Steroid 17β-estradiol Research Articles

Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay.

Mixture effects of 3 model endocrine disruptors, prochloraz, ketoconazole, and genistein, on steroidogenesis were tested in the adrenocortical H295R cell line. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and 17β-estradiol) were analyzed using gas chromatography and tandem mass spectrometry (GC-MS/MS) to investigate the effects throughout the steroidogenic pathway. Current modeling approaches often rely on models assuming compounds acting independently and that the individual effects in some way can be summarized to predict a mixture effect. In H295R cells with an intact steroidogenic pathway, such assumptions may not be feasible. The purpose of this study was therefore to evaluate whether effects of a mixture with differing modes of action followed or deviated from additivity (concentration addition) and whether the H295R cell line was suitable for evaluating mixture toxicity of endocrine disruptors with different modes of action. The compounds were chosen because they interfere with steroidogenesis in different ways. They all individually decrease the concentrations of the main sex steroids downstream but exert different effects upstream in the steroidogenic pathway. Throughout the study, we observed lowest observed effect concentrations of mixtures at levels 2 to 10 times higher than the predicted EC(50), strongly indicating antagonistic effects. The results demonstrate that chemical analysis combined with the H295R cell assay is a useful tool also for studying how mixtures of endocrine disruptors with differing modes of action interfere with the steroidogenic pathway and that existing models like concentration addition are insufficient in such cases. Furthermore, for end points where compounds exert opposite effects, no relevant models are available.

Abstract 36: Administration of 17β-Estradiol in C57Bl/6N Female Mice Leads to Cardiac Atrophy and Dysfunction via a β-Catenin Mechanism

The steroid hormone 17β-estradiol (E2) regulates several biological processes. In contrast to its anti-hypertrophic effects under pressure overload, we recently found that E2 induced physiological hypertrophic growth in healthy C57Bl/6J mice but not C57Bl/6N mice. Here, we aimed at the characterization of the effects of E2 in C57Bl/6N mice and tested the hypothesis that β-catenin mediates these E2 effects. Following ovariectomy, 2-month-old C57Bl/6N wild-type and cardiac-specific β-catenin-deleted (β-cat Δex2-6 ) mice were randomized to an E2-containing or soy-free (control, CON) diet ( n = 7-13/group). Cardiac function was examined by echocardiography following established procedures. The 3-month physiological dose of E2 led to a higher relative uterus weight compared with CON ( P < 0.001) in both WT and β-cat Δex2-6 mice. The relative heart weight was significantly reduced by E2 compared with CON in WT mice ( P < 0.001), while there was no significant effect in β-cat Δex2-6 mice. Cardiomyocyte cross-sectional area was also significantly decreased by E2 ( n = 5-7/group; P < 0.001) compared with CON in WT mice, while there was no significant effect in β-cat Δex2-6 mice. Echocardiography revealed a significant decrease in septum width ( P < 0.001) and posterior wall thickness ( P < 0.01) in E2 treated WT mice compared with CON, while there was no significant effect in β-cat Δex2-6 mice ( n = 8/group). These E2-induced structural changes in WT mice were accompanied by a significant decrease in cardiac function, namely a 23% decrease in fractional shortening compared with CON ( P < 0.05), while there was no significant effect in β-cat Δex2-6 mice. Immunoblotting revealed a significant increase in the levels of the ubiquitin ligase and key regulator of proteasome-dependent protein degradation muscle-specific RING finger protein 1 (MuRF1) by E2 compared with CON in WT mice ( P < 0.05), while there was no significant effect in β-cat Δex2-6 mice. Although we hypothesized increased autophagic activity, we found no effect on the autophagy-related protein LC3 in WT or β-cat Δex2-6 mice. In conclusion, our surprising findings show that E2 leads to cardiac atrophy and dysfunction in C57Bl/6N mice via a β-catenin mechanism seemingly in an autophagy-independent manner.

