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The objective of this study was to examine the effect of 11 organochlorine pesticides on human and rat 17β-Hydroxysteroid dehydrogenase 1 (17β-HSD1) in human placental and rat ovarian microsome and on estradiol production in BeWo cells. The results showed that the IC 50 values for endosulfan, fenhexamid, chlordecone, and rhothane on human 17β-HSD1 were 21.37, 73.25, 92.80, and 117.69 μM. Kinetic analysis revealed that endosulfan acts as a competitive inhibitor, fenhexamid as a mixed/competitive inhibitor, chlordecone and rhothane as a mixed/uncompetitive inhibitor. In BeWo cells, all insecticides except endosulfan significantly decreased estradiol production at 100 μM. For rats, the IC 50 values for dimethomorph, fenhexamid, and chlordecone were 11.98, 36.92, and 109.14 μM. Dimethomorph acts as a mixed inhibitor, while fenhexamid acts as a mixed/competitive inhibitor. Docking analysis revealed that endosulfan and fenhexamid bind to the steroid-binding site of human 17β-HSD1. On the other hand, chlordecone and rhothane binds to a different site other than the steroid and NADPH-binding site. Dimethomorph binds to the steroid/NADPH binding site, and fenhexamid binds to the steroid binding site of rat 17β-HSD1. Bivariate correlation analysis showed a positive correlation between IC 50 values and LogP for human 17β-HSD1, while a slight negative correlation was observed between IC 50 values and the number of HBA. ADMET analysis provided insights into the toxicokinetics and toxicity of organochlorine pesticides. In conclusion, this study identified the inhibitory effects of 3–4 organochlorine pesticides and binding mechanisms on human and rat 17β-HSD1, as well as their impact on hormone production. [Display omitted] • Endosulfan inhibits human 17β-HSD1. • Endosulfan binds to steroid-bind site of human 17β-HSD1. • Endosulfan is a competitive inhibitor of human 17β-HSD1. • Species-dependent inhibition of organochlorines on human and rat 17β-HSD1. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) describes a group of clinically common autoimmune diseases characterized by chronic intestinal inflammation, with gender differences in prevalence. Estrogen has been previously shown to exert anti-inflammatory action in IBD development, however, the mechanisms remain obscure. Recent research has revealed that myeloid-derived suppressor cells (MDSCs) play a protective role in IBD pathogenesis. To investigate the molecular mechanisms of estrogen steroid 17β-estradiol (E2) in IBD progression, we established IBD mouse models (DNB-induced) with or without prior ovariectomy (OVX) and E2 implantation. We found that OVX led to worse IBD symptoms and reduced MDSCs frequency, whereas E2 significantly alleviated these effects in vivo. Moreover, in vitro experiments showed that E2 promoted the proliferation and immunosuppressive function of MDSCs through phosphorylation of Stat3 and p65. Mechanistically, E2-mediated Stat3/p65 phosphorylation depends on the interaction between HOTAIR, a long non-coding RNA that are well-known in MDSCs proliferation, and Stat3/p65 respectively. In conclusion, our study revealed that E2 promotes the expansion and immunosuppressive function of MDSCs, and thus diminished the occurrence and development of IBD. ● We found that estrogen can alleviate the development of IBD. ● We demonstrated that estrogen promots MDSCs expansion and function by upregulates STAT3, NF-κB, and iNOS via lncRNA HOTAIR. ● We showed new molecular mechanisms on estrogen slows IBD progression via MDSCs, which provides clinical implications for IBD. [ABSTRACT FROM AUTHOR]

Estrogens regulate important processes in reproductive, skeletal, cardiovascular, and central nervous systems that impact women's overall health. Understanding endogenous and exogenously administered estrogen metabolism is vital to determining therapeutic estrogen levels. The present review provides an overview of estrogen metabolites formed in non-pregnant and pregnant women, and those resulting from exogenous estrogen administration. There are four principal endogenous estrogens: estrone (E 1), estradiol (E 2), estriol (E 3), and estetrol (E 4). E 4 , which is produced only in pregnancy, has emerged recently as an estrogen with significant therapeutic potential. E 1 , E 2 , and E 3 undergo extensive metabolism primarily through phase I (hydroxylation, oxidation, reduction) and phase II (primarily conjugation) reactions, whereas E 4 undergoes only phase II reactions. Exogenous estrogens commonly used for menopausal treatment and/or contraception, including micronized E 2 , conjugated equine estrogens, and ethinyl estradiol, also undergo phase I and phase II reactions, but differ widely in the types of metabolites formed. The mechanisms by which estrogen metabolites are formed and their excretion in urine, bile, and feces, are still poorly understood. We highlight areas that require further research to foster a better understanding of how estrogen metabolism impacts dosing of oral estrogens for therapeutic use, as well as the physiological regulation of endogenous estrogens. • Estrogens play an important role in health and disease. • Understanding metabolism is vital to determine therapeutic estrogen levels. • E 1 , E 2 , E 3 , conjugated equine estrogens, ethinyl estradiol undergo extensive metabolism. • Estetrol undergoes a distinctive metabolic pathway. • Regulation of these metabolic processes is poorly understood. [ABSTRACT FROM AUTHOR]

Endometriosis was claimed to negatively affect the intrafollicular environment, hindering oocyte competence. Previous studies evaluated expression levels of cytochrome P450 aromatase (CYP19A) in granulosa and cumulus oophorus cells collected from endometriosis women, but results are controversial. To further investigate the intrafollicular environment whose alteration may potentially disturb ovarian steroidogenesis in endometriosis, gene expression of CYP19A and of its upstream enzymes, StAR and 3βHSD was assessed in luteinized granulosa cells isolated from follicular fluids (FF) collected during Assisted Reproduction Technology (ART) procedures in women with stage III-IV disease and from subjects without the condition. In a subgroup of patients, cumulus oophorus cells (COCs) were also assessed for CYP19A, StAR and 3βHSD gene expression. No difference in mRNA expression of CYP19A1 , StAR and 3βHSD in both granulosa cells and COCs was observed between the two groups of patients. No significant difference was also found between estradiol FF levels detected in endometriosis patients (median=873, IQR=522–1221 ng/ml)) and control patients (median=878, IQR=609–1137 ng/ml). To gain more insight into the intrafollicular regulation of CYP19A in patients with endometriosis, associations between expression of the analyzed genes, systemic and follicular 17β-estradiol levels and ART outcomes were assessed. While in the control group, levels of CYP19A1, StAR and 3βHSD transcripts significantly correlated with follicular estradiol levels (adjusted R² of 0.60), no significant association was detected in affected women (adjusted R² of 0.23). After stratification of the populations based on the presence of the disease, CYP19A1 expression was shown to correlate with the number of oocytes retrieved [β:− 1.214;95%CI: − 2.085 - (−0.343); p = 0.007] in the control group while this association was not present in patients with endometriosis [β:− 0.003; 95%CI:− 0.468–0.461; p = 0.988)]. These results do not support data from the literature indicating a reduced aromatase expression in granulosa cells of affected women, but they highlight a potential subtle mechanism affecting the ovulation process in these women. • No gene expression change in ovarian steroidogenic enzymes for endometriosis. • No estradiol follicular fluid level differences in endometriosis vs. control patients. • CYP19A1 expression correlated with oocytes retrieved in control, not in endometriosis. [ABSTRACT FROM AUTHOR]

Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish gper1 and gper1-like genes suggest their duplication may have not resulted from the teleost-specific whole genome duplication. We confirmed that both sbsGper isoforms activate the cAMP signalling pathway and respond differentially to distinct estrogenic compounds. Therefore, as observed for nuclear estrogen receptors, both sbsGpers duplicates retain estrogenic activity although they differ in their specificity and potency (Gper1 being more potent and more specific than Gper1-like), suggesting a more conserved role for Gper1 than for Gper1-like. In addition, Gpers were able to respond to estrogenic environmental pollutants known to interfere with estrogen signalling, such as the phytoestrogen genistein and the anti-depressant fluoxetine, a point that can be taken into account in aquatic environment pollution screenings and chemical risk assessment, complementing previous assays for sea bass nuclear estrogen receptors. • Fish Gper duplicates did not originate from the TSGD. • gper1-like sequences are less conserved among fish species than gper1 genes. • Both seabass Gper1 and Gper1-like are functional, activated by estrogens and pollutants. • Different ligand activation suggests different roles between sbsGper1 and sbsGper1-like. • In vitro testing of Esr/Gpers activation pathways is suggested for chemical hazard assessment. [ABSTRACT FROM AUTHOR]

Aldosterone synthase (CYP11B2) represents a promising drug target because its genetic dysregulation is causally associated with cardiovascular disease, its autonomous activity leads to primary aldosteronism, and its deficiency leads to salt wasting syndromes. The serendipitous discovery that the dextro-rotatory stereoisomer of the racemic aromatase (CYP19A1) inhibitor CGS16949A mediates potent CYP11B2 inhibition led to the purification and clinical development of dexfadrostat phosphate. To characterize the pharmacophore of dexfadrostat phosphate, structure-based enzyme coordination with CYP11B2, CYP11B1 and CYP19A1 was combined with steroid turnover upon in vitro and clinical treatment. Dexfadrostat, but not its 5S-enantiomer (5S-fadrozole), precisely coordinates with the catalytic heme moiety in the space of the CYP11B2 substrate binding pocket forming a tight and stable complex. Conversely, neither rigid nor flexible docking led to a plausible coordination geometry for dexfadrostat in steroid 11β-hydroxylase (CYP11B1 – orthologue to CYP11B2) or in CYP19A1. The inhibitory preference of dexfadrostat was confirmed in vitro using an adrenal cortex-derived cell line. Dexfadrostat phosphate treatment of healthy subjects in the context of a clinical phase 1 study led to a dose-dependent decrease in urinary aldosterone secretion, accompanied by an increase in urinary corticosterone and deoxycorticosterone metabolites. Increased urinary corticosterone metabolites are indicative of CYP11B2 (18-oxidase) inhibition with clinical features reminiscent of patients with inborn corticosterone methyloxidase type II deficiency. An off-target effect on CYP19A1 was not observed as indicated by no clinical changes in testosterone and estradiol levels. Therefore, dexfadrostat exhibits the ideal structural features for binding and catalytic inhibition of CYP11B2 but not CYP11B1. Clinically, treatment with dexfadrostat phosphate leads to suppression of aldosterone levels by inhibiting predominantly one or both final CYP11B2-mediated reactions. • Aldosterone synthase inhibition is a new strategy to control hyperaldosteronism. • Dexfadrostat aligns with the catalytic moiety of CYP11B2, but not CYP11B1 or CYP19A1. • In vitro and clinically, dexfadrostat inhibits CYP11B2 but not CYP11B1 or CYP19A1. • Inhibiting the final CYP11B2 reactions leads to dose-dependent aldosterone reduction. [ABSTRACT FROM AUTHOR]

