Free E2 Research Articles

Physiological declines in sex steroid exposure in relation to changes in body composition in healthy men - a prospective cohort study.

Androgen levels decline from early adulthood and decreases are steeper in men with increasing body mass index. It is, however, unclear to what extent changes in other indices of body composition and metabolism associate with changes in sex steroid levels in healthy men. Therefore, this study investigated longitudinal changes in body composition and metabolic health in relation to sex steroid levels in healthy adult men. Longitudinal, population-based study. 676 healthy men aged 24-46 years were measured at baseline and after ± 12 years. Serum sex hormone binding globulin (SHBG) was measured using immuno-assay, testosterone (T), estradiol (E2), dihydrotestosterone using LC-MS/MS, free T and E2 (cFE2) and homeostasis model for insulin resistance (HOMA-IR) were calculated. Grip strength was measured by hand grip dynamometry. Body composition was determined using dual-energy x-ray absorptiometry and peripheral quantitative computed tomography. Mean fat mass (FM), lean mass (LM) and HOMA-IR increased (all p<0.001). Decreasing androgen and SHBG levels were associated with increasing FM, whereas decreasing (cF)E2 levels were associated with decreasing FM (all p<0.005). Decreasing (cF)E2 levels and increasing SHBG levels associated with decreasing LM (all p<0.002). Changes in sex steroid levels and HOMA-IR or grip strength were not interrelated. Aging leads to increases in fat mass indices and insulin resistance, whereas changes in parameters of lean mass are less unequivocal. In healthy adult men physiological changes in sex steroid exposure clearly correlate with changes in adiposity but not so with lean mass, insulin resistance or grip strength.

Understanding the female athlete: molecular mechanisms underpinning menstrual phase differences in exercise metabolism.

Research should equitably reflect responses in men and women. Including women in research, however, necessitates an understanding of the ovarian hormones and menstrual phase variations in both cellular and systems physiology. This review outlines recent advances in the multiplicity of ovarian hormone molecular signaling that elucidates the mechanisms for menstrual phase variability in exercise metabolism. The prominent endogenous estrogen, 17-β-estradiol (E2), molecular structure is bioactive in stabilizing plasma membranes and quenching free radicals and both E2 and progesterone (P4) promote the expression of antioxidant enzymes attenuating exercise-induced muscle damage in the late follicular (LF) and mid-luteal (ML) phases. E2 and P4 bind nuclear hormone receptors and membrane-bound receptors to regulate gene expression directly or indirectly, which importantly includes cross-regulated expression of their own receptors. Activation of membrane-bound receptors also regulates kinases causing rapid cellular responses. Careful analysis of these signaling pathways explains menstrual phase-specific differences. Namely, E2-promoted plasma glucose uptake during exercise, via GLUT4 expression and kinases, is nullified by E2-dominant suppression of gluconeogenic gene expression in LF and ML phases, ameliorated by carbohydrate ingestion. E2 signaling maximizes fat oxidation capacity in LF and ML phases, pending low-moderate exercise intensities, restricted nutrient availability, and high E2:P4 ratios. P4 increases protein catabolism during the luteal phase by indeterminate mechanisms. Satellite cell function supported by E2-targeted gene expression is countered by P4, explaining greater muscle strengthening from follicular phase-based training. In totality, this integrative review provides causative effects, supported by meta-analyses for quantitative actuality, highlighting research opportunities and evidence-based relevance for female athletes.

The relationship between offspring's 2D:4D ratio and postpartum maternal circulating testosterone, estradiol, and their indices in a Ghanaian population.

