kinase (AMPK) is a major regulator of cellular energetics which plays key role in acute metabolic response and in long-term adaptation to stress. Recent works have also suggested non-metabolic effects.MethodsTo decipher AMPK roles in the heart, we generated a cardio-specific inducible model of gene deletion of the main cardiac catalytic subunit of AMPK (Ampkα2) in mice. This allowed us to avoid the eventual impact of AMPK-KO in peripheral organs.ResultsCardio-specific Ampkα2 deficiency led to a progressive left ventricular systolic dysfunction and the development of cardiac fibrosis in males. We observed a reduction in complex I-driven respiration without change in mitochondrial mass or in vitro complex I activity, associated with a rearrangement of the cardiolipins and reduced integration of complex I into the electron transport chain supercomplexes. Strikingly, none of these defects were present in females. Interestingly, suppression of estradiol signaling by ovariectomy partially mimicked the male sensitivity to AMPK loss, notably the cardiac fibrosis and the rearrangement of cardiolipins, but not the cardiac function that remained protected.ConclusionOur results confirm the close link between AMPK and cardiac mitochondrial function, but also highlight links with cardiac fibrosis. Importantly, we show that AMPK is differently involved in these processes in males and females, which may have clinical implications for the use of AMPK activators in the treatment of heart failure.

17-β estradiol signalling affects cardiovascular and cancer pathogenesis by regulating the crosstalk between transcription factors and EC-miRNAs

17β-estradiol (E2) or the bioactive form of estrogen has wide range of impact in regulating human physiology. It not only regulates organ development but also controls immune responses. E2 impacts the recruitment of immune cells to endothelium by regulating the expression of pattern recognition receptors. As endothelium is central to human physiology, it becomes a good target for many cancer and cardiovascular diseases. A broad range of genes get dysregulated during the course of pathophysiological events and many of them are regulated via E2 signalling. Recent evidence shows that E2 regulate the expression of many endothelial cell specific microRNAs (EC-miRNA), thereby affecting expression and stability of many factors including estrogen receptor alpha. In this study, we analysed two distinct datasets of different endothelial cell models to discern a set of genes that potentially play very crucial role in regulating general physiological as well as pathophysiological events. GEO2R and Cytoscape were used to analyze the datasets GSE51535 and GSE16683. DAVID and ClueGO functional annotation of the genes revealed association with genetically associated disease classes such as cancer and cardiovascular. KEGG pathways, Reactome pathways and Wiki-pathways were used for pathway enrichment analysis. One of the enriched pathways includes the synthesis of pri-miRNA by RNA polymerase II. Endothelial cell specific miRNA and target gene association revealed key miRNAs and their involvement in regulatory pathways. Overall, our study highlights the complexity of miRNA mediated E2 regulation of genes in endothelial cells and provides potential target genes for therapeutics in cancer and cardiovascular pathophysiological conditions.

176-OR: STIM1 Deletion Leads to Loss of Beta-Cell Identity in High-Fat Diet-Fed Female Mice

STIM1 is an endoplasmic reticulum (ER) calcium (Ca2+) sensor that replenishes ER Ca2+ stores in a process known as store operated calcium entry (SOCE). Previously, we have shown that SOCE is impaired in the diabetic pancreatic β cell; however, the consequences of reduced β cell SOCE have not been well studied. To this end, mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ) were generated by crossing STIM1flox/flox with Ins-Cre mice and fed a high fat diet (HFD) beginning at 8 wk of age. Male STIM1Δβ mice did not show changes in glucose tolerance or body composition. In contrast, female HFD-fed STIM1Δβ mice were heavier (25.1 vs. 28.3 g, p<0.05), had increased fat mass (4.9 vs. 8.2g, P<0.01), and exhibited significant glucose intolerance (AUC IPGTT 22461 vs. 29245 mg/dL*min; p<0.0001) with impaired insulin secretion by in-vivo GSIS (p<0.05) without differences insulin tolerance, energy expenditure or food intake. Bulk RNA-sequencing of wild-type and STIM1Δβ female islets revealed reduced expression of MafA, UCN3, and Ins1 and Ins2 in STIM1Δβ islets, indicating potential loss of β cell identity. Pathway analysis showed modulation of pathways related to 17-beta estradiol (E2) signaling, with downregulation of a G-protein coupled estrogen receptor 1 (GPER), while differentially expressed genes were enriched for functions related to apoptosis, lipid metabolism, and epithelial cell differentiation. Consistently, STIM1Δβ female mice had lower β cell mass (2.984 mg vs. 1.349 mg, p<0.05) and higher alpha cell mass (0.1822 mg vs. 0.4463 mg, p<0.05). Proteomics analysis performed in STIM1-deficient INS-1 cells revealed significantly increased glucagon expression. Further, lower mobilization of intracellular cAMP levels was noted in response to treatment with E2 or G1, an agonist of GPER. These findings suggest a role for STIM1 in the maintenance of pancreatic β cell identity and indicate a potential interaction between SOCE and E2 signaling in female mice. Disclosure P. Sohn: None. P. Krishnan: None. C. Lee: None. T. Kono: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc. Funding National Institutes of Health (5T32DK064466-17, 5F30DK123996); U.S. Department of Veterans Affairs (2-I01BX001733); National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK093954-06A1); Indiana University School of Medicine; Gates Millennium Scholars Program