Estetrol (E 4) has emerged as a novel and highly promising estrogen for therapeutic use. E 4 is a weak natural estrogen produced only in pregnancy. Because of its novelty, there is considerable interest by clinicians in how it is produced in pregnancy. Although the fetal liver plays a key role in its production, the placenta is also involved. A current view is that estradiol (E 2) formed in the placenta enters the fetal compartment and is then rapidly sulfated. E 2 sulfate then undergoes 15α-/16α-hydroxylation in the fetal liver thereby forming E 4 sulfate (phenolic pathway). However, another pathway involving 15α,16α-dihydroxy-DHEAS formed in the fetal liver and converted to E 4 in the placenta also plays a significant role (neutral pathway). It is not known which pathway predominates, but both pathways appear to be important in E 4 biosynthesis. In this commentary, we summarize the well-established pathways in the formation of estrogens in the nonpregnant and pregnant female. We then review what is known about the biosynthesis of E 4 and describe the 2 proposed pathways involving the fetus and placenta. • Estetrol (E 4) biosynthesis in pregnancy is usually attributed to a phenolic pathway. • This pathway involves E 4 formation in fetal liver from placental estradiol. • A neutral pathway contributes significantly to E 4 biosynthesis as well. • It involves placental aromatization of fetal 15α-/16α-hydroxylated androgens. [ABSTRACT FROM AUTHOR]

• Continuous administration of a COC, composed of EE and DRSP, causes hyperinsulinemia in female mice. • This effect was due to the chronic actions of EE and DRSP on pancreatic β-cells, ledding to insulin hypersecretion. • COC-treated mice had reduced insulin clearance that contributed to IR downregulation in the liver. • Our study may provide a warning that the prolongation of COC treatment may cause insulin resistance. Oral contraception is the most commonly used interventional method in the world. However, several women employ the continuous use of these hormones to avoid pre- and menstruation discomforts. Some studies indicate that oral contraceptives are associated with disturbances in glycemia and the effects of the use of a continuous regime are poorly elucidated. Herein, we evaluated the effects of the continuous administration of a combined oral contraceptive (COC) composed by ethinyl estradiol (EE) and drospirenone (DRSP) on glucose homeostasis in female mice. Adult Swiss mice received 0.6 μg EE and 60 μg DRSP (COC group) or vehicle [control (CTL)] daily by gavage for 35 days. COC treatment had no effect on body weight or adiposity, but increased uterus weight and induced hepatomegaly. Importantly, COC females displayed normal glycemia and glucose tolerance, but hyperinsulinemia and lower plasma C-peptide/insulin ratio, indicating reduced insulin clearance. Furthermore, COC mice displayed reduced protein content of the β subunit of the insulin receptor (IRβ) in the liver. Additionally, pancreatic islets isolated from COC mice secreted more insulin in response to increasing glucose concentrations. This effect was associated with the activity of steroid hormones, since IN S-1 E cells incubated with EE plus DRSP also secreted more insulin. Therefore, we provide the first evidence that the continuous administration of EE and DRSP lead to hyperinsulinemia, due to enhancement of insulin secretion and the reduction of insulin degradation, which possibly lead to the down-regulation of hepatic IRβ. These findings suggest that the continuous administration of COC could cause insulin resistance with the prolongation of treatment. [ABSTRACT FROM AUTHOR]

• ERα, ERβ1, ERβ2 and GPER are all involved in the regulation of vitellogenesis. • ERα is the central factor, acting interactively with ERβ1, ERβ2 and GPER in zebrafish. • GPER gets involved in vitellogenesis directly and interactively with ERα, ERβ1 and ERβ2 in zebrafish. Estrogens exert their biological functions through the estrogen receptors (ERs). In zebrafish, three nuclear estrogen receptors (nERs) named ERα, ERβ1 and ERβ2 and one membrane-bound G protein-coupled estrogen receptor (GPER) are identified. Vitellogenin (Vtg) is predominantly expressed in liver and strongly response to the stimulation of estrogen. It has been proposed that all three nERs are functionally involved in vitellogenesis and ERα may act as the major mediator in teleost. However, the role of GPER and its interaction with nERs in this process are not yet defined in teleost species. In the present study, we provide genetic evidence for the functional significance of ERα that the expression of Vtg genes (vtg1 , vtg2 , vtg3) and their response to estradiol stimulation were significantly decreased in esr1 mutant zebrafish. Activation of ERβ1 and ERβ2 induced Vtg expression through ERα. Moreover, the involvement of GPER in vitellogenesis and its interaction with nERs in zebrafish were firstly proposed in this work. Activation of GPER induced Vtg genes expression while inhibition of GPER significantly attenuated the estrogenic effect on Vtg. Both treatments altered the expression levels of nERs, suggesting GPER acts interactively with nERs. Collectively, the involvement of both nERs and GPER in regulation of vitellogenesis is demonstrated. ERα is the central factor, acting interactively with ERβ1, ERβ2 and GPER, and GPER regulates vitellogenesis directly and interactively with nERs. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • 17β-estradiol accelerates the process of oocyte meiosis resumption via GPR30. • 17β-estradiol-GPR30 mediated meiosis resumption ascribe downregulation of gap junction permeability in goat COCs. • GPR30-mediated gap junction permeability downregulation resulted from GPR30-MAP kinase-dependent phosphorylation of Cx43. Abstract Estrogen plays a critical role in the regulation of gap junctions between oocytes and granulosa cells in mammalian ovaries. G protein-coupled receptor 30 (GPR30) was identified as a membrane estrogen receptor, mediating rapid, nongenomic signaling events that might be responsible for the regulation of oocyte meiosis resumption and gap junction intercellular communications (GJICs). The present study aimed to determine the expression and localization of GPR30 and its role in oocyte meiotic progression and GJICs in goat cumulus-oocyte complexes (COCs). Immunofluorescence experiments revealed that GPR30 was primarily located in the plasma membrane of cumulus cells and oocytes in goats. 17β-estradiol could promote oocyte meiotic progression, which was blocked by G15 (a selective GPR30 antagonist) but not ICI182780 (a nuclear estrogen receptor inhibitor) in the early stage of in vitro culture. The effect of 17β-estradiol on the GJICs was quantified by lucifer yellow (LY) microinjection and calcein-AM fluorescent dye diffusion. 17β-estradiol treatment of goat COCs resulted in rapid downregulation of GJICs. The transfer of calcein from cumulus cells to oocytes could be significantly inhibited by carbenoxolone (a known gap junction blocker), 17β-estradiol or G1 (a GPR30 agonist), and this inhibition could be reversed by G15 but not ICI182780, indicating that GPR30 mediates the effect of 17β-estradiol on the rapid downregulation of GJICs. 17β-estradiol also stimulated the serine 368 phosphorylation of connexin 43 (Cx43) when COCs were in vitro cultured for 4 h, 6 h, and 8 h. More importantly, 17β-estradiol or G1 could separately promote the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and Cx43 significantly when COCs were cultured for 4 h. Furthermore, both ERK1/2 and Cx43 phosphorylation could be inhibited by G15 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or by the ERK1/2 inhibitor PD98059, indicating that EGFR-ERK1/2 signaling was involved in these events. These results supported the hypothesis that GPR30 mediated 17β-estradiol-stimulated meiotic resumption and GJIC reduction in goat COCs. Thus, the present study provides novel insights into elucidating the mechanisms for steroid hormone action in the regulation of oocyte maturation. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Chitosan-nanoconjugated eurycomanone nanoparticles were fabricated and characterized. • Short-term and long-term experiments were conducted to evaluate the effect of nano-formulation on reproductive output in catfish. • High and sustained mRNA expression was detected in the chitosan nanoconjugated treatments. • The effect of chitosan conjugated eurycomanone was also evaluated in the primary cell culture. Abstract The study was undertaken to explore the molecular mechanism of eurycomanone, a major compound of Eurycoma longifolia plant in increasing the reproductive processes in the male fish model. Chitosan-nanoconjugated eurycomanone nanoparticles with a significant particle size [130 nm (CED1); 144.1 nm (CED2)] and stable zeta potentials (+49.1 mV and +30 mV) were synthesized and evaluated against naked eurycomanone (ED1 and ED2). In present study, short-term and long-term experiments were conducted to evaluate the effect of nano-formulation on expression of endocrine-related genes, circulating hormone concentrations (Follicle stimulating hormone, FSH; luteinizing hormone, LH; progesterone, testosterone and 17-β estradiol) and reproductive capacity of male Clarias magur. In short-term experiment, the sampling of tissues was done on hourly basis after injection of eurycomanone either alone or with chitosan and long-term experiment was carried for 21 days and in this the injection was repeated after 7 days and 14 days. Treatments CED1 and CED2 showed controlled and sustained surge of the transcript level of selected genes (except aromatase) and serum hormones (except 17β-estradiol) compared to ED1 and ED2 groups. The transcript levels of aromatase and serum 17β-estradiol hormone showed the declining trend in the chitosan conjugated groups. The gonadosomatic index (GSI), reproductive capacity, intracellular calcium and selenium and cellular structure of testes were improved in CED1 and CED2 groups compared to other treatments. Furthermore, the effect of chitosan conjugated eurycomanone was evaluated in primary testicular cells and an increase in the mRNA expression level of endocrine-related genes was detected. This is the first report of the use of chitosan conjugated eurycomanone and present study elucidates the molecular mechanism of eurycomanone in increasing the reproductive output in animals. [ABSTRACT FROM AUTHOR]