The 2D:4D ratio is influenced by prenatal testosterone (PT) and estrogen (PE) exposure in utero. This study sought to determine whether evidence of Manning's hypothesis can still be observed even in the postpartum period. We hypothesize that the offspring 2D:4D ratios will be inversely correlated with maternal postpartum circulating testosterone but positively correlated with estradiol. This study was conducted between December 2020 and April 2021 and was cross-sectional in nature. There were 272 mother-offspring pairs; the mothers were aged between 18 and 36 years while the median (IQR) age of their offspring was 111 (44-210) days. Offspring right (2D:4DR) and left (2D:4DL) digit ratios were measured using computer-assisted analysis. Sampling was done at 111 (44-210) days postpartum and blood was analyzed for total testosterone (TT), estradiol (E2) and sex hormone-binding globulins using the enzyme-linked immunosorbent assay technique. The 2D:4DR of sons was significantly lower compared to daughters (p=.031). Mothers with sons had significantly increased levels of serum TT (p=.001) while mothers with daughters had significantly increased levels of E2 (p=.000). As hypothesized, the maternal serum free testosterone (FT%) was inversely correlated with their daughters' (r=-0.320, p=.003), and also with their sons' (r=-0.213, p=.047), 2D:4DL. Unexpectedly, daughters' 2D:4DL was inversely correlated with maternal circulating free E2 (r=-0.255, p=.015). In humans, evidence of the relationship between maternal testosterone levels and their offspring's 2D:4D ratio may persist even into the postpartum period.

Bisphenol A substitutes and sex hormones in children and adolescents

Bisphenol F (BPF) and bisphenol S (BPS) are increasingly used as bisphenol A (BPA) substitutes in consumer products. Little is known about the effects of BPA substitutes on reproductive endocrine function in children and adolescents. Thus, we conducted a cross-sectional study to examine the associations of BPA, BPF, and BPS with sex steroid hormones among 6-19-year old participants. Included were 1317 participants with information on BPA, BPF, BPS, and serum sex hormones [total testosterone (TT), estradiol (E2), and sex hormone binding globulin (SHBG)]. Multiple linear regression accounting for complex survey design was used to assess the association between bisphenols and sex hormones by sex-age groups. Exposure-response (ER) relationships were examined via restricted cubic splines. Significant association with BPF or BPS was sporadic, but BPA presented inverse association with the free androgen index (FAI, calculated as the ratio of TT to SHBG) and E2 and positive association with SHBG and TT/E2 in female adolescents. Further exploration of ER relationships showed that BPA (P for non-linearity = 0.03), BPF (P for non-linearity = 0.005), and BPS (P for non-linearity = 0.08) had a U-shaped relationship with FAI among female adolescents. Additionally, an inverse U-shaped curve was observed for BPA (P for non-linearity = 0.03), BPS (P for non-linearity = 0.01), and BPF (P for non-linearity = 0.01) with SHBG. The associations were virtually nonsignificant among males. Our study demonstrated that BPS and BPF may possess similar endocrine interrupting abilities as BPA.

Metabolism of testosterone during weight loss in men with obesity

ObjectiveMen with obesity often have low total and, with increasing adiposity, also low free testosterone (T) levels, which can partially restore during weight loss. Although this is partly explained by lower sex hormone binding globulin (SHBG) production and hypothalamic-pituitary downregulation, it is still not unravelled whether changes in androgen metabolism contribute to this phenomenon. Therefore, early changes in urinary excretion of T and its metabolites, during weight loss, in men with obesity are investigated. DesignLongitudinal study. MethodsFourteen men with obesity (age 52(45–60)years, BMI 42.6(41.8–44.8)kg/m²) underwent gastric bypass surgery (GBS). Before surgery and 3 weeks, 6 weeks, 6 months and 1 year thereafter, 24 h urine and fasting serum samples were collected. Serum T and estradiol (E2) levels were analyzed using LC-MS/MS and urinary metabolites of T with GC-MS/MS. ResultsAlready three weeks after GBS, serum SHBG and total T levels increased and remained increased as compared to baseline (all,p < 0.0125). Gonadotropins and (free) E2 levels were unchanged, serum E2/T ratio decreased (p < 0.0125). Total amount of urinary T increased non-significantly with mean increases of 53 % one year after GBS (p = 0.026). Urinary E2/T, estrone/T, 3α-androstanediol/T and androsterone/T ratios decreased after GBS (p < 0.0125). ConclusionsRestoration of circulating T levels during weight loss in this population is not only brought about by normalization of circulating SHBG levels, but increased production of and alterations in T metabolism also contribute. More specifically, relative decreases in aromatization and lower 5α-reductase activity might also be involved in restoring T levels in men with obesity.

