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Introduction: It is becoming more evident that hormone balance is not only vital to a healthy lifestyle, but necessary for health, vitality and longevity. Men and women alike suffer from [...]

Many tumors contain heterogeneous populations of cells, only some of which exhibit increased tumorigenicity and resistance to anticancer therapies. Evidence suggests that these aggressive cancer cells, often termed 'cancer stem cells' or 'cancer stem-like cells' (CSCs), rely upon developmental signaling pathways that are important for survival and expansion of normal stem cells. Here we report that, in analogy to embryonic mammary epithelial biology, estrogen signaling expands the pool of functional breast CSCs through a paracrine FGF/FGFR/Tbx3 signaling pathway. Estrogen or FGF9 pretreatment induced CSC properties of breast cancer cell lines and freshly isolated breast cancer cells, whereas cotreatment of cells with tamoxifen or a small molecule inhibitor of FGFR signaling was sufficient to prevent the estrogen-induced expansion of CSCs. Furthermore, reduction of FGFR or Tbx3 gene expression was able to abrogate tumorsphere formation, whereas ectopic Tbx3 expression increased tumor seeding potential by 100fold. These findings demonstrate that breast CSCs are stimulated by estrogen through a signaling pathway that similarly controls normal mammary epithelial stem cell biology. doi: 10.1073/pnas.1007863107

Whether prepubertal estrogen production impacts on the timing of puberty is not clear. We aimed to investigate prepubertal 24-h estrogen excretion levels and their association with early and late pubertal markers. Daily urinary excretion rates of estrogens of 132 healthy children, who provided 24-h urine samples 1 and 2 yr before the start of the pubertal growth spurt [age at takeoff (ATO)], were quantified by stable isotope dilution/GC-MS. E-sum3 (estrone + estradiol + estriol) was used as a marker for potentially bioactive estrogen metabolites and E-sum5 (E-sum3 + 16-epiestriol + 16-ketoestradiol) for total estrogen production. Pubertal outcomes were ATO, age at peak height velocity (APHV), duration of pubertal growth acceleration (APHV-ATO), age at Tanner stage 2 for pubic hair (PH2), genital (G2, boys) and breast (B2, girls) development, and age at menarche. Prepubertal urinary estrogen excretions (E-sum3 and E-sum5) were not associated with ATO, APHV, and age at PH2 but with duration of pubertal growth acceleration (P < 0.01) in both sexes. Girls with higher E-sum3 reached B2 0.9 yr (P = 0.04) and menarche 0.3 yr earlier (P = 0.04) than girls with lower E-sum3. E-sum3 was not associated with age at G2 in boys (P = 0.6). For most pubertal variables, the associations with E-sum3 were stronger than with E-sum5. In conclusion, prepubertal estrogens may not be critical for the onset of the pubertal growth spurt but are correlated with its duration in both boys and girls. Prepubertal estrogen levels may already predict the timing of girls' menstruation and breast development but do not appear to affect sexual maturation in boys. sex differentiation; developmental biology; pediatrics; steroids doi: 10.1152/ajpendo.00374.2010.

Estrogen receptor [alpha](ER[alpha]) plays an important role in the onset and progression of breast cancer, whereas p53 functions as a major tumor suppressor. We previously reported that ER[alpha] binds to p53, resulting in inhibition of transcriptional regulation by p53. Here, we report on the molecular mechanisms by which ER[alpha] suppresses p53's transactivation function. Sequential ChIP assays demonstrated that ER[alpha] represses p53-mediated transcriptional activation in human breast cancer cells by recruiting nuclear receptor corepressors (NCoR and SMRT) and histone deacetylase 1 (HDAC1). RNAi-mediated down-regulation of NCoR resulted in increased endogenous expression of the cyclin-dependent kinase (CDK)-inhibitor [p21.sup.Waf1/cip1] (CDKNIA) gene, a prototypic transcriptional target of p53. While 17[beta]-estradiol (E2) enhanced ER[alpha] binding to p53 and inhibited p21 transcription, antiestrogens decreased ER[alpha] recruitment and induced transcription. The effects of estrogen and antiestrogens on p21 transcription were diametrically opposite to their known effects on the conventional ERE-containing ER[alpha] target gene, pS2/TFF1. These results suggest that ER[alpha] uses dual strategies to promote abnormal cellular proliferation: enhancing the transcription of ERE-containing proproliferative genes and repressing the transcription of p53-responsive antiproliferative genes. Importantly, ER[alpha] binds to p53 and inhibits transcriptional activation by p53 in stem/progenitor cell-containing murine mammospheres, suggesting a potential role for the ER-p53 interaction in mammary tissue homeostasis and cancer formation. Furthermore, retrospective studies analyzing response to tamoxifen therapy in a subset of patients with ER-positive breast cancer expressing either wild-type or mutant p53 suggest that the presence of wild-type p53 is an important determinant of positive therapeutic response. nuclear receptor corepressor | mammary epithelial cells | mammospheres | tumor suppressor protein | tamoxifen therapy doi/ 10.1073/pnas.1009575107

Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ER[alpha] also enhances insulin synthesis in cultured islets. Whether ERa stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERa knockout mouse (PER[alpha][KO.sup.-/-]) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and [beta] cells. Whereas 17[beta]-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERcdox/lox mice, these E2 effects are abolished in PER[alpha][KO.sup.-/-] mice. We find that E2-activated ER[alpha] increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ER[alpha] eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ER[alpha] interacts with the tyrosine kinase Src, which activates extracellular signal-regulated [kinases.sub.1/2], to increase nuclear localization and binding to the insulin promoter of the transcription factor NeuroD1. This study supports the importance of ERa in [beta] cells as a regulator of insulin synthesis in vivo. diabetes | islet doi/ 10.1073/pnas.0914501107

Estrogen receptors (ERs) play a pivotal role in adipogenesis; therefore, compounds targeting ERs may also affect fat formation. Recent studies have shown that the Dioscorea plant (commonly called yam) exhibits an antiobesity effect on rodents. However, the active compounds and underlying mechanisms responsible for this effect are not yet fully understood. We evaluated the effects of pseudoprotodiocsin (PPD), a steroid saponin from Dioscorea nipponica Makino (a type of Dioscorea), on adipogenesis and the mechanisms underlying this effect. Treatment with PPD at the onset of adipogenic differentiation resulted in significantly decreased adipogenesis in both in vitro and in vivo experimental systems. An increased amount of ERa mRNA, protein, and the accumulation of ERa in the nucleus were also observed. However, the expression pattern of ER[beta] was not altered. Furthermore, the antiadipogenic effect of PPD was found to be ER dependent. It was also accompanied by the decreased expression of several genes involved in adipogenesis, including lipoprotein lipase (LPL), leptin, CCAAT/ enhancer-binding-protein-[alpha] (C/EBP[alpha]), and peroxisome proliferator-activated receptor--[gamma] (PPAR[gamma]), as well as the increased expression of some negative factors of adipogenesis, including preadipocyte factor 1 (Pre-1), GATA-binding protein 2 (GATA-2), GC-induced leucinezipper protein (GILZ), and C/EBP homologous protein (CHOP-10). In addition to its estrogenic action, PPD also abolished the p38 mitogen-activated protein kinase (p38 MAPK) activation. Our results suggest that PPD inhibits adipogenesis in an ER-dependent manner and induces the expression of ERa. These findings may provide a lead toward a novel agent that can be used to treat obesity. obesity; Dioscorea nipponica Makino; steroid saponin doi: 10.1152/ajpcell.00538.2009.

