Steroid Estrogens Research Articles

Methodological Recommendations for Menstrual Cycle Research in Sports and Exercise.

The aim of this review is to provide methodological recommendations for menstrual cycle research in exercise science and sports medicine based on a review of recent literature. Research in this area is growing but often reports conflicting results, and it is proposed that some of this may be explained by methodological issues. This review examined the menstrual cycle verification methods used in recent literature on exercise performance over the menstrual cycle identified through a literature search of PubMed and SportDiscus from 2008 until 2018. Potential changes over the menstrual cycle are likely related to hormone fluctuations; however, only 44% of the selected studies measured the actual concentrations of the female steroid hormones estrogen and progesterone. It was shown that the likely inclusion of participants with anovulatory or luteal phase-deficient cycles in combination with small participant numbers has affected results in recent menstrual cycle research and, consequently, our understanding of this area. To improve the quality of future menstrual cycle research, it is recommended that a combination of three methods is used to verify menstrual cycle phase: the calendar-based counting method combined with urinary luteinizing hormone surge testing and the measurement of serum estrogen and progesterone concentrations at the time of testing. A strict luteal phase verification limit of >16 nmol·L for progesterone should be set. It is also recommended that future research should focus on the inclusion of the late follicular estrogen peak. It is envisaged that these methodological recommendations will assist in clarifying some of the disagreement around the effects of the menstrual cycle on exercise performance and other aspects of exercise science and sports medicine.

Predictive role of spectral slope ratio towards 17α-ethynylestradiol photodegradation sensitized by humic acids

Humic acids (HAs) have been shown to dominate the photodegradation of steroid estrogens in natural waters. Nevertheless, how the photosensitizing ability of HAs relates to their structural and optical characteristics remains largely unknown. In this study, 17α-ethynylestradiol (EE2) was selected as a model compound to study to what extent easily-measurable characteristics of HAs might be used to predict their photosensitization potency. HAs were extracted from sediments of two different sources, and then subjected to structural and optical properties characterization using elemental analyzer, UV–vis spectroscopy and fluorescence spectroscopy. Photochemical experiments show that the HAs from the two sources can effectively meditate EE2 photodegradation. Although with drastically different structural and optical properties, the photosensitizing ability of these HAs towards EE2 can be well described by simple linear regressions using a spectroscopic index, the spectral slope ratio (SR). This optical indicator is correlated with various physicochemical properties of HAs, including the molecular weight, lignin content, charge-transfer interaction potential, photobleaching extent and sources. No universal prediction model could be established for predicting EE2 photodegradation kinetics on the basis of SR, but in specific waters SR could be a powerful indictor for predicting the EE2 photodegradation sensitized by HAs.

Abstract 2947: Discovering novel secondary drug combinations in sex hormone-related cancers using large-scale pharmacogenomics databases through computational modeling and molecular genetics

Abstract Response to drugs is heterogeneous - not all patients respond equally well to treatment and those who do often acquire resistance over the course of treatment. Therefore, predicting the efficacy of anti-cancer drugs and identifying secondary regimens to circumvent drug resistance is essential to prevent delay in the selection of more effective alternative strategies. Epidemiologic studies have demonstrated a tendency for common cancers to aggregate in families. For example, breast, prostate and ovarian cancers are sometimes grouped together owing to shared etiology, common genetic susceptibility regions and similar effects of sex hormones (estrogen or testosterone) exposure on their progression. Our objective is to use the vast array of human cancer cell lines in Genomics of Drug Sensitivity in Cancer (GDSC1000), the largest public database of drug sensitivity in human cancer cell lines - 265 anti-cancer drugs in 1074 cell lines (224,510 IC50 values), to apply a computational prediction algorithm that will identify successful secondary drug combinations in sex-hormone related cancers resistant to standard-of-care drugs. We used a greedy algorithm-based set-covering computational optimization method followed by a regularization technique to seek all secondary drugs that could kill maximum number of cancer cell lines resistant to the standard-of-care drug in a sequential manner ordered by the number of cell lines killed. A greedy algorithm constructs a solution to an optimization problem piece by piece through a sequence of choices to find the overall, or globally, optimal solution. For the purpose of this study, we used 136 potentially sex-hormone related cancer subtypes: breast (n=52), cervix (n=14), endometrium (n=11), ovary (n=45), prostate (n=8), testis (n=3), vulva (n=3). To validate our in silico prediction results, we treated the drug-resistant cancer cell lines with the predicted best secondary drugs, alone or as combination, and performed in vitro chemo-sensitivity assays to evaluate the effect of combination treatments using Chou-Talalay’s combination index (CI) method and the isobologram algorithm. Furthermore, we performed gene expression profiling (GEP) analysis using llumina’s next-generation sequencing system to identify differentially expressed (DE) genes and pathways underlining novel secondary therapies in chemotherapy-resistant cancers. Our preliminary data suggests promising early breakthrough and supports our hypothesis that the predicted drug combinations can circumvent drug resistance in human cancers - which we will be presenting at the annual meeting. This study is part of our unique research endeavor that aims to create a universally applicable software application for predicting novel secondary therapies in drug-resistant cancers for any cancer type and any test drug. Citation Format: Suman Mazumder, Li Chen, Ujjal K. Mukherjee, Amit K. Mitra. Discovering novel secondary drug combinations in sex hormone-related cancers using large-scale pharmacogenomics databases through computational modeling and molecular genetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2947.

