Increasing evidence has associated the exposure of endocrine-disrupting chemicals (EDCs) with the cardiovascular (CV) system. This exposure is particularly problematic in a sensitive window of development, pregnancy. Pregnancy exposome can affect the overall health of the pregnancy by dramatic changes in vascular physiology and endocrine activity, increasing maternal susceptibility. Moreover, fetoplacental vascular function is generally altered, increasing the risk of developing pregnancy complications (including cardiovascular diseases, CVD) and predisposing the foetus to adverse health risks later in life. Thus, our review summarizes the existing literature on exposures to EDCs during pregnancy and adverse maternal health outcomes, focusing on the human placenta, vein, and umbilical artery associated with pregnancy complications. The purpose of this review is to highlight the role of fetoplacental vasculature as a model for the study of human cardiovascular endocrine disruption. Therefore, we emphasize that the placenta, together with the umbilical arteries and veins, allows a better characterization of the pregnant woman's exposome. Consequently, it contributes to the protection of the mother and foetus against CV disorders in life.

The main estrogens: estradiol, estrone, and their acyl-esters have been studied essentially related to their classical estrogenic and pharmacologic functions. However, their main effect in the body is probably the sustained control of core energy metabolism. Estrogen nuclear and membrane receptors show an extraordinary flexibility in the modulation of metabolic responses, and largely explain gender and age differences in energy metabolism: part of these mechanisms is already sufficiently known to justify both. With regard to energy, the estrogen molecular species act essentially through four key functions: (1) Facilitation of insulin secretion and control of glucose availability; (2) Modulation of energy partition, favoring the use of lipid as the main energy substrate when more available than carbohydrates; (3) Functional protection through antioxidant mechanisms; and (4) Central effects (largely through neural modulation) on whole body energy management. Analyzing the different actions of estrone, estradiol and their acyl esters, a tentative classification based on structure/effects has been postulated. Either separately or as a group, estrogens provide a comprehensive explanation that not all their quite diverse actions are related solely to specific molecules. As a group, they constitute a powerful synergic action complex. In consequence, estrogens may be considered wardens of energy homeostasis.

Voltage-gated calcium channels are key regulators of brain function, and their dysfunction has been associated with multiple conditions and neurodegenerative diseases because they couple membrane depolarization to the influx of calcium-and other processes such as gene expression-in excitable cells. L-type calcium channels, one of the three major classes and probably the best characterized of the voltage-gated calcium channels, act as an essential calcium binding proteins with a significant biological relevance. It is well known that estradiol can activate rapidly brain signaling pathways and modulatory/regulatory proteins through non-genomic (or non-transcriptional) mechanisms, which lead to an increase of intracellular calcium that activate multiple kinases and signaling cascades, in the same way as L-type calcium channels responses. In this context, estrogens-L-type calcium channels signaling raises intracellular calcium levels and activates the same signaling cascades in the brain probably through estrogen receptor-independent modulatory mechanisms. In this review, we discuss the available literature on this area, which seems to suggest that estradiol exerts dual effects/modulation on these channels in a concentration-dependent manner (as a potentiator of these channels in pM concentrations and as an inhibitor in nM concentrations). Indeed, estradiol may orchestrate multiple neurotrophic responses, which open a new avenue for the development of novel estrogen-based therapies to alleviate different neuropathologies. We also highlight that it is essential to determine through computational and/or experimental approaches the interaction between estradiol and L-type calcium channels to assist these developments, which is an interesting area of research that deserves a closer look in future biomedical research.

The estrogen receptor (ER) exists in two forms known as ERalpha and ERbeta. Currently, a clinical role has only been established for ERalpha. The primary use of ERalpha in breast cancer is for predicting likely response to hormone treatment. Patients with breast cancers expressing ERalpha are approximately seven to eight times more likely to benefit from endocrine therapy than ERalpha-negative patients. For the initial three to five years after primary diagnosis, ERalpha-positive patients generally have a better outcome than ERalpha-negative patients. Overall, however, the prognostic value of ERalpha is relatively weak and only of limited value in the clinically important subgroup of patients with lymph node-negative disease. Further work is required to establish if ERbeta has a clinical role in breast cancer.