Highlights • 1,25D 3 increases mtDNA content and attenuates BPA-induced mtDNA deletion. • 1,25D 3 inhibits BPA-induced decrease in COXI expression and 17β-estradiol secretion. • 1,25D 3 increases mitochondrial biogenesis-related proteins by PI3K-Akt signalling. • 1,25D 3 increases ATP production and cellular oxygen consumption rate. Abstract Bisphenol A (BPA), an endocrine-disrupting chemical, is capable of producing reproductive toxicity. BPA results in mitochondrial DNA (mtDNA) deletion and mitochondrial dysfunction; however, the effect of BPA on the mitochondria of ovarian granulosa cells is not clear. Further, 1,25-dihydroxyvitamin D 3 (1,25D 3) may play a role in reproduction, because its receptor, VDR, contributes to the inhibition of oxidative stress and predominantly exists in the nuclei of granulosa cells. Hence, the role of 1,25D 3 in BPA-mediated effects on mitochondrial function was examined in this study. Primary rat granulosa cells treated with BPA, 1,25D 3 , or both were subjected to molecular/biochemical assays to measure cell survival, mtDNA content, mtDNA deletion, superoxide dismutase activity, levels of proteins related to mitochondrial biogenesis, and mitochondrial function. We found that cell viability was dose-dependently reduced and reactive oxygen species (ROS) levels were increased by BPA treatment. BPA administration elevated Mn-superoxide dismutase (MnSOD) expression but negatively regulated total SOD activity. 1,25D 3 treatment alone increased 17β-estradiol secretion, ATP production, and cellular oxygen consumption. In cells treated with both agents, 1,25D 3 enhanced BPA-induced MnSOD protein upregulation and blocked the BPA-mediated decline in total SOD activity. Furthermore, 1,25D 3 attenuated BPA-mediated mtDNA deletion but showed no effect on BPA-induced increases in mtDNA content. Although BPA had no influence on the levels of peroxisome proliferator-activated receptor-γ coactivator-1 α, nuclear respiratory factor-1, mitochondrial transcription factor A, or cytochrome c oxidase subunit IV, 1,25D 3 plus BPA markedly increased mitochondrial biogenesis-related protein expression via the PI3K-Akt pathway. Moreover, BPA-mediated negative regulation of cytochrome c oxidase subunit I levels and 17β-estradiol secretion was attenuated by 1,25D 3 pre-treatment. Our results suggest that 1,25D 3 attenuates BPA-induced decreases in 17β-estradiol and that treatment with 1,25D 3 plus BPA regulates granulosa cell mitochondria by elevating mitochondrial biogenesis-related protein levels. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • In adipocytes 3T3-L1, a IL6 treatment induces a profile of mitochondrial dysfunction and inflammation. • In 3T3-L1, E2 ameliorates the adverse effects of inflammation on mitochondrial function through the combined activation of both GPER and ERα. • The effects improving the mitochondrial function of estradiol in adipocytes 3T3-L1 through ERα and GPER is dependent on protein kinase A (PKA). Abstract Obesity is associated with inflammation, dysregulated adipokine secretion, and disrupted adipose tissue mitochondrial function. Estradiol (E2) has been previously reported to increase mitochondrial function and biogenesis in several cell lines, but neither the type of oestrogen receptor (ERα, ERβ and GPER) involved nor the mechanism whereby such effects are exerted have been fully described. Considering the anti-inflammatory activity of E2 as well as its effects in enhancing mitochondrial biogenesis, the aim of this study was to investigate the contribution of ERα, ERβ, and GPER signaling to the E2-mediated enhancement of adipocyte mitochondrial function in a pro-inflammatory situation. 3T3-L1 cells were treated for 24 h with ER agonists (PPT, DPN, and G1) and antagonists (MPP, PHTPP, and G15) in the presence or absence of interleukin 6 (IL6), as a pro-inflammatory stimulus. Inflammation, mitochondrial function and biogenesis markers were analyzed. To confirm the involvement of the PKA pathway, cells were treated with a GPER agonist, a PKA inhibitor, and IL6. Mitochondrial function markers were analyzed. Our results showed that activation of ERα and GPER, but not ERβ, was able to counteract the proinflammatory effects of IL6 treatment, as well as mitochondrial biogenesis and function indicators. Inhibition of PKA prevented the E2- and G1-associated increase in mitochondrial function markers. In conclusion E2 prevents IL6 induced inflammation in adipocytes and promotes mitochondrial function through the combined activation of both GPER and ERα. These findings expand our understanding of ER interactions under inflammatory conditions in female rodent white adipose tissue. [ABSTRACT FROM AUTHOR]

Highlights • A GC–MS/MS based method for quantification of sex steroids in children is presented. • Adrostenedione, dihydrotestosterone, testosterone, estrone, estradiol were analyzed. • The GC–MS/MS- based method was sensitive enough for use in pediatric applications. • Reference intervals were established in relation to sex and pubertal maturation. Abstract Background Androgen and estrogen determinations serve as important diagnostic markers in a variety of clinical conditions. However, one challenge is to enhance assay sensitivity for determination in the lowest range, such as in prepubertal children. We here present a recently developed gas chromatography–tandem mass spectrometry (GC–MS/MS) method for determination of androstenedione (A 4), dihydrotestosterone (DHT), testosterone (T), estrone (E 1), and estradiol (E 2) in children, which we have compared with the sensitive radioimmunoassays; E 2 extraction-RIA and T-RIA. Methods Steroids were extracted in ethyl acetate n-hexane solution from serum spiked with isotopically labeled internal standard and derivatized sequentially with pentafluorobenzyl bromide, pentafluorobenzyl hydroxylamine and pentafluoropropionic acid anhydride and analyzed by GC–MS/MS using a triple quadrupole mass spectrometer operated in negative chemical ionization mode. Leftover routine samples (n = 414) were used to evaluate the concordance between GC–MS/MS and RIAs and the validity of GC–MS/MS for pediatrics; of these samples, 101 were from seemingly healthy children. Pubertal stage was recorded for reference interval evaluation. Results Lower limit of detection for A 4 , T, DHT, E 1 , and E 2 were 0.1 nmol/L, 0.1 nmol/L, 27 pmol/L, 9 pmol/L, and 2 pmol/L, respectively. Good agreement was found between GC–MS/MS and T-RIA (r = 0.98) as well as between GC–MS/MS and E 2 extraction-RIA (r = 0.98, for E 2 concentrations above 14 pmol/L). In boys, T and DHT increased significantly from prepuberty throughout pubertal development, and in girls the same increase was observed for E 1 and E 2. The greatest increase in A 4 for both genders, as well as E 1 and E 2 in boys and T and DHT in girls, occurred in mid to late puberty. Conclusions We report the development of a GC–MS/MS method sensitive enough to accurately determine serum levels of androgens and estrogens in children. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Reasons for limited effects of sulfatase inhibition in breast cancer was analyzed. • DHT Restoration improves anti-proliferative effects of sulfatase inhibition. • The promising combined treatment for ER + breast cancer has been proposed. Abstract Steroid sulfatase is detectable in most hormone-dependent breast cancers. STX64, an STS inhibitor, induced tumor reduction in animal assay. Despite success in phase І clinical trial, the results of phase II trial were not that significant. Breast Cancer epithelial cells (MCF-7 and T47D) were treated with two STS inhibitors (STX64 and EM1913). Cell proliferation, cell cycle, and the concentrations of estradiol and 5α-dihydrotestosterone were measured to determine the endocrinological mechanism of sulfatase inhibition. Comparisons were made with inhibitions of reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs). Proliferation studies showed that DNA synthesis in cancer cells was modestly decreased (approximately 20%), accompanied by an up to 6.5% in cells in the G0/G1 phase and cyclin D1 expression reduction. The concentrations of estradiol and 5α-dihydrotestosterone were decreased by 26% and 3% respectively. However, supplementation of 5α-dihydrotestosterone produced a significant increase (approximately 35.6%) in the anti-proliferative effect of sulfatase inhibition. This study has clarified sex-hormone control by sulfatase in BC, suggesting that the different roles of estradiol and 5α-dihydrotestosterone can lead to a reduction in the effect of sulfatase inhibition when compared with 17β-HSD7 inhibition. This suggests that combined treatment of sulfatase inhibitors with 17β-HSD inhibitors such as the type7 inhibitor could hold promise for hormone-dependent breast cancer. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Estradiol dimer inhibits tubulin assembly in vitro. • Estradiol dimer reversibly disrupts microtubule structures in U2OS cells. • Estradiol dimer inhibits microtubule dynamics in U2OS cells. • Estradiol dimer shows cytotoxic activities on cancer cell lines, arrests cells in the G2/M phase of the cell cycle and attenuates DNA/RNA synthesis. • Estradiol dimer has no effect on the transactivation by estrogen receptor α. Abstract Microtubule dynamics is one of the major targets for new chemotherapeutic agents. This communication presents the synthesis and biological profiling of steroidal dimers based on estradiol, testosterone and pregnenolone bridged by 2,6-bis(azidomethyl)pyridine between D rings. The biological profiling revealed unique properties of the estradiol dimer including cytotoxic activities on a panel of 11 human cell lines, ability to arrest in the G2/M phase of the cell cycle accompanied with the attenuation of DNA/RNA synthesis. Thorough investigation precluded a genomic mechanism of action and revealed that the estradiol dimer acts at the cytoskeletal level by inhibiting tubulin polymerization. Further studies showed that estradiol dimer, but none of the other structurally related dimeric steroids, inhibited assembly of purified tubulin (IC 50 , 3.6 μM). The estradiol dimer was more potent than 2-methoxyestradiol, an endogenous metabolite of 17β-estradiol and well-studied microtubule polymerization inhibitor with antitumor effects that was evaluated in clinical trials. Further, it was equipotent to nocodazole (IC 50 , 1.5 μM), an antimitotic small molecule of natural origin. Both estradiol dimer and nocodazole completely and reversibly depolymerized microtubules in interphase U2OS cells at 2.5 μM concentration. At lower concentrations (50 nM), estradiol dimer decreased the microtubule dynamics and growth life-time and produced comparable effect to nocodazole on the microtubule dynamicity. In silico modeling predicted that estradiol dimer binds to the colchicine-binding site in the tubulin dimer. Finally, dimerization of the steroids abolished their ability to induce transactivation by estrogen receptor α and androgen receptors. Although other steroids were reported to interact with microtubules, the estradiol dimer represents a new structural type of steroid inhibitor of tubulin polymerization and microtubule dynamics, bearing antimitotic and cytotoxic activity in cancer cell lines. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • 17β-Estradiol acutely increases hTREK1 channel activity through hGPER. • The potentiation is pertussis toxin-sensitive involving the action of Gβγ subunits. • The effect involves cAMP-PKA pathway downregulation and action of phosphatases. • Serines 315 and 348 in hTREK1 carboxy terminal are targeted for dephosphorylation. Abstract TWIK-related potassium channel 1 (TREK1), a two-pore domain potassium channel, is modulated by various hormones and neurotransmitters by activation of membrane receptor - coupled second messengers. 17β-estradiol is a neuromodulator capable of regulating several cellular processes including the activity of ion channels, in a rapid and non-genomic manner. The G protein-coupled estrogen receptor (GPER) is known to facilitate rapid actions of 17β-estradiol, though its role in modulation of ion channels is not widely explored. Several studies have shown both TREK1 and 17β-estradiol to be neuromodulatory but the interaction between them is not known. In the present study, using single channel cell-attached patch clamp electrophysiology in HEK293 cells, we show that 17β-estradiol increases the activity of hTREK1 channel by acting through hGPER and increasing the channel opening probability within minutes. The potentiation induced by 17β-estradiol is pertussis toxin – sensitive involving action of Gβγ subunits while the inhibitory effect of cAMP-PKA pathway on TREK1 is reduced. Protein phosphatases were also found to be important for the action of 17β-estradiol, which in concert with reduced activity of PKA, may alter the phosphorylation state of the channel and thus increase channel activity. Mutational studies revealed the serines at positions 315 and 348 in the C-terminal domain of hTREK1 to be the target sites for dephosphorylation induced by 17β-estradiol action through hGPER. Elucidation of the pathway for the potentiating action of 17β-estradiol via hGPER on hTREK1 channel activity will help us understand better one of the many possible neuroprotective mechanisms of 17β-estradiol and hTREK1 channel. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Estrogen typically inhibits Runx2 in osteoblast progenitors. • Estrogen does not inhibit Runx2 at particular genes and concentrations. • Bimodal effect of increasing estrogen concentrations on osteoblast differentiation. • SERMs poorly mimic the bimodal effect of estrogen in pre-osteoblasts. • SERMs closely mimic estrogen in pre-osteoclasts. Abstract Estrogens attenuate bone turnover by inhibiting both osteoclasts and osteoblasts, in part through antagonizing Runx2. Apparently conflicting, stimulatory effects in osteoblast lineage cells, however, sway the balance between bone resorption and bone formation in favor of the latter. Consistent with this dualism, 17ß-estradiol (E2) both stimulates and inhibits Runx2 in a locus-specific manner, and here we provide evidence for such locus-specific regulation of Runx2 by E2 in vivo. We also demonstrate dual, negative and positive, regulation of Runx2-driven alkaline phosphatase (ALP) activity by increasing E2 concentrations in ST2 osteoblast progenitor cells. We further compared the effects of E2 to those of the Selective Estrogen Receptor Modulators (SERMs) raloxifene (ral) and lasofoxifene (las) and the phytoestrogen puerarin. We found that E2 at the physiological concentrations of 0.1–1 nM, as well as ral and las, but not puerarin, antagonize Runx2-driven ALP activity. At ≥10 nM, E2 and puerarin, but not ral or las, stimulate ALP relative to the activity measured at 0.1–1 nM. Contrasting the difference between E2 and SERMs in ST2 cells, they all shared a similar dose-response profile when inhibiting pre-osteoclast proliferation. That ral and las poorly mimic the locus- and concentration-dependent effects of E2 in mesenchymal progenitor cells may help explain their limited clinical efficacy. [ABSTRACT FROM AUTHOR]