Spontaneous changes in dissolved organic matter affect the bio-removal of steroid estrogens

Microbial action is the main pathway removing steroid estrogens (SEs) from both aerobic and anaerobic natural waters. The rate is influenced by other active substances present, particularly dissolved organic matter (DOM). DOM in natural surface waters has unstable components which undergo spontaneous photochemical oxidation, biological oxidation, chemical oxidation changes. How these changes influence the biosorption and bio-removal of SEs was the subject of this research. Photo oxidation-induced DOM increased the proportion of the fluorescence in area V, but biological oxidation and chemical oxidation caused fluorescence area V to decrease. All three oxidation processes can reduce the proportions of molecular weight (MW) > 5 kg·mol−1 and increase the proportions of MW < 5 kg·mol−1. Both the electron transfer capacity decreased monotonically with photo oxidation and chemical oxidation ageing, but biological oxidation ageing increased them. 17β-estradiol (E2) was the SEs used in the experiments. In aerobic conditions, fresh river humic acids (RHA) and aged RHA had the stronger mediating effect on the rate of E2 bio-removal under aerobic conditions. Its greater effectiveness was probably related to its binding with E2. Binding, biosorption of E2 and bio-removal of E2 were strongly positively correlated with the elemental C (R > 0.8, p ≤ 0.01) and SUVA254 (R > 0.8, p ≤ 0.01) by correlation matrix. Besides, fresh river fulvic acids (RFA) and aged RFA had the bigger mediating effect on E2 bio-removal under anaerobic conditions, and this imply that changes in aged DOM affected by other electron transfer groups in an anaerobic water environment. In anaerobic conditions, biosorption of E2 and binding action could cluster together with SUVA254, p(v), and 1 kg·mol−1 < MW < 5 kg·mol−1 by redundancy analysis, and but bio-removal of E2 could be well polymerized with EAC, EDC, p(iv), and MW > 5 kg·mol−1.

Enhanced adsorption of steroid estrogens by one-pot synthesized phenyl-modified mesoporous silica: Dependence on phenyl-organosilane precursors and pH condition

In this study, the phenyl-modified mesoporous materials were successfully synthesized using phenyl-organosilanes (trimethoxyphenylsilane and triethoxyphenylsilanea) by one-pot co-condensation method for the removal of estrone (E1), 17β-estradiol (E2), and 17α-ethinyl estradiol (EE2). Both the triethoxyphenylsilane-modified material (20%EtPh-MCM-41) and trimethoxyphenylsilane-modified material (20%MePh-MCM-41) could rapidly achieve equilibrium in 30 min at low adsorbent dosage of 0.025 g L−1. But the different hydrolysable groups of trimethoxyphenylsilane and triethoxyphenylsilane led to the discrepancies in physicochemical properties of the 20%EtPh-MCM-41 and 20%MePh-MCM-41, and thus affected adsorption performance. The 20%EtPh-MCM-41 exhibited the faster estrogen adsorption rates expressed in pseudo-second-order kinetic constant than the 20%MePh-MCM-41 due to the more hydrophobicity. Conversely, the 20%MePh-MCM-41 had much more estrogen adsorption capacities than the 20%EtPh-MCM-41 because of the more available adsorption sites. The addition of the phenyl-organosilane improved estrogen adsorption by π-π and hydrophobic interactions, and the Langmuir-model-based maximum adsorption amounts could reach 99.02, 83.47, and 53.60 mg g−1 for EE2, E2, and E1, respectively. But excessive concentration of phenyl-organosilane decreased adsorption capacities due to poor pore structure. Alkaline solution, which induced estrogen deprotonation and negative surface charge of absorbents, inhibited estrogen adsorption by electrostatic repulsion and the decreased hydrophobic interaction, but acidic and neutral solutions, ionic strength, and humic acid did not significantly affect estrogen removal. This work not only showed the high potential of trimethoxyphenylsilane-modified MCM-41 used in water purification for steroid estrogens, but also demonstrated the suitable selection of organosilane precursors was key in producing favorable materials with designed functionality.