Effects of selective estrogen receptor alpha and beta modulators on prepulse inhibition in male mice

Multiple lines of evidence suggest that the sex steroid hormone 17-β estradiol (E2) plays a protective role in schizophrenia. Systemic E2 enhances prepulse inhibition (PPI) of the acoustic startle reflex, an operational measure of sensorimotor gating known to be impaired in schizophrenia and related disorders. However, the relative contribution of different estrogen-receptor (ER) isoforms in these associations still awaits examination. The present study explored the effects of ER-α and ER-β stimulation or blockade on PPI and their functional relevance in an amphetamine-induced PPI deficiency model in male mice. Prior to the assessment of PPI, C57BL/6N male mice were injected with the ER-α agonist 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), the ER-α antagonist 1,3-bis (4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy) phenol]-1N-pyrozole dihydrochloride (MPP), the ER-β agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN), or the ER-β antagonist 4-[2-phenyl-5,7-bis (trifluoromethyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PHTPP), with or without concomitant amphetamine treatment. Acute pharmacological stimulation and blockade of ER-α, respectively, led to a dose-dependent increase and decrease in basal PPI. In contrast, acute treatment with preferential ER-β modulators spared PPI under basal conditions. Pretreatment with either ER-α or ER-β agonist was, however, effective in blocking amphetamine-induced PPI disruption. Our study demonstrates that activation of either ER isoform is capable of modulating dopamine-dependent PPI levels. At the same time, our results suggest that endogenous ER-α signaling may be more relevant than ER-β in the regulation of sensorimotor gating under basal conditions.

The effects of salinity on reproductive performance and plasma levels of sex steroids in Caspian kutumRutilus frisii kutum

Sexually mature kutum, Rutilus frisii kutum, captured from its natural habits, the Caspian Sea and the Khoushkrood River, reared at 0.5 g L−1 and 8–13 g L−1 for approximately 1 year in experimental condition, for assessing the effect of salinity on reproduction. Plasma concentrations of sex steroid hormones (17β-estradiol, testosterone and 17α-hydroxyprogestrone) were measured in the three stages of gametogenesis. Female kutum held at <0.5 g L−1 or 8–13 g L−1 had no ovulated oocytes in their ovaries. In contrast, males held in captivity were spermiated, similar to their wild counterparts. The average sperm volume of males held at <0.5 g L−1 (2.36 ± 0.46 mL) was lower than males held at 8–13 g L−1 (3.65 ± 0.73 mL) at the end of the experimental period. The highest concentration of testosterone was observed in mid-gametogenesis in wild fish that was significantly higher than the concentration seen in fish held in either <0.5 g L−1 or 8–13 g L−1. Female kutum showed suppressed steroid hormones in captivity, resulting in failure in the gonad development. However, male kutum adapt well to captivity and showed synchrony in steroid hormone variations with the wild fish, resulting in the testicular development. Results of this study also indicate that salinity plays a minor, but vital, role in reproduction of kutum, a factor that needs to be considered for keeping broodstock of brackish water fish species like kutum.

Co-treatment with the non-steroidal anti-androgen drug, flutamide and the natural estrogen, 17β-estradiol does not lead to additive reproductive impairment in juvenile Murray rainbowfish (Melanotaenia fluviatilis).

The aim of this study was to investigate if the anti-androgen, flutamide, and the estrogen, 17β-estradiol work together to feminize juvenile Murray rainbowfish (Melanotaenia fluviatilis). Fish (60 days post-hatch) were exposed to 25 ng/L 17β-estradiol (E2), 25 µg/L flutamide (Flu low), 250 µg/L flutamide (Flu high), E2 + Flu low and E2 + Flu high. After 35 days of exposure, concentrations of sex steroid hormones, 17β-estradiol and 11-keto testosterone (11-KT), were determined in the head; and vitellogenin (VTG) concentration was measured in the tail. The abdomens were used for histological investigation of the gonads. Treatment with E2 + Flu high resulted in reduction in body weights and lengths in males and condition factor in females. Intersex was noted in Flu high and E2 + Flu high treatments. Exposures to E2 and/or Flu (low and high) resulted in precocious oocyte development but inhibited sperm development. The 17β-estradiol levels decreased significantly in the heads of both sexes after exposures to E2 and/or Flu (high and low). Flu high and E2 alone increased the 11-KT levels in both sexes. However, E2 + Flu low decreased 11-KT levels in males and increased them in females. Flutamide (low and high) induced VTG protein in the tails of both sexes. In males, VTG was not induced in the tail after exposure to E2. No significant effect of flutamide on E2-induced VTG concentration was noted. We conclude that co-treatment with flutamide and 17β-estradiol does not lead to additive reproductive impairment in juvenile Murray rainbowfish.