PIK3CA mutations are reported to be present in approximately 25% of breast cancer (BC), particularly the estrogen receptor--positive (ER+) and HER2-overexpressing (HER2+) subtypes, making them one of the most common genetic aberrations in BC. In experimental models, these mutations have been shown to activate AKT and induce oncogenic transformation, and hence these lesions have been hypothesized to render tumors highly sensitive to therapeutic PI3K/ mTOR inhibition. By analyzing gene expression and protein data from nearly 1,800 human BCs, we report that a PIK3CA mutation-associated gene signature (PIK3CA-GS) derived from exon 20 (kinase domain) mutations was able to predict PIK3CA mutation status in two independent datasets, strongly suggesting a characteristic set of gene expression-induced changes. However, in ER+/HER2-BC despite pathway activation, PIK3CA mutations were associated with a phenotype of relatively low mTORC1 signaling and a good prognosis with tamoxifen monotherapy. The relationship between clinical outcome and the PIK3CA-GS was also assessed. Although the PIK3CA-GS was not associated with prognosis in ER- and HER2+ BC, it could identify better clinical outcomes in ER+/HER2--disease. In ER+ BC cell lines, PIK3CA mutations were also associated with sensitivity to tamoxifen. These findings could have important implications for the treatment of PIK3CA-mutant BCs and the development of PI3K/mTOR inhibitors. gene expression profiling | PI3 kinase doi/ 10.1073/pnas.0907011107

At 2 years of age, 100% (23/23) of ER[[beta].sup.-/-] female mice have developed large pituitary and ovarian tumors. The pituitary tumors are gonadotropin-positive and the ovarian tumors are sex cord (less differentiated) and granulosa cell tumors (differentiated and estrogen secreting). No male mice had pituitary tumors and no pituitary or ovarian tumors developed in ER[[alpha].sup.-/-] mice or in ER[alpha][[beta].sup.-/-] double knockout mice. The tumors have high proliferation indices, are ER[alpha]positive, ER[beta]-negative, and express high levels of nuclear phospho-SMAD3. Mice with granulosa cell tumors also had hyperproliferative endometria. The cause of the pituitary tumors appeared to be excessive secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus resulting from high expression of NPY. The ovarian phenotype is similar to that seen in mice where inhibin is ablated. The data indicate that ER[beta] plays an important role in regulating GnRH secretion. We suggest that in the absence of ER[beta], the proliferative action of FSH/SMAD3 is unopposed and the high proliferation leads to the development of ovarian tumors. The absence of tumors in the ER[alpha][[beta].sup.-/-] mice suggests that tumor development requires the presence of ER[alpha]. estrogen receptor [beta] (ER[beta]) | pituitary | gonadotropin releasing hormone (GnRH) | follicle stimulating hormone (FSH) | TGF[beta] doi/10.1073/pnas.1002029107

The primordium that generates the embryonic posterior lateral line of zebrafish migrates from the head to the tip of the tail along a trail of SDF1-producing cells. This migration critically depends on the presence of the SDF1 receptor CXCR4 in the leading region of the primordium and on the presence of a second SDF1 receptor, CXCR7, in the trailing region of the primordium. Here we show that inactivation of the estrogen receptor ESR1 results in ectopic expression of cxcr4b throughout the primordium, whereas ESR1 overexpression results in a reciprocal reduction in the domain of cxcr4b expression, suggesting that ESR1 acts as a repressor of cxcr4b. This finding could explain why estrogens significantly decrease the metastatic ability of ESR-positive breast cancer cells. ESR1 inactivation also leads to extinction of cxcrTb expression in the trailing cells of the migrating primordium; this effect is indirect, however, and due to the down-regulation of cxcr7b by ectopic SDF1/CXCR4 signaling in the trailing region. Both ESR1 inactivation and overexpression result in aborted migration, confirming the importance of this receptor in the control of SDFl-dependent migration. collective cell migration | CXCR7 | FGF | SDF1 | Wnt doi/10.1073/pnas.0909998107

The need to segregate poultry based on sex is driven by sex-related differences in growth rate, market age, management practices, and nutritional requirements. Each day, poultry industry staff globally would ideally like to determine the sex of >150 million newly hatched birds. Currently, this can be done only manually at the hatchery, which is a virtually impossible undertaking. It is becoming more difficult each year to conduct manual sexing because this skill is disappearing from the workforce, is becoming unaffordable to the industry, and is encumbered by such negative effects as repetitive motion disorder. Automated sex sorting of eggs before hatching could resolve many, if not all, of these problems. We have developed a facile, rapid, and low-cost yeast-based assay that distinguishes male from female embryonated eggs before hatching based on the estrogen concentration of their allantoic fluid. Herein, we describe this novel sex-sorting technology, which we believe offers the potential to standardize and automate sex sorting in the poultry industry. Key words: allantoic fluid, embryonated egg, estrogen conjugate, sex sorting doi: 10.2527/jas.2009-2212

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor-[beta] (ER[beta]) in BPH and PCa. ER[beta] agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNF[alpha] knock-out mice fail to respond to ER[beta] agonist, demonstrating the requirement for TNF[alpha] signaling. In human tissues, ER[beta] agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the [CD133.sup.+] enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ER[beta] causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ER[beta] agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ER[beta] agonist for treatment of PCa and/or BPH with or without androgen withdrawal. castration | steroid receptors | selective estrogen receptor modulators www.pnas.org/cgi/doi/10.1073/pnas.0905524107

p53 is a master regulatory, sequence-specific transcription factor that directly controls expression of over 100 genes in response to various stress signals. Transactivation is generally considered to occur through p53 binding to a consensus response element (RE) composed of two 5'-RRRCWWGYYY-3' decamers. Recently, studying the human angiogenesis-related gene FLT1 we discovered that p53 can mediate limited transactivation at a noncanonical 1/2 site and could synergize with the estrogen receptor (ER) acting in cis at a nearby ER 1/2 site. To address the generality of concerted transactivation by p53 and ER, the 1/2 site in the FLT1 promoter was replaced with a variety of 1/2 sites, as well as canonical weak and strong p53 REs of human target genes. The p53 transactivation of all tested sequences was greatly enhanced by ligand-activated ER acting in cis. Furthermore, enhanced transactivation extends to several cancer-associated p53 mutants with altered function, suggesting ER-dependent mutant p53 activity for at least some REs. The enhanced transactivation was also found with p63 and p73. We propose a general synergistic relationship between p53 family and ER master regulators in transactivation of p53 target canonical and noncanonical REs, which might be poorly responsive to p53 on their own. This relationship greatly expands the transcriptional master network regulated by p53 in terms of genes affected and levels of expression and has implications for the appearance and possible treatments of cancer. FLT1 | noncanonical response element | half-site | transcriptional synergy doi/ 10.1073/pnas.0909129107

MYCN, a proto-oncogene normally expressed in the migrating neural crest, is in its amplified state a key factor in the genesis of human neuroblastoma (NB). However, the mechanisms underlying MYCN-mediated NB progression are poorly understood. Here, we present a MYCN-induced miRNA signature in human NB involving the activation and transrepression of several miRNA genes from paralogous clusters. Several family members derived from the miR-17~92 cluster, including miR-18a and miR-19a, were among the up-regulated miRNAs. Expression analysis of these miRNAs in NB tumors confirmed increased levels in MYCN-amplified samples. Specifically, we show that miR-18a and miR-19a target and repress the expression of estrogen receptor-[alpha] (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. Immunohistochemical staining demonstrated ESR1 expression in human fetal sympathetic ganglia, suggesting a role for ESR1 during sympathetic nervous system development. Concordantly, lentiviral restoration of ESR1 in NB cells resulted in growth arrest and neuronal differentiation. Moreover, lentiviral-mediated inhibition of miR-18a in NB cells led to severe growth retardation, outgrowth of varicosity-containing neurites, and induction of neuronal sympathetic differentiation markers. Bioinformatic analyses of microarray data from NB tumors revealed that high ESR1 expression correlates with increased event-free survival in NB patients and favorable disease outcome. Thus, MYCN amplification may disrupt estrogen signaling sensitivity in primitive sympathetic cells through deregulation of ESR1, thereby preventing the normal induction of neuroblast differentiation. Collectively, our findings demonstrate the molecular consequences of abnormal miRNA transcription in a MYCN-driven tumor and offer unique insights into the pathology underlying MYCN-amplified NB. oncogene | embryonic development | pediatric tumor | transcription factor | hormone receptor doi/ 10.1073/pnas.0913517107