Intranasal 17β-Estradiol Modulates Spatial Learning and Memory in a Rat Model of Surgical Menopause.

Exogenously administered 17β-estradiol (E2) can improve spatial learning and memory, although E2 also exerts undesired effects on peripheral organs. Clinically, E2 has been solubilized in cyclodextrin for intranasal administration, which enhances brain-specific delivery. Prior work shows that the cyclodextrin structure impacts region-specific brain distribution of intranasally administered small molecules. Here, we investigated (1) cyclodextrin type-specific modulation of intranasal E2 brain distribution, and (2) cognitive and peripheral tissue effects of intranasal E2 in middle-aged ovariectomized rats. First, brain and peripheral organ distribution of intranasally administered, tritiated E2 was measured for E2 solubilized freely or in one of four cyclodextrin formulations. The E2-cyclodextrin formulation with greatest E2 uptake in cognitive brain regions versus uterine horns was then compared to free E2 on learning, memory, and uterine measures. Free E2 improved spatial reference memory, whereas E2-cyclodextrin impaired spatial working memory compared to their respective controls. Both E2 formulations increased uterine horn weights relative to controls, with E2-cyclodextrin resulting in the greatest uterine horn weight, suggesting increased uterine stimulation. Thus, intranasal administration of freely solubilized E2 is a strategic delivery tool that can yield a cognitively beneficial impact of the hormone alongside decreased peripheral effects compared to intranasal administration of cyclodextrin solubilized E2.

Poly(lactic-co-glycolic Acid) Nanoparticle Encapsulated 17β-Estradiol Improves Spatial Memory and Increases Uterine Stimulation in Middle-Aged Ovariectomized Rats

Hormone therapy that contains 17β-estradiol (E2) is used commonly for treatment of symptoms associated with menopause. E2 treatment has been shown to improve cognitive function following the decrease in ovarian hormones that is characteristic of menopause. However, once in circulation, the majority of E2 is bound to serum hormone binding globulin or albumin, becoming biologically inactive. Thus, therapeutic efficacy of E2 stands to benefit from increased bioavailability via sustained release of the hormone. Here, we focus on the encapsulation of E2 within polymeric nanoparticles composed of poly(lactic-co-glycolic) acid (PLGA). PLGA agent encapsulation offers several delivery advantages, including improved bioavailability and sustained biological activity of encapsulated agents. We hypothesized that delivery of E2 from PLGA nanoparticles would enhance the beneficial cognitive effects of E2 relative to free E2 or non-hormone loaded nanoparticle controls in a rat model of menopause. To test this hypothesis, spatial learning and memory were assessed in middle-aged ovariectomized rats receiving weekly subcutaneous treatment of either oil-control, free (oil-solubilized) E2, blank (non-hormone loaded) PLGA, or E2-loaded PLGA. Unexpectedly, learning and memory differed significantly between the two vehicle control groups. E2-loaded PLGA nanoparticles improved learning and memory relative to its control, while learning and memory were not different between free E2 and its vehicle control. These results suggest that delivery of E2 from PLGA nanoparticles offered cognitive benefit. However, when evaluating peripheral burden, E2-loaded PLGA was found to increase uterine stimulation compared to free E2, which is an undesired outcome, as estrogen exposure increases uterine cancer risk. In sum, a weekly E2 treatment regimen of E2 from PLGA nanoparticles increased cognitive efficacy and was accompanied with an adverse impact on the periphery, effects that may be due to the improved agent bioavailability and sustained biological activity offered by PLGA nanoparticle encapsulation. These findings underscore the risk of non-specific enhancement of E2 delivery and provide a basic framework for the study and development of E2's efficacy as a cognitive therapeutic with the aid of customizable polymeric nano-carriers.

Association of phthalate exposure and endogenous hormones with self-reported sleep disruptions: results from the Midlife Women's Health Study.