Conversion of Testosterone to Estradiol is Necessary for the Development of Long‐term Facilitation in Adult Male Rats

Phrenic long-term facilitation (pLTF) and ventilatory long-term facilitation (vLTF) are well-defined models of respiratory neuroplasticity. In each model, acute intermittent hypoxia (AIH) stimulates persistent increases in respiratory neural drive or minute ventilation respectively. Recent findings from our laboratory indicate that sex hormones are necessary for the induction of respiratory neuroplasticity in both male and female rats. For example, AIH induced pLTF and vLTF only develop during the proestrus phase of the estrous cycle in female rats, notable for high levels of circulating 17β-estradiol, the most neuroactive form of estrogen. Removal of the ovaries (ovariectomy, OVX), the primary source of circulating sex hormones in females, also eliminates the development of pLTF and vLTF. Plasticity can be restored in OVX females with estradiol supplementation supporting a key role for estradiol signaling in development of respiratory plasticity. Testosterone, the principle sex hormone in males, is also important for development of respiratory plasticity. Male rats demonstrate reduced pLTF magnitude with aging, in correlation with reduced circulating testosterone levels, and removal of the gonads (gonadectomy, GDX) eliminates AIH-induced pLTF. Testosterone replacement is sufficient to restore pLTF in GDX male rats, but requires the activity of aromatase, the enzyme responsible for converting testosterone to estradiol, suggesting that estradiol may also play a key role in development of respiratory neuroplasticity in male rats. It is unknown whether conversion of testosterone to estradiol is necessary for development of AIH-induced plasticity in gonadally-intact male rats. To address this question, we used systemic injections of Letrozole, an aromatase inhibitor, as a first step towards determining the necessity of testosterone conversion to estradiol for development of AIH-induced respiratory neuroplasticity in gonadally-intact adult male rats. We hypothesized that aromatization of testosterone to estradiol would be necessary for induction of both AIH-induced vLTF and pLTF. Letrozole (1mg/kg), or a vehicle control, was administered subcutaneously over four days. On day three, vLTF was measured via whole-body plethysmography. On the fourth day, pLTF was quantified via phrenic nerve recordings. Researchers were blinded to treatment for all experiments and data analysis. Preliminary findings suggest that daily Letrozole injections impaired development of AIH-induced vLTF and pLTF, supporting our hypothesis. These data provide further evidence that estradiol plays a critical role in the development of respiratory neuroplasticity in both males and females.

Arsenic-induced uterine apoptotic damage is protected by ethyl acetate fraction of Camellia sinensis (green tea) via Bcl-2-BAX through NF-κB regulations in Wistar rats.

The non-invasive treatment strategy is indispensable to overcome the side effects of conventional treatment with chelating agents against arsenic. Presence of catechins and flavonoids inCamellia sinensis have potential antioxidant properties and other beneficial effects. The aim of the study was to explore the curative potential role of Camellia sinensis against uterine damages produced by sodium arsenite in mature albino rats. A dose of 10 mg ofCamellia sinensis ethyl acetate (CS-EA) fraction/100 gm body weight was provided to the sodium arsenite-treated rats (10 mg/Kg body weight). LC-MS analysis was used for the detection of active component in CS-EA fraction. Enzymatic antioxidants analysis carried out by reproducible native gel technique. Hormones and some pro and anti-inflammatory markers were detected by ELISA, PCR, and western blot techniques respectively. Immunostaining was performed for the detection of estradiol receptor alpha. LC-MS analysis of CS-EA fraction ensured the presence of active tea polyphenol and tea catechin of which highest peak of epigallocatechin-3 gallate (EGCG) was obtainedin this study. Significant elevations of lipid peroxidation end products followed by the diminution of antioxidant enzymes activities werenoted in arsenicated rats which were capably retrieved by the treatmentof CS-EA fraction. Post-treatment with CS-EA fraction meaningfully improved gonadotrophins and estradiol signalling in association with a highly expressing estradiol receptor-α (ERα) in the ovary and uterus followed by the maintenance of normal utero-ovarian histoarchitecture in arsenic fed rats. CS-EA fractioned treated group overturned the sodium arsenite driven higher expression of pro-inflammatory cytokines and proapoptotic markers along with a low level of anti apoptotic Bcl-2 expression and comparatively lower NF-κB signalling in the uterus via regulating IKK β kinase mostly by EGCG of CS-EA fraction. However, ethyl acetate fractionof Camellia sinensis played a critical role in minimizing arsenic-mediated uterine hypo-function.

