Steroid 17β-estradiol Research Articles

Circulating Sex Hormones Modulate Vascular Contractions and Acute Response to 17β-Estradiol in Rat Mesenteric Arteries

Aims: We investigated how modification of levels of the sex hormones 17β-estradiol and testosterone affects vascular contraction and nongenomic vascular effects of 17β-estradiol. Methods: Male and female rats were treated with vehicle, 17β-estradiol (25 µg/kg/day) or testosterone (1 mg/kg/day) for 14 consecutive days after sham gonadectomy or gonadectomy was performed. Isometric tensions were then measured from mesenteric arteries of each group of rats. Results: Contraction to phenylephrine was increased in mesenteric arteries from rats with or without gonadectomy treated with testosterone for 14 days compared to their intact controls. Contraction to phenylephrine was reduced in mesenteric arteries of rats with or without gonadectomy treated with 17β-estradiol for 14 days compared to their intact controls. Incubation of mesenteric arteries with 17β-estradiol (1 nmol/l) for 30 min reduced contraction to phenylephrine in mesenteric arteries of rats that were treated with testosterone for 14 days. This acute incubation of 17β-estradiol had no effect on arteries from rats that were treated with 17β-estradiol for 14 days. The acute effect of 17β-estradiol (1 nmol/l) is preserved in arteries without endothelium. Conclusion: Our results suggest that 14 days’ testosterone treatment enhances while 14 days’ 17β-estradiol treatment suppresses contraction as well as the nongenomic effects of 17β-estradiol in the vascular smooth muscles.

Distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals in wild fish species from Dianchi Lake, China

The distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals (EDCs) were studied in various tissues of wild fish species from Dianchi Lake, China. In muscle tissue, 4-tert-octylphenol, 4-cumylphenol, 4-nonlyphenol and bisphenol A were detected in fish from each sampling site, with maximal concentrations of 4.6, 4.4, 18.9 and 83.5ng/g dry weight (dw), respectively. Steroids (estrone, 17β-estradiol 17α-ethynylestradiol and estriol) were found at lower levels (<11.3ng/g dw) and less frequently in muscle samples. The highest concentrations of steroids and phenols were found in liver, followed by those in gill and the lowest concentration was found in muscle. The field bioconcentration factors (BCFs) of phenols were calculated in fish species ranged from 18 to 97. Moreover, the measured tissue concentrations were utilized in order to estimate water concentration of steroids (4.4–18.0ng/L). These results showed that steroidal and phenolic EDCs were likely ubiquitous contaminants in wild fish.

In vitro inhibition of porcine cytochrome P450 by 17β-estradiol and 17α-estradiol

Sexually mature pigs are known to possess high concentrations of testicular steroids, which have been shown to change the activities of cytochrome P450 in vitro. The aim of the present study was to evaluate the regulation of CYP1A and CYP2E1 activity by the steroids dihydrotestosterone (DHT), 3β-androstenol, 17β-estradiol and 17α-estradiol. Catalytic activities of 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD) were used as markers of CYP1A activities, while p-nitrophenol hydroxylase (PNPH) was used as a marker of CYP2E1 activities. Of the steroids tested, only 17β-estradiol and 17α-estradiol inhibited EROD and MROD activities. This inhibition was observed when a steroid concentration of 100 µM was used, while lower concentrations showed no inhibitory effect. PNPH activities were inhibited only by 100 µM of 17β-estradiol. The significance of these results in vivo is unknown because inhibition was only found when concentrations of estrogens higher than physiological levels were used. Nevertheless, the results provided further evidence on the important role of estrogens in regulation of porcine cytochrome P450 activities.