The present study tested the hypotheses that 1) nitric oxide (NO) is involved in attenuated responses to ANG II in female mice, and 2) there is differential expression of neuronal NO synthase (nNOS) in the subfornical organ (SFO) and paraventricular nucleus (PVN) in response to systemic infusions of ANG II in males vs. females. Aortic blood pressure (BP) was measured in conscious mice with telemetry implants. [N.sup.G]-nitro-L-arginine methyl ester (L-NAME; 100 [micro]g x [kg.sup.-1] [day.sup.-1]), an inhibitor of NOS, was administrated into the lateral cerebral ventricle for 14 days before and during ANG II pump implantation. Central infusion of L-NAME augmented the pressor effects of systemic ANG II in females ([DELTA]21.5 [+ or -] 2.2 vs. [DELTA]9.2 [+ or -] 1.5 mmHg) but not in males ([DELTA]29.4 [+ or -] 2.5 vs. [DELTA]30.1 [+ or -] 2.5 mmHg). Central administration of [N.sup.5]-(1-imino-3-butenyl)-L-ornithine (L-VNIO), a selective nNOS inhibitor, also significantly potentiated the increase in BP induced by ANG II in females ([DELTA]17.5 [+ or -] 3.2 vs. [DELTA]9.2 [+ or -] 1.5 mmHg). In gonadectomized mice, central L-NAME infusion did not affect the pressor response to ANG II in either males or females. Ganglionic blockade after ANG II infusion resulted in a greater reduction in BP in central L-NAME- or L-VNIO-treated females compared with control females. Western blot analysis of nNOS protein expression indicated that levels were ~12-fold higher in both the SFO and PVN of intact females compared with those in intact males. Seven days of ANG II treatment resulted in a further increase in nNOS protein expression only in intact females (PVN, to ~51-fold). Immunohistochemical studies revealed colocalization of nNOS and estrogen receptors in the SFO and PVN. These results suggest that NO attenuates the increase in BP induced by ANG II through reduced sympathetic outflow in females and that increased nNOS protein expression associated with the presence of female sex hormones plays a protective role against ANG II-induced hypertension in female mice. sex hormone; nitric oxide/nitric oxide synthase; blood pressure doi: 10.1152/ajpheart.00502.2009.

Sympathetic baroreflex sensitivity (BRS) and muscle sympathetic nerve activity (MSNA) responses during early follicular (EF) and midluteal (ML) phases of the menstrual cycle are controversial. We hypothesize an augmented sympathetic BRS and MSNA response to orthostatic stress during the ML phase of the menstrual cycle. MSNA, mean arterial pressure (MAP), and heart rate (HR) were recorded during progressive lower body negative pressure (LBNP) (-5, -10, -15, -20, -30, and -40 mmHg; 3 min/stage) in 13 healthy, eumenorrheic women (age 21 [+ or -] 1 yr). Sympathetic BRS was assessed by examining relations between spontaneous fluctuations of diastolic arterial pressure and MSNA at rest and during progressive LBNP. Plasma estradiol (42 [+ or -] 6 vs. 112 [+ or -] 12 pg/ml; P < 0.01) and progesterone (2 [+ or -] 0 vs. 10 [+ or -] 2 ng/ml; P < 0.04) were elevated during the ML phase. Resting MSNA (8 [+ or -] 1 vs. 11 [+ or -] 1 bursts/min), MAP (79 [+ or -] 2 vs. 78 [+ or -] 2 mmHg), and HR (58 [+ or -] 2 vs. 60 [+ or -] 2 beats/min) were not different during EF and ML phases. MSNA and HR increased during progressive LBNP (P < 0.001), and the increases in MSNA burst frequency (bursts/min) and HR were similar during both phases. In contrast, increases in total MSNA (arbitrary units) during progressive LBNP were augmented during the ML phase (P < 0.04), but this response does not appear to be linked to differences in sympathetic BRS. Progressive LBNP did not change MAP during either phase. Our results demonstrate an augmentation of the MSNA response to progressive LBNP during the ML phase of the menstrual cycle. These findings suggest that hormonal fluctuations of eumenorrheic women may influence sympathoexcitation during an orthostatic challenge, but not through sympathetic baroreflex-mediated pathways. muscle sympathetic nerve activity; arterial blood pressure; lower body negative pressure; baroreflex; estrogen

Glucose uptake and homeostasis are regulated mainly by skeletal muscle (SM), white adipose tissue (WAT), pancreas, and the liver. Participation of estradiol in this regulation is still under intense investigation. We have demonstrated that, in SM of male mice, expression of the insulin-regulated glucose transporter (GLUT)4 is reduced by estrogen receptor (ER)[beta] agonists. In the present study, to investigate the relative contributions of ER[alpha] and ER[beta] in glucose homeostasis, we examined the effects of tamoxifen (Tam) on GLUT4 expression in SM and WAT in wild-type (WT) and ER-/- mice. ER[beta]-/- mice were characterized by fasting hypoglycemia, increased levels of SM GLUT4, pancreatic islet hypertrophy, and a belated rise in plasma insulin in response to a glucose challenge. ER[alpha]-/- mice, on the contrary, were hyperglycemic and glucose intolerant, and expression of SM GLUT4 was markedly lower than in WT mice. Tam had no effect on glucose tolerance or insulin sensitivity in WT mice. In ER[alpha]-/- mice, Tam increased GLUT4 and improved insulin sensitivity, i.e., it behaved as an ER[beta] antagonist in SM but had no effect on WAT. In ER[beta]-/- mice, Tam did not affect GLUT4 in SM but acted as an ER[alpha] antagonist in WAT, decreasing GLUT4. Thus, in the interplay between ER[alpha] and ER[beta], ER[beta]-mediated repression of GLUT4 predominates in SM but ER[alpha]-mediated induction of GLUT4 predominates in WAT. This tissue-specific difference in dominance of one ER over the other is reflected in the ratio of the expression of the two receptors. ER[alpha] predominates in WAT and ER[beta] in SM. estrogen receptor-[alpha]; estrogen receptor-[beta]; glucose transporter 4; tamoxifen

We have applied surface plasmon resonance (SPR) spectroscopy to study the cooperative interactions of estrogen receptor a (ERa) and transcription factor Sp1 with a composite DNA element, containing an estrogen response element (ERE) half-site upstream of two adjacent Sp1 sites (+571 ERE/Sp1 composite site in promoter A of the human PR gene). Using nuclear extracts of MCF-7 breast cancer cells as sample, we have shown that Sp1 is associated with Sp1-binding sites only, whereas ERa can be recruited to DNA both through direct binding to the ERE half-site and/or through protein-protein interactions with DNA-bound Sp1. The ERE half-site and the proximal Sp I site are only 4 bp apart, and our data suggests that one transcription factor bound to DNA constitutes a sterical hindrance of the accessibility of the binding site for the other transcription factor. Our data confirms previous observations that ERa increases the amount of Sp1 recruited to the composite binding site in a dose-dependent manner. Using recombinant proteins, we have unambiguously proved the formation of a ternary complex of ERa/Sp1--composite DNA, for which previously published electrophoretic mobility shift assay (EMSA) results are contradictive. With this study, we have demonstrated that the solid--liquid-phase SPR assay is a powerful alternative for studying multiprotein--DNA interactions and is superior to the EMSA experiments as it is capable of real-time measurements, can quantify the amount of protein bound, and can capture transient and weak binding interactions. The comprehensive characterization of the synergistic interactions between ERa--DNA, Sp1--DNA, and ER[alpha]--Sp1 contributes to the understanding of how ERa and Sp1 influence and activate gene transcription.