Digit ratio (2D:4D) does not correlate with daily 17β-estradiol and progesterone concentrations in healthy women of reproductive age.

Second-to-fourth digit ratio (2D:4D) is proposed as a proxy for the prenatal balance of sex hormones, is related to hormone-dependent characteristics in adult life, and is a possible predictor of disease later in life. Here, we studied the relationship between 2D:4D and ovarian steroid hormones (17β-estradiol and progesterone) among women of reproductive age. From 186 healthy premenopausal women, aged 24-37 years, we collected saliva samples daily during the entire menstrual cycle. Data on reproductive and lifestyle characteristics were collected via questionnaires, and anthropometric measurements were performed. No statistically significant relationships were detected between adult women's sex hormone concentrations (17β-estradiol and progesterone) during the menstrual cycle and 2D:4D, in either left or right hand, when controlling for size at birth, body mass index, and physical activity. This study shows, for the first time in a large sample of women of reproductive age, that 2D:4D is not a predictor of adult women's sex hormone concentration. The lack of relationship may be because 2D:4D might be genetically determined and is not related to maternal nutritional environment during fetal development. These results support the hypothesis that, in contrast to the nutritional quality of the fetal environment, the fetal hormonal environment (reflected by 2D:4D) does not determine reproductive physiology in later life.

Animal Manure Separation Technologies Diminish the Environmental Burden of Steroid Hormones

Newly developed treatment technologies are capable of separating livestock manure into a liquid fraction and a solid fraction using sedimentation, mechanical, and/or chemical methods. These technologies offer a potential means of distributing nutrients to agricultural lands without the unwanted environmental risks associated with the release of steroid hormones to adjacent waterways. To assess the potential benefit of these technologies in reducing the level of release of steroid hormones to adjacent waterways, distribution profiles of nine steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol, and 17β-estradiol) were determined in raw swine manure, and in solid and liquid fractions separated from ten full-scale manure separation systems. Steroid hormone concentrations, normalized for nitrogen content, were significantly higher in separated solids than in liquids. If separated liquids are applied instead of raw manure, steroid hormone loading can be reduced by a factor of 2 at a constant nitrogen fertilization level.

Sterile ‘Judas’ carp—Surgical sterilisation does not impair growth, endocrine and behavioural responses of male carp

Use of ‘Judas’ fish to betray the locations of conspecifics is a powerful tool in management of invasive pest fish but poses a risk of contributing to recruitment. Our aim therefore was to generate surgically sterilised male common carp (Cyprinus carpio) and test whether they readily assimilate into wild populations, retain sexual behaviour and successfully betray the locations of feral carp. Male common carp were surgically sterilised (n=44) adopting a two-point nip technique, using either a haemoclip, suture or electro cautery to tie each of the testicular ducts about 2.5cm cranial to urogenital sinus—retaining all of the glandular testis tissue. Observed survival (95%) and success (>70%) rates were relatively high. Plasma steroids (11-keto testosterone and 17β-estradiol) were quantified by immunoassay. A subset of sterile and control male fish (n=7 each) were implanted with radio-transmitters and released into Lake Sorell (50km2) and their ability to betray the location of feral carp was assessed by radio tracking and targeted fishing. There was a statistically significant difference in 11-keto testosterone and 17β-estradiol levels over time (P<0.05), but not between the sterile and control groups within each sampling time (P>0.05), implying that surgery did not compromise the animals physiologically. The sterile Judas fish integrated well into the population—behaving similarly to control Judas males and assisted in the capture of feral carp. The study marks a significant breakthrough in the management of this pest fish with potential adoption to the management of other pest fish globally.