Gonadal Stage and Sex Steroid Correlations in Siberian Sturgeon, Acipenser baeri, Habituated to a Semitropical Environment

Stages of gonadal development, in association with plasma concentrations of the sex steroids 17β-estradiol (E2), progesterone (P4), testosterone (T), and 11-ketotestosterone (11-KT), were investigated for a single time point during a natural breeding season in 7-yr-old Siberian sturgeon, Acipenser baeri Brandt, exposed lifelong to a warmwater environment. Among females, examination of gonadal tissue showed variation in ovarian stage, with 12.5, 47.5, 22.5, and 17.5% of females found at Stages 2 (previtellogenic), 3 (early vitellogenic), 4 (mid-vitellogenic), and 5 (migratory nucleus), respectively. Although patterns varied among the hormones, plasma concentrations of E2, T, and 11-KT became increasingly elevated in females as maturation progressed. On the basis of histological criteria, males were classified as either premeiotic (quiescent) or meiotic and 50% of the males sampled were found at each stage. Significant elevations in circulating concentrations of plasma E2 and T were observed in meiotic versus premeiotic males, and there was a rise in plasma 11-KT concentration that approached significance (P = 0.056).

Interaction of gonadal steroids and the glucocorticoid corticosterone in the regulation of the L‐type Ca2+ current in rat left ventricular cardiomyocytes

Gonadal steroids as well as glucocorticoids have been shown to regulate the cardiac L-type Ca(2+) current (I(CaL) ). Herein, we compare the effects of the gonadal steroids testosterone and 17β-estradiol with the glucocorticoid corticosterone on I(CaL) , and investigate the interaction between the gonadal steroids and corticosterone. Myocytes were isolated from the left ventricular free wall of female and male Wistar rats and investigated using the ruptured-patch whole-cell patch-clamp technique. In myocytes isolated from female rats, 24 h incubation with 100 nm testosterone led to a 33% increase in I(CaL) compared with control (-8.8 ± 0.5 pA pF(-1) , n = 25 vs. -6.6 ± 0.4 pA pF(-1) , n = 26, P < 0.01, V(Pip) = 0 mV). Incubation with 1 μm corticosterone resulted in a 79% increase in I(CaL) (-11.8 ± 0.7 pA pF(-1) , n = 29, P < 0.001). However, the combination of testosterone and corticosterone did not have any additional effect compared with corticosterone alone (-11.7 ± 0.6 pA pF(-1) , n = 25, ns). In cardiomyocytes from male rats, I(CaL) was not affected by testosterone, whereas the effect of corticosterone was preserved (P < 0.05). 24 h incubation with 17β-estradiol increased I(CaL) by 32% from -7.6 ± 0.5 pA pF(-1) (n = 15) to 10.0 ± 0.9 pA pF(-1) (n = 15, P < 0.05). 17β-estradiol did not exert an additional effect upon co-incubation with corticosterone and did not have an effect on I(CaL) in cardiomyocytes from female rats. Higher concentrations of the gonadal steroids did not result in increased effects. When compared with corticosterone, the in vitro effects of the gonadal steroids are small. However, under conditions in which I(CaL) is not fully activated by glucocorticoids, gonadal steroids may significantly contribute to I(CaL) regulation.

Estrogenic Activity of Chemical Constituents from Tephrosia candida

In a continued investigation of medicinal plants from the genus Tephrosia, phytochemical analysis of a methylene chloride-methanol (1:1) extract of the air-dried aerial parts of Tephrosia candida afforded two new 8-prenylated flavonoids, namely, tephrocandidins A (1) and B (2), a new prenylated chalcone, candidachalcone (3), a new sesquiterpene (4), and a previously reported pea flavonoid phytoalexin, pisatin (5). The structures of 1-4 were established by spectroscopic methods, including HREIMS, and 1H, 13C, DEPT, HMQC, and HMBC NMR experiments. The most potent estrogenic activity of these isolated natural products in an estrogen receptor (ERα) competitive-binding assay was for 3, which exhibited an IC50 value of 80 μM, compared with 18 nM for the natural steroid 17β-estradiol. Results were interpreted via virtual docking of isolated compounds to an ERα crystal structure.