Estrogen ([E.sub.2]) is reported to regulate skeletal muscle mass and contractile function; whether [E.sub.2] exerts its effects through estrogen receptor-[alpha] (ER[alpha]) or -[beta] (ER[beta]) is unclear. We determined the effect of ER[alpha] or ER[beta] elimination on muscle mass and contractile function in multiple muscles of the lower limb, muscles with different locomotor tasks and proportions of fiber types I and II: soleus (Sol), plantaris (Plan), tibialis anterior (TA), and gastrocnemius (Gast) in mature female mice. To determine [E.sub.2] elimination effects on muscle, we also used aromatase (Ar) knockout (KO) and wild-type (WT) mice. ER[alpha] and ArKO body weights were ~10 and 20% higher than WT. Although muscle mass tended to show a commensurate increase in both groups, only the TA was significantly larger in ER[alpha] (P < 0.05). Ratios of muscle mass to body mass revealed significantly lower values for Gast and TA in ArKO mice (P < 0.05). Tetanic tension ([P.sub.o]) per calculated anatomical cross-sectional area (aCSA) in ER[alpha] KO was lower in TA and Gast than in WT. Lower [P.sub.o]/aCSA in ER[alpha] KO Gast and TA was also supported histologically by significantly less [P.sub.o]/fiber areas (P < 0.05). ArKO mice also had lower [P.sub.o]/aCSA in Gast and TA compared with WT. ER[beta] KO and WT mice were comparable in all measures. Our results support the hypothesis that [E.sub.2] effects on skeletal muscle are mediated in part via the ER[alpha] but that [E.sub.2] effects may be mediated via more than one mechanism or receptor. muscle mass; peak tetanic tension; fiber area; myosin protein

Colony-stimulating factor-1 (CSF1) is one of two cytokines required for normal osteoclastogenesis. There are two major isoforms of CSF1, the cell-surface or membrane-bound isoform (mCSF1) and soluble CSFl (sCSF1). Whether these isoforms serve nonredundant functions in bone is unclear. To explore this question, we generated transgenic mice expressing human sCSF1, human mCSF1, or both (s/mCSF1) in osteoblasts using the 2.3-kb rat [alpha]I-collagen promoter. Bone density determined by peripheral quantitative computed tomography was significantly reduced in mCSF1, sCSF1, and s/mCSFl transgenic mice compared with wild-type animals. When analyzed by sex, sCSF1, and s/mCSF1, female animals but not mCSF1 female mice were found to have greater bone loss than their male littermates (-20 vs. -9.2%; P < 0.05 for sCSF1 and -21.6 vs. -11.2% for s/mCSF1; P < 0.01). By breeding CSF1 isoform-selective transgenic mice to an op/op background, mice were generated in which a single CSF1 isoform was the only source of the cytokine ([sCSF1.sup.op/op] and [mCSF1.sup.op/op]). Unlike osteoblast-targeted overexpression of mCSF1, selective transgenic expression of sCSF1 did not completely correct the op/op phenotype in 5-mo-old animals. Interestingly, compared with sham-ovariectomized mice of the same genotype, ovariectomy in [sCSF1.sup.op/op] mice led to a greater loss of spinal bone mineral density (22.1%) than was seen in either [mCSF1.sup.op/op] mice (12.9%) or in wild-type animals (10.9%). Our findings support the conclusion that sCSFI and mCSF1 serve nonredundant functions in bone and that sCSFI may play a role in mediating estrogen-deficiency bone loss. osteoclastogenesis; estrogen deficiency; membrane-bound colony-stimulating factor

17[beta]-Estradiol ([E.sub.2]) exerts inhibitory actions on the nitric oxide pathway in rat adult pituitary glands. Previously, we reported that in vivo [E.sub.2] acute treatment had opposite effects on soluble guanylyl cyclase (sGC) subunits, increasing [[alpha].sub.1]-and decreasing [[beta].sub.1]-subunit protein and mRNA expression and decreasing sGC activity in immature rats. Here we studied the [E.sub.2] effect on sGC protein and mRNA expression in anterior pituitary gland from adult female rats to address whether the maturation of the hypothalamus-pituitary axis influences its effects and to corroborate whether these effects occur in physiological conditions such as during estrous cycle. [E.sub.2] administration causes the same effect on sGC as seen in immature rats, and these effects are estrogen receptor dependent. These results suggest that [E.sub.2] is the main effector of these changes. Since the sGC [alpha]-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC [alpha]-subunit are expressed. Here we show for the first time that sGC[[alpha].sub.2] and sGC[[alpha].sub.2] inhibitory ([[alpha].sub.2i]) isoforms are expressed in this gland, but only sGC[[alpha].sub.2i] mRNA increased after [E.sub.2] acute treatment. Finally, to test whether [E.sub.2] effects take place under a physiological condition, sGC subunit expression was monitored over estrous cycle, sGC[[alpha].sub.1], -[[beta].sub.1], and -[[alpha].sub.2i] fluctuate along estrous cycle, and these changes are directly related with [E.sub.2] level fluctuations rather than to NO level variations. These findings show that [E.sub.2] physiologically regulates sGC expression and highlight a novel mechanism by which [E.sub.2] downregulates sGC activity in rat anterior pituitary gland. estrogen; soluble guanylyl cyclase; inhibitory subunit

It is now accepted that estrogens not only stimulate lactotrope proliferation but also sensitize anterior pituitary cells to proapoptotic stimuli. In addition to their classical mechanism of action through binding to intracellular estrogen receptors (ERs), there is increasing evidence that estrogens exert rapid actions mediated by cell membrane-localized ERs (mERs). In the present study, we examined the involvement of membrane-initiated steroid signaling in the proapoptotic action of estradiol in primary cultures of anterior pituitary cells from ovariectomized rats by using estren, a synthetic estrogen with no effect on classical transcription and a cell-impermeable 17[beta]-estradiol conjugate ([E.sub.2]-BSA). Both compounds induced cell death of anterior pituitary cells after 60 min of incubation as assessed by flow cytometry and the [3-(4,5-dimethylthiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4- sulfophe-nyl)-2H-tetrazolium assay. Estren, [E.sub.2], and [E.sub.2]-BSA induced apoptosis of lactotropes and somatotropes as evaluated by the deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and immuno-detection of prolactin (PRL) and growth hormone (GH). The proapoptotic effect of [E.sub.2]-BSA was abrogated by ICI-182,780, an antagonist of ERs. The expression of membrane-associated ER[alpha] was observed in PRL-and GH-bearing cells. Our results indicate that estradiol is able to exert a rapid apoptotic action in anterior pituitary cells, especially lactotropes and somatotropes, by a mechanism triggered by mERs. This mechanism could be involved in anterior pituitary cell turnover. estrogen; membrane receptors; pituitary; lactotropes; somatotropes; apoptosis