Sex hormones change visceral pigmentation in Eupemphix nattereri (Anura): effects in testicular melanocytes and hepatic melanomacrophages

Melanomacrophages and melanocytes are able to modify pigment intensity on the body surface as a response to temperature change, hibernation, hypoxia, infection, oxidative stress and exogenous use of hormones. This study evaluated the effect of the sex hormones 17β-estradiol and testosterone cypionate on the hepatic melanomacrophages and testicular melanocytes of Eupemphix nattereri under hyperestrogenic and hyperandrogenic conditions, eight days after daily hormonal administration as well as after a 15-day recovery period. Animals were injected subcutaneously with 17β-estradiol, testosterone cypionate or the carrier medium (as control). Histological analysis was used to examine the effect of the treatments. The results show that sex hormones altered the pigmentation of hepatic melanomacrophages and testicular melanocytes. Both liver melanomacrophages and testicular melanocytes in E. nattereri males displayed increased pigmentation after testosterone treatment, while estradiol decreased liver pigmentation only. This study describes, for the first time, the effects of sex hormones on visceral pigmentation of anura, showing the responsiveness of melanocytes and melanomacrophages to sex hormones.

Brain nonapeptide and gonadal steroid responses to deprivation of heterosexual contact in the black molly.

ABSTRACTFish may respond to different social situations with changes in both physiology and behaviour. A unique feature of fish is that social interactions between males and females strongly affect the sexual characteristics of individuals. Here we provide the first insight into the endocrine background of two phenomena that occur in mono-sex groups of the black molly (Poecilia sphenops): masculinization in females and same-sex sexual behaviour, manifested by gonopodial displays towards same-sex tank mates and copulation attempts in males. In socially controlled situations, brain neurohormones impact phenotypic sex determination and sexual behaviour. Among these hormones are the nonapeptides arginine vasotocin (AVT) and isotocin (IT), counterparts of the well-known mammalian arginine vasopressin and oxytocin, respectively. To reveal potential hormone interactions, we measured the concentrations of bioactive AVT and IT in the brain, along with those of the sex steroids 17β-estradiol and 11-ketotestosterone in the gonads, of females, masculinized females, males displaying same-sex sexual behaviour and those who did not. These data were supplemented by morphological and histological analyses of the gonads. Correlations between brain nonapeptides and gonadal steroids strongly suggest a cross talk between hormonal systems. In the black molly, the masculinization process was associated with the production of brain AVT and gonadal steroids, whereas same-sex sexual behaviour involves both brain nonapeptides, but neither of the sex steroids. This study extends current knowledge of endocrine control of phenotypic sex and sexual behaviour in fish and for the first time links brain nonapeptides with the occurrence of male-male sexual behaviour in lower vertebrates.

In vitro changes in secretion activity of rat ovarian fragments induced by molybdenum.

The aim of this in vitro study was to examine the secretion activity (progesterone, 17beta-estradiol and insulin-like growth factor-I) of rat ovarian fragments after molybdenum (Mo) addition. Rat ovarian fragments were incubated with ammonium molybdate (NH(4))(6)Mo(7)O(24).4H(2)O at the doses 90, 170, 330 and 500 microg.ml(-1) for 24 h and compared with control group without Mo addition. Release of progesterone (P(4)), estradiol (17beta-estradiol) and insulin-like growth factor I (IGF-I) by ovarian fragments was assessed by radioimmunoassay (RIA). Data show that P(4) release by ovarian fragments was not affected by (NH(4))(6).Mo(7)O(24).4H(2)O addition at all the doses used (90-500 microg.ml(-1)). However, addition of ammonium molybdate was found to cause a significant (P<0.05) dose-dependent decrease (at the doses 90, 170 and 500 microg.ml(-1)) in release of 17beta-estradiol by ovarian fragments in comparison to control. Also, addition of ammonium molybdate significantly (P<0.05) inhibited IGF-I release at all the doses (90-500 microg.ml(-1)) used in the study. Results suggest ammonium molybdate induced inhibition in the release of growth factor IGF-I and its dose-dependent effect on secretion of steroid hormone 17beta-estradiol but not progesterone. These data contribute to new insights regarding the mechanism of action of Mo on rat ovarian functions.

The sensitive period for male-to-female sex reversal begins at the embryonic stage in the Nile tilapia and is associated with the sexual genotype.