Biomphalaria alexandrina: a model organism for assessing the endocrine disrupting effect of 17β-estradiol.

A wide range of endocrine disruptor compounds are routinely discharged to the ecosystem. Water contaminated with these compounds has a potential effect on the reproductive physiology of aquatic organisms as well as humans. In the present study, we tested the effect of the steroid estrogen, 17β-estradiol, on Biomphalaria alexandrina, a snail species that is widely distributed in Egypt and that acts as an intermediate host for the human blood fluke, Schistosoma mansoni. The effects of exposure to 0.3mg/L and 1mg/L 17β-estradiol on fecundity (MX) and reproductive rate (R0) of B. alexandrina were recorded. In addition, levels of steroid sex hormones and antioxidants in the hemolymph and ovotestis (OT) of exposed snails were measured. Histopathological changes in the OT of B. alexandrina were also investigated. Exposure to 0.3mg/L and 1mg/L 17β-estradiol caused a significant increase in the number of egg masses per snail after 3weeks and 1week of exposure for the two tested concentrations compared with unexposed controls. An increase in the levels of progesterone hormone was recorded in the hemolymph of exposed snails in comparison with unexposed controls. Additionally, levels of the antioxidant enzyme glutathione (GSH) were increased in the hemolymph and OT tissues of snails after 2 and 4weeks of exposure. Histopathological sections in the OT revealed an increase in the oocyte and a decrease in the sperm densities after 2weeks and this effect was restored to normal conditions after 4weeks of exposure to both tested concentrations. The current results indicate that B. alexandrina is sensitive to 17β-estradiol and can therefore be used as bioindicator and model organism for the assessment of water pollution with endocrine disruptor compounds.

Monitoring estrogenic activities of waste and surface waters using a novel in vivo zebrafish embryonic (EASZY) assay: Comparison with in vitro cell-based assays and determination of effect-based trigger values

This study reports the use of the recently developed EASZY assay that uses transgenic cyp19a1b-GFP zebrafish (Danio rerio) embryos to assess in vivo estrogenic activity of 33 surface (SW) and waste water (WW) samples collected across Europe that were previously well-characterized for estrogen hormones and in vitro estrogenic activity. We showed that 18 out of the 33 SW and WW samples induced estrogenic responses in the EASZY assay leading to a significant and concentration-dependent up-regulation of the ER-regulated cyp19a1b gene expression in the developing brain. The in vivo 17β-estradiol-equivalents (EEQs) were highly correlated with, both, the chemical analytical risk quotient (RQ) based on steroidal estrogen concentrations and EEQs reported from five different in vitro reporter gene assays. Regression analyses between the vitro and in vivo effect concentrations allowed us to determine an optimal cut-off value for each in vitro assay, above which in vivo responses were observed. These in vitro assay-specific effect-based trigger values (EBTs), ranging from 0.28 to 0.58 ng EEQ/L define the sensitivity and specificity of the individual in vitro assays for predicting a risk associated with substances acting through the same mode of action in water samples. Altogether, this study demonstrates the toxicological relevance of in vitro-based assessment of estrogenic activity and recommends the use of such in vitro/in vivo comparative approach to refine and validate EBTs for mechanism-based bioassays.