Endothelium-derived hyperpolarizing factor (EDHF) plays an important role in the regulation of vascular microcirculatory tone. This study explores the role of estrogen in controlling EDHF-mediated vasodilation of uterine resistance arteries of the rat and also analyzes the contribution of endothelial cell (EC) [Ca.sup.2+] signaling to this process. A parallel study was also performed with mesenteric arteries to provide comparison with a nonreproductive vasculature. Mature female rats underwent ovariectomy, with one half receiving 17 [beta]-estradiol replacement (OVX+E) and the other half serving as estrogen-deficient controls (OVX). Uterine or mesenteric resistance arteries were harvested, cannulated, and pressurized. Nitric oxide and prostacyclin production were inhibited with 200 [micro]M [N.sup.G]-nitro-L-arginine and 10 [micro] M indomethacin, respectively. ACh effectively dilated the arteries preconstricted with phenylephrine but failed to induce dilation of vessels preconstricted with high-[K.sup.+] solution. ACh [EC.sub.50] values were decreased by estrogen replacement by five- and twofold in uterine and mesenteric arteries, respectively. As evidenced by fura-2-based measurements of EC cytoplasmic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), estrogen replacement was associated with increased basal and ACh-stimulated EC [[[Ca.sup.2+]].sub.i] rise in uterine, but not mesenteric, vessels. These data demonstrate that EDHF contributes to endothelium-dependent vasodilation of uterine and mesenteric resistance arteries and that estrogen controls EDHF-related mechanism(s) more efficiently in reproductive vs. nonreproductive vessels. Enhanced endothelial [Ca.sup.2+] signaling may be an important underlying mechanism in estrogenic modulation of EDHF-mediated vasodilation in small resistance uterine arteries. endothelium-derived hyperpolarizing factor; acetylcholine; fura-2; high potassium

In vitro studies suggest that the membrane G protein-coupled receptor GPR30 is a functional estrogen receptor (ER). The aim of the present study was to determine the possible in vivo role of GPR30 as a functional ER primarily for the regulation of skeletal parameters, including bone mass and longitudinal bone growth, but also for some other well-known estrogen-regulated parameters, including uterine weight, thymus weight, and fat mass. Three-month-old ovariectomized (OVX) GPR30-deficient mice ([GPR30.sup.-/-]) and wild-type (WT) mice were treated with either vehicle or increasing doses of estradiol ([E.sub.2]; 0, 30, 70, 160, or 830 ng x [mouse.sup.-1] x [day.sup.-1]). Body composition [bone mineral density (BMD), fat mass, and lean mass] was analyzed by dual-energy-X ray absorptiometry, while the cortical and trabecular bone compartments were analyzed by peripheral quantitative computerized tomography. Quantitative histological analyses were performed in the distal femur growth plate. Bone marrow cellularity and distribution were analyzed using a fluorescence-activated cell sorter. The estrogenic responses on most of the investigated parameters, including increase in bone mass (total body BMD, spine BMD, trabecular BMD, and cortical bone thickness), increase in uterine weight, thymic atrophy, fat mass reduction, and increase in bone marrow cellularity, were similar for all of the investigated [E.sub.2] doses in WT and [GPR30.sup.-/-] mice. On the other hand, [E.sub.2] treatment reduced longitudinal bone growth, reflected by decreased femur length and distal femur growth plate height, in the WT mice but not in the [GPR30.sup.-/-] mice compared with vehicle-treated mice. These in vivo findings demonstrate that GPR30 is not required for normal estrogenic responses on several major well-known estrogen-regulated parameters. In contrast, GPR30 is required for a normal estrogenic response in the growth plate. estrogen receptor; growth; bone

Environmental contaminants that mimic native estrogens (i.e., environmental estrogens) are known to significantly impact a wide range of vertebrate species and have been implicated as a source for increasing human male reproductive deficiencies and diseases. Despite the widespread occurrence of environmental estrogens and recognized detrimental effects on male vertebrate reproduction, no specific mechanism has been determined indicating how reduced fertility and/or fecundity is achieved. Previous studies show that male rainbow trout, Oncorhynchus mykiss, exposed to the environmental estrogen 17[alpha]-ethynylestradiol (EE2) before gamete formation and fertilization produce progeny with significantly reduced embryonic survival. To determine whether this observed decrease results from sperm chromosome alterations during spermatogenesis, male rainbow trout were exposed to 10 ng of EE2/I for 50 days. After exposure, semen was collected and sperm aneuploidy levels analyzed with two chromosome markers by fluorescent in situ hybridization. In vitro fertilizations were also conducted by using control and exposed sperm crossed to eggs from an unexposed female for offspring analysis. Evaluations for nucleolar organizer region number and karyotype were performed on developing embryos to determine whether sperm aneuploidy translated into embryonic aneuploidy. Results conclusively show increased aneuploid sperm formation due to EE2 exposure. Additionally, embryonic cells from propagated progeny of individuals possessing elevated sperm aneuploidy display high levels of embryonic aneuploidy. This study concludes that EE2 exposure in sexually developing male rainbow trout increases levels of aneuploid sperm, providing a mechanism for decreased embryonic survival and ultimately diminished reproductive success in EE2 exposed males. aneuploidy | EE2 | xenoestrogens

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-[alpha] or ER-[beta], and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-[alpha] agonist propylpyrazole triol (PPT) and/or the selective ER-[beta] agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings (n = 3-10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine ([10.sup.-8]M - [10.sup.-5]M) and hypoxia ([Po.sub.2] 35-45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine ([10.sup.-8]M - [10.sup.-4]M) and sodium nitroprusside ([10.sup.-9]M - [10.sup.-5]M). PPT or DPN ([10.sup.-9]M - 5 x [10.sup.-5]M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor [N.sup.[omega]]-nitro-t.-arginine methyl ester (L-NAME) ([10.sup.-4]M). Selective ER-[alpha] activation (PPT, 5 x [10.sup.-5]M) rapidly ( propylpyrazole triol; diarylpropiolnitrile; phenylephrine; hypoxic pulmonary vasoconstriction; nongenomic effects

Many drugs and chemicals exert their biological effect by modulating protein--protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor [alpha] ligand binding domain (hER[alpha] LBD). Nanoelectro-spray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hER[alpha] LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein--protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.

The 'healthy cell bias of estrogen action' hypothesis examines the role that regulating mitochondrial function and bioenergetics play in promoting neural health and the mechanistic crossroads that lead to divergent outcomes following estrogen exposure. Estrogen-induced signaling pathways in hippocampal and cortical neurons converge upon the mitochondria to enhance aerobic glycolysis coupled to the citric acid cycle, mitochondrial respiration and ATP generation. Convergence of estrogen-induced signaling onto mitochondria is also a point of vulnerability when activated in diseased neurons which exacerbates degeneration through increased load on dysregulated calcium homeostasis. As the continuum of neurological health progresses from healthy to unhealthy so too do the benefits of estrogen or hormone therapy. The healthy cell bias of estrogen action hypothesis provides a lens through which to assess disparities in outcomes across basic and clinical science and on which to predict outcomes of estrogen interventions for sustaining neurological health and preventing age-associated neurodegenerative diseases such as Alzheimer's.

The high-fat diet (HFD)-fed mouse is a model of obesity, impaired glucose tolerance, and insulin resistance. The main objective of this study was to elucidate the molecular mechanisms underlying the antidiabetogenic and weight-lowering effects of 17[beta]-estradiol ([E.sub.2]) in this mouse model. C57BL/6 female mice (8 wk old) were fed on a HFD for 10 mo. [E.sub.2], given daily (50 [micro]g/kg sc) during the last month of feeding, decreased body weight and markedly improved glucose tolerance and insulin sensitivity. Plasma levels of insulin, leptin, resistin, and adiponectin were decreased. We demonstrated that [E.sub.2] treatment decreased the expression of genes encoding resistin and leptin in white adipose tissue (WAT), whereas adiponectin expression was unchanged. Furthermore, in WAT we demonstrated decreased expression levels of sterol regulatory element-binding protein 1c (SREBP1c) and its lipogenic target genes, such as fatty acid synthase and stearoyl-CoA desaturase 1 (SCD1). In the liver, the expression levels of transcription factors such as liver X receptor [alpha] and SREBP1c were not changed by [E.sub.2] treatment, but the expression of the key lipogenic gene SCD1 was reduced. This was accompanied by decreased hepatic triglyceride content. Importantly, [E.sub.2] decreased the hepatic expression of glucose-6-phosphatase (G-6-Pase). We conclude that [E.sub.2] treatment exerts antidiabetic and antiobesity effects in HFD mice and suggest that this is related to decreased expression of lipogenic genes in WAT and liver and suppression of hepatic expression of G-6-Pase. Decreased plasma levels of resistin probably also play an important role in this context. glucose tolerance; insulin sensitivity; obesity; lipogenic genes; glucose-6-phosphatase