Estrogen receptors (ERα and β) and androgen receptor (AR) regulate various critical processes during spermatogenesis. Since spermatogenesis is very sensitive to hormonal stimuli and perturbations, it is important to understand the regulation of expression of these receptors by sex steroid hormones. Although many studies have reported deregulation of steroid receptors on endocrine disruption, there is no consensus on the regulation of their expression by steroid hormones during spermatogenesis, and a lack of clear understanding of the mechanism of regulation. Here, we evaluated the receptor expressions in a well-established exogenous estradiol administration model. We then investigated the mechanisms by which the individual receptors regulate their expression by binding to the respective hormone response elements upstream of these receptor genes. By further employing in vitro and in vivo models of ER and AR stimulation or antagonism, we delineated their regulation in a receptor subtype-specific manner. Our results indicate that ERα positively regulates expression of both the ERs; whereas, ERβ and AR negatively regulate expression of both ERβ and AR by direct binding to upstream regulatory regions. The perturbations in the levels of steroid receptors could be an important factor contributing to spermatogenic defects and male sub-fertility after estradiol and ER agonist treatment. Our study delineates the direct contribution of the individual steroid receptors in the regulation of their own expression. [ABSTRACT FROM AUTHOR]

Aromatase (encoded by Cyp19a1) in the ovarian follicular cells catalyzes the production of estradiol from testosterone, which plays important roles in the ovarian development of vertebrates. In the present study, the interaction of Dmrt1, Foxl2, and Nr5a1a on the regulation of cyp19a1a transcription in ovarian follicles was examined in a teleost, the ricefield eel Monopterus albus. The expression of dmrt1a , foxl2 , and nr5a1a was detected in ovarian follicular cells together with cyp19a1a at the mRNA and/or protein levels. Sequence analysis identified one conserved Foxo binding site in the proximal promoter region of ricefield eel cyp19a1a. Transient transfection assay showed that Foxl2 may bind to the conserved Foxo site to activate cyp19a1a transcription and act synergistically with Nr5a1a. Mutation of either the conserved Nr5a1 site or Foxo site abolished or significantly decreased the synergistic effects of Nr5a1a and Foxl2 on cyp19a1a transcription. The sequence between Region III and I-box of Nr5a1a was critical to this synergistic effect. Dmrt1a modulated the Foxl2- and Nr5a1a-induced activation of cyp19a1a transcription and their synergistic effects in a biphasic manner, with inhibitory roles observed at lower doses (10–50 ng) but release of the inhibition or even potentiating effects observed at higher doses (100–200 ng). Collectively, data of the present study suggest that the interaction of Dmrt1a, Foxl2, and Nr5a1a in the ovarian follicular cells may facilitate the adequate expression of cyp19a1a and the production of estradiol, and contribute to the development and maturation of ovarian follicles in ricefield eels and other vertebrates as well. • cyp19a1a , nr5a1a , foxl2 , and dmrt1a were expressed in follicular cells. • Foxl2 up-regulated cyp19a1a transcription via the conserved Foxo site. • Nr5a1a and Foxl2 bound to cyp19a1a promoter to exert their full synergism. • The sequence between Region III and I-box of Nr5a1a was key to the synergism. • Dmrt1a abolished the synergism of Nr5a1a and Foxl2 on c yp19a1a transcription. [ABSTRACT FROM AUTHOR]

Cytochrome P450 aromatase (AROM) catalyzes the biosynthesis of estrogen from androgen. Previously crystal structures of human AROM in complex with the substrate androstenedione, and inhibitors exemestane, as well as the newly designed steroidal compounds, have been reported. Here we report the first crystal structure of testosterone complex of human placental AROM. Testosterone binds at the androgen-specific heme distal pocket. The polar and hydrophobic interactions with the surrounding residues resemble the interactions observed for other ligands. The heme proximal region comprises the intermolecular interface in AROM, and also the putative interaction surface of its redox partner cytochrome P450 reductase. Unreported previously, the proximal region is characterized by a large surface cavity, unlike most known P450’s. Using five best X-ray data sets from androstenedione and testosterone complexes of AROM, we now unequivocally show the presence of an unexplained ligand electron density inside the proximal cavity. The density is interpreted as ordered five ethylene glycol units of polyethylene glycols used as a solvent for steroids and also in crystallization. Interestingly, polyethylene glycol exhibits weak inhibition of AROM enzyme activity in a time dependent manner. Besides its critical role in the redox partner coupling and electron transfer process, the proximal cavity possibly serves as the interaction site for other molecules that may have regulatory effects on AROM activity. In addition, the new data also reveal a previously unidentified water channel linking the active site to the lipid interface. The channel could be the predicted passage for water molecules involved in catalysis. [ABSTRACT FROM AUTHOR]

We report the synthesis and detailed biological study of the synthetic brassinosteroid analog 2α,3α-dihydroxy-6-oxo-5α-androstan-17β-yl N-(tert -butoxycarbonyl)-D,L-valinate (BR4848). The panel of cancer cell lines was used for characterization of its antiproliferative activity, yet had no adverse effects in normal human fibroblasts. In HeLa cells, BR4848-induced apoptosis was accompanied by increase of apoptotic subG 1 cells, PARP-1 and caspase-7 fragmentation, downregulation of Bcl-2 and Mcl-1, an increase in caspase activity and G 2 /M phase cell cycle arrest. Antiproliferative properties of BR4848 were exhibited by inhibition of phosphorylation of Akt, Erk1/2 and FAK. Furthermore, the developed analog exhibited in vitro antiangiogenic activity in human umbilical vein endothelial cells (HUVECs). BR4848-induced apoptosis accompanied with G 2 /M arrest was detected in endothelial cells. BR4848 also inhibited adhesion, tube formation and migration of endothelial cells by inhibition of FAK, Erk 1/2, CDK5, VEGFR2, TNFα-stimulated production of IL-6, angiopoietin-2 and Jagged1. Finally, BR4848 did not modulate the activity nor nuclear translocation of any of the steroid receptors (ERα, ERβ, AR, MR and PR) included in reporter cell-based assays, which excludes the genomic activity of steroid receptors as a contributing factor to the observed biological activities of BR4848. [ABSTRACT FROM AUTHOR]

Endometrial cancer (EC) is the most common gynecologic malignancy prevailing after menopause. Defining steroid profiles may help predict the risk of recurrence after hysterectomy, which remains limited due to the lack of reliable markers. Adrenal precursors, androgens, parent estrogens and catechol estrogen metabolites were measured by mass spectrometry (MS) in preoperative serums and those collected one month after hysterectomy from 246 newly diagnosed postmenopausal EC cases. We also examined the associations between steroid hormones and EC status by including 110 healthy postmenopausal women. Steroid concentrations were analyzed in relation to clinicopathological features, recurrence and overall survival (OS). The mean follow-up time was 65.5 months and 26 patients experienced relapse after surgery for a recurrence incidence of 10.6% (6.4% Type I and 29.5% Type II). Recurrence and OS were related to a more aggressive disease but not linked to body mass index. Preoperative levels of estriol (E 3 ) and estrone-sulfate (E 1 -S) were inversely associated with recurrence in a multivariate logistic regression analysis (Hazard ratios (HRs) of 0.31, P = 0.039 and 3.01, P = 0.024; respectively). All circulating steroids declined considerably after surgery almost reaching those of healthy women, except 4-methoxy-E 2 (4MeO-E 2 ) for which postoperative levels increased by 35% and were associated to a 68% decreased risk of recurrence (HR = 0.32, P = 0.015). Women diagnosed with both histological types of EC present significantly higher levels of steroids, in support of their mitogenic effects. The estrogen precursor E 1 -S, the anticancer metabolite 4MeO-E 2 , and E 3 that exert mixed antagonist and agonist estrogenic activities and immunological effects, are potential independent prognostic factors. [ABSTRACT FROM AUTHOR]

The numerous estrogen functions reported across vertebrates have been classically explained by their binding to specific transcription factors, the nuclear estrogen receptors (ERs). Rapid non-genomic estrogenic responses have also been recently identified in vertebrates including fish, which can be mediated by membrane receptors such as the G protein-coupled estrogen receptor (Gper). In this study, two genes for Gper, namely gpera and gperb , were identified in the genome of a teleost fish, the European sea bass. Phylogenetic analysis indicated they were most likely retained after the 3R teleost-specific whole genome duplication and raises questions about their function in male and female sea bass. Gpera expression was mainly restricted to brain and pituitary in both sexes while gperb had a widespread tissue distribution with higher expression levels in gill filaments, kidney and head kidney. Both receptors were detected in the hypothalamus and pituitary of both sexes and significant changes in gpers expression were observed throughout the annual reproductive season. In female pituitaries, gpera showed an overall increase in expression throughout the reproductive season while gperb levels remained constant. In the hypothalamus, gpera had a higher expression during vitellogenesis and decreased in fish entering the ovary maturation and ovulation stage, while gperb expression increased at the final atresia stage. In males, gpers expression was constant in the hypothalamus and pituitary throughout the reproductive cycle apart from the mid- to late testicular development stage transition when a significant up-regulation of gpera occurred in the pituitary. The differential sex, seasonal and subtype-specific expression patterns detected for the two novel gper genes in sea bass suggests they may have acquired different and/or complementary roles in mediating estrogens actions in fish, namely on the neuroendocrine control of reproduction 2 Protein and gene nomenclature followed that recommended by genenames.org and used for fish at http://zfin.org/ ; in this abbreviation list for each case protein abbreviation is presented first followed by the corresponding gene abbreviation. . [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the combinatory effects of an isoflavone (ISO)-rich diet and exercise on fat mass and lipid metabolism in ovariectomized (OVX) rats. Therefore the female Wistar rats were sedentary, performed an intense treadmill uphill running, received ISOs, or a combination of ISOs and running after ovariectomy. The exercise reduced visceral fat mass, adipocyte size and serum leptin in Sham animals and antagonized the increases of these parameters induced by OVX. ISOs reduced OVX induced increase of serum leptin. The combination of training and ISOs was most effective in reducing serum triglyceride levels. In OVX rats the training stimulated the expression of genes associated with fatty acid synthesis (SREBP-1c and FAS) in adipose tissue, soleus muscle, liver and genes associated with fatty acid oxidation (PPARδ and PGC-1α) in adipose tissue. ISOs stimulated the expression of SREBP-1c and FAS in soleus muscle and PGC-1α in adipose tissue, whereas suppressed hepatic SREBP-1c and FAS expression. Strong additive effects of ISOs combined with the training were observed for PPARδ and PGC-1α expressions in soleus muscle. In conclusion our results demonstrate that both the training and ISOs affect fat mass and fatty acid metabolism in OVX rats. The training seems to have a higher impact than ISO exposure in regulating gene expression in adipose tissue. However, the strongest effects for several of the addressed parameters could be observed in the combination group especially in the soleus muscle. Therefore a combination of training and an ISO-rich diet may have beneficial effects on fatty acid metabolism and could be a concept for the prevention of obesity in postmenopausal females. [ABSTRACT FROM AUTHOR]