Effect of biochar amendment on sorption-desorption and dissipation of 17α‑ethinylestradiol in sandy loam and clay soils

Animal manure application in agricultural land has caused the release of steroid estrogens in the soil environment and further movement to aquatic systems. The objective of this study was to investigate the effects of biochar addition on sorption-desorption and dissipation behaviors of 17α‑ethinylestradiol (EE2) in two different textured soils. A Commerce sandy loam and a Shakey clay were selected and subjected to sterilization. Soil samples with and without sterilization were reacted with a series of EE2 solutions of different concentrations for sorption followed by desorption and quantification using HPLC-MS/MS. Long-term dissipation of EE2 in the same soils was also evaluated over a 30-d incubation. Biochar amendment increased the maximum EE2 sorption capacity but decreased its water desorption in both sandy loam and clay soils. On other hand, biochar addition increased the Koc in the clay soil which had low EE2 sorption efficiency but decreased Koc in the sandy loam which had high EE2 sorption efficiency. Biochar did significantly increase both desorbable and non-extractable fractions of EE2, while it reduced the bioavailability of EE2 to microbial degradation. The dissipation of EE2 in non-sterilized soils fit to the first-order kinetic model, whereas it was better described by zero-order kinetic for sterilized soil. Biochar increased the half-life of EE2 dissipation in non-sterilized Commerce sandy loam soil by 48% (from 3.63 to 5.37 d) and in non-sterilized Sharkey clay soil by 67% (from 2.28 to 3.81 d). Overall, this study demonstrated positive impacts of biochar on the retention of estrogen hormones in soils.

Arterial Smooth Muscle Cell PAQR7 Activation Attenuates Myogenic Tone In Pressurized Cerebral Arteries

Sex steroid hormones (estrogen, progesterone) are regulators of arterial contractility. Specifically, progesterone exerts rapid, nongenomic vasodilation via membrane receptors termed membrane progesterone receptors (mPR) belonging to the GPCR-like family of Progestin & Adiponectin Q Receptors (PAQR). Moreover, PAQR7 (mPRα) is reportedly expressed in smooth muscle cells (SMC) of human conduit vessels and has a putative role in vasodilation. However, the expression and function of PAQR7 in pressurized resistance size arteries, arteries that regulate local blood flow and blood pressure, have not been investigated. PURPOSE: To examine the expression and physiological function of PAQR7 in cerebral resistance size arteries. METHODS: RT-PCR was performed on whole cerebral arteries and isolated arterial SMCs to detect PAQR7 mRNA expression. Western blotting was performed on whole cerebral arteries to examine PAQR7 protein expression. Pressurized artery myography was performed on endothelium-intact and -denuded cerebral arteries to examine the role of SMC PAQR7 in vasodilation using the PAQR7-specific agonist, Org OD 02-0. RESULTS: PAQR7 mRNA (whole artery and isolated arterial SMCs) and protein were detected in resistance size cerebral arteries (n=4). Interestingly, there were no significant differences in vasodilation between endothelium-intact and -denuded arteries at any concentration (10-9M to 10-5M, n=4). CONCLUSION: These data suggest that arterial smooth muscle PAQR7 activation elicits vasodilation in pressurized resistance size arteries.

Group-Targeting Detection of Total Steroid Estrogen Using Surface-Enhanced Raman Spectroscopy.

Steroid estrogens, including 17β-estradiol (TE2), estrone (TE1), and ethinyl estradiol (TEE2), which are the strongest endocrine disruptors and coexist in the environment, seriously harm the health of organisms; thus, the monitoring of total steroid estrogens (TEs) has attracted growing attention. Herein, a method based on surface-enhanced Raman spectroscopy (SERS) group-targeting detection is established to detect TEs in natural water for the first time. The TEs response detection range and detection limit were 0.01-50 nM and 5 pM, respectively. An anti-interference ability was observed: even if coexisting interfering species were present in the system at 100-fold the concentration of estrogens, the detection error of the method was less than 0.276. In addition, the association constants between the aptamers and TE1, TE2, and TEE2 were similar, and therefore, the recognition of TE1, TE2, and TEE2 by the aptamers was consistent. Furthermore, the interaction sites A44, T72, and G69 between the aptamers and TE1, TE2, and TEE2 were investigated by molecular docking. On this basis, the estrogens in environmental water samples, including animal farm wastewater, maternity hospital wastewater, surface water from near an animal farm, and surface water from near a maternity hospital, were successfully determined.