Follicle-stimulating hormone and estradiol (E2) have been associated with sleep in midlife women, however, few studies have examined the association of other hormones or environmental chemical exposure such as phthalates, with self-reported sleep quality. We assessed the relationship of self-reported sleep with hormones and phthalates. In total, 762 women (aged 45-54 y, 459 premenopausal, and 303 perimenopausal) from the Midlife Women's Health Study answered self-reported questions regarding the frequency of sleep disturbances, insomnia, and restless sleep. Serum E2, progesterone, testosterone, serum hormone binding globulin, free E2 index, free testosterone index, E2:progesterone, and E2:testosterone were measured. Summary measures of phthalate mixtures, including the phthalates from plastic sources (sumPLASTIC), personal care products (sumPCP), di-(2-ethyhexyl) phthalate (sumDEHP), anti-androgenic phthalates (sumAA), and all phthalate metabolites measured (sumALL), were calculated from urinary phthalate metabolites. Ordinal logistic regression was used to fit each outcome sleep measure with all hormones and summary phthalates. Progesterone and testosterone were significantly negatively associated with the frequency of sleep disturbances and insomnia. Free testosterone index was also negatively associated with insomnia frequency. E2:progesterone was positively associated with frequency of sleep disturbances and restless sleep in self-reported nonsmokers. SumPCP and sumALL were significantly negatively associated with frequency of sleep disturbances, insomnia, and restless sleep. SumDEHP and sumPLASTIC were negatively associated with insomnia frequency. Further, the direction of association between phthalates and sleep appears to be dependent on the quartile of phthalate exposure. All significant associations between phthalates and sleep were in self-reported nonsmokers or former smokers. Our study supports previous literature that hormones beyond follicle-stimulating hormone and E2 are associated with sleep disruptions in menopause. Further, we are among the first to show that phthalate exposure is associated with sleep disruptions in midlife women.

Photoelectrochemical competitive immunosensor for 17β-estradiol detection based on ZnIn2S4@NH2-MIL-125(Ti) amplified by PDA NS/Mn:ZnCdS

A competitive-type PEC immunosensor for 17β-estradiol (E2) detection was successfully fabricated using ZnIn2S4@NH2-MIL-125(Ti) composite as matrix. The excellent PEC behavior of ZnIn2S4@NH2-MIL-125(Ti) composite could be attributed to that the Ti4+-Ti3+ intervalence cycles in the titanium oxo-cluster of NH2-MIL-125(Ti) as well as the matching energy level between ZnIn2S4 and NH2-MIL-125(Ti) promote the migration and separation of photocarrier. Besides, polydopamine (PDA) with abundant amino- and quinone-groups was selected to further improve the PEC signals and capture antibody, which implement through the covalent bonding of PDA and BSA-E2 or carboxyl-group functionalized Mn:ZnCdS QDs in the competitive-type strategy. Concretely, the quinone functional groups in PDA film was applied to immobilize BSA-E2 through Michael reactions, and the PDA nanosphere loaded Mn:ZnCdS quantum dot (PDA NS/Mn:ZnCdS QDs) was used as antibodies’ labels to amplify PEC signals. After PDA NS/Mn:ZnCdS-anti-E2 immobilized on the modified electrode, a remarkable increase of photocurrent signal was observed owing to the specific bonding of antigen and antibody. Based on the competitive binding of PDA NS/Mn:ZnCdS-anti-E2 with either free E2 or bovine serum albumin (BSA)-E2 causing the change of the photocurrent signal, the standard sample free E2 could be accuracy detect. Under optimal conditions, the competitive-type PEC immunosensor exhibited the linear range from 0.0005 ng/mL to 20 ng/mL and a limit detection of 0.3 pg/mL (S/N = 3). Meanwhile, the acceptable stability, selectivity and reproducibility of the proposed PEC immunosensing platform indicating the promising detection of small molecular environmental pollutants.

Circulating androgen levels before and after oophorectomy in premenopausal and postmenopausal women