In this study, we sought to determine the mechanism of early sex reversal in a teleost by applying 4 hr feminization treatments to XY (17α-ethynylestradiol 2000 μg L(-1) ) and YY (6500 μg L(-1) ) Nile tilapia embryos on the first day post-fertilization (dpf). We then searched for changes in the expression profiles of some sex-differentiating genes in the brain (cyp19a1b, foxl2, and amh) and in sex steroids (testosterone, 17β-estradiol, and 11-ketotestosterone) concentrations during embryogenesis and gonad differentiation. No sex reversal was observed in YY individuals, whereas sex-reversal rates in XY progeny ranged from 0-60%. These results, together with the clearance profile of 17α-ethynylestradiol, confirmed the existence of an early sensitive period for sex determination that encompasses embryonic and larval development and is active prior to any sign of gonad differentiation. Estrogen treatment induced elevated expression of cyp19a1b and higher testosterone and 17β-estradiol concentrations at 4 dpf in both XY and YY individuals. foxl2 and amh were repressed at 4 dpf and their expression levels were not different between treated and control groups at 14 dpf, suggesting that foxl2 did not control cyp19a1b in the brains of tilapia embryos. Increased cyp19a1b expression in treated embryos could reflect early brain sexualization, although this difference alone cannot account for the observed sex reversal as the treatment was ineffective in YY individuals. The differential sensitivity of XY and YY genotypes to embryonic induced-feminization suggests that a sex determinant on the sex chromosomes, such as a Y repressor or an X activator, may influence sex reversal during the first steps of tilapia embryogenesis.

Cofactor-free light-driven whole-cell cytochrome P450 catalysis.

Cytochromes P450 can catalyze various regioselective and stereospecific oxidation reactions of non-functionalized hydrocarbons. Here, we have designed a novel light-driven platform for cofactor-free, whole-cell P450 photo-biocatalysis using eosin Y (EY) as a photosensitizer. EY can easily enter into the cytoplasm of Escherichia coli and bind specifically to the heme domain of P450. The catalytic turnover of P450 was mediated through the direct transfer of photoinduced electrons from the photosensitized EY to the P450 heme domain under visible light illumination. The photoactivation of the P450 catalytic cycle in the absence of cofactors and redox partners is successfully conducted using many bacterial P450s (variants of P450 BM3) and human P450s (CYPs 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) for the bioconversion of different substrates, including marketed drugs (simvastatin, lovastatin, and omeprazole) and a steroid (17β-estradiol), to demonstrate the general applicability of the light-driven, cofactor-free system.

Occurrence of selected pharmaceuticals in the principal sewage treatment plants in Rome (Italy) and in the receiving surface waters.

This paper provides data on the occurrence of selected human pharmaceuticals (carbamazepine, clofibric acid, diclofenac, fenofibrate, fenoprofen, gemfibrozil, ibuprofen, ketoprofen, and naproxen) including steroid hormones (17β-estradiol, 17α-ethinylestradiol, and estrone) in influents/effluents to/from the four principal wastewater treatment plants (WWTPs) serving the city of Rome (Italy), in two different sampling campaigns. Target compounds were also analyzed in the receiving River Tiber and River Aniene. Analytical determination was carried out by LC-MS/MS after sample cleanup and concentration by off-line solid-phase extraction (SPE). The aim of the study was to increase the information currently available on the presence and persistence of pharmaceuticals in Italian urban wastewaters and to evaluate the environmental impact of the pharmaceutical residues discharged through effluents into the receiving rivers. Results indicated that after the treatment processes, most of pharmaceuticals were not completely eliminated, as average removal efficiencies were in the 14-100% wide range during both sampling periods, with higher yields in spring than in winter. Levels detected in overall samples ranged from 5 to 2,230 ng/L in influents and from 5 to 1,424 ng/L in effluents. Carbamazepine, diclofenac, ibuprofen, and gemfibrozil showed the highest persistence to removal. Concentrations in the receiving waters were about one order of magnitude lower than in effluents, with a tendency to increase progressively through the urban tract of the river. Finally, an environmental risk analysis showed that carbamazepine, gemfibrozil, and estrone can pose a high risk at the concentrations detected in effluents and a medium risk in rivers, highlighting their potential hazard for the health of the aquatic ecosystem.