EFFECT OF EXPOSURE TO ELECTROMAGNETIC RADIATION ON SEX STEROIDS AND SYSTEMIC &amp; LOCAL UTERINE REDOX STATUS DURING EARLY AND LATE PREGNANCY IN RATS

Background: pregnant women are at high risk of exposure to environmental EMR because of their higher oxygen consumption and amniotic fluid-induced ROS, which is associated with adverse pregnancy outcomes. Objective: The present study was designed to demonstrate the effects of EMR exposure at 900, 1800, and 2450 MHz on the female sex hormones (estrogen, progesterone) and systemic and local uterine redox status during early and late stages of pregnancy in adult albino rats. Design: The present study was carried out on 88 weaned albino female rats were divided into four equal groups: control group: rats were not exposed to EMR, group2: rats were exposed to 900 MHz EMR, group3: rats were exposed to 1800 MHz EMR (emitted from mobile phones), and group 4: rats were exposed to 2450 MHz EMR (emitted from Wi-Fi). After induction of pregnancy each group was subdivided into two equal groups: early pregnant and late pregnant subgroups. At the end of each experimental period samples were collected for estimation of serum estradiol & progesterone and plasma and local oxidative stress markers.Results: The present study showed that EMR exerted a significant frequency and duration dependent reduction in serum estrogen, progesterone, plasma TAS, vitamin C and uterine GSH which was accompanied by significant increase in plasma and uterine MDA. Conclusion: Exposure to EMR induced progressive reduction in sex steroid hormones, and imbalance in oxidative/antioxidative stress parameters in pregnant rats. These effects were dependent on the frequency and the duration of the EMR

The Role of Sexual Hormones on the Enteroinsular Axis.

Sex steroid estrogens, androgens, and progesterone, produced by the gonads, which have long been considered as endocrine glands, are implicated in sexual differentiation, puberty, and reproduction. However, the impact of sex hormones goes beyond these effects through their role on energy metabolism. Indeed, sex hormones are important physiological regulators of glucose homeostasis and, in particular, of the enteroinsular axis. In this review, we describe the roles of estrogens, androgens, and progesterone on glucose homeostasis through their effects on pancreatic α- and β-cells, as well as on enteroendocrine L-cells, and their implications in hormonal biosynthesis and secretion. The analysis of their mechanisms of action with the dissection of the receptors implicated in the several protective effects could provide some new aspects of the fine-tuning of hormonal secretion under the influence of the sex. This knowledge paves the way to the understanding of transgender physiology and new potential therapeutics in the field of type 2 diabetes.

A combined FSTRA-shotgun proteomics approach to identify molecular changes in zebrafish upon chemical exposure

The fish short-term reproduction assay (FSTRA) is a common in vivo screening assay for assessing endocrine effects of chemicals on reproduction in fish. However, the current reliance on measures such as egg number, plasma vitellogenin concentration and morphological changes to determine endocrine effects can lead to false labelling of chemicals with non-endocrine modes- of-action. Here, we integrated quantitative liver and gonad shotgun proteomics into the FSTRA in order to investigate the causal link between an endocrine mode-of-action and adverse effects assigned to the endocrine axis. Therefore, we analyzed the molecular effects of fadrozole-induced aromatase inhibition in zebrafish (Danio rerio). We observed a concentration-dependent decrease in fecundity, a reduction in plasma vitellogenin concentrations and a mild oocyte atresia with oocyte membrane folding in females. Consistent with these apical measures, proteomics revealed a significant dysregulation of proteins involved in steroid hormone secretion and estrogen stimulus in the female liver. In the ovary, the deregulation of estrogen synthesis and binding of sperm to zona pellucida were among the most significantly perturbed pathways. A significant deregulation of proteins targeting the transcriptional activity of estrogen receptor (esr1) was observed in male liver and testis. Our results support that organ- and sex-specific quantitative proteomics represent a promising tool for identifying early gene expression changes preceding chemical-induced adverse outcomes. These data can help to establish consistency in chemical classification and labelling.