Vitamin D is essential for mineral homeostasis and contributes to bone metabolism by stimulating osteoblast differentiation of marrow stromal cells (MSCs). In this study, we used MSCs from pre-pubertal girls and boys to test the hypothesis that 1α,25(OH) 2 D and 17β-estradiol have synergistic effects on these MSCs, and what mechanism is involved. With IRB approval, we isolated MSCs from discarded excess iliac marrow graft from children undergoing alveolar cleft repair. Plasma was available from 8 female (9.3 ± 0.2 years) and 8 male (9.6 ± 0.1 years) subjects for hormone assays [25(OH)D, total testosterone, 17β-estradiol, estrone, DHEA-S, Growth Hormone, IGF-I]. RT-PCR was used for gene expression. Alkaline phosphatase (ALP) activity was used to measure osteoblast differentiation at day 7; alizarin red was used to measure matrix mineralization at day 21. All subjects were pre-pubertal based on their hormone levels. Serum 25(OH)D levels ranged from 13.1 to 26.4 ng/mL, with 75% below 20 ng/mL. Constitutive gene expression of VDR and ERα, β varied from subject to subject with no association with sex or serum chemistries. In osteoblastogenic medium, 1α,25(OH) 2 D 3 (10 nM) increased ALP activity by 36% (p < 0.05) in MSCs; 10 nM of E2 was not stimulatory but the combination of 1α,25(OH) 2 D 3 and E2 increased ALP 151% (p < 0.05 vs. control) and by 84.5% (p < 0.05 vs. 1α,25(OH) 2 D3 alone). The combination of 1α,25(OH) 2 D 3 and E2 significantly increased mineralization 11-fold, compared with either agent alone. Twenty-four hour treatment with 1α,25(OH) 2 D 3 (10 nM) or E2 (10 nM) upregulated each other’s receptor by as much as 5.8-fold for ERα and 2.9-fold for the VDR. In summary, 1α,25(OH) 2 D3 stimulated osteoblast differentiation and matrix mineralization with MSCs from pre-pubertal subjects, with a synergistic effect of E2, mediated by upregulated receptor levels, at least in part. These studies add new information about the regulation of human osteoblast differentiation, effects of 1α,25(OH) 2 D 3 and E2 on MSCs, and the importance of vitamin D for skeletal health. [ABSTRACT FROM AUTHOR]

To further investigate the interaction between vitamin D system and estrogen-mimetic compounds in the human vasculature we studied the effect of the “less- calcemic” analog of 1,25(OH) 2 D 3 (1,25D); JK 1624F 2 -2 (JKF) in the presence of selective estrogen modulator femarelle (F), the phytoestrogen daidzein (D) and estradiol-17b (E 2 ) on 3 [H] thymidine incorporation (DNA synthesis) and creatine kinase specific activity (CK) in human umbilical artery vascular smooth muscle cells (VSMC). F, D and E 2 , stimulated DNA synthesis at low concentrations, and inhibited it at high concentrations. All estrogen-related compounds increased CK dose- dependently. Daily treatment with JKF (1 nM for 3 days) resulted in decreased DNA synthesis, increased CK and up- regulation of the stimulation of DNA synthesis by low estrogen-related hormones whereas D- and E 2 - mediated inhibition of cell proliferation was abolished by JKF. In contrast, inhibition of cell proliferation by F could not be blocked by JKF. JKF also up-regulated the stimulatory effects on CK by F, E 2 and D. VSMC expressed Estrogen Receptor (ER)a and ERb mRNA at a relative ratio of 2.7:1.0, respectively. JKF pretreatment increased ERa (∼50%) and decreased ERb (∼25%) expression. E 2 did not affect ERs whereas both D and F up-regulated ERb (∼100%) and ERa (∼50%). Additionally, JKF increased the intracellular competitive binding of F (from ∼70 to ∼310%), of D (from ∼60 to ∼250%) and of E 2 from (from ∼ 70 to ∼320%). F reciprocally modulated the vitamin D system by up-regulating VDR- and 25 hydroxyy vitamin D 1-a hydroxylase (1OHase) mRNA expression (∼120%). F also stimulated 1OHase activity as indicated by an increase in the production of 1, 25D (∼250%). A similar increase was elicited by D (∼90%) but not by E 2 . In conclusion, F has unique effects on human VSMC in that it can sustain inhibition of cell growth even in the presence of the vitamin D analog JKF. That JKF increases ER expression and F increased the endogenous production of 1, 25D and VDR expression offer new opportunities to modulate VSMC growth. Whether or not these mutual effects of F and JKF can be exploited to promote vascular health, particularly in estrogen-deficient states (e.g., menopause) is under investigation. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) is involved in several physiological events that resulted in a growing interest in its modulation. Moreover, the uterine levels of anandamide (AEA), the major endocannabinoid, must be tightly regulated to create proper embryo implantation conditions. However, there are no evidences about the regulation of AEA in uterus by estrogen. Thus, the aim of this study is to elucidate whether estradiol benzoate (EB) and tamoxifen (TAM) administration to ovariectomized (OVX) rats can induce changes in the expression of cannabinoid receptors and AEA-metabolic enzymes in uterus by evaluating gene transcription and protein levels by qPCR, Western blot and immunohistochemistry. Moreover, the plasmatic and uterine levels of AEA and of prostaglandin E 2 (PGE 2 ) and prostaglandin F 2 α (PGF 2α ), the major cyclooxygenase-2 (COX-2) products, were determined by UPLC–MS/MS. The immunohistochemistry showed that cannabinoid receptors, as well as AEA-metabolic enzymes are mainly located in the epithelial cells of both lumen and glands and, to a lesser extent, in the muscle cells. Moreover, EB administration to OVX rats significantly increased CB1, CB2, NAPE-PLD, FAAH and COX-2 expression and transcription. These effects were absent in TAM and TAM + EB treatments showing that this response is estrogen receptor dependent. Additionally, although uterine levels of AEA remained unchanged in EB or TAM treated animals, they showed a rise with EB treatment in plasma. The latter also produced a decrease in uterine PGE 2 levels. In summary, these data collectively indicate that the expression of ECS components, as well as, the AEA and PGE 2 levels in rat uterus is modulated by EB. Thus, estradiol may have a direct regulatory role in the modulation of ECS in female reproductive tissues. [ABSTRACT FROM AUTHOR]

Although ERα activation properties have been intensively studied, this is not the case for their repressive properties. In this report, the ERα ligand binding domain (LBD) is shown to interact both with a deacetylase function and with HDAC1 and HDAC3. Ligands do not affect binding to the deacetylase activity or to HDAC1. In distinction, E2 reduced LBD binding to HDAC3, whereas Tmx had no effect. Knock-down of either HDAC1 or 3 led to increased transcriptional activity by both HDACs, presumably by decreased repression. In distinction, only HDAC3 knock-down led to increased activity in the presence of Tmx. In summary, ERα differentially interacts with HDACs 1 and 3 to regulate transcriptional activity. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an apicomplexan parasite that infects most species of warm-blooded animals, including humans, and causes abortions and severe damage to the fetal central nervous system. During pregnancy, the prevalence of toxoplasmosis increases throughout the second and third quarter of gestation, while the hormones progesterone and estradiol simultaneously increase. Thus, it has been suggested that these hormones could affect parasite reproduction. This study was mainly focused on an estradiol regulatory factor-Hydroxysteroid dehydrogenase (HSD) gene in T. gondii . Our data showed that estradiol promoted Pru (Type II) and VEG (Type III) infection and thus significantly contributed to the pathogenicity of T. gondii in mice. Subsequently, we found that this phenomenon may relate to the interplay of T. gondii and estradiol. We reported that estradiol can enter T. gondii tachyzoites. Bioinformatics analysis showed that T. gondii may have a residual estradiol metabolism-related gene HSD . To verify the gene function, HEK293T cells were transiently transfected with Tg-HSD and gene expression was induced. Then, HPLC (high-performance liquid chromatography) analysis showed that Tg-HSD can efficiently transform estrone into estradiol. Moreover, Tg-HSD −overexpressing parasites showed significantly enhanced pathogenicity and upregulation of estradiol levels in mice. In conclusion, estradiol can promote T. gondii infection in vitro and in vivo , and this may be related to its Tg- HSD gene. [ABSTRACT FROM AUTHOR]

The objective is to compare the effect of intravaginal dehydroepiandrosterone (DHEA, prasterone), conjugated equine estrogens (CEE) and estradiol (E 2 ) on moderate to severe dyspareunia and/or vaginal dryness. In a review of available data, independent prospective, randomized, double-blind and placebo-controlled Phase III 12-week clinical trials involved daily administration of 6.5 mg (0.50%) prasterone, daily (21 days on/7 days off) 0.3 mg CEE, twice weekly 0.3 mg CEE or 10 μg E 2 daily for 2 weeks followed by twice weekly for 10 weeks. Vulvovaginal atrophy (VVA) symptoms were evaluated by questionnaires. The total severity score of dyspareunia decreased from baseline by 1.27 to 1.63 units with prasterone treatment, 1.4 with CEE and 1.23 in one statistically significant study with E 2 (combined symptoms). Decreases over placebo ranged from 0.35 to 1.21 with prasterone, 0.7 to 1.0 with CEE and 0.33 for the E 2 study. The total decreases in vaginal dryness severity ranged from 1.44 to 1.58 units for prasterone, 1.1 unit for CEE and 1.23 unit for E 2 . The decreases over placebo of vaginal dryness intensity ranged from 0.30 to 0.43 unit for prasterone, 0.40 unit for CEE and 0.33 for the E 2 study with combined symptoms. Daily 6.5 mg (0.50%) prasterone appears to be at least as efficacious as 0.3 mg CEE or 10 μg E 2 for treatment of the VVA symptoms. In summary, the beneficial effects on the VVA symptomatology can be obtained by the addition of a small amount of intravaginal prasterone to compensate for the low serum concentration of prasterone observed in the majority of women after menopause without concerns about systemic effects. [ABSTRACT FROM AUTHOR]