The steroid hormone 17beta-estradiol regulates the normal function and development of the mammalian nervous system. Many of estradiol's effects are mediated via the nuclear hormone estrogen receptors ERalpha and ERbeta. In addition to regulating estrogen-responsive gene expression, estradiol also acts in an immediate and cell-specific fashion to regulate various intracellular signal transduction pathways. The goal of this review is to develop a contextual framework to understand the generalized function of estrogen during development of brain regions not known to be sexually specialized. However, it is first important to build this framework on the more well-developed foundation of estrogen's gonad-driven sex-specific actions. As a result, a discussion of known and proposed mechanisms of estrogen actions in reproductive and other tissues will be presented. Building upon this information, a review of our research group's recent in vitro and in vivo studies that have focused on elucidating the mechanisms of estrogen actions in neurons of the non-sexually specialized cerebellum will be presented. While the full spectrum of estrogen action during normal cerebellar development remains unresolved, results of recent studies have revealed a pathologic role for estrogen and estrogen receptors in medulloblastoma, common pediatric brain tumors that arise from cerebellar granule cell-like precursors. The potential use of anti-estrogen signaling agents as adjuvant therapy for medulloblastoma is proposed based on those finding.

Many natural and synthetic compounds present in the environment exert a number of adverse effects on the exposed organisms, leading to endocrine disruption, for which they were termed endocrine disrupting chemicals (EDCs). A decrease in reproduction success is one of the most well-documented signs of endocrine disruption in fish. Estrogens are steroid hormones involved in the control of important reproduction-related processes, including sexual differentiation, maturation and a variety of others. Careful spatial and temporal balance of estrogens in the body is crucial for proper functioning. At the final step of estrogen biosynthesis, cytochrome P450 aromatase, encoded by the cyp19 gene, converts androgens into estrogens. Modulation of aromatase CYP19 expression and function can dramatically alter the rate of estrogen production, disturbing the local and systemic levels of estrogens. In the present review, the current progress in CYP19 characterization in teleost fish is summarized and the potential of several classes of EDCs to interfere with CYP19 expression and activity is discussed. Two cyp19 genes are present in most teleosts, cyp19a and cyp19b, primarily expressed in the ovary and brain, respectively. Both aromatase CYP19 isoforms are involved in the sexual differentiation and regulation of the reproductive cycle and male reproductive behavior in diverse teleost species. Alteration of aromatase CYP19 expression and/or activity, be it upregulation or downregulation, may lead to diverse disturbances of the above mentioned processes. Prediction of multiple transcriptional regulatory elements in the promoters of teleost cyp19 genes suggests the possibility for several EDC classes to affect cyp19 expression on the transcriptional level. These sites include cAMP responsive elements, a steroidogenic factor 1/adrenal 4 binding protein site, an estrogen-responsive element (ERE), half-EREs, dioxin-responsive elements, and elements related to diverse other nuclear receptors (peroxisome proliferator activated receptor, retinoid X receptor, retinoic acid receptor). Certain compounds including phytoestrogens, xenoestrogens, fungicides and organotins may modulate aromatase CYP19 activity on the post-transcriptional level. As is shown in this review, diverse EDCs may affect the expression and/or activity of aromatase cyp19 genes through a variety of mechanisms, many of which need further characterization in order to improve the prediction of risks posed by a contaminated environment to teleost fish population.