Objective: This study aimed to determine the effect of oophorectomy on baseline serum levels of androgens and estrogens in premenopausal and postmenopausal women.Methods: Fourteen premenopausal and 10 postmenopausal women underwent total hysterectomy and bilateral oophorectomy for benign disease of the uterus. Serum levels of dehydroepiandrosterone sulfate (DHEAS), androstenedione (A), testosterone (T), dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol-17β-glucuronide (3α-diol G), estrone (E1), estradiol (E2), and sex hormone-binding globulin (SHBG) were measured by highly specific immunoassays prior to surgery and 2 weeks afterward. Free T and free E2 were calculated. Differences were determined between preoperative (preop) and postoperative (postop) samples, and between premenopausal and postmenopausal women.Results: In premenopausal women, postop levels of total and free T, DHT, and total and free E2 decreased significantly from preop. Postop levels of DHEAS, A, 3α-diol G, and SHBG were decreased, but not significantly different from preop. In postmenopausal women, postop levels of total and free T and total and free E2 decreased significantly from preop, but there was little change in the other compounds. Significant differences in the mean change from baseline between premenopausal and postmenopausal women were observed only for E1 and total and free E2.Conclusion: The significant decrease in serum T in postmenopausal women following oophorectomy adds to the evidence that the postmenopausal ovary continues to produce T.

A surface plasmon resonance based inhibition immunoassay for measurement of steroid hormones

Quantitative measurement of small-molecule metabolites is now emerging as an effective way to link the metabolite profile to disease state. Surface plasmon resonance (SPR) is a sensing platform that has demonstrated applicability for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. Herein, we utilized an indirect detection format and developed an inhibition immunoassay for the quantitative measurement of 17β-estradiol (E2) using SPR. One competitor, BSA-E2 conjugate, was immobilized to the SPR chip via the reaction between the primary amino group of the conjugate and the succinimide group (NHS) introduced by the formation of a thiol-NHS monolayer on gold surface. Free E2 molecules compete with BSA-E2 on chip surface for binding sites provided by a monoclonal anti-E2 antibody. It was found the binding affinity of the antibody to BSA-E2 conjugate increases with decreasing surface coverage of BSA-E2 conjugate. Under optimal conditions, a sigmoidal calibration curve with a negative slope and a dynamic range from 10 pM to 2 nM was generated. The detection limit of the immunoassay is estimated to be 0.3 pM. Moreover, the immunoassay exhibits high specificity for E2 detection using estrone (E1) as a potential interference.

Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats

The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17β-estradiol (free E2). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats.

Low Aromatase Activity and Estradiol/Sex Hormone Binding Globulin Ratio are Associated with Total Hip Bone Mineral Density and the Presence of Osteoporosis: A Study in Chinese Postmenopausal Women.

Several groups have reported the important role of estradiol (E2) and testosterone (T) in postmenopausal osteoporosis (PMOP). Because aromatase catalyzes the conversion of T to E2, the purpose of this study was to determine the influence of aromatase activity on the bone mineral density (BMD) in postmenopausal women. A total of 344 postmenopausal women were selected for this study. Serum E2, T, sex hormone-binding globulin (SHBG), calcium (Ca), alkaline phosphatase (ALP), C-terminal telopeptide of type I collagen (CTX), and procollagen type I amino-terminal propeptide (PINP) were examined. The E2/T was positively associated with total hip BMD and PINP (p<0.05). When E2/T was divided into quartiles, participants in lower quartiles of E2/T were likely to have higher PINP and lower BMD (p<0.05). The prevalence of osteoporosis significantly increased as E2/T ratio decreased. The receiver operating characteristic (ROC) curves were constructed for serum E2, free E2 index (FEI), and E2/T, to assess their diagnostic accuracy in PMOP. The overall area under the curve (AUC) were 0.83 (95% CI=0.77-0.88) for E2, 0.87 (95% CI=0.82-0.92) for FEI, and 0.89 (95% CI=0.85-0.94), respectively. In conclusion, the study suggests that in postmenopausal women, aromatase activity could be an important determinant of skeletal health. The women with lower aromatase activity may have greater likelihood of PMOP and the E2/T was expected to be a valuable indicator for the prediction of PMOP and to monitor the process of osteoporosis.

High estradiol exposure disrupts the reproductive cycle of the clam Ruditapes decussatus in a sex-specific way.