Neuroglobin, estrogens, and neuroprotection

Globins have been found in glial cells and neurons of invertebrates and vertebrates. The first nerve globin has been recognized in the nerve cord of the polychaete annelid Aphrodite aculeata in 1872. In some invertebrates, the nerve globin reaches a millimolar concentration which is likely sufficient to sustain the aerobic metabolism and thus the excitability of the nervous system. In 2000, the first vertebrate nerve globin, named neuroglobin (Ngb), has been identified in neuronal tissues of mice and humans. In contrast to invertebrate nerve globins, the concentration of Ngb, the prototype of vertebrate nerve globins, is low (μM), reaching a maximum of 100 μM in retina cells. Therefore, Ngb appears unlikely to act primarily as an O₂ buffer and to facilitate O₂ diffusion to the mitochondria. Indeed, Ngb has been hypothesized to catalyze the formation/decomposition of reactive nitrogen and/or oxygen species and to be part of intracellular signaling pathways enhancing cell survival. Here, we report that neuronal Ngb levels are strongly induced by the steroid hormone 17β-estradiol. Furthermore, Ngb participates to mechanisms involved in 17β-estradiol-induced protective effects against H₂O₂ -induced neurotoxicity.

Revealing genetic relationships between compounds affecting boar taint and reproduction in pigs1

Boar taint is characterized by an unpleasant taste or odor in intact male pigs and is primarily attributed to increased concentrations of androstenone and skatole and to a lesser extent by increased indole. The boar taint compounds skatole and indole are produced by gut bacteria, metabolized in the liver, and stored in the fat tissue. Androstenone, on the other hand, is synthesized in the testis along with testosterone and estrogens, which are known to be important factors affecting fertility. The main goal of this study was to investigate the relationship between genetic factors involved in the primary boar taint compounds in an attempt to discover ways to reduce boar taint without decreasing fertility-related compounds. Heritabilities and genetic correlations between traits were estimated for compounds related to boar taint (androstenone, skatole, indole) and reproduction (testosterone, 17β-estradiol, and estrone sulfate). Heritabilities in the range of 0.47 to 0.67 were detected for androstenone concentrations in both fat and plasma, whereas those for skatole and indole were slightly less (0.27 to 0.41). The genetic correlations between androstenone in plasma and fat were extremely high (0.91 to 0.98) in Duroc and Landrace. In addition, genetic correlations between androstenone (both plasma and fat) and the other sex steroids (estrone sulfate, 17β-estradiol, and testosterone) were very high, in the range of 0.80 to 0.95. Furthermore, a genome-wide association study (GWA) and a combined linkage disequilibrium and linkage analysis (LDLA) were conducted on 1,533 purebred Landrace and 1,027 purebred Duroc to find genome regions involved in genetic control of the boar taint compounds androstenone, skatole, and indole, and sex hormones related to fertility traits. Up to 3,297 informative SNP markers were included for both breeds, including SNP from several boar taint candidate genes. From the GWA study, we found that altogether 27 regions were significant at a genome-wide level (P < 0.05) and an additional 7 regions were significant at a chromosomal level. From the LDLA study, 7 regions were significant on a genome-wide level and an additional 7 regions were significant at a chromosomal level. The most convincing associations were obtained in 6 regions affecting skatole and indole in fat on chromosomes 1, 2, 3, 7, 13, and 14, 1 region on chromosome 6 affecting androstenone in plasma only, and 5 regions on chromosomes 3, 4, 13, and 15 affecting androstenone, testosterone, and estrogens.

Prevention of Amyloid-Beta Oligomer-Induced Neuronal Death by EGTA, Estradiol, and Endocytosis Inhibitor