Estrogen can significantly influence CD16 expression and alter monocytic cytokine release upon CD16 receptor activation. However, the function of the estrogen receptor (ER) alpha and beta in this response is unclear. To test whether estrogen binds ERalpha and/or ERbeta to affect CD16 expression, monocytic cells were treated with and without physiological levels of 17beta-estradiol and various doses of the ERalpha and ERbeta antagonist fulvestrant followed by measurement of CD16 transcript levels. To determine how estrogen induced changes in TNF-alpha and IL-1beta release due to CD16 activation we quantitated the amount of cytokines after treatment with estrogen, fulvestrant and antibodies that specifically bind and activate the CD16 receptor. Interaction of ERalpha and the CD16 promoter was then determined by chromatin immunoprecipitation. Furthermore, specific promoter elements utilized by estrogen to control CD16 expression were mutated and expression from a luciferase reporter quantitated after transfection. Using the luciferase reporter construct containing a wild type CD16 promoter, the role of ERalpha and ERbeta in the estrogen response was tested by treating transfected monocytes with an ERalpha specific agonist or an ERbeta specific agonist and measuring expression. Our results show that CD16 transcript levels significantly decreased in monocytic cells due to estrogen and that the observed decrease in message was blocked by the antagonist fulvestrant. Estrogen reduced CD16 expression and decreased TNF-alpha and IL-1beta release upon CD16 activation but the administration of fulvestrant blocked this decrease. ERalpha was found to interact with a region 5' of the CD16 gene in the presence of estrogen, and site-directed mutational analysis of this region indicated the necessity for an estrogen response element in modulating estrogen effects on CD16 expression. Moreover, both an ERalpha and an ERbeta agonist reduced expression of the CD16 reporter construct suggesting both receptors can play a role in CD16 regulation. In conclusion, CD16 expression can be altered by the activity of ERalpha or ERbeta and our results also show that ERalpha can associate with a region within the CD16 promoter that is important in production of transcript.

In addition to cellular responses that are elicited by steroids involving the modulation of transcription in the nucleus, it is now generally accepted that additional phenomena occur that do not depend on the genome. However, there is a puzzling variety of candidate receptors described in the literature.

Estrogen is known to stimulate breast cancer development in humans. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The mechanism by which estrogen induces tumour regression in breast cancer is still unknown. We found that under low growth-stimulated conditions, high concentrations of 17-beta-estradiol (estradiol) induces apoptosis and concomitantly increases phosphorylation of c-jun in estrogen receptor (ER)-positive breast cancer cell line, MCF-7, but not in ER-negative breast cancer cell line MDA-MB 231 suggesting an ER-mediated event. Interestingly, when the c-jun NH2-terminal kinase (JNK) signalling pathway was disrupted by the JNK inhibitor SP600125, the ability of estradiol to inhibit the growth of MCF-7 cells and to induce apoptosis was completely blocked. These data suggest that JNK plays a central role in mediating the anticancer effect of high concentrations of estradiol in MCF-7 cells. Our data showing the apoptotic effect of estradiol in low growth-stimulated conditions suggest potential implications for the pharmacological control of breast cancer with high dose estrogen in postmenopausal women. Furthermore, our results indicate that augmenting JNK activity could be an efficient novel approach for treating breast cancer.

Estrogens have multiple influences on immune functions. Estrogen receptors (ERs) have two distinct subtypes - alpha and beta. To explore the specific roles of each ER subtype in estrogen-mediated immunomodulation, we investigated the effects of ER subtype-selective agonists on immune functions in ovariectomized Balb/c mice. Treatment with ERalpha-selective agonist propyl pyrazole triol (PPT) caused thymic atrophy and significant changes in thymic CD4/CD8 phenotypic profile. In contrast, ERbeta-selective agonist diarylpropionitrile (DPN) alone had no effect on thymic weight, cellularity or CD4/CD8 phenotype expression. When coadministered with PPT, DPN partially antagonized PPT-evoked decrease in thymic cellularity and also partially attenuated PPT-induced shifts in thymic T-cell phenotype. These results indicate that ERalpha plays a predominant role in estrogen-induced thymic atrophy and ERbeta activation may partially down-regulate ERalpha-mediated effects on thymic cellularity and T-cell phenotype expression. In addition, PPT administration induced a reduction in the percentage of mature B cells in the spleen, and enhanced IFN-gamma production but suppressed IL-6 production from in vitro Con A-stimulated splenocytes as estradiol (E(2)) did, whereas DPN treatment had no effects either alone or with PPT, suggesting ERalpha mediates these estrogen actions. Treatment with PPT or DPN did not augment anti-DNP antibody production after DNP-KLH immunization as E(2) did, implying that not merely one ER signaling pathway is involved in mediating estrogen's effects on specific humoral immune responses. Our study further indicates ER subtype-selective agonists provide a novel approach to explore each ER subtype-mediated immunomodulation.