Bivalve species may be susceptible to environmental estrogenic compounds including estradiol (E2). However, they are able to biotransform the hormone quite readily and inactivate its estrogenic action. To study the long-term effects of elevated free E2 tissue levels, we transiently exceeded the biotransformation capacity of the clam Ruditapes decussatus by exposing them with high E2 concentrations (400ng/L) and subsequently study the consequences on gametogenesis during the following reproductive cycle. Exposure to 400ngE2/L led to a significant increase in tissue free E2 levels, which reached 10-50ng E2Eq/gww. No deleterious effect on gonado-somatic index (GSI), condition index (CI), or ability to respond to the stress on stress test could be detected after a month of exposure, suggesting the absence of negative effects on the clam's health. However, a marked increase in gametogenesis could be observed in both sexes during the exposure. Subsequent transplantation of the clams in the field allowed the normal development of the male clams and maturation of the gonads without any detrimental effect observed after 4months. In contrast, in early July, all female clams formerly exposed to E2 showed lower health status, and only ovaries with atretic oocytes while all control and indigenous females were normal and mature. These results show a sex-specific effect of high E2 exposure and suggest either a direct or indirect role for E2 in R. decussatus' reproduction.

Is Estradiol a Biomarker of Type 2 Diabetes Risk in Postmenopausal Women?

Cross-sectional studies have suggested an association between the main female hormone, 17β-estradiol (E2), and the risk of type 2 diabetes (T2D) in postmenopausal women (1,2). Even prospective studies have found that among postmenopausal women not using hormone therapy, higher circulating levels of total and free E2 were strongly and prospectively associated with increased long-term risk of T2D, independently of adiposity or insulin resistance (3,4). In this issue of Diabetes , Muka et al. (5) examine the association among sex hormone–binding globulin (SHBG)—a protein that binds and carries E2 in the blood and limits its bioavailability—sex hormones, and T2D risk in a large population-based sample of healthy postmenopausal women who were free of T2D at baseline, with a median follow-up of 11 years. They report that lower levels of SHBG (which translate into higher bioavailable E2) and higher concentrations of total E2 are associated with increased risk of T2D, independently of established risk factors, including BMI and insulin resistance. In contrast, they observed no association between testosterone and T2D risk. Their findings are reinforced by a systematic meta-analysis of 13 studies with 14,902 participants who included 1,912 case subjects with T2D, confirming that lower SHBG and increased total E2 are robust risk markers of T2D in postmenopausal women. Of …

Estrogen receptor α and β are involved in the activation of lordosis behavior in estradiol-primed rats

The present study was designed to assess the participation of estrogen receptors alpha (ERα) and beta (ERβ) in the short-term facilitation of lordosis behavior in ovariectomized (ovx), estradiol (E2) primed rats. In experiment 1, dose response curves for PPT and DPN (ERα and ERβ agonists, respectively) facilitation of lordosis behavior (lordosis quotient and lordosis score) were established by infusing these agonists into the right lateral ventricle (icv) in female rats injected 40h previously with 5μg of E2 benzoate. PPT doses of 0.08 and 0.4ng produced high lordosis quotients starting at 30min and continuing at 120 and 240min post-injection. DPN induced high levels of lordosis behavior at all times tested. However, the intensity of lordosis induced by both agonists was weak. In experiment 2, we tested the involvement of each ER in facilitation of lordosis by icv infusion of MPP (ERα-selective antagonist) or PHTPP (ERβ-selective antagonist) prior to infusion of 2ng of free E2. Icv infusion of either MPP or PHTPP 30min before free E2 significantly depressed E2 facilitation of lordosis. The results suggest that both forms of ER are involved in the short-latency facilitation of lordosis behavior in E2-primed rats.

Mussels (Mytilus spp.) display an ability for rapid and high capacity uptake of the vertebrate steroid, estradiol-17β from water