L-type voltage-gated [Ca.sup.2+] channels (VGCC) play an important role in dendritic development, neuronal survival, and synaptic plasticity. Recent studies have demonstrated that the gonadal steroid estrogen rapidly induces [Ca.sup.2+] influx in hippocampal neurons, which is required for neuroprotection and potentiation of LTP. The mechanism by which estrogen rapidly induces this [Ca.sup.2+] influx is not clearly understood. We show by electrophysiotogical studies that extremely low concentrations of estrogens acutely potentiate VGCC in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, in a manner that was estrogen receptor (ER)-independent. Equilibrium, competitive, and whole-cell binding assays indicate that estrogen directly interacts with the VGCC. Furthermore, a L-type VGCC antagonist to the dihydropyridine site displaced estrogen binding to neuronal membranes, and the effects of estrogen were markedly attenuated in a mutant, dihydropyridine-insensitive L-type VGCC, demonstrating a direct interaction of estrogens with L-type VGCC. Thus, estrogen-induced potentiation of calcium influx via L-type VGCC may link electrical events with rapid intracellular signaling seen with estrogen exposure leading to modulation of synaptic plasticity, neuroprotection, and memory formation. estrogen receptors | signaling | estradiol | memory

Cortical information storage requires combined changes in connectivity and synaptic strength between neurons, but the signaling mechanisms underlying this two-step wiring plasticity are unknown. Because acute 17[beta]-estradiol (E2) modulates cortical memory, we examined its effects on spine morphogenesis, AMPA receptor trafficking, and GTPase signaling in cortical neurons. Acute E2 application resulted in a rapid, transient increase in spine density, accompanied by temporary formation of silent synapses through reduced surface GluR1. These rapid effects of E2 were dependent on a Rap/AF-6/ERK1/2 pathway. Intriguingly, NMDA receptor (NMDAR) activation after E2 treatment potentiated silent synapses and elevated spine density for as long as 24 h. Hence, we show that E2 transiently increases neuronal connectivity by inducing dynamic nascent spines that 'sample' the surrounding neuropil and that subsequent NMDAR activity is sufficient to stabilize or 'hold' E2-mediated effects. This work describes a form of two-step wiring plasticity relevant for cortical memory and identifies targets that may facilitate recovery from brain injuries.

It has been shown that reactive oxygen species (ROS) contribute to the central effect of ANG II on blood pressure (BP). Recent studies have implicated an antihypertensive action of estrogen in ANG II-infused female mice. The present study used in vivo telemetry recording and in vitro living mouse brain slices to test the hypothesis that the central activation of estrogen receptors in male mice inhibits ANG II-induced hypertension via the modulation of the central ROS production. In male wild-type mice, the systemic infusion of ANG II induced a significant increase in BP ([DELTA]30.1 [+ or -] 2.5 mmHg). Either central infusion of Tempol or 17[beta]-estradiol ([E.sub.2]) attenuated the pressor effect of ANG II ([DELTA]10.9 [+ or -] 2.3 and [DELTA]4.5 [+ or -] 1.4 mmHg), and the protective effect of [E.sub.2] was prevented by the coadministration of an estrogen receptor, antagonist ICI-182780 ([DELTA]23.6 [+ or -] 3.1 mmHg). Moreover, the ganglionic blockade on day 7 after the start of ANG II infusions resulted in a smaller reduction of BP in central Tempol- and in central [E.sub.2]-treated males, suggesting that estrogen inhibits the central ANG II-induced increases in sympathetic outflow. In subfornical organ slices, the application of ANG II resulted in a 21.5 [+ or -] 2.5% increase in ROS production. The coadministration of irbesartan, an ANG II type 1 receptor antagonist, or the preincubation of brain slices with Tempol blocked ANG II-induced increases in ROS production (-1.8 [+ or -] 1.6% and -1.0 [+ or -] 1.8%). The ROS response to ANG II was also blocked by [E.sub.2] (-3.2 [+ or -] 2.4%). The results suggest that the central actions of [E.sub.2] are involved in the protection from ANG II-induced hypertension and that estrogen modulation of the ANG II-induced effects may involve interactions with ROS production. sex hormone; blood pressure; oxidative stress; subfornical organ

The influence of sexual category as a modifier of cellular function is underinvestigated. Whether sex differences affect estrogen-mediated mitochondrial cytoprotection was determined using cell cultures of normal human lens epithelia (nHLE) from postmortem male and female donors. Experimental indicators assessed included differences in estrogen receptor-[beta] (ER[beta]) isoform expression, receptor localization in mitochondria, and estrogen-mediated prevention of loss of mitochondrial membrane potential using the potentiometric fluorescent compound JC-1 after nHLE were exposed to peroxide. The impact of wild-type ER[beta] (wtER[beta]) was also assessed using wtER[beta]1 siRNA to suppress expression. A triple-primer PCR assay was employed to determine the proportional distribution of the receptor isoforms (wtER[beta]1, -[beta]2, and -[beta]5) from the total ER[beta] message pool in male and female cell cultures. Irrespective of sex, nHLE express wtER[beta]1 and the ER[beta]2 and ER[beta]5 splice variants in similar ratios. Confocal microscopy and immunofluorescence revealed localization of the wild-type receptor in peripheral mitochondrial arrays and perinuclear mitochondria as well as nuclear staining in both cell populations. The ER[beta]2 and ER[beta]5 isoforms were distributed primarily in the nucleus and cytosol, respectively; no association with the mitochondria was detected. Both male and female nHLE treated with [E.sub.2] (1 [micro]M) displayed similar levels of protection against peroxide-induced oxidative stress. In conjunction with acute oxidative insult, RNA suppression of wtER[beta]1 elicited the collapse of mitochondrial membrane potential and markedly diminished the otherwise protective effects of [E.sub.2]. Thus, whereas the estrogen-mediated prevention of mitochondrial membrane permeability transition is sex independent, the mechanism of estrogen-induced mitochondrial cytoprotection is wtER[beta]1 dependent.

Evidence suggests that both the arterial baroreflex and vestibulosympathetic reflex contribute to blood pressure regulation, and both auto nomic reflexes integrate centrally in the medulla cardiovascular center. A previous report indicated increased sympathetic baroreflex sensitivity during the midluteal (ML) phase of the menstrual cycle compared with the early follicular (EF) phase. On the basis of this finding, we hypothesize an augmented vestibulosympathetic reflex during the ML phase of the menstrual cycle. Muscle sympathetic nerve activity (MSNA). mean arterial pressure (MAP), and heart rate responses to head-down rotation (HDR) were measured in 10 healthy females during the EF and ML phases of the menstrual cycle. Plasma estradiol ([DELTA]72 [+ or -] 13 pg/ml, P < 0.01) and progesterone ([DELTA]8 [+ or -] 2 ng/ml, P < 0.01) were significantly greater during the ML phase compared with the EF phase. The menstrual cycle did not alter resting MSNA, MAP, and heart rate (EF: 13 [+ or -] 3 bursts/min, 80 [+ or -] 2 mmHg, 65 [+ or -] 2 beats/min vs. ML: 14 [+ or -] 3 bursts/min, 81 [+ or -] 3 mmHg, 64 [+ or -] 3 beats/min). During the EF phase, HDR increased MSNA ([DELTA]3 [+ or -] 1 bursts/min, P < 0.02) but did not change MAP or heart rate ([DELTA]0 [+ or -] 1 mmHg and [DELTA]1 [+ or -] 1 beats/min). During the ML phase, HDR increased both MSNA and MAP ([DELTA]4 [+ or -] 1 bursts/min and [DELTA]3 [+ or -] 1 mmHg, P < 0.04) with no change in heart rate ([DELTA]0 [+ or -] 1 beats/min). MSNA and heart rate responses to HDR were not different between the EF and ML phases, but MAP responses to HDR were augmented during the ML phase (P < 0.03). Our results demonstrate that the menstrual cycle does not influence the vestibulosympathetic reflex but appears to alter MAP responses to HDR during the ML phase. muscle sympathetic nerve activity; arterial blood pressure: otolith stimulation; head-down rotation; estrogen doi: 10.1152/ajpendo.00056.2008.