Following our previous demonstration that p,p'-DDE (dichlorodiphenylchloroethylene), at environmentally relevant concentrations, can rapidly increase intracellular calcium [Ca2+]i concentrations in human granulosa-lutein cells, we examined whether other pesticides, such as Kepone, o,p-DDE and methoxychlor, have similar effects. Cultured human granulosa-lutein cells were loaded with Fura-2 AM, and changes in [Ca2+]i concentrations within small areas of single cells were studied with a dynamic digital Ca2+ imaging system. Kepone, at concentrations of 0.2-2 nmol/ml, consistently increased [Ca2+]i concentrations 2-6 times higher than baseline values within minutes of exposure. Methoxychlor at concentrations of 2.8-280 nmol/ml failed to alter [Ca2+]i levels consistently in cells from 10 patients. However, at 0.28 and 1.4 nmol/ml, increases in [Ca2+]i concentrations could be elicited by methoxychlor. The isomer o,p-DDE at 3 nmol/ml increased [Ca2+]i in granulosa cells of 11/20 patients. Pertussis toxin treatment inhibited the [Ca2+]i increases induced by estradiol, p,p'-DDE, o,p-DDE and methoxychlor, but not by Kepone or progesterone, indicating that Kepone and progesterone may act through an insensitive G protein-coupled receptor. The [Ca2+]i increases induced by Kepone also occurred in Ca2+-free medium, suggesting that [Ca2+]i mobilization occurred from the smooth endoplasmic reticulum. Thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca2+ pump, also stimulated [Ca2+]i increases but did not inhibit the Ca2+ response to all the pesticides. These results demonstrate that pesticides can have a rapid effect on human granulosa-lutein cells, and a nongenomic mechanism of action is suggested.

Several species of Ferula genus have been used in folk medicine in digestive disorders, rheumatism, headache, arthritis, and as tranquilizers, antispasmodic and aphrodisiac. From the dry and powdered roots of Ferula hermonis Boiss was extracted the oxygenated sesquiterpene 1,5-trans-daucane type: ferutinine (1). The structure of (1) was established by spectroscopic methods as: IR, (1)H RMN, (13)C RMN, COSY, HMBC, HMQC, NOESY, EIMS, and CIMS. The possible signaling pathway of ferutinin (1) in nervous tissue in vitro was assessed and the results showed that this compound is able to increase nitric oxide synthase activity and inositol monophosphate accumulation (49%, each) in the median eminence of the rat brain, suggesting that compound (1) is associated to the activation of phosphoinositide breakdown and nitric oxide production (NO), the last is a gaseous intercellular messenger known to play a broad role in human biology from homeostasis to pathology.

As phytoestrogens are postulated as being neuroprotectants, we assessed the hypothesis that dietary isoflavone-type phytoestrogens are neuroprotective against ischemic stroke. Transient focal cerebral ischemia (90 min) was induced by middle cerebral artery occlusion (MCAO) following the intraluminal thread technique, both in rats fed with soy-based diet and in rats fed with isoflavone-free diet. Cerebro-cortical laser-Doppler flow (cortical perfusion, CP), arterial blood pressure, core temperature, PaO2, PaCO2, pH and glycemia were measured before, during and after MCAO. Neurological examination and infarct volume measurements were carried out 3 days after the ischemic insult. Dietary isoflavones (both glycosides and aglycones) were measured by high-performance liquid chromatography. Neither pre-ischemic, intra-ischemic nor post-ischemic CP values were significantly different between the soy-based diet and the isoflavone-free diet groups. Animals fed with the soy-based diet showed an infarct volume of 122 +/- 20.2 mm3 (19 +/- 3.3% of the whole ipsilateral hemisphere volume). In animals fed with the isoflavone-free diet the mean infarct volume was significantly higher, 191 +/- 26.7 mm3 (28 +/- 4.1%, P < 0.05). Neurological examination revealed significantly higher impairment in the isoflavone-free diet group compared with the soy-based diet group (3.3 +/- 0.5 vs. 1.9 +/- 0.5, P < 0.05). These results demonstrate that dietary isoflavones improve stroke outcome after transient focal cerebral ischemia in such a way that a higher dietary isoflavone content results in a lower infarct volume and a better neurological status.