MON-337 A Case Study: Steroid and Gonadotropin Hormones Secretion Pattern in Ovarian Cancer: 10-Year Follow-Up

Background: The ovaries in females maintain the health of the reproductive system. The ovaries secrete two main hormones, estrogen and progesterone. In ovarian cancer (Granulosa cell tumor) the secretion pattern of these hormones changes and few data are available that clearly describes the secretion of these hormones. Clinical case: In may 2007, in Port Harcourt, Rivers State, South of Nigeria, a 46years old woman was diagnosed with tumor of the ovaries (Granulosa cell tumor)), stage 11 after presenting features of ovarian cancer, that includes infertility and 9 months secondary amenorrhea The study investigated the sex hormones and gonadotropin hormones secretion pattern before the surgery and post surgery chemotherapeutic treatment for about 10years. Because of her interest in still getting pregnant, she resisted the surgery for about six months. Within this period the samples were collected thrice for the sex hormones (estrogen, progesterone, and testosterone), Inhibin B and gonadotropic hormones (FSH and LH). After Salpingo-oophorectomy was performed, the samples for the hormones were thereafter collected twice every year till August 2017 when she died at the age of 57years. Enzyme Linked Immunosorbent Assay (ELISA) method was used for the determination of the hormones. The result showed a Mean S.D serum estradiol level of 386.55 35.62 pg/ml, progesterone level of 3.26 0.44ng/ml, testosterone level of 0.62 0.24 ng/ml, Inhibin B = 145.90 20.05 pg/ml. The serum FSH was 1.95 0.84miu/ml, LH = 4.07 1.55 miu/l at preoperatomy. 3 months after the surgery, the sex hormones reduced significantly (P<0.05) by about 92.1%, the serum inhibin B reduced to 34.7pg/ml and the FSH increased by about 23.50% with no statistical difference in the values for LH, with a value of 2.97 miu/ml. The sex hormones secretion increased by 9.34% by the 8th to the 10th year before her death without any significant change in the gonadotropic hormones. The origin of the slight increase in the sex hormones secretion is unclear, but should be attributed to its secretion from other sources (Fat cells and adrenal) apart from the ovary. Conclusion: The hormone studies in a case of granulosa cell tumor of the ovary was established pre-and postoperatively. The sex hormones were elevated and gonadotropic hormones reduced significantly before surgery. Postoperative sex hormones secretion showed undetected levels, the slight increase in the sex hormone levels after about 6 years postoperative were not statistically significant (P>0.05).

SUN-192 Functional Interactions between Estrogen and Relaxin Signaling in the Myometrium

The complex interplay between the steroid hormones progesterone and estrogen (E2), acting through their cognate nuclear receptors, coordinates uterine myometrial contractility. Upon binding E2, estrogen receptor alpha (ERα) binds to regulatory elements across the genome, and promotes the assembly of transcriptional enhancers. The E2-induced ERα (i.e., cistrome) is plastic, and can be reconfigured in response to activation by other cellular signaling pathways. In addition to steroid hormones, the peptide hormone relaxin (Rln), acting via its G-protein coupled receptor, Rxfp1, has numerous, well described roles in the physiology of pregnancy. Interestingly, many of the effects of Rln require, or are augmented by E2 signaling through ERα. However, the extent, and molecular mechanisms by which cross-talk between E2 and Rln is achieved to ensure myometrial function is unknown. We hypothesized that Rln signaling in the myometrium alters or regulates a parturition-specific ERα cistrome that results in a cell-specific and dynamic altered transcriptome that controls downstream biological events. We used myometrial tissue from ovariectomized (ovx) mice treated with E2+Rln or either hormone alone, or from pregnant mice with or without a targeted deletion of Rxfp1. We generated high quality cistromic (ChIP-seq) and transcriptomic (RNA-seq) data sets in the ovx mouse model. The RNA-seq data sets have uncovered a unique transcriptome signature regulated by combined actions of E2 and Rln in the myometrium. In addition, we observed that many of the ERα binding sites were depleted in the presence of Rln, although histone modification associated with active enhancers (i.e., H3K27ac, and H3K4me1) were still enriched at those sites. The reduction in ERα binding is not explained by a reduction in ERα protein, as total ERα protein does not change across these treatments. In contrast, specific phosphorylation sites on ERα regulated by kinases were changed across these treatments. We also observed increased Erk1/2 activation with E2+Rln in the ovx mouse system compared to either hormone alone. Consistent with this observation, myometrial Erk1/2 activation is elevated in late pregnancy in wild type mice, but not in the Rxfp1 KO mouse. Current studies are underway to assess Erk1/2 signaling as a potential downstream mediator of Rln that alters the ERα activity, and also chromatin accessibility to direct the E2/ERa transcriptome signature for appropriate uterine function. This work is supported by theGreenCenterTrainingPrograminReproductiveBiologyScience Research.