17β-estradiol attenuates ketamine-induced neuroapoptosis and persistent cognitive deficits in the developing brain

Previous studies have demonstrated that the commonly used anesthetic ketamine can induce widespread neuroapoptosis in the neonatal brain and can cause persistent cognitive impairments as the animal matures. Therefore, searching for adjunctive neuroprotective strategies that inhibit ketamine-induced neuroapoptosis and persistent cognitive impairments is highly warranted. The primary goal of this study was to investigate the protective effect of 17β-estradiol against ketamine-induced neuroapoptosis and persistent cognitive impairments in adult rats. Starting from postnatal day 7, Sprague-Dawley male rat pups were given a daily administration of ketamine (75mg/kg, i.p.) or 17β-estradiol (600μg/kg, s.c.) in combination with ketamine (75mg/kg, i.p.). The animals were treated for three consecutive days. 24h after the last injection, the rats were decapitated, and the prefrontal cortex (PFC) was isolated to detect neuroapoptosis by cleaved caspase-3 immunohistochemistry and by using the TUNEL assay. The neuroactive steroid 17β-estradiol was quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The protein levels of BDNF and pAkt were measured by western blot analysis. At two months of age (60 days), the learning and memory abilities were tested using the Morris water maze. The results showed that ketamine triggered significant neuroapoptosis in the neonatal PFC accompanied by the downregulation of 17β-estradiol, BDNF and pAkt. The co-administration of 17β-estradiol with ketamine attenuated these changes. Moreover, 17β-estradiol significantly reversed the learning and memory deficits observed at 60 days of age. In brief, our present data demonstrate that 17β-estradiol attenuates ketamine-induced neuroapoptosis and reverses long-term cognitive deficits in developing rats and thus may be a potential therapeutic and neuroprotective method for the treatment of neurodevelopmental disorders.This article is part of a Special Issue entitled SI: Brain and Memory.

Ubiquitin-activating enzyme is necessary for 17β-estradiol-induced breast cancer cell proliferation and migration.

The sex steroid hormone 17β-estradiol (E2) regulates breast cancer (BC) cell proliferation and migration through the activation of a plethora of signal transduction cascades (e.g., PI3K/AKT activation) starting after E2 binding to the estrogen receptor alpha (ERα). The activity of the ubiquitin (Ub)-system modulates many physiological processes (e.g., cell proliferation and migration), and recently, a specific inhibitor (Pyr-41) of the Ub-activating enzyme (E1), which works as the activator of the Ub-based signaling, has been identified to prevent the functions of the Ub-system. Here, by using Pyr-41, we studied the involvement of the Ub-system in E2-induced signaling to proliferation and migration of BC cells. Our data indicate that E1 activity is involved in the E2:ERα signaling important for cell proliferation and migration through the modulation of the E2-evoked activation of the PI3K/AKT and the p38/MAPK pathways. These discoveries indicate a new molecular circuitry that can be further explored to define new opportunities for BC treatment.

Minireview: Steroid-regulated paracrine mechanisms controlling implantation.

Implantation is an essential process during establishment of pregnancy in mammals. It is initiated with the attachment of the blastocyst to a receptive uterine epithelium followed by its invasion into the stromal tissue. These events are profoundly regulated by the steroid hormones 17β-estradiol and progesterone. During the past several years, mouse models harboring conditional gene knockout mutations have become powerful tools for determining the functional roles of cellular factors involved in various aspects of implantation biology. Studies using these genetic models as well as primary cultures of human endometrial cells have established that the estrogen receptor α, the progesterone receptor, and their downstream target genes critically regulate uterine growth and differentiation, which in turn control embryo-endometrial interactions during early pregnancy. These studies have uncovered a diverse array of molecular cues, which are produced under the influence of estrogen receptor α and progesterone receptor and exchanged between the epithelial and stromal compartments of the uterus during the progressive phases of implantation. These paracrine signals are critical for acquisition of uterine receptivity and functional interactions with the embryo. This review highlights recent work describing paracrine mechanisms that govern steroid-regulated uterine epithelial-stromal dialogue during implantation and their roles in fertility and disease.