BACKGROUND AND OBJECTIVE. Alzheimer's disease is a progressive neurodegenerative disease that is biochemically characterized by the accumulation of amyloid beta (Aβ) peptides in the brain. The current hypothesis suggests that Aβ oligomers rather than fibrillar aggregates are the most toxic species of Aβ though the mechanisms of their neurotoxicity are unclear. The authors have previously shown that small Aβ(1-42) oligomers at around 1 µM concentration caused rapid (in 24 h) neuronal death in cerebellar granule cell (CGC) cultures. In this study, we aimed to investigate whether protracted (up to 7 days) incubation of CGC cultures with lower submicromolar concentration of various aggregates of Aβ(1-42) had an effect on viability of neurons. In order to get some insight into the mechanism of Aβ-induced cell death, we also sought to determine whether extracellular Ca(2+) and process of endocytosis contributed to Aβ oligomer-induced neurotoxicity and whether pharmacological interventions into these processes would prevent Aβ oligomer-induced cell death. MATERIAL AND METHODS. Primary cultures of CGC were treated with various aggregate forms of Aβ(1-42). Cell viability was assessed by fluorescent microscopy using propidium iodide and Hoechst 33342 staining. RESULTS. Exposure of neurons to 500 nM Aβ(1-42) oligomers for 72-168 h caused extensive neuronal necrosis. Lower concentrations (100-250 nM) were not toxic to cells during 7 days of incubation. Aβ(1-42) monomers and fibrils had no effect on neuronal viability even after 7 days of incubation. Treatment of neurons with EGTA, steroid hormone 17β-estradiol, and methyl-β-cyclodextrin significantly reduced Aβ(1-42) oligomers-induced neuronal death. CONCLUSIONS. The results show that submicromolar concentrations of Aβ(1-42) oligomers were highly toxic to neurons during protracted incubation inducing neuronal necrosis that can be prevented by chelating extracellular Ca(2+) with EGTA, inhibiting endocytosis with methyl-β-cyclodextrin, or by estradiol, which may protect against mitochondrial permeability transition pore opening.

Modulation of innate immunity in human pancreatic islets infected with enterovirus in vitro

Present knowledge of innate immunity in infected cells relies on studies of cell lines and animal models. In this study, primary human pancreatic islets of Langerhans were used to study virus-host interactions in a model of the possible induction of type 1 diabetes by enterovirus (EV). Human islets were infected with a strain of EV isolated at onset of type 1 diabetes, or exposed to synthetic dsRNA (poly(I:C)), used commonly to mimic viral infection. Induction of innate immunity and the effect of the female sex hormone 17β-estradiol, known to have cell-protective effects, on islet chemokine secretion were investigated. 17β-Estradiol reduced EV-but not poly(I:C)-induced IP-10/CXCL10 secretion from human islets, suggesting that separate signaling pathways of the innate immune response are triggered by EV and poly(I:C), respectively. Infection with EV increased the gene-expression of toll-like receptor 3, interferon-β, and the intracellular helicase MDA5, involved in antiviral innate immunity, multi-fold over time, whereas poly(I:C) increased the expression of these genes transiently. The induced expression pattern was similar in all donors, but the expression levels varied greatly. Pre-exposure to poly(I:C) blocked viral replication in islets from 56% of the donors. These data provide insight on the innate immune responses induced by EV in human islets, and show that this can be modulated by 17β-estradiol, and suggest an important difference between virus- and poly(I:C)-induced signaling.

Photodegradation of 17β-estradiol in aquatic solution under solar irradiation: Kinetics and influencing water parameters

Photodegradation of the natural steroid 17β-estradiol (E2), an endocrine disrupting hormone which is commonly released into aquatic environments, was investigated under simulated sunlight (290–700 nm) using a solar simulator in the presence of several natural water constituents including NO 3 −, Fe 3+, HCO 3 −, humic acid and turbidity. The E2 degradation followed pseudo-first-order kinetics, with the rate constant decreasing slightly with increasing initial constituent concentration while increasing with the square root of solar intensity in the region of 25–100 mW cm −2. The rate of mineralization based on the total organic carbon (TOC) reduction was always lower than E2 degradation, although the TOC of the solution decreased steadily with irradiation time. In the presence of NO 3 −, Fe 3+, and humic acid, the photodegradation rate increased significantly, attributed to photosensitization by the reactive species, while HCO 3 − slowed down the degradation rate because of OH scavenging. Turbidity also reduced the photodegradation of E2 by decreasing light transmittance due to attenuation. The solution pH also had a considerable effect on the rate with maximum degradation occurring around a neutral pH of 7.