The ecdysteroids (Ec), invertebrate steroid hormones, elicit genomic but also non-genomic effects. By analogy to vertebrates, non-genomic responses towards Ec may be mediated not only by distinct membrane-integrated but also by membrane-associated receptors like the classical nuclear ecdysteroid receptor (EcR) of arthropods. This is supported by a comparison of physiological properties between invertebrate and vertebrate steroid hormone systems and recent findings on the subcellular localization of EcR. The measured or predicted high degree of conformational flexibility of both Ec and the ligand binding domain (LBD) of EcR give rise to a conformational compatibility model: the compatibility between conformations of the cognate receptor's ligand binding domain and structures or conformations of the ligand would determine their interaction and eventually the initiation of genomic versus non-genomic pathways. This model could also explain why specific non-genomic effects are generally not observed with non-steroidal agonists of the bisacylhydrazine group.

Recent gene microarray studies have illustrated heterogeneity in gene expression changes not only between estrogens and selective estrogen receptor modulators (SERMs), but also across different SERM molecules. In ovariectomized rats, this phenomenon was observed with respect to a number of genes involved in collagen turnover and extracellular matrix (ECM) integrity in the uterus and vaginal tissues. Preliminary mechanistic data suggest that these effects on ECM integrity may have relevance in the context of the effect of estrogens and some SERMs to increase the risk of pelvic organ prolapse and the incidence of urinary incontinence in postmenopausal women. Given the pivotal role of ECM integrity and collagen turnover in other tissues and disease states, these processes may provide a fruitful target for future research into the mechanisms for the heterogeneous pharmacology of estrogens and SERMs across different cell types and target tissues.

The higher cardiovascular risk in men and post-menopausal women implies a protective action of estrogen. A large number of experimental studies have provided strong support to this concept. However, the recent clinical trials with negative outcomes regarding hormone replacement therapy call for "post hoc" reassessment of existing information, models, and research strategies as well as a summary of recent findings. Sex steroid hormones, in particular estrogen, regulate numerous processes that are related to the development and progression of cardiovascular disease through a variety of signaling pathways. Use of genetically modified models has resulted in interesting information on diverse actions mediated by steroid receptors. By focusing on experimental findings, we have reviewed hormonal, cellular, and signaling mechanisms responsible for sex dimorphism and actions of hormone replacement therapy and addressed current limitations and future directions of experimental research.

Hypothalamic luteinizing hormone-releasing hormone neurons (LHRH) form the final pathway for the central control of reproduction through the release of LHRH into the pituitary-hypothalamic system. We previously found that LHRH-producing GT1-7 cells respond to acetylcholine (ACh) with an increase in intracellular calcium ([Ca2+]i) through activation of muscarinic receptors. This effect is acutely modulated by 17beta-estradiol in a manner compatible with specific membrane binding sites. Because increasing evidence suggests that second messengers are involved in the rapid action of estradiol, the aim of the present study was to identify the pathway underlying estrogen actions on ACh-induced Ca2+ signals. 8-Bromoguanosine 3',5'-cyclic monophosphate (10 microm) and C-type natriuretic peptide (10 microm) mimicked the effect of estradiol. On the contrary, neither dibutyryl cAMP (100 microm), forskolin (100 nm or 10 microm), or sodium nitroprusside (10 microm) induced any modification of [Ca2+]i in response to ACh. The effect of estradiol on calcium transients was totally blocked by two different cGMP-dependent protein kinase (PKG) inhibitors. In addition, phosphorylation of inositol 1,4,5-triphosphate (IP3) receptor was rapidly induced by estradiol but totally blocked when the cells were pretreated with a PKG inhibitor. We conclude that physiological concentrations of estradiol reduce ACh-induced Ca2+ transients via a mechanism involving a membrane-associated guanylate cyclase, which finally induces a PKG-dependent IP3 receptor phosphorylation that modifies calcium release from the endoplasmic reticulum.