A preliminary risk assessment of potential exposure to naturally occurring estrogens from Beijing (China) market milk products

This study was conducted to determine the occurrence of the natural steroid hormones estrone (E1), 17α-estradiol (αE2), 17β-estradiol (βE2) and estriol (E3) in 38 commercial milk samples obtained from markets in Beijing, China. Liquid Chromatography coupled with tandem mass spectrometry (LC–MS/MS) was employed to determine estrogens levels. The concentrations of E1, αE2, βE2 and E3 in different milk products varied from 0–146.12ng/L, 0–70.12ng/L, 0–31.85ng/L to 0–2.18ng/L, respectively. We compared exposures to estrogens through milk consumption with acceptable daily intakes (ADIs) and threshold for toxicological concern (TTC) to determine whether estrogen intakes from milk consumption are larger or smaller than the toxicity-based benchmarks. The combined margin of safety MOS (MOST) for total estrogens are about 72–99, 118–161, 539–1104, for 2–4, 4–7year-old residential children, and adults, respectively. The lowest MOST for children of 2–4years old result from comparing total of estrogens with the lowest TTC value (0.15μg/person/day) (MOS=3.5). The MOS values suggest that the individual and total estrogens that may present in milk are not causing a health risk for the local residents, including young children.

Inhibition by Female Sex Hormones of Collagen Gel Contraction Mediated by Retinal Pigment Epithelial Cells

Collagen contraction mediated by retinal pigment epithelial (RPE) cells contributes to the pathogenesis of proliferative vitreoretinopathy (PVR). We examined the effects of sex hormones on this process. Mouse RPE cells were cultured in a type I collagen gel and exposed to 17β-estradiol, progesterone, or dehydro-epiandrosterone. Collagen contraction induced by transforming growth factor-β2 (TGF-β2) was determined by measurement of gel diameter. Expression of α-smooth muscle actin (α-SMA), as well as phosphorylation of Smad2 and myosin light chain (MLC), was examined by immunoblot analysis. Matrix metalloproteinase (MMP) release was evaluated by gelatin zymography. Fibronectin and interleukin-6 secretion was measured with immunoassays. The female sex hormones 17β-estradiol and progesterone inhibited TGF-β2-induced collagen contraction mediated by RPE cells, whereas the male sex hormone dehydro-epiandrosterone had no such effect. The TGF-β2-induced release of MMP-2 and MMP-9 from RPE cells was also inhibited by 17β-estradiol and progesterone, and the MMP inhibitor GM6001 attenuated TGF-β2-induced collagen contraction. Expression of the mesenchymal markers α-SMA and fibronectin, interleukin-6 release, and Smad2 and MLC phosphorylation induced by TGF-β2 were all inhibited by 17β-estradiol and progesterone. Immunohistochemical analysis also detected nuclear immunoreactivity for estrogen and progesterone receptors in proliferative fibrocellular membranes of PVR patients. Female sex hormones inhibited TGF-β2-induced collagen contraction mediated by RPE cells. This action appeared to be mediated through inhibition both of MMP, α-SMA, and fibronectin expression as well as of Smad2 and MLC phosphorylation. Female sex hormones might thus prove effective for the treatment of PVR.

Lysosomal function is involved in 17β-estradiol-induced estrogen receptor α degradation and cell proliferation.

The homeostatic control of the cellular proteome steady-state is dependent either on the 26S proteasome activity or on the lysosome function. The sex hormone 17β-estradiol (E2) controls a plethora of biological functions by binding to the estrogen receptor α (ERα), which is both a nuclear ligand-activated transcription factor and also an extrinsic plasma membrane receptor. Regulation of E2-induced physiological functions (e.g., cell proliferation) requires the synergistic activation of both transcription of estrogen responsive element (ERE)-containing genes and rapid extra-nuclear phosphorylation of many different signalling kinases (e.g., ERK/MAPK; PI3K/AKT). Although E2 controls ERα intracellular content and activity via the 26S proteasome-mediated degradation, biochemical and microscopy-based evidence suggests a possible cross-talk among lysosomes and ERα activities. Here, we studied the putative localization of endogenous ERα to lysosomes and the role played by lysosomal function in ERα signalling. By using confocal microscopy and biochemical assays, we report that ERα localizes to lysosomes and to endosomes in an E2-dependent manner. Moreover, the inhibition of lysosomal function obtained by chloroquine demonstrates that, in addition to 26S proteasome-mediated receptor elimination, lysosome-based degradation also contributes to the E2-dependent ERα breakdown. Remarkably, the lysosome function is further involved in those ERα activities required for E2-dependent cell proliferation while it is dispensable for ERα-mediated ERE-containing gene transcription. Our discoveries reveal a novel lysosome-dependent degradation pathway for ERα and show a novel biological mechanism by which E2 regulates ERα cellular content and, as a consequence, cellular functions.