Six experiments were carried out to define the optimum conditions for investigating the dynamics of uptake and metabolism of tritiated E2 from water by adult blue mussels, Mytilus spp. Optimum uptake was achieved using 400mL aerated sea water animal−1 and an incubation period of no more than 24h. The pattern of disappearance conformed closest to an inverse hyperbolic curve with the percentage of radiolabel that could be measured in the water reaching an asymptote that was on average 50% of the original. This apparent inability of the animals to absorb all the radiolabel was investigated further. Solvent partition and chromatography revealed that, after 24h, c. 60% of the radiolabel still present in the water was composed of water soluble conjugates, c. 25% was composed of tritiated water and only 15% ran on and around the chromatographic position of E2. The major water soluble constituent was identified by chromatography and mass-spectrometry as 1,3,5(10)-estratriene-3,17β-diol 3-sulfate (estradiol 3-S). The clearance rate of radiolabel was 46.9±1.8mLanimal−1h−1. This was not significantly affected by the addition of as much as 25μgL−1 cold E2 to the water, demonstrating that mussels have a large capacity for E2 uptake. A new procedure involving solvent partition was developed for separating the free, esterified and sulfated forms of E2 present in the flesh of mussels. This involved extracting the soft tissue with organic solvents and then treating a portion of dried extract with a combination of heptane (dissolved fatty acid esters of E2) and 80% ethanol (dissolved free and sulfated E2). The latter fraction was further partitioned between water (sulfate) and diethyl ether (free steroid). This procedure was much cheaper and less time-consuming than chromatography. Approximately 80% of the radioactivity that was taken up by the animals was present in the form of ester. Moreover, E2 was the only steroid identified after saponification of these esters. Of the remaining radioactivity, c. 10% was in the form of unidentified free steroids and c. 10% was estradiol 3-S. In order to determine how rapidly mussels were able to depurate tritiated E2 and its metabolites, two experiments were carried out. Animals from the first experiment purged up to 63% of radioactivity in 20days under flow-through conditions; whereas animals from the second experiment released only 16% of radioactivity in 10days under semi-static conditions. The ratios of the different forms of E2 did not change substantially during the course of depuration.

Steroid Probes Conjugated with Protein-Protected Gold Nanocluster: Specific and Rapid Fluorescence Imaging of Steroid Receptors in Target Cells.

Steroid ligands can easily diffuse through the cell membrane and this property makes it feasible to be used for in-situ staining of the nuclear receptors. However, nonspecific binding of the internalized ligand probe with the cellular components has caused serious interferences for the detection of receptor-expressing cells. We report a novel gold nanocluster (AuNC)-conjugated estrogen probe that can eliminate nonspecific internalization and accelerate nuclear localization to achieve selective and rapid detection of estrogen receptors (ERs) in live cells. The AuNC, protected by bovine serum albumin (BSA), BSA-AuNCs, was prepared by the synthesis and confirmed to be 1.9nm in core size and 18 nm in diameter. Ethinyl estradiol was used as the precursor of 17β-estradial (E2) to conjugate with BSA-protected AuNCs via polyethylene glycol linker (E2-PEG/BSA-AuNCs) or to conjugate with Cy3 dyes (E2-Cy3). The conjugated probe was determined to contain five E2 molecules per BSA-AuNC by mass spectrometry and exhibit an emission maximum of around 640 nm, which was not altered by E2 conjugation indicating that the structural integrity of BSA-AuNCs was conserved. E2-PEG/BSA-AuNCs probes were quickly internalized by MCF-7 (ER+) cells and localized to the nuclei in 2 h. Such internalization was sensitive to competition by free E2 and was rarely detected in the controls using either non-conjugated BSA-AuNCs in MCF-7 (ER+) cells or E2-PEG/BSA-AuNCs in MDA-MB-231 (ER-) cells. In contrast to the high specificity of E2-PEG/BSA-AuNCs probe, the uptake of E2-Cy3 probe could not differentiate between MCF-7(ER+) and MDA-MB-231(ER-) cells during the early phases of the treatment. Moreover, nuclear targeting by E2-Cy3 was three times slower than that by the E2-PEG/BSA-AuNC probe. Such accelerated nuclei targeting was consistent with the enhanced cell viability by conjugating E2 with BSA-AuNC. In conclusion, the E2-PEG/BSA-AuNC probes are promising candidates that can be used for the detection of ER+ tumor tissues and the same strategy can be applied to fabricate other steroid probes.