SUN-004 Unique Cell Fates and Metastatic Phenotypes Are Activated by Progesterone Receptor (PR) Isoform-specific Gene Expression in Fallopian Tube Models of HGSC

High grade serous ovarian carcinoma (HGSC), the most prevalent and aggressive form of ovarian cancer, contains abundant receptors for the ovarian steroid hormones, estrogen (ER; 76%) and progesterone (PR; 35%). These receptors are known to contribute to breast and reproductive cancer development. Our understanding, however, of the mechanisms of PR isoform action in the initiation and progression of ovarian cancer is limited. HGSC is thought to originate from the fallopian tube epithelium (FTE), and accumulating evidence suggests that serous tubal intraepithelial carcinomas (STICs) are precursor lesions to most HGSCs. Our IHC analysis of normal FTE and STICs showed expression of both total PR and activated phopho-Ser294 PR (p-PR). Interestingly, STIC lesions had greater intensity of focal nuclear p-PR, indicative of highly active transcriptional complexes. To investigate PR isoform signaling in early stage HGSC, we utilized p53-dominant negative mutant FTE cells to generate stable cell lines expressing either the PR-A or PR-B isoform. Progestin (progesterone; R5020) treatment of 2D adherent cultures revealed functional PR signaling through MAPK-dependent p-PR as well as isoform specific expression of PR target genes encoding adhesion molecules (e.g. HEF1), cell cycle regulators (e.g. FOXO1), and glucocorticoid signaling proteins (e.g. CRISPLD2, NDRG1). Progestin treatment also dramatically inhibited proliferation which was recovered following growth factor stimulus, suggesting that PR signaling may promote cell quiescence (G0). To mimic dissemination of FTE cells from STICs, a 3D spheroid model was established. Interestingly, we observed that PR expression and activation greatly increased the formation, size and number of total spheroids compared to PR negative controls. Cells within these spheroids are predominantly Ki67-negative indicating that they are likely non-proliferative and potentially G0-arrested cells. When the spheroids were embedded into collagen, to mimic tumor cell invasion into the peritoneum, only PR+ spheroids exhibited invasive behavior. Additionally, PR-B+ spheroids were more invasive than PR-A+ spheroids; progestin treatment during 3D formation dramatically increased spheroid invasion. Notably, this is the opposite of our results in 2D cultures, where PR-A+ FTE cells display greater proliferative and migratory phenotypes relative to PR-B+ FTE cells. Taken together, our data suggest that activation of PR signaling promotes and enhances the formation and survival of non-adherent 3D FTE structures. Such effects of progesterone are predicted to influence the shedding, aggregation and dissemination of early STIC lesions that form ovarian and peritoneal metastases. Our findings demonstrate the importance of understanding the impact of steroid hormone receptors, including PR isoforms, on early ovarian cancer development.

14C-Labelling of the natural steroid estrogens 17α-estradiol, 17β-estradiol, and estrone.

The worldwide environmental occurrence of natural steroid estrogens has drawn increasing concerns. However, the fate of the estrogens, especially the α-isomer of estradiol, in the environmental matrices is still obscure. Using 14C-radioactively labelled forms of these estrogens can facilitate and is sometimes a prerequisite for studying their transformation and residual distribution in the environment, but the availability of labelled compounds (owing to commercially high prices or unavailable) hampers such studies. Here we developed simple and stable methods to synthesize 14C-labelled estradiol isomers and estrone using relatively low-priced [carboxyl-14C]-labelled sodium acetate as a precursor. The radiochemical syntheses started from an enol lactone, which was prepared from nandrolone by oxidation to open the A-ring followed by recyclization. Inversion of the 17β-hydroxyl group into its 17α-form was achieved via the Walden inversion using the Mitsunobu reaction. [3-14C]-17β-estradiol, [3-14C]-17α-estradiol, and [3-14C]-estrone were synthesized in five, six, and seven steps with an overall radiochemical yield of 17.4%, 16.2%, and 13.9%, respectively. The synthesized 14C-labelled compounds provide materials for studying the fate and behavior of estrogens in complex environmental matrixes and for further synthesis of their 14C-labelled sulfate and glucuronide conjugates.