Occurrence of endocrine-disrupting chemicals in riverine sediments from the Pearl River Delta, China

The endocrine-disrupting chemicals (EDCs) was investigated in 28 riverine sediments from the Pearl River system, China and analyzed by an ultrasonication extraction and GC–MS method. The concentrations of 4-tert-octylphenol (OP), 4-nonylphenol (NP), and bisphenol A (BPA) in the sediments were in the ranges of <2.0–210, 107–16198 and <1.7–430ng/g dw, respectively. The steroid estrogens estrone (E1) and 17β-estradiol (E2) in the sediments ranged from <1.3 to 10.9ng/gdw and from <0.9 to 2.6ng/gdw, respectively. The spatial distribution of these chemicals was related to the discharge of domestic and industrial wastewater along the rivers. The positive correlation between EDCs and total organic carbon indicates that sedimentary organic carbon is an important factor in controlling the distributions of EDCs. Compared with other previous studies, the ZR and DR rivers from the PRD were heavily contaminated by APs and BPA.

Effect of sublethal hexavalent chromium exposure on the pituitary‐ovarian axis of a teleost, Channa punctatus (Bloch)

Exposure to sublethal hexavalent chromium (as 2 and 4 mg L⁻¹ potassium dichromate for 1 and 2 months) during late preparatory to mid prespawning phase of annual reproductive cycle severely affected the pituitary-ovarian axis of a teleost Channa punctatus. Gonadosomatic index (GSI), ovarian histopathology, immunocytochemistry of the pituitary gonadotrophs (LHβ-immunoreactive cells), and serum 17β-estradiol level revealed distinct dose and duration-dependent effects. Gonadosomatic index was declined. Diameter of ovary as well as ovarian follicles was reduced along with a distinct variation in the percentage of follicles. The greater percentage of previtellogenic follicles (stage I) and decline in vitellogenic ones (stage II and stage III) compared to control indicated the arrest of follicular development. The percentage of atretic follicles was also increased indicating toxic impact of metal on ovary. Decreased serum level of ovarian steroid 17β-estradiol further indicated the same. Chromium interference of the pituitary was demonstrated as atrophy and less immunointensity of LH-immunoreactive gonadotrophs. Thus, the hexavalent chromium impaired the pituitary-ovarian axis affecting at the sites of both pituitary and ovary.

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

BACKGROUND, GOALS, AND SCOPE: In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline. A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and "negative" chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria. With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production. With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2). Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.

Assessing the estrogenic potency in a Portuguese wastewater treatment plant using an integrated approach

The estrogenic potency of a wastewater treatment plant (WWTP) was evaluated using chemical and biological analyses, which showed that after the station treatment processes some of the selected endocrine disruptor compounds (EDCs) were still present in the treated effluent (e.g., bisphenol A, alkylphenols, estrone). Thus, the most common endocrine EDCs were identified and quantified and the overall estrogenicity of the treated effluent assessed by integrating the results. Male goldfish ( Carassius auratus) were used as biological indicators in a 28-day experiment. Vitellogenin (Vtg), gonadosomatic and hepatosomatic indices, steroids (17β-estradiol and 11-ketotestosterone) and histopathology were biomarkers used in fish to evaluate WWTP treated effluent estrogenicity, in combination with instrumental analyses. The results showed a significant increase ( P < 0.01) in plasma and liver Vtg, which were significantly correlated ( r = 0.66; P < 0.01). The gonadosmatic index was significantly ( P < 0.01) reduced in exposed fish. The steroid analyses revealed significant elevations in 17β-estradiol and depressed 11-ketotestosterone concentrations. The histological examinations show changes in exposed fish gonads, such as regressed testes and in some cases (43% to 75%) the development of ovo-testis in fish exposed to 50% and 100% treated effluent.