Resistin is known as an adipocyte-specific secretory hormone that can cause insulin resistance and decrease adipocyte differentiation. It can be regulated by sexual hormones. Whether environmental estrogens regulate the production of resistin is still not clear. Using 3T3-L1 adipocytes, we found that octylphenol upregulated resistin mRNA expression in dose- and time-dependent manners. The concentration of octylphenol that increased resistin mRNA levels by 50% was ~100 nM within 6 h of treatment. The basal halt-life of resistin mRNA induced by actinomycin D was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin mRNA degradation. In addition, octylphenol stimulated resistin protein expression and release. The basal half-life of resistin protein induced by cycloheximide was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin protein degradation. While octylphenol was shown to increase activities of the estrogen receptor (ER) and MEK1, signaling was demonstrated to be blocked by pretreatment with either ICI-182780 (an ER[alpha] antagonist) or U-0126 (a MEK1 inhibitor), in which both inhibitors prevented octylphenol-stimulated phosphorylation of ERK. These results imply that ER[alpha] and ERK are necessary for the octylphenol stimulation of resistin mRNA expression. Moreover, U-0126 antagonized the octylphenol-increased resistin protein expression and release. These data suggest that the way octylphenol signaling increases resistin protein levels is similar to that by which it increases resistin mRNA levels; it is likely mediated through an ERK-dependent pathway. In vivo, octylphenol increased adipose resistin mRNA expression and serum resistin and glucose levels, supporting its in vitro effect. environmental hormone; adiponectin; leptin; nonylphenol; bisphenol A

C-reactive protein (CRP) is a widely used, sensitive biomarker of inflammation. Studies conducted among users of exogenous hormones suggest that estrogen increases CRP, whereas progesterone decreases CRP. Examinations of CRP in normally cycling women suggest the opposite: CRP is negatively associated with endogenous estrogen and positively associated with endogenous progesterone. This work evaluates the association between menstrual cycle-related hormone changes and events (menstruation and ovulation) and CRP. Eight female subjects gave urine and blood samples from twelve days across the menstrual cycle, for a total of eleven cycles. Blood samples were assayed for CRP; urine samples for [beta]-follicle stimulating hormone [beta]FSH), pregnanediol 3-glucuronide (PDG), and estrone glucuronide (E1G). Ovulation day was estimated using hormone levels. Presence or absence of menses was reported by subjects. Analyses were conducted with random-effects linear regression. All cycles were ovulatory; day of ovulation was identified for nine cycles. A ten-fold increase in progesterone was associated with a 23% increase in CRP (P = 0.01), a ten-fold increase in estrogen was associated with a 29% decrease in CRP (P = 0.05), and menses was associated with a 17% increase in CRP (P = 0.18); no association between ovulation or FSH and CRP was found. Hormone changes across the menstrual cycle should be controlled for in future studies of inflammation in reproductive-age women. KEY WORDS inflammation; estrogen; progesterone; biomarker

Here, we demonstrate that a single biochemical assay is able to predict the tissue-selective pharmacology of an array of selective estrogen receptor modulators (SERMs). We describe an approach to classify estrogen receptor (ER) modulators based on dynamics of the receptor-ligand complex as probed with hydrogen/deuterium exchange (HDX) mass spectrometry. Differential HDX mapping coupled with cluster and discriminate analysis effectively predicted tissue-selective function in most, but not all, cases tested. We demonstrate that analysis of dynamics of the receptor-ligand complex facilitates binning of ER modulators into distinct groups based on structural dynamics. Importantly, we were able to differentiate small structural changes within ER ligands of the same chemotype. In addition, HDX revealed differentially stabilized regions within the ligand-binding pocket that may contribute to the different pharmacology phenotypes of the compounds independent of helix 12 positioning. In summary, HDX provides a sensitive and rapid approach to classify modulators of the estrogen receptor that correlates with their pharmacological profile. discriminate analysis | hydrogen/deuterium exchange | mass spectrometry

Osteoblasts respond in direct and indirect ways to estrogens, and age-dependent changes in hormone levels and bone health can be limited by focused hormone replacement therapy. In this study, we report the release and isolation of an estrogen receptor agonist from osteoblast cultures. This entity reprises many aspects of estradiol activity in isolated osteoblasts, but differs from authentic estradiol by several biochemical and physical criteria. At levels that occur in conditioned medium from differentiating osteoblast cultures, the agonist directly drives gene expression through estrogen-sensitive response elements, activates the obligate osteoblast transcription factor Runx2, and potently enhances Smad-dependent gene expression in response to TGF-[beta], but exhibits relatively lesser suppressive effects on gene expression through C/EBP and AP-1-binding protein transcription factors. Estrogen receptor agonist activity is resistant to heating at 100[degrees]C and separable from the bulk of the remaining alcohol- and hexane-soluble molecules by C18 chromatography. MS and molecular fragmentation analyses predict a Mr of 415.2 to 437.2. Therefore, in addition to earlier studies showing that osteoblasts readily respond to and metabolize various sex steroid-like substrates, we find that they also generate a potent estrogen receptor agonist during differentiation in vitro. Changes in the availability of a molecule like this within bone may relate to differences in skeletal integrity with aging or metabolic disease. intracrine | selective estrogen receptor modulator | steroid

Clinical trials revealed that estrogen may result in cardiovascular risk in patients with coronary heart disease, despite earlier studies demonstrating that estrogen provided cardiovascular protection. It is possible that the preexisting condition of hypertension and the ability of estrogen to activate the renin-angiotensin system could confound its beneficial effects. Our hypothesis is that the attenuation of estrogen to agonist-induced vasoconstrictor responses through the activation of nitric oxide (NO) synthase (NOS) is impaired by hypertension. We investigated the effects of 17[beta]-estradiol ([E.sub.2]) replacement in normotensive Sprague-Dawley (SD) and (mRen2)27 hypertensive transgenic (TG) rats on contractile responses to three vasoconstrictors, angiotensin II (ANG II), serotonin (5-HT), and phenylephrine (PE), and on the modulatory role of vascular NO to these responses. The aorta was isolated from ovariectomized SD and TG rats treated chronically with 5 mg [E.sub.2] or placebo (P). The isometric tension of the aortic rings was measured in organ chambers, and endothelial NOS (eNOS) in the rat aorta was detected using Western blot analysis. [E.sub.2] treatment increased eNOS expression in the SD and TG aorta and reduced ANG II- and 5-HT- but not PE-induced contractions in SD and TG rats. The inhibition of NOS with [N.sup.[omega]]-nitro-L-arginine methyl ester enhanced ANG II-, 5-HT-, and PE-induced contractions in P-treated and ANG II and PE responses in [E.sub.2]-treated SD and TG rats. Only the responses to 5-HT were augmented in hypertensive rats. In conclusion, this study shows that the preexisting condition of hypertension augmented the vascular responsiveness of 5-HT, whereas the attenuation of estrogen by ANG II and 5-HT vascular responses was not impaired by hypertension. The adrenergic agonist was unresponsive to estrogen treatment. The contribution of NO as a factor contributing to the relative refractoriness of the vascular responses is dependent on the nature of the vasoconstrictor and/or the presence of estrogen. angiotensin II; serotonin; phenylephrine