Gamma amino-butyric acid and the control of GnRH secretion in sheep

The release of GnRH from nerve terminals in the median eminence into the portal vessels is influenced by factors in the internal and external environment of the animal. In the former category are the gonadal steroid hormones oestrogen and progesterone which alter the characteristics of GnRH secretion during the oestrous and seasonal cycles. These cannot exert their actions directly on the GnRH neurones as they do not possess hormone receptors. Therefore, some other steroid-sensitive neuronal system must relay this information to the GnRH neurones. Gamma amino-butyric acid (GABA) neurones are good candidates for this role as they contain steroid hormone receptors and synapse on GnRH neurones. Recent studies in ewes have sought to identify a role for GABA in mediating the actions of both oestrogen and progesterone on GnRH release. The technique of microdialysis was used to monitor GABA concentrations in areas containing GnRH cell bodies during the oestrogen-induced surge of GnRH and during progesterone negative feedback. Concentrations of this inhibitory neurotransmitter have been shown to fall in the former situation where GnRH release is being stimulated, but to be increased when progesterone is depressing GnRH release. GABA may also be important in mediating the seasonal switch in the negative feedback actions of oestradiol. During the anoestrous season, when oestradiol is a potent inhibitor of GnRH secretion, specific GABA receptor antagonists can stimulate neurohormone release, an action that is not observed in the breeding season when oestrogen is much less potent.

Integrated Assessment of Wastewater Reuse, Exposure Risk, and Fish Endocrine Disruption in the Shenandoah River Watershed.

Reuse of municipal and industrial wastewater treatment plant (WWTP) effluent is used to augment freshwater suppliesglobally. The Shenandoah River Watershed (U.S.A.) was selected to conduct on-site exposure experiments to assess endocrine disrupting characteristics of different source waters. This investigation integrates WWTP wastewater reuse modeling, hydrological and chemical characterization, and in vivo endocrine disruption bioassessment to assess contaminant sources, exposure pathways, and biological effects. The percentage of accumulated WWTP effluent in each river reach (ACCWW%) was used to predict environmental concentrations for consumer product chemicals (boron), pharmaceutical compounds (carbamazepine), and steroidal estrogens (estrone, 17-β-estradiol, estriol, and 17-α-ethinylestradiol). Fish endocrine disruption was evaluated using vitellogenin induction in adult male or larval fathead minnows. Water samples were analyzed for >500 inorganic and organic constituents to characterize the complex contaminant mixtures. Municipal ACCWW% at drinking water treatment plant surface water intakes ranged from <0.01 to 2.0% under mean-annual streamflow and up to 4.5% under mean-August streamflow. Measured and predicted environmental concentrations resulted in 17-β-estradiol equivalency quotients ranging from 0.002 to 5.0 ng L-1 indicating low-to-moderate risk of fish endocrine disruption. Results from the fish exposure experiments showed low (0.5- to 3.2-fold) vitellogenin induction in adult males.

Isoflavones.

Phytoestrogens are naturally occurring nonsteroidal phenolic plant compounds that, due to their molecular structure and size, resemble vertebrate steroids estrogens. This review is focused on plant flavonoids isoflavones, which are ranked among the most estrogenic compounds. The main dietary sources of isoflavones for humans are soybean and soybean products, which contain mainly daidzein and genistein. When they are consumed, they exert estrogenic and/or antiestrogenic effects. Isoflavones are considered chemoprotective and can be used as an alternative therapy for a wide range of hormonal disorders, including several cancer types, namely breast cancer and prostate cancer, cardiovascular diseases, osteoporosis, or menopausal symptoms. On the other hand, isoflavones may also be considered endocrine disruptors with possible negative influences on the state of health in a certain part of the population or on the environment. This review deals with isoflavone classification, structure, and occurrence, with their metabolism, biological, and health effects in humans and animals, and with their utilization and potential risks.