QSAR-assisted design of an environmental catalyst for enhanced estrogen remediation

A quantitative structure–activity relationship (QSAR) was used to streamline re-design of a model environmental catalyst, horseradish peroxidase (HRP), for enhanced reactivity towards a target pollutant, steroid hormone 17β-estradiol. This QSAR, embodying relationship between reaction rate and intermolecular binding distance, was used in silico to screen for mutations improving enzyme reactivity. Eight mutations mediating significant reductions in binding distances were expressed in Saccharomyces cerevisiae, and resulting recombinant HRP strains were analyzed to determine Michaelis–Menten parameters during reaction with the target substrate. Enzyme turnover rate, ln( k CAT), exhibited inverse relationship with model-predicted binding distances ( R 2 = 0.81), consistent with the QSAR. Additional analysis of native substrate degradation by selected mutants yielded unexpected increases in ln( k CAT) that were also inversely correlated ( R 2 = 1.00) with model-predicted binding distances. This suggests that the mechanism of improvement comprises a nonspecific “opening up” of the active site such that it better accommodates environmental estrogens of any size. The novel QSAR-assisted approach described herein offers specific advantages compared to conventional design strategies, most notably targeting an entire class of pollutants at one time and a flexible hybridization of benefits associated with rational design and directed evolution. Thus, this approach is a promising tool for improving enzyme-mediated environmental remediation.

Amyloid Precursor Protein (APP) mRNA Level is Higher in the Old Mouse Cerebral Cortex and is Regulated by Sex Steroids

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid beta peptides, which are produced by the proteolytic cleavage of amyloid precursor protein (APP). As the incidence of AD is higher in females and sex steroids are implicated in this disease, we have examined the effect of sex steroids (testosterone and 17β-estradiol) on the expression of APP mRNA and protein in the cerebral cortex of adult and old mice of both sexes. Northern blot analysis detected APP mRNA as a single 3.5-kb band and its level is increased in old as compared to adult. Following gonadectomy, its level was upregulated in female mice but downregulated in male mice. Supplementation with testosterone or estradiol decreased its levels in female mice of both ages. Testosterone supplementation increased the mRNA levels in both adult and old male mice. Estrogen supplementation decreased its level in adult but increased in old male mice. Western blot analysis detected APP specific bands ranging from 95 to 125 kD. The level of 95 kDa band representing APP695 protein showed difference in levels with age or hormone treatment. These results provide evidence for increase in APP mRNA level in the cerebral cortex of old mice and its regulation by sex steroids during aging.

Reproductive cycle in female Waigieu seaperch ( Psammoperca waigiensis) reared under different salinity levels and the effects of dopamine antagonist on steroid hormone levels

Waigieu seaperch ( Psammoperca waigiensis) is a tropical marine finfish species, which may inhabit wide range of salinity during the entire life cycle. Regardless of the wide salinity tolerance, little is known about how salinity may influence the reproductive endocrinology of this important tropical species. In the present study, we investigated the seasonal variations in steroid hormone levels, oocyte maturation (OM) and ovulation in fish reared under different salinity levels. In addition, we investigated the effects a dopamine antagonist (domperidone: DOM) during the peak spawning period. Mature brood fish at 3 years old were cultured in four different salinities of 5, 10, 20 and 30‰ (part per thousand, ppt) from March to December 2007. Blood samples were collected monthly and key steroid hormones (testosterone (T), 11-ketotestosterone (11-KT), 17β-estradiol (E2) and progesterone (P)) levels in female fish plasma were analyzed using enzyme immunoassay method. OM, ovulation and several reproductive performance indexes were evaluated twice per month during the breeding season. Plasma hormone analysis showed significant differences in fish groups cultured in different salinities during the seasonal cycle. The gonadosomatic index (GSI) gradually increased from March, peaking in July at 10 and 30‰, and then decreased thereafter until December. Plasma steroid levels (E2, T, 11-KT and P) were observed in variable concentrations during the spawning period and showed monthly fluctuations that were apparently salinity dependent during on and off-seasons. Interestingly, 11-KT levels were found in small amounts in female fish and peaked in September at salinities of 10 and 20‰. DOM exposure produced significant differences in steroid hormone levels that were apparently dependent on dose, exposure time and the individual hormone. The present study indicated that holding Waigieu seaperch brood fish in different salinities during the breeding season produced significant effects on gonadal development and spawning incidences. The first spawning was observed on 8th of April for all groups after 100% water exchange at night. No significant differences of absolute and relative fecundity were observed between groups. Fish held at 10 ppt salinity during the breeding season spawned naturally, but maturity and spawning rates were reduced and no hatching was observed at the same salinity. Fish holding at 5‰ resulted to 100% mortality in brood fish. This indicated that salinity limits for oocyte maturation or ovulation and subsequent reduction in spawning, but embryonic development requires a higher salinity. The effect of salinity on embryo development and hatching rate suggests that in breeding season the brood fish spawned in areas where the salinity and other parameters are favorable.