Obesity, characterized by enhanced food intake (hyperphagia) and reduced energy expenditure that results in the accumulation of body fat, is a major risk factor for various diseases, including diabetes, cardiovascular disease, and cancer. In the United States, more than half of adults are overweight, and this number continues to increase. The adipocyte-secreted hormone leptin and its downstream signaling mediators play crucial roles in the regulation of energy balance. Leptin decreases feeding while increasing energy expenditure and permitting energy-intensive neuroendocrine processes, such as reproduction. Thus, leptin also modulates the neuroendocrine reproductive axis. The gonadal steroid hormone estrogen plays a central role in the regulation of reproduction and also contributes to the regulation of energy balance. Estrogen deficiency promotes feeding and weight gain, and estrogen facilitates, and to some extent mimics, some actions of leptin. In this review, we examine the functions of estrogen and leptin in the brain, with a focus on mechanisms by which leptin and estrogen cooperate in the regulation of energy homeostasis. obesity; metabolism; signal transducer and activator of transcription 3

There are two major subtypes of human breast cancers: the luminal, estrogen, and progesterone receptor-positive, cytokeratin 18-positive (E[R.sup+]P[R.sup+]CK[18.sup+]) subtype, and the basal E[R.sup-]P[R.sup-]CK[18.sup-]CK[5.sup+] subtype. Tumor-initiating cells (CD[44.sup+]) have been described for human breast cancers; whether these are common to the two subtypes is unknown. We have identified a rare population of cells that are both CD[44.sup+] and E[R.sup-]P[R.sup-]CK[5.sup+] in luminal-like E[R.sup+]P[R.sup+] T47D human breast tumor xenografts. The tumor-isolated CD[44.sup+] cell fraction was highly enriched for clonogenic (in vitro culture) and tumorigenic (in vivo reimplantation) cells compared with the CD[44.sup-] cell fraction. Rare E[R.sup-]P[R.sup-]CK[5.sup+] cells were present within CD[44.sup+]-derived colonies. Tumor-isolated cells placed in minimal media also contained rare E[R.sup-]P[R.sup-] CK[5.sup+] cells at early time points ( cancer stem cell | cytokeratin 5 | estrogen receptor | progesterone receptor

August-Copenhagen-Irish (ACI) rats are unique in that the ovaryintact females develop high incidence of mammary cancers induced solely by hormones upon prolonged exposure to high levels of estrogen alone. Studies have also shown that such prolonged exposure to high-dose estrogen results in human-like aneuploid mammary cancers in ovary-intact ACI rats. To determine the role of progesterone in mammary carcinogenesis, six-week-old intact and ovariectomized ACI rats were continuously exposed to low- and high-dose estrogen alone, progesterone alone, low-dose estrogen plus progesterone, and ovariectomized ACI rats with high-dose estrogen plus progesterone. Also, ovariectomized ACI rats were treated with high-dose estrogen plus progesterone plus testosterone to determine the role of the androgen, testosterone, if any, in hormonal mammary carcinogenesis. The results indicate that continuous exposure to high, but not low, concentrations of estrogen alone can induce mammary carcinogenesis in intact but not in ovariectomized rats. Mammary carcinogenesis in ovariectomized ACI rats requires continuous exposure to high concentrations of estrogen and progesterone. The addition of testosterone propionate does not affect tumor incidence in such rats. These results suggest that both ovarian hormones estrogen and progesterone are necessary for mammary carcinogenesis induced solely by hormones in ovariectomized ACI rats. Our results are in agreement with the Women's Health Initiative studies, where treatment of postmenopausal women with estrogen (ERT) alone did not increase the risk of breast cancer, but estrogen and progesterone (HRT) did.

Estrogen signaling is mediated by estrogen receptors a (ER[alpha]) and [beta] (ER[beta]). Although a consensus has now been reached concerning many physiological functions of ERa, those of ER[beta] are still controversial: When housed and examined in two distant laboratories, mice originating from the same initial ER[beta] mutant exhibited widely different phenotypes, which were themselves different from the phenotype of another ER[beta] mutant previously generated in our laboratory. Because, in addition to a knockout insertion in exon 3, all these mouse mutants displayed alternative splicing transcripts, we have now constructed a ER[beta] mouse mutant ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]) in which exon 3 was cleanly deleted by Cre/LoxP-mediated excision and was devoid of any transcript downstream of exon 3. Both females and males were sterile. The histology of the ovary was mildly affected, and no histological defects were detected in other organs, neither in females nor in males. Our present results, which are in contrast with previously published data, suggest that, with the notable exception of male and female reproduction, ER[beta] is not required in the mouse for the development and homeostasis of the major body systems. aging | ER[beta] function | ER[beta] knockout | estrogen signaling | histopathology

Experimental evidence for the physiological effects of secondhand smoke (SHS) is limited, although it affects millions of people globally and its prevalence is increasing, despite currently adopted antismoking measures. Also, scarce evidence suggests that the effects of SHS may be more pronounced in men. We conducted a randomized single-blind crossover study to investigate the sex-specific SHS effects in a controlled simulated bar/restaurant environment on gonadal and thyroid hormones, inflammatory cytokines, and vascular function. Twenty-eight (women = 14) nonsmoking adults underwent a 1-h exposure to moderate SHS and a 1-h control trial. Serum and urine cotinine, gonadal and thyroid hormones, inflammatory cytokines, heart rate, and arterial blood pressure were assessed before exposure and immediately after in both trials. Results showed that testosterone (P = 0.019) and progesterone (P < 0.001) in men and 1713-estradiol (P = 0.001) and progesterone (P < 0.001) in women were significantly decreased after SHS. In men, SHS was accompanied by increased free thyroxine (P < 0.001), triiodothyronine (P = 0.020), and decreased the triiodothyronine-to-free thyroxine ratio (P = 0.033). In women, significant SHS-induced change was observed only in free thyroxine (P = 0.010), with considerable sex variation in free thyroxine and triiodothyronine and a decrease in luteinizing hormone (P = 0.026) and follicle-stimulating hormone (P < 0.001). After SHS, IL-1 [3 (P = 0.001) and systolic blood pressure (P = 0.040) were increased in men but not women. We concluded that a 1-h SHS exposure at bar/ restaurant levels is accompanied by decrements in gonadal hormones in both sexes and marked increases in thyroid hormone secretion, IL-1[beta] production, and systolic blood pressure in men. environmental tobacco smoke; cotinine; estrogen; cytokines

Progesterone and estrogens play key roles in regulating various physiological phenomena related to normal growth, development, and reproduction of domestic animals. This review focuses on the mechanisms by which progesterone and estrogens regulate the reproductive processes in these animals. The majority of research on the actions of progesterone and estrogens on the reproductive systems of cattle, sheep, and pigs has been genomic in nature and represents attempts to better understand how these steroids regulate gene expression. Results of recent research suggest that progesterone and estrogens can alter target cell responses nongenomically via membrane receptors. The characteristics of membrane receptors for progesterone and estrogen in various cell types are described and the intracellular signal pathways defined. Estrogens acting via membrane receptors can suppress LH secretion by gonadotropes and stimulate rapid increases in uterine blood flow. Progesterone acting via a membrane receptor has been shown to inhibit binding of oxytocin to oxytocin receptors in isolated endometrial plasma membranes and stimulate capacitation of spermatozoa. Results of research suggest that progesterone and estrogens can act nongenomically to alter target cell responses in domestic animals. The biological implications of this mode of action in these animals are discussed. Key words: estradiol-17[beta], luteinizing hormone, membrane receptor, oxytocin, progesterone, uterus

BACKGROUND: Male reproductive tract abnormalities such as hypospadias and cryptorchidism, and testicular cancer have been proposed to comprise a common syndrome together with impaired spermatogenesis with a common etiology resulting [...]

Spine density in the hippocampus changes during the estrus cycle and is