Conventional hormone therapy (HT) containing estrogens such as ethinylestradiol (EE) have been associated with an increased risk of breast cancer and cardiovascular disease resulting in women seeking safer alternatives that are claimed to have fewer health risks. One such alternative gaining popularity, is custom-compounded bioidentical (b)HT formulations containing bioidentical estradiol (bE 2 ) and estriol (bE 3 ). However, the preparation of these custom-compounded estrogens is not regulated, and depending on the route of synthesis, steroid mixtures with differing activities may be produced. Thus, an investigation into the activities of estrogens prepared by custom-compounded pharmacies is warranted. The aim of this study was therefore to directly compare the pharmacological properties of bE 2 and bE 3 of unknown purity relative to commercially available, pure E 2 , E 3 and estrone (E 1 ) standards as well as synthetic EE used in conventional HT via the human estrogen receptor (ER)-α and −β. We determined precise equilibrium dissociation constants (K d or K i values) and showed that bE 2 and bE 3 display similar binding affinities to the E 2 and E 3 standards, while EE had a higher affinity for ERα, and E 1 a lower affinity for ERβ. Furthermore, all the estrogens display similar agonist efficacies, but not potencies, for transactivation on a minimal ERE-containing promoter via the individual ER subtypes. Although E 2 and E 3 were equally efficacious and potent on the endogenous ERE-containing pS2 promoter in the MCF-7 BUS breast cancer cell line co-expressing ERα and ERβ, E 1 was less efficacious and potent than E 2 . This study is the first to demonstrate that the bioidentical estrogens, commercially available estrogen standards and synthetic EE are full agonists for transrepression on both minimal and endogenous NFκB-containing promoters. Moreover, we showed that these estrogens all increase proliferation and anchorage-independent growth of MCF-7 BUS cells to a similar extent, suggesting that custom-compounded bHT may in fact not be a safer alternative to conventional HT. Furthermore, our results showing that E 3 and E 1 are not weak estrogens, and that E 3 does not antagonize the activity of E 2 , suggest that the rationale behind the use of E 3 and E 1 in custom-compounded bHT formulations should be readdressed. Taken together, the results indicating that there is mostly no difference between the custom-compounded bioidentical estrogens, commercially available estrogen standards and synthetic EE, at concentrations reflecting serum levels in women using estrogen-containing HT, suggest that there is no clear advantage in choosing bHT above conventional HT. [ABSTRACT FROM AUTHOR]

Estradiol (E 2 ) has many important actions in the tissues of the oral cavity. Disruption of E 2 metabolism or alterations in systemic E 2 concentrations have been associated with compromised periodontal health. In many instances such changes occur secondarily to the well characterised effects of E 2 on bone physiology −especially maintenance of bone mineral density (BMD). Despite these important epidemiological findings, little is known about the mechanism of action of E 2 in oral tissues or the expression and function of oestrogen receptor (ER) isoforms in these tissues. We have isolated human dental pulp cells (hDPCs), which are able to differentiate towards an osteogenic lineage under appropriate culture conditions. We show that hDPCs express ERα, ERβ1, ERβ2 and the cell membrane associated G protein-coupled ER (GPR30). Following osteogenic differentiation of hDPCs, ERβ1 and ERβ2 were up regulated approximately 50-fold while ERα and GPR30 were down regulated, but to a much lesser degree (approximately 2-fold). ERβ was characterised as a 59 kDa protein following Western blot analysis with validated antibodies and ERβ was detected in both nuclear and cytoplasmic cell compartments following immunofluorescence (IF) and immunohistochemical (IHC) analysis of cultured cells. Furthermore isoform specific antibodies detected both ERβ1 and ERβ2 in DPC cultures and in situ analysis of ERβ expression in decalcified tooth/pulp sections identified the odontoblast layer of pulp cells juxtaposed to the tooth enamel as strongly reactive for both ERβ isoforms. Finally the use of isoform specific agonists identified ERβ as the main receptor responsible for the pro-osteogenic effect of oestrogenic hormones in this tissue. Our data suggest that oestrogens stimulated osteogenic differentiation in hDPCs and that this action is mediated principally through the ERβ isoform. These findings may have important consequences for the investigation and treatment of oral and periodontal pathologies which are associated with imbalances in oestrogen concentrations and action. [ABSTRACT FROM AUTHOR]

Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops ( Humulus lupulus ) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50 mg/kg body weight of MNU on postnatal days PND 50 and 52. 28 days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140 days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E 2 -benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms. [ABSTRACT FROM AUTHOR]

Celecoxib is known to alter the preferred position of SULT2A1-catalyzed sulfonation of 17β-estradiol (17β-E2) and other estrogens from the 3- to the 17-position. Understanding the effects of celecoxib on estrogen sulfonation is of interest in the context of the investigational use of celecoxib to treat breast cancer. This study examined the effects on celecoxib on cytosolic sulfotransferases in human and rat liver and on SULT enzymes known to be expressed in liver. Celecoxib’s effects on the sulfonation of several steroids catalyzed by human liver cytosol were similar but not identical to those observed previously for SULT2A1. Celecoxib was shown to inhibit recombinant SULT1A1-catalyzed sulfonation of 10 nM estrone and 4 μM p -nitrophenol with IC 50 values of 2.6 and 2.1 μM, respectively, but did not inhibit SULT1E1-catalyzed estrone sulfonation. In human liver cytosol, the combined effect of celecoxib and known SULT1A1 and 1E1 inhibitors, quercetin and triclosan, resulted in inhibition of 17β-E2-3-sulfonation such that the 17-sulfate became the major metabolite: this is of interest because the 17-sulfate is not readily hydrolyzed by steroid sulfatase to 17β-E2. Investigation of hepatic cytosolic steroid sulfonation in rat revealed that celecoxib did not stimulate 17β-E2 17-sulfonation in male or female rat liver as it does with human SULT2A1 and human liver cytosol, demonstrating that rat is not a useful model of this effect. In silico studies suggested that the presence of the bulky tryptophan residue in the substrate-binding site of the rat SULT2A homolog instead of glycine as in human SULT2A1 may explain this species difference. [ABSTRACT FROM AUTHOR]

Peroxiredoxin 3 (PRX3) plays a role as a regulator of the adipocyte mitochondrial function due to its antioxidant activity. We have previously reported the existence of a sexual dimorphism in the mitochondrial oxidative stress status of many rat tissues such as white (WAT) and brown (BAT) adipose tissues. The aim was to elucidate whether sex hormones may play a role in PRX3 expression in the adipose tissues of rats. In in vivo experiments, male and female standard diet fed rats, high fat diet (HFD) fed rats and rosiglitazone-supplemented HFD (HDF + Rsg) fed rats, as well as ovariectomized (OVX) and 17beta-estradiol-supplemented OVX (OVX + E2) female rats were used. 3T3-L1 adipocytes and brown adipocyte primary culture were used to study the roles of both E2 and testosterone in in vitro experiments. PRX3 levels were greater in the WAT of female rats than in males. This sexual dimorphism disappeared by HFD feeding but was magnified with Rsg supplementation. PRX3 sexual dimorphism was not observed in BAT, and neither HFD nor ovariectomy modified PRX3 levels. Rsg increased Prx3 expression in the BAT of both sexes. In vitro studies supported the results obtained in vivo and confirmed the contribution of E2 to sex differences in WAT Prx3 expression. Finally, we reported an E2 upregulation of both PRX3 and thioredoxin 2 (TRX2) in WAT but not in BAT that could play a key role in the sex dimorphism reported in the antioxidant defence of WAT in order to palliate the detrimental effect of the oxidative stress. [ABSTRACT FROM AUTHOR]

Butyric acid (BA), one of the short chain fatty acids (SCFAs), has positive actions on the metabolism, inflammation, etc. However, whether it influences the reproductive physiology and if so the detail mechanism involved has not yet been determined. In this study, the porcine granulosa cells (PGCs) were treated with gradient concentrations of BA. After 24 h culture, 0.05 mM BA significantly stimulated the progesterone (P 4 ) secretion ( P < 0.05), 5 mM and 10 mM BA significantly inhibited the P 4 secretion ( P < 0.05). Simultaneously, BA up-regulated the estradiol (E 2 ) secretion in a dose dependent manner, 5 mM and 10 mM BA significantly promoted the E 2 level ( P < 0.05). In addition, 10 mM BA significantly promoted the G-protein-coupled receptor 41/43 mRNA ( P < 0.05). Interestingly, 5 mM BA treatment significantly down-regulated cyclic adenosine monophosphate (cAMP) content ( P < 0.05), steroidogenic acute regulatory (StAR), steroidogenic factor 1 (SF1), P450scc in the mRNA and/or protein level ( P < 0.05), and these actions were reversed by cAMP activator forskolin (FK). Moreover, the co-treatment of 5 mM BA and bupivacaine (BPC, the cAMP inhibitor) significantly accumulated the inhibition action of BPC on cAMP, the secretion of P 4 , and the abundance of StAR mRNA ( P < 0.05), inhibited the up-regulation of 5 mM BA on the E 2 secretion ( P < 0.05). Further, the Global Proteome and KEGG pathway analysis found that 5 mM BA significantly up-regulated the I3LM80 proteins ( P < 0.05), which is involved in the steroid biosynthesis signaling pathway. 5 mM BA significantly decreased the F2Z5G3 protein level ( P < 0.05), and the cAMP signaling pathway. In conclusion, present findings for the first time demonstrated that BA could regulate the P 4 and E 2 hormone synthesis in PGCs via the cAMP signaling pathway. [ABSTRACT FROM AUTHOR]

17beta-hydroxysteroid dehydrogenase type 7 (17β-HSD7) promotes breast cancer cell growth via dual-catalytic activity by modulating estradiol and DHT. Here, we clarified the expression pattern of 17β-HSD7 in postmenopausal luminal A type breast cancer with The Cancer Genome Atlas (TCGA) cohort. The impact of 17β-HSD7 inhibition on the proteome of MCF-7 cells was investigated and on cell apoptosis was revealed. MCF-7 cells were treated with an efficient inhibitor of 17β-HSD7 (INH7) or with vehicle, and a differential proteomics study was performed using two-dimensional (2D) gel electrophoresis followed by mass spectrometry and ingenuity pathway analysis (IPA). Cell apoptosis was analyzed by flow cytometry, followed by reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot to investigate the expression of apoptosis-related genes. Our data showed 17β-HSD7 is amplified in primary and progressive breast cancer, inhibition of 17β-HSD7 in MCF-7 cells modulated 104 proteins primarily involved in cell death/survival, cell growth and DNA processing. The expression of 78 kDa glucose-regulated protein (GRP78) and anti-apoptosis factor Bcl-2 were significantly suppressed via 17β-HSD7 inhibition with INH7, consequently induced MCF-7 cell apoptosis. However, INH7 treatment of T47D, another widely used epithelial ER+ breast cancer cell line, led to an up-regulation of GRP78 expression, resulting in a limited increase in apoptosis. These results suggest cell-specific effects of INH7 in the breast cancer, which is interesting for further study. An combinatory effect on apoptosis by INH7 and Letrozole (aromatase inhibitor) was further demonstrated in MCF-7. Down-regulation of GRP78 via 17β-HSD7 inhibition enhances cell apoptosis in response to Letrozole. This study highlights GRP78 as a key regulator related to 17β-HSD7 inhibition and effect. Taken together, results from the present study suggest a hypothesis that inhibition of 17β-HSD7 would be a complementary strategy to Letrozole by suppression of GRP78 in ER+ breast cancer. However, from a research perspective, further studies have to be carried out with more breast cancer cell lines as well as in vivo model to assess the efficacy of inhibitor combination. [ABSTRACT FROM AUTHOR]