Previous studies have shown that the progesterone metabolite, 5alpha-pregnane-3,20-dione (5alphaP), exhibits mitogenic and metastatic activity in breast cell lines and that specific, high affinity receptors for 5alphaP are located in the plasma membrane fractions of tumorigenic (ER/PR-positive) MCF-7 cells. The aim of this study was to determine the effects of the mitogenic (estradiol; 5alphaP) and anti-mitogenic (3alpha-hydroxy-4-pregnen-20-one, 3alphaHP; 20alpha-hydroxy-4-pregnen-3-one, 20alphaHP) endogenous steroid hormones on 5alphaP receptor (5alphaP-R) numbers and on cell proliferation and adhesion of MCF-7 and MCF-10A cells. Exposure of MCF-7 cells for 24h to estradiol or 5alphaP resulted in significant (p < 0.05-0.001) dose-dependent increases in 5alphaP-R levels. Conversely, treatment with 3alphaHP or 20alphaHP resulted in significant (p < 0.05-0.01) dose-dependent decreases in 5alphaP-R levels. Treatment with one mitogenic and one anti-mitogenic hormone resulted in inhibition of the mitogen-induced increases, whereas treatment with two mitogenic or two anti-mitogenic hormones resulted in additive effects on 5alphaP-R numbers. Treatments with cycloheximide and actinomycin D indicate that changes in 5alphaP-R levels depend upon transcription and translation. The non-tumorigenic breast cell line, MCF-10A, was also shown to posses specific, high affinity plasma membrane receptors for 5alphaP that were up-regulated by estradiol and 5alphaP and down-regulated by 3alphaHP. Estradiol binding was demonstrated in MCF-10A cell membrane fractions and may explain the estradiol action in these cells that lack intracellular ER. In both MCF-7 and MCF-10A cells, the increases in 5alphaP-R due to estradiol or 5alphaP, and decreases due to 3alphaHP or 20alphaHP correlate with respective increases and decreases in cell proliferation as well as detachment. These results show distribution of 5alphaP-R in several cell types and they provide further evidence of the significance of progesterone metabolites and their novel membrane-associated receptors in breast cancer stimulation and control. The findings that 3alphaHP and 20alphaHP down-regulate 5alphaP-R and suppress mitogenic and metastatic activity suggest that these endogenous anti-mitogenic progesterone metabolites deserve considerations in designing new breast cancer therapeutic agents.

Estrogen is traditionally associated with females but is also present in males, and influences aspects of brain chemistry and brain morphology in males, females and also during prenatal development. Humans as well as animals are additionally exposed to environmental products that mimic estrogen activity, also known as endocrine disrupters (EDCs). This hypothesis article investigates the role of estrogen (and also EDCs) in the brain and how it influences the Ca2+ pathway. Ca2+ and its movement in and out of the cell is an extremely important ion controlling normal cell physiology. Any dysfunction in the movement from outside to inside the cell or between organelles may have fundamentally negative effects and the disturbance may even lead to apoptosis and/or necrosis. Therefore we consider whether estrogen and EDCs may alter the Ca2+ physiology and whether these changes may be one of the main causes of interference in physiology that is seen when humans and animals are exposed to EDCs. We come to the conclusion that on a molecular level Ca2+ and Ca2+ fluxes ([Ca2+]i, endocrine disrupting chemicals, redox modulation, mitochondria and cytochrome c followed by apoptosis, necrosis or most likely aponecrosis may contribute to chemical-mediated developmental toxicity. Similarly, we hypothesize that calcium-mediated aponecrosis do not only play a central role in the pathophysiology of estrogenic chemical-induced neurotoxicity, but can contribute to chemical-mediated developmental toxicity in general, thereby affecting almost all cells and organs of the living organism.