Transcriptome Analysis During Follicle Development in Turkey Hens With Low and High Egg Production.

Low and high egg producing hens exhibit gene expression differences related to ovarian steroidogenesis. High egg producing hens display increased expression of genes involved in progesterone and estradiol production, in the granulosa layer of the largest follicle (F1G) and small white follicles (SWF), respectively, whereas low egg producing hens display increased expression of genes related to progesterone and androgen production in the granulosa (F5G) and theca interna layer (F5I) of the fifth largest follicle, respectively. Transcriptome analysis was performed on F1G, F5G, F5I, and SWF samples from low and high egg producing hens to identify novel regulators of ovarian steroidogenesis. In total, 12,221 differentially expressed genes (DEGs) were identified between low and high egg producing hens across the four cell types examined. Pathway analysis implied differential regulation of the hypothalamo-pituitary-thyroid (HPT) axis, particularly thyroid hormone transporters and thyroid hormone receptors, and of estradiol signaling in low and high egg producing hens. The HPT axis showed up-regulation in high egg producing hens in less mature follicles but up-regulation in low egg producing hens in more mature follicles. Estradiol signaling exclusively exhibited up-regulation in high egg producing hens. Treatment of SWF cells from low and high egg producing hens with thyroid hormone in vitro decreased estradiol production in cells from high egg producing hens to the levels seen in cells from low egg producing hens, whereas thyroid hormone treatment did not impact estradiol production in cells from low egg producing hens. Transcriptome analysis of the major cell types involved in steroidogenesis inferred the involvement of the HPT axis and estradiol signaling in the regulation of differential steroid hormone production seen among hens with different egg production levels.

Dysfunctional Eating Behaviour and Leptin in Middle-Aged Women: Role of Menopause and a History of Anorexia Nervosa.

Weight gain is common as women approach mid-life. Reduced levels of leptin, an anorexigenic hormone, may facilitate this. Studies in middle-aged women with obesity have shown that dysfunctional eating behaviour, such as restrained eating, is linked to lower leptin. Furthermore, states of low oestradiol signalling, as are found in post-menopause or anorexia nervosa, have been found to impact leptin levels. The aim of this study was to investigate, for the first time, how different aspects of dysfunctional eating, menopausal status, and a history of anorexia nervosa relate to leptin levels in normal-weight middle-aged women. A total of N=57 women were recruited. Thirty-one were post-menopausal, and 27 had a history of anorexia nervosa. Dysfunctional eating behaviour was measured by the Three-Factor Eating Questionnaire, which contains three subscales: susceptibility/responsiveness to hunger, restraint, and disinhibition. Body composition was assessed by bioelectrical impedance analysis. A fasting blood sample was obtained to determine leptin. Controlling for age, body mass index, and fat mass, susceptibility/responsiveness to hunger was positively associated with leptin (β=0.267, p = 0.031), whereas restrained eating (β=-0.183, p=0.079) and a history of anorexia nervosa (β=-0.221, p=0.059) were, by trend, negatively associated with leptin. Neither disinhibited eating nor menopausal status was related to leptin. Leptin may decline as a response to repeated states of a negative energy balance. A possible implication is that mid-life weight management should avoid extreme changes in eating behaviour and instead focus on the macronutrient composition of diet and physical activity. Further, longitudinal enquiries are warranted to investigate these relationships.