This study addresses first the role of human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) in breast cancer (BC) cells. The enzyme has a high estrone-activating activity that is subject to strong substrate inhibition as shown by enzyme kinetics at the molecular level. We used BC cells to verify this phenomenon in living cells: estrone concentration increase did reduce the reaction with 0.025 to 4 μM substrate. Moreover, 5α-dihydrotestosterone (DHT) demonstrated some inhibition of estrogen activation at both the molecular and cellular levels. The presence of DHT did not change the tendency toward substrate inhibition for estrone conversion, but shifted the inhibition toward higher substrate concentrations. Moreover, a binding study demonstrated that both DHT and dehydroepiandrosterone (DHEA) can be bound to the enzyme, thereby supporting the multi-specificity of 17β-HSD1. We then followed the concentrations of estradiol and performed q-RT-PCR measurements of reductive 17β-HSDs after 17β-HSD1 inhibition. The estradiol decrease by the 17β-HSD1 inhibition was demonstrated lending support to this observation. Knockdown and inhibition of 17β-HSD1 produced reduction in estradiol levels and the down-regulation of another reductive enzyme 17β-HSD7, thus “amplifying” the reduction of estradiol by the 17β-HSD1 modulation itself. The critical positioning of 17β-HSD7 in sex-hormone-regulation as well as the mutual regulation of steroid enzymes via estradiol in BC, are clearly demonstrated. Our study demonstrates that fundamental enzymological mechanisms are relevant in living cells. Moreover, further enzyme study in cells is merited to advance biological and medical research. We also demonstrated the central role of 17β-HSD7 in sex-hormone conversion and regulation, supporting it as a novel target for estrogen-dependent (ER+) BC. [ABSTRACT FROM AUTHOR]

Sulfated steroid hormones, such as dehydroepiandrosterone sulfate or estrone-3-sulfate, have long been regarded as inactive metabolites as they cannot activate classical steroid receptors. Some of them are present in the blood circulation at quite high concentrations, but generally sulfated steroids exhibit low membrane permeation due to their hydrophilic properties. However, sulfated steroid hormones can actively be imported into specific target cells via uptake carriers, such as the sodium-dependent organic anion transporter SOAT, and, after hydrolysis by the steroid sulfatase (so-called sulfatase pathway), contribute to the overall regulation of steroid responsive organs. To investigate the biological significance of sulfated steroid hormones for reproductive processes in humans and animals, the research group “Sulfated Steroids in Reproduction” was established by the German Research Foundation DFG (FOR1369). Projects of this group deal with transport of sulfated steroids, sulfation of free steroids, desulfation by the steroid sulfatase, effects of sulfated steroids on steroid biosynthesis and membrane receptors as well as MS-based profiling of sulfated steroids in biological samples. This review and concept paper presents key findings from all these projects and provides a broad overview over the current research on sulfated steroid hormones in the field of reproduction. [ABSTRACT FROM AUTHOR]

Obesity and ageing are associated with lower serum testosterone levels in men. How fat distribution or adipose tissue metabolism, independent of genetic factors and age, are related to sex steroid metabolism is less clear. We studied the associations between adiposity and serum sex hormone concentrations, and mRNA expression of genes regulating sex hormone metabolism in adipose tissue in young adult male monozygotic (MZ) twin pairs. The subjects [n = 18 pairs; mean age, 32 years; individual body mass indexes (BMIs) 22–36 kg/m 2 ] included 9 male MZ twin pairs discordant for BMI [intra-pair difference (Δ) in BMI ≥3 kg/m 2 ]. Sex steroid concentrations were determined by liquid chromatography–tandem mass spectrometry, body composition by dual-energy X-ray absorptiometry and magnetic resonance imaging, and mRNA expressions from subcutaneous adipose tissue by Affymetrix. In BMI-discordant pairs (mean ΔBMI = 5.9 kg/m 2 ), serum dihydrotestosterone (DHT) was lower [mean 1.9 (SD 0.7) vs . 2.4 (1.0) nmol/l, P = 0.040] and mRNA expressions of DHT-inactivating AKR1C2 ( P = 0.021) and cortisol-producing HSD11B1 ( P = 0.008) higher in the heavier compared to the leaner co-twins. Serum free 17 β -estradiol (E2) was higher [2.3 (0.5) vs . 1.9 (0.5) pmol/l, P = 0.028], and in all twin pairs, serum E2 and estrone concentrations were higher in the heavier than in the leaner co-twins [107 (28) vs . 90 (22) pmol/l, P = 0.006; and 123 (43) vs . 105 (27) pmol/l, P = 0.025]. Within all twin pairs, i.e. independent of genetic effects and age, 1) the amount of subcutaneous fat inversely correlated with serum total and free testosterone, DHT, and sex hormone-binding globulin (SHBG) concentrations ( P < 0.01 for all), 2) intra-abdominal fat with total testosterone and SHBG ( P < 0.05), and 3) liver fat with SHBG ( P = 0.006). Also, 4) general and intra-abdominal adiposity correlated positively with mRNA expressions of AKR1C2 , HSD11B1 , and aromatase in adipose tissue ( P < 0.05). In conclusion, acquired adiposity was associated with decreased serum DHT and increased estrogen concentrations, independent of genetic factors and age. The reduction of DHT could be linked to its increased degradation (by AKR1C2 and HSD11B1) and increased estrogen levels to increased adiposity-related expression of aromatase in adipose tissue. [ABSTRACT FROM AUTHOR]

17β-estradiol (17β-E 2) has been implicated in activating autophagy by upregulating SIRT3 (Sirtuin 3) expression, thereby inhibiting the senescence of vascular endothelial cells. Herein, we further examined the molecular mechanisms that regulate SIRT3 expression in 17β-E 2 -induced autophagy. Reverse-transcription-polymerase chain reaction was employed to measure the expression of plasmacytoma variant translocation 1 (PVT1), microRNAs (miRNAs), and SIRT3, and the dual-luciferase assay was used to determine their interaction. Electron microscopy observes autophagosomes, green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) staining, and immunoblot analysis with antibodies against LC3，beclin-1, and P62 were conducted to measure autophagy. Cellular senescence was determined using immunoblot analysis with anti-phosphorylated retinoblastoma and senescence-associated β-galactosidase staining. Women with higher estrogen levels (during the 10–13th day of the menstrual cycle or premenopausal) exhibit markedly higher serum levels of PVT1 than women with lower estrogen levels (during the menstrual period or postmenopausal). The dual-luciferase assay showed that PVT1 acts as a sponge for miR-31, and miR-31 binds to its target gene, SIRT3. The 17β-E 2 treatment increased the expression of PVT1 and SIRT3 and downregulated miR-31 expression in human umbilical vein endothelial cells (HUVECs). Consistently, PVT1 overexpression suppresses miR-31 expression, promotes 17β-E2-induced autophagy, and inhibits H 2 O 2 -induced senescence. miR-31 inhibitor increases SIRT3 expression and leads to activation of 17β-E 2 -induced autophagy and suppression of H 2 O 2 - induced senescence. Conclusion : Our findings demonstrated that 17β-E 2 upregulates PVT1 gene expression and PVT1 functions as a sponge to inhibit miR-31, resulting in the upregulation of SIRT3 expression and activation of autophagy and subsequent inhibition of H 2 O 2 -induced senescence in HUVECs. • 17β-E 2 is associated with an increase in PVT1 expression, then PVT1 binds directly to miR-31 and down-regulates miR-31 expression, while miR-31 binds to SIRT3 and up-regulates SIRT3 expression. • 17β-E 2 increases the expression of lncRNA PVT1 and PVT1 acts as a sponge to suppress the function of miR-31, which leads to the upregulation of SIRT3 expression. [ABSTRACT FROM AUTHOR]

Human placental estrone (E1)/dehydroepiandrosterone (DHEA) sulfatase (human placental steroid sulfatase; hSTS) is an integral membrane protein of the endoplasmic reticulum. This Ca2+-dependent enzyme catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, which then act as precursors for the biosynthesis of 17β-estradiol (E2) and dihydrotestosterone (DHT), respectively, the most potent forms of estrogens and androgens. hSTS is a key enzyme for the local production of E2 and DHT in the breast and the prostate. The enzyme is known to be responsible for maintaining high levels of estrogens in the breast tumor cells. The crystal structure of hSTS purified from human placenta has previously been reported at 2.6 Å resolution. Here we present the structure of hSTS determined at the superior 2.0 Å resolution bringing new clarity to the atomic architecture of the active site. The molecular basis of catalysis and steroid-protein interaction are revisited in light of the new data. We also reexamine the enzyme's quaternary association and its implication on the membrane integration. A secondary ligand binding pocket at the intermolecular interface and adjacent to the active site access channel, buried into the gill of the mushroom-shaped molecule, has been identified. Its role as well as that of a phosphate ion bound to an exposed histidine side chain are examined from the structure-function perspective. Higher resolution data also aids in the tracing of an important loop missing in the previous structure. • New clarity to the atomic structure of human placental steroid sulfatase. • Molecular basis of catalysis and steroid-protein interaction at a higher resolution. • Quaternary association and its implication on the membrane integration. • A secondary ligand site, a phosphate binding site, and their roles from structural perspective. [ABSTRACT FROM AUTHOR]

Steroid hormones, estrogen and androgen, control transcription in various reproductive and non-reproductive tissues. Both hormones are known to be important for control of sperm release from the seminiferous epithelium (spermiation), a process characterized by extensive remodeling of actin filaments and endocytosis. Earlier studies with an estrogen (E2)-induced rat model of spermiation failure revealed genes involved in actin remodeling (Arpc1b and Evl) and endocytosis (Picalm, Eea1, and Stx5a) to be differentially regulated. Further, among these genes, Arpc1b and Evl were found to be estrogen-responsive whereas Eea1 and Stx5a were androgen-responsive and Picalm was responsive to both hormones in seminiferous tubule cultures. Yet, the mechanism by which these genes are regulated by estrogen and androgen in the testis was unclear. Here, we report the presence of a functional estrogen response element (ERE) upstream of Arpc1b and Evl genes and androgen response element (ARE) upstream of Picalm, Eea1, and Stx5a genes. Chromatin immunoprecipitation in control versus E2-treated testes revealed significant changes in estrogen receptor beta (ERβ) recruitment along with coregulators to the EREs upstream of Arpc1b and Evl genes and androgen receptor (AR) at AREs upstream of Picalm, Eea1, and Stx5a genes. Enrichment patterns of these EREs/AREs with coregulators, activating and repressing histone modifications along with RNA polymerase II recruitment, correlated with the observed expression patterns of these genes upon E2 treatment. Taken together, our results reveal direct targets of estrogen and androgen in the testes and provide insights into transcriptional control of sperm release by the two steroid hormones. [ABSTRACT FROM AUTHOR]