Effects of Modest Weight Gain on Estrogens in Healthy Men

BackgroundDecreases in estrogen are shown to increase body fat in men. However, obese men have high levels of estrogen and the effects of weight gain on estrogen have not been previously investigated. The objective of this study was to investigate the relationship between regional fat depots and estrogen during modest weight in healthy men.MethodsWe investigated the effects of overfeeding in 25 healthy men (30 ± 7 years, 24.7 ± 3.1 kg/m2). Changes in regional fat depot during weight gain were assessed using dual‐energy X‐ray absorptiometry and abdominal computed tomography scans. Participants gained approximately 5% body weight in 8 weeks. Estrone (E1), 17β‐estradiol (E2) and sex hormone binding globulin (SHBG) were measured at baseline and after weight gain.ResultsOverfeeding was accompanied by 3.9 ± 1.3 kg weight gain. These increases in weight resulted from increases in total body fat mass (18.3 ± 8.5 vs. 21.7 ± 8.8 kg, p&lt;0.001) while the fat free mass did not change (58.4 ± 8.6 vs. 58.9 ± 8.7 kg, p=0.15). E1 (32.28 ± 14.25 vs. 33.44 ± 12.29 pg/mL, p=0.56) and E2 (25.04 ± 8.3 vs. 26.52 ± 7.8 pg/mL, p=0.28) did not change after weight gain. However, weight gain resulted in decreased SHBG (22.8 ± 10.5 vs. 20.8 ± 11.1 nmol/l, p=0.036) and increased E2/SHBG ratio (1.4 ± 1.0 vs 1.63 ± 1.0, p=0.027). The changes in E2/SHBG ratio, a surrogate marker of bioavailable E2, was positively associated with increases in abdominal fat mass (p=0.048, rho=0.39) and increases in visceral fat mass (p=0.049, rho=0.39).ConclusionModest weight gain in healthy men does not alter circulating E1 and E2 levels. However weight gain results in decreased SHBG and increased free E2 (E2/SHBG ratio). The increased E2/SHBG ratio suggesting a pro‐estrogen effect is associated with increases in visceral fat mass during weight gain. Our study therefore suggests a complex relationship between abdominal fat gain and free E2 in men which may conceivably predispose to feminizing characteristics in obese men.Support or Funding InformationPS is supported by AHA Grant 11SDG7260046 and NIH grant DK81014. NC is supported by AHA grant AHA 13POST16420009. MDJ is supported by NIH grants DK45343 and DK40484. VKS is supported by NIH Grants HL73211, and DK81014. This study was made possible by a grant from NCRR (1UL1 RR024150).

Distinguishing characteristics of metabolically healthy versus metabolically unhealthy obese adolescent girls with polycystic ovary syndrome

To investigate the key physical, metabolic, hormonal and cardiovascular characteristics of metabolically healthy obese (MHO) versus unhealthy obese (MUHO) girls with polycystic ovary syndrome (PCOS). Cross-sectional study. Research center. Seventy obese girls with PCOS were divided into 19 MHO and 51 MUHO based on cutoff points for invivo insulin sensitivity (within and < 2 SDs of the mean of the insulin sensitivity of the normal-weight girls, respectively). None. Body composition, abdominal fat, invivo insulin sensitivity and secretion (hyperinsulinemic-euglycemic and hyperglycemic clamps respectively), hormonal profile, and cardiovascular disease risk markers. MUHO-PCOS girls had higher waist circumference, visceral adipose tissue, leptin, and free testosterone, lower SHBG and E2, higher non-high-density lipoprotein (HDL) cholesterol and atherogenic lipoprotein particle concentrations, smaller HDL particle size, and higher high-sensitivity C-reactive protein compared with MHO-PCOS girls. Hepatic and peripheral insulin sensitivity were lower with higher first- and second-phase insulin secretion, but β-cell function relative to insulin sensitivity was lower in MUHO versus MHO. Pair matching of MHO and MUHO regarding age and body mass index revealed similar findings. MUHO-PCOS girls had larger visceral adiposity, lower insulin sensitivity and β-cell function, worse hormonal profile, and severely atherogenic lipoprotein concentrations compared with MHO-PCOS girls. MHO-PCOS girls have favorable physical, metabolic, hormonal, and cardiovascular disease (CVD) characteristics and lower risk biomarkers for type 2 diabetes compared with their MUHO-PCOS peers. A greater understanding of the contrast in this risk phenotype in obese girls with PCOS may have important implications for therapeutic interventions, their outcomes, and their durability.