Persistent metabolites of pesticides such as p,p'-DDE, at environmentally relevant concentrations, have been shown to have a rapid effect on intracellular calcium [Ca2+]i concentrations in human granulosa-lutein cells. Since endocrine disrupting substances can be transferred from the maternal circulation to the fetus the present study examined whether the pesticides, kepone, o,p-DDE, p,p'-DDE and methoxychlor, could alter cytoplasmic calcium [Ca2+]cyt concentrations in human umbilical vein endothelial (HUVE) cells. Cultured HUVE cells were loaded with Fura-2 AM and changes in [Ca2+]cyt of single cells were studied using a dynamic digital Ca2+ imaging system. Kepone and methoxychlor consistently increased [Ca2+]cyt concentrations, similar to the effects of estradiol and progesterone. p,p'-DDE increased [Ca2+]cyt concentrations in 80% of experiments whereas o,p-DDE stimulated its increases in 42%. Estrone, estriol, pregnenolone and cortisol were not effective. These results demonstrate that pesticides can have a rapid effect on HUVE cells probably through a nongenomic mechanism of action.

There are two estrogen receptor (ER) subtypes in fish, Esr1 and Esr2 (formerly ERalpha and ERbeta), and in some species the Esr2 subtype has two forms, Esr2b (formerly ERbeta1) and Esr2a (formerly ERbeta2 or ERgamma). There is little information, however, on the different characteristics and functional significance of the two receptor subtypes in fish, and this is especially relevant for understanding the disruption of ER signaling by chemicals with estrogenic activity. In this study, the full-length cDNAs for esr1 (3167 base pairs [bp]) and esr2b (2318 bp), and a partial-length (267 bp) cDNA for esr2a, were cloned and characterized in fathead minnow (fhm; Pimephales promelas), and their patterns of expression established during development and in adults. Real-time polymerase chain reaction revealed some clear distinctions in the ontogenic and tissue expression of fhm esr1, esr2b, and esr2a, suggesting different functions for each ER subtype. Fhm ERs were expressed in brain, pituitary, liver, gonad, intestine, and gill of male and female fish, esr2b and esr2a were also expressed in muscle. Fhm esr1 and esr2b were expressed predominantly in the liver, whereas fhm esr2a was expressed predominantly in intestine and was lowest expressed in liver. Responses of the different hepatic ERs in male fathead minnow exposed to 100 ng estradiol/L differed, with a significant induction (5-fold) of fhm esr1 but no effect on esr2b or esr2a expression, suggesting different mechanisms of regulation for the different ERs. The detailed characterization of ERs in fathead minnow provides the foundation for understanding the molecular basis of estrogenic disruption in fish.

The present study examined whether the sex steroids, estradiol and progesterone, could alter cytoplasmic calcium concentrations ([Ca(2+)](cyt)) in human granulosa-lutein cells.

Human granulosa cells were obtained at the time of oocyte retrieval for IVF and cultured for 3-7 days. Cells were loaded with Fura-2 AM and changes in [Ca(2+)](cyt) of single cells were studied using a dynamic digital Ca(2+) imaging system.

Both estradiol and progesterone stimulated elevations of [Ca(2+)](cyt) in Ca(2+)-containing medium within seconds of exposure of the granulosa-lutein cells to the steroid, but only estradiol caused an increase in [Ca(2+)](cyt) in Ca(2+)-free medium. Both ICI-182780 and RU 486 stimulated [Ca(2+)](cyt) increases and inhibited the effects of estradiol and progesterone, respectively. Tamoxifen also induced transient increases in [Ca(2+)](cyt) concentrations but inhibited the effects of both estradiol and progesterone. The inhibitory effects of tamoxifen, ICI-182780 and RU 4486 on [Ca(2+)](cyt) responses to estradiol and progesterone could be reversed with higher concentrations of estradiol and progesterone, respectively. The [Ca(2+)](cyt) effects induced with tamoxifen could not be eliminated by prior treatment with RU 486 or ICI-182780.

These results provide strong evidence that both estradiol and progesterone as well as the steroid antagonists, tamoxifen, RU 486 and ICI-182780, can act on human granulosa-lutein cells through a non-genomic mechanism.