Abstract PS19-06: Role of the calcium-sensing receptor (CaSR) in invasive lobular breast carcinoma metastasis to the ovary

Abstract Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, after the more common invasive ductal carcinoma (IDC). ILC accounts for 10 to 15% of all invasive breast carcinomas, affecting approx. 26,000-40,000 women in 2020 in the US alone. Despite 95% of ILC being Luminal A conferring patients a favorable prognosis, patients with ILC have poorer long-term outcomes when compared to patients with Luminal A IDC. ILC spread to common sites of ER+ breast cancer metastasis such as the bones, but are also three times more likely to spread to the ovaries, peritoneum, and gastrointestinal tract compared to IDC, and these unique aspects of metastases remain poorly understood. To better understand metastasis to the ovary we performed DNA and RNA sequencing of 11 pairs of primary breast tumor and ovarian metastasis as well as 13 orphan breast cancer ovarian metastases. Our cohort was enriched for lobular histology, with 13 samples originating from ILC, 6 from IDC and 6 from mixed ILC/IDC. We found mutations in CDH1 (43%), PIK3CA (40%), and FOXA1 (29%) highlighting the enrichment in ILC cases present in our cohort. Gene expression analysis lead to identification of 874 differentially expressed between primary tumors and ovarian metastases. We identified the calcium-sensing receptor CaSR as one of the top upregulated in ovarian metastases compared to primary tumor in 10 out of the 11 paired samples. Other genes highly expressed in ovarian metastases included TAC3, GLRA2, ALLC, MUC19, CHRNA2, PIP, CST4, and TEX15. Pathways analyses showed an enrichment of signaling through glutamate receptor and glycine receptor families. To assess the contribution of CaSR to breast cancer cell proliferation and metastatic properties, and due to the absence of breast cancer cell lines expressing CaSR, we generated CaSR overexpression models using lentiviral infection in MDA-MB-134, MDA-MB-330, BCK4 and SUM44PE ILC cell lines. While overexpression of CaSR alone did not confer a cell growth advantage or migratory ability to the cell lines, stimulation with calcium or a calcium mimetic resulted in enhanced migration in transwell assays in 3 of the 4 cell lines. Scratch assays further confirmed the stimulation of cell migration in cells with CaSR overexpression in the presence of calcium. Cell migration in CaSR overexpression models could be stimulated with the calcimimetic R568, and inhibited by the calcilytics NPS2143, and F-actin staining confirmed the need to activate the receptor to enhance migratory properties. Our studies further revealed that the induced migratory properties of CaSR overexpressing cells required estradiol and ER signaling, and that migration could be blocked with ER inhibitors such as ICI 182,780 and tamoxifen. Western blotting data revealed that the enhanced cell migratory properties of CaSR overexpressing cells was via activation of the MEK/ERK pathway and migration could be inhibited using specific small molecule pathway inhibitors.Altogether, our study provides insight on the potential mechanism by which upregulation of the CaSR supports breast cancer ovarian metastasis. We hope that these studies will not only deepen our understanding of ILC ovarian metastasis but will eventually lead to the development of more effective therapies and improve the outcome of patients with this understudied type of breast cancer. Citation Format: Ye Qin, Laura Savariau, Ahmed Mohammed Basudan, Osama Shah, Zheqi Li, Tiantong Liu, Nilgun Tasdemir, Lan Coffman, Esther Elishaev, Jennifer M Atkinson, Peter Lucas, Adrian V Lee, Steffi Oesterreich. Role of the calcium-sensing receptor (CaSR) in invasive lobular breast carcinoma metastasis to the ovary [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-06.

Norepinephrine Regulation of Ventromedial Hypothalamic Nucleus Astrocyte Glycogen Metabolism.

The catecholamine norepinephrine (NE) links hindbrain metabolic-sensory neurons with key glucostatic control structures in the brain, including the ventromedial hypothalamic nucleus (VMN). In the brain, the glycogen reserve is maintained within the astrocyte cell compartment as an alternative energy source to blood-derived glucose. VMN astrocytes are direct targets for metabolic stimulus-driven noradrenergic signaling due to their adrenergic receptor expression (AR). The current review discusses recent affirmative evidence that neuro-metabolic stability in the VMN may be shaped by NE influence on astrocyte glycogen metabolism and glycogen-derived substrate fuel supply. Noradrenergic modulation of estrogen receptor (ER) control of VMN glycogen phosphorylase (GP) isoform expression supports the interaction of catecholamine and estradiol signals in shaping the physiological stimulus-specific control of astrocyte glycogen mobilization. Sex-dimorphic NE control of glycogen synthase and GP brain versus muscle type proteins may be due, in part, to the dissimilar noradrenergic governance of astrocyte AR and ER variant profiles in males versus females. Forthcoming advances in the understanding of the molecular mechanistic framework for catecholamine stimulus integration with other regulatory inputs to VMN astrocytes will undoubtedly reveal useful new molecular targets in each sex for glycogen mediated defense of neuronal metabolic equilibrium during neuro-glucopenia.