17beta-hydroxysteroid dehydrogenase type 5 (17β-HSD5) is an important enzyme associated with sex steroid metabolism in hormone-dependent cancer. However, reports on its expression and its prognostic value in breast cancer are inconsistent. Here, we demonstrate the impact of 17β-HSD5 expression modulation on the proteome of estrogen receptor-positive (ER+) breast cancer cells. RNA interference technique (siRNA) was used to knock down 17β-HSD5 gene expression in the ER+ breast cancer cell line MCF-7 and the proteome of the 17β-HSD5-knockdown cells was compared to that of MCF-7 cells using two-dimensional (2-D) gel electrophoresis followed by mass spectrometry analysis. Ingenuity pathway analysis (IPA) was additionally used to assess functional enrichment analyses of the proteomic dataset, including protein network and canonical pathways. Our proteomic analysis revealed only four differentially expressed protein spots (fold change > 2, p < 0.05) between the two cell lines. The four spots were up-regulated in 17β-HSD5-knockdown MCF-7 cells, and comprised 21 proteins involved in two networks and in functions that include apoptosis inhibition, regulation of cell growth and differentiation, signal transduction and tumor metastasis. Among the proteins are nucleoside diphosphate kinase A (NME1), 78 kDa glucose-regulated protein (GRP78) and phosphoglycerate kinase 1 (PGK1). We also showed that expression of 17β-HSD5 and that of the apoptosis inhibitor GRP78 are strongly but negatively correlated. Consistent with their opposite regulation, GRP78 knockdown decreased MCF-7 cell viability whereas 17β-HSD5 knockdown or inhibition increased cell viability and proliferation. Besides, IPA analysis revealed that ubiquitination pathway is significantly affected by 17β-HSD5 knockdown. Furthermore, IPA predicted the proto-oncogene c-Myc as an upstream regulator linked to the tumor-secreted protein PGK1. The latter is over-expressed in invasive ductal breast carcinoma as compared with normal breast tissue and its expression increased following 17β-HSD5 knockdown. Our present results indicate a 17β-HSD5 role in down-regulating breast cancer development. We thus propose that 17β-HSD5 may not be a potent target for breast cancer treatment but its low expression could represent a poor prognosis factor. [ABSTRACT FROM AUTHOR]

Recent reports show that, in patients treated with finasteride for male pattern hair loss, persistent side effects including sexual side effects, depression, anxiety and cognitive complaints may occur. We here explored the psychiatric and andrological features of patients affected by post-finasteride syndrome (PFS) and verified whether the cerebrospinal fluid (CSF) and plasma levels of neuroactive steroids (i.e., important regulators of nervous function) are modified. We found that eight out of sixteen PFS male patients considered suffered from a DSM-IV major depressive disorder (MDD). In addition, all PFS patients showed erectile dysfunction (ED); in particular, ten patients showed a severe and six a mild-moderate ED. We also reported abnormal somatosensory evoked potentials of the pudendal nerve in PFS patients with severe ED, the first objective evidence of a neuropathy involving peripheral neurogenic control of erection. Testicular volume by ultrasonography was normal in PFS patients. Data obtained on neuroactive steroid levels also indicate interesting features. Indeed, decreased levels of pregnenolone, progesterone and its metabolite (i.e., dihydroprogesterone), dihydrotestosterone and 17beta-estradiol and increased levels of dehydroepiandrosterone, testosterone and 5alpha-androstane-3alpha,17beta-diol were observed in CSF of PFS patients. Neuroactive steroid levels were also altered in plasma of PFS patients, however these changes did not reflect exactly what occurs in CSF. Finally, finasteride did not only affect, as expected, the levels of 5alpha-reduced metabolites of progesterone and testosterone, but also the further metabolites and precursors suggesting that this drug has broad consequence on neuroactive steroid levels of PFS patients. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion of organic compounds, abundant in exhaust fumes and cigarette smoke. They act by binding to the aryl hydrocarbon receptor (AHR) which induces expression of phase 1 and phase 2 enzymes in the liver. PAH induced AHR activation may also lead to adverse effects by modulating other pathways, for example estrogen receptor (ER) signaling in the female reproductive tract. We have investigated the effects of the PAH 3-methylcholanthrene (3-MC) on 17β-estradiol (E2) dependent signaling in the uterus of ovariectomized rats to characterize the cross talk between AHR and ER on an mRNA transcriptome wide scale. A standard three day uterotrophic assay was performed in young adult Lewis rats. Treatment induced effects were analyzed using histology, immunohistochemistry and gene expression analysis by microarray and qPCR . 3-MC shows broad E2 antagonistic effects on uterine mRNA transcription of the vast majority of E2 regulated genes, significantly altering prostaglandin biosynthesis, complement activation, coagulation pathways and other inflammatory response pathways. The regulation of ER expression in the uterus, but not the regulation of E2 metabolism in the liver, was identified as a potentially important factor in mediating this general antiestrogenic effect. The regulation of prostaglandin biosynthesis by E2 is important for inflammation-like events during pregnancy including the initiation of birth. Our results suggest that adverse effects of PAHs on prostaglandin related pathways are likely caused by the interference with E2 signaling, specifically by inhibiting the E2 mediated downregulation of PGF2α. Characterization of the generalized antagonistic effect of 3-MC on E2 dependent signaling in the rat uterus thus contributes to a better understanding of molecular mechanisms of the toxicity of PAHs in female reproductive organs. [ABSTRACT FROM AUTHOR]

The majority of breast cancer cases are estrogen receptor positive (ER + ). Although, third-generation aromatase inhibitors (AIs) are used as first-line treatment in post-menopausal women, they cause endocrine resistance and bone loss, which limits their success. Therefore, there is a demand to discover new potent molecules, with less toxicity that can circumvent these drawbacks. Our group has previously demonstrated that new 7α-substituted steroidal molecules, 7α-(2ξ,3ξ-epoxypropyl)androsta-1,4-diene-3,17-dione (3) , 7α-allylandrost-4-ene-3,17-dione (6) , 7α-allylandrost-4-en-17-one (9) , 7α-allyl-3-oxoandrosta-1,4-dien-17β-ol (10) and 7α-allylandrosta-1,4-diene-3,17-dione (12) are potent AIs in placental microsomes. In this work, it was investigated their anti-aromatase activity and in vitro effects in sensitive and resistant breast cancer cells. All the steroids efficiently inhibit aromatase in breast cancer cells, allowing to establish new structure-activity relationships for this class of compounds. Moreover, the new AIs can inhibit breast cancer cell growth, by causing cell cycle arrest and apoptosis. The effects of AIs 3 and 12 on sensitive cells were dependent on aromatase inhibition and androgen receptor (AR), while for AI 9 and AI 10 were AR- and ER-dependent, respectively. In addition, it was shown that all the AIs can sensitize resistant cancer cells being their behavior similar to the sensitive cells. In summary, this study contributes to the understanding of the structural modifications in steroidal scaffold that are translated into better aromatase inhibition and anti-tumor properties, providing important information for the rational design/synthesis of more effective AIs. In addition, allowed the discovery of new potent 7α-substituted androstane molecules to inhibit tumor growth and prevent endocrine resistance. [ABSTRACT FROM AUTHOR]

As the knowledge on the estrogenic system in the brain grows, the possibilities to modulate it in order to afford further neuroprotection in brain damaging disorders so do it. We have previously demonstrated the ability of the selective estrogen receptor modulator, bazedoxifene (BZA), to reduce experimental ischemic brain damage. The present study has been designed to gain insight into the molecular mechanisms involved in such a neuroprotective action by investigating: 1) stroke-induced apoptotic cell death; 2) expression of estrogen receptors (ER) ERα, ERβ and the G-protein coupled estrogen receptor (GPER); and 3) modulation of MAPK/ERK1/2 and PI3K/Akt signaling pathways. For comparison, a parallel study was done with 17β-estradiol (E2)-treated animals. Male Wistar rats subject to transient right middle cerebral artery occlusion (tMCAO, intraluminal thread technique, 60 min), were distributed in vehicle-, BZA- (20.7 ± 2.1 ng/mL in plasma) and E2- (45.6 ± 7.8 pg/mL in plasma) treated groups. At 24 h from the onset of tMCAO, RT-PCR, Western blot and histochemical analysis were performed on brain tissue samples. Ischemia-reperfusion per se increased apoptosis as assessed by both caspase-3 activity and TUNEL-positive cell counts, which were reversed by both BZA and E2. ERα and ERβ expression, but not that of GPER, was reduced by the ischemic insult. BZA and E2 had different effects: while BZA increased both ERα and ERβ expression, E2 increased ERα expression but did not change that of ERβ. Both MAPK/ERK1/2 and PI3K/Akt pathways were stimulated under ischemic conditions. While BZA strongly reduced the increased p-ERK1/2 levels, E2 did not. Neither BZA nor E2 modified ischemia-induced increase in p-Akt levels. These results show that modulation of ERα and ERβ expression, as well as of the ERK1/2 signaling pathway accounts, at least in part, for the inhibitory effect of BZA on the stroke-induced apoptotic cell death. This lends mechanistic support to the consideration of BZA as a potential neuroprotective drug in acute ischemic stroke treatment. [ABSTRACT FROM AUTHOR]

17β-estradiol (E2) and progesterone (P) are neuroprotective factors in the brain preventing neuronal death under different injury paradigms. In previous studies, we demonstrated that both steroids dampen neuronal damage, improve local energy metabolism and attenuate pro-inflammatory responses. MicroRNAs (miRNAs) are small regulators of distinct target genes on the RNA level. Their expression patterns are misbalanced in several neurological disorders. To explore the regulatory mechanisms of steroid hormones on selected miRNAs and their validated targets in ischemia, we used the transient middle cerebral artery occlusion (tMCAO) model. 12-week old male rats were subjected to 2 h tMCAO and expression patterns of miR‐223, miR‐200c, miR‐375, miR‐199 and miR‐214 (all −3p) were determined. Using semi-quantitative real time PCR, we examined the role of E2 or P as regulatory factors for miRNAs and theirs target genes. Besides miR-375, all mentioned miRNAs showed a steady increase with a peak at 72 h post tMCAO, whereas highest levels of miR-375 were detected at 12 h post tMCAO. E2 or P selectively dampened miR-223 and miR-214 but further boosted miR-375 levels. With respect to the miR-223 regulated target genes NR2B and GRIA2 which both decreased after tMCAO, E2 and P application reversed this effect. Further, steroid treatment inhibited the hypoxia-induced increase of the miR-375 target genes Bcl-2 and RAD1. These findings provide new insights into the regulatory role of neuroprotection mediated by sex steroids in the brain. Both hormones are capable of influencing the expression of miRNAs which are relevant during neuropathological processes. Thereby, E2 and P indirectly control pro-apoptotic and −inflammatory gene translation and provide a mechanism to dampen explosive tissue damage. [ABSTRACT FROM AUTHOR]