Given the suspected effects of estrogens on breast cancer, xenoestrogenic insecticides may be a risk factor. Studies of the weak xenoestrogen, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), have failed to demonstrate a causal relationship, though another estrogenic organochlorine insecticide, dieldrin, belonging to the cyclodiene family, has recently been linked to breast cancer. Other cyclodienes such as heptachlor epoxide (HE) and oxychlordane (OC) present in breast tissue have not been evaluated as rigorously, presumably due to their lower concentration and lower recovery using solvent extraction procedures. We used sparging extraction coupled with gas chromatography to determine the levels of HE, OC, and DDE in adipose tissue within breast biopsies in a series of 34 women evaluated for breast abnormality. Of the three insecticides tested, only HE (p=0.007) was positively associated with prevalence of breast cancer in the biopsies. In rapid, non-genomic studies using isolated human leukocytes, flow cytometric methods were used to measure HE-induced oxidants and DNA damage. These studies indicated that HE, at concentrations similar to those in breast biopsies, induced an inverted-U increase in intracellular oxidants and DNA strand breaks [both blocked by specific nitric oxide- (NO-) synthesis blockade withL: -NMMA] in human polymorphonuclear leukocytes (PMNs). HE-treated PMNs also induced damage to surrounding lymphocytes in mixed-leukocyte incubations (also inhibited by NO blockade). The HE-induced changes in NO were inhibited by 17beta-estradiol-(17beta-E2) receptor antagonists and were mimicked by similar concentrations of 17beta-E2. The addition of tumor necrosis factor-alpha (TNF-alpha) increased intracellular oxidants and DNA damage and shifted the responses to lower HE concentrations. This study, along with others, suggests that HE-induced NO production may contribute to initiation, promotion, and progression of cancer.

Mammalian spermatogenesis is a complex hormone-dependent developmental program in which a myriad of events must take place to ensure that germ cells reach their proper stage of development at the proper time. Many of these events are controlled by cell type- and stage-specific transcription factors. The regulatory mechanisms involved provide an intriguing paradigm for the field of developmental biology and may lead to the development of new contraceptives an and innovative routs to treat male infertility. In this review, we address three aspects of the genetic regulatory mechanism that drive spermatogenesis. First, we detail what is known about how steroid hormones (both androgens and estrogens) and their cognate receptors initiate and maintain mammalian spermatogenesis. Steroids act through three mechanistic routes: (i) direct activation of genes through hormone-dependent promoter elements, (ii) secondary transcriptional responses through activation of hormone-dependent transcription factors, and (iii) rapid, transcription-independent (nonclassical) events induced by steroid hormones. Second, we provide a survey of transcription factors that function in mammalian spermatogenesis, including homeobox, zinc-finger, heat-shock, and cAMP-response family members. Our survey is not intended to cover all examples but to give a flavor for the gamut of biological roles conferred by transcription factors in the testis, particularly those defined in knockout mice. Third, we address how testis-specific transcription is achieved. In particular, we cover the evidence for and against the idea that some testis-specific genes are transcriptionally silent in somatic tissues as a result of DNA methylation.

Although testosterone (T) may decrease anxiety and enhance cognitive performance, its mechanisms are not well understood. The authors hypothesized that if T's effects are mediated in part through actions of its 5alpha-reduced, nonaromatizable metabolite dihydrotestosterone (DHT) and/or its 3alpha-hydroxysteroid dehydrogenase reduced metabolite 3alpha-androstanediol (3alpha-diol) in the hippocampus, then T, DHT, and 3alpha-diol administration should produce similar behavioral effects concomitant with elevating T metabolites in the hippocampus. Gonadectomized male rats administered T, DHT, or 3alpha-diol via Silastic capsules or intrahippocampal infusions had greater analgesia (tail flick, paw lick), less anxiety behavior (plus-maze, open field, defensive freezing), and better learning (inhibitory avoidance) compared with vehicle control rats. Only 3alpha-diol levels in the hippocampus were consistently elevated in conjunction with these behavioral effects.