Female and Male Sex Hormones Differentially Regulate Expression ofIfi202, an Interferon-Inducible Lupus Susceptibility Gene within theNba2Interval

Increased expression of IFN-inducible Ifi202 gene in certain strains of female mice is associated with susceptibility to systemic lupus erythematosus (SLE). Although, the development of SLE is known to have a strong sex bias, the molecular mechanisms remain unknown. Here we report that in vivo treatment of orchiectomized (NZB x NZW)F(1) male mice with the female sex hormone 17beta-estradiol significantly increased steady-state levels of Ifi202 mRNA in splenic cells, whereas treatment with the male hormone dihydrotestosterone decreased the levels. Moreover, increased expression of Ifi202 in B6.Nba2 B cells and reduced expression in T cells were associated with increased levels of estrogen receptor-alpha (ERalpha) and androgen receptor, respectively. Furthermore, the steady-state levels of Ifi202 mRNA were higher in splenic cells from C57BL/6, B6.Nba2, NZB, and (NZB x NZW)F(1) female mice as compared with males. 17beta-estradiol treatment of B cells and WT276 cells increased Ifi202 mRNA levels, whereas treatment with dihydrotestosterone decreased the levels. Interestingly, overexpression of ERalpha in WT276 cells increased the expression of Ifi202 and stimulated the activity of the 202-luc-reporter through the c-Jun/AP-1 DNA-binding site. Accordingly, ERalpha preferentially associated with the regulatory region of the Ifi202 gene in female B6.Nba2 B cells than in males. Furthermore, Ifi202 mRNA levels were detectable in splenic cells of wild-type (Esr1(+/+)), but not null (Esr1(-/-)), (NZB x NZW)F(1) female mice. Collectively, our observations demonstrate that the female and male sex hormones differentially regulate the expression of Ifi202, thus providing support for the role of Ifi202 in sex bias in SLE.

Simple methodology coupling microwave-assisted extraction to SPE/GC/MS for the analysis of natural steroids in biological tissues: Application to the monitoring of endogenous steroids in marine mussels Mytilus sp.

A simple analytical procedure for the simultaneous determination of eight endogenous steroids (testosterone, androstenedione, 17β-estradiol, estrone, pregnenolone, progesterone, dihydroandrostenedione, and dihydrotestosterone) in aquatic molluscs by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC–MS) has been developed. After a microwave-assisted extraction, samples were further extracted and purified using two successive SPE (EnviChrom-P and NH 2) cartridges. Steroids were derivatized with a mixture of N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA)/mercaptoethanol/ammonium iodide (NH 4I) and determined by GC–MS in selective ion monitoring mode. Recoveries were in the range 85–114%, although slightly lower for dihydrotestosterone, and the repeatability of the procedure, expressed as the coefficient of variation, was lower than 16%. The limits of detection determined in digestive glands of mussels were in the range 0.1–0.4 ng g −1 wet weight for all the steroids. The developed procedure was then applied to the monitoring of steroid profiles in the digestive glands of mussels from the Arcachon Bay (France) during two reproductive cycles. In parallel, two physiological parameters (lipid content and the condition index of mussels) were also monitored, as well as the seawater temperature and salinity. Only progesterone and pregnenolone were detected in the digestive glands of mussels, and the seasonal variations of progesterone levels seemed to be related to the spawning periods of Mytilus sp. in the Bay. The current challenge for the determination of natural steroids in aquatic invertebrates is also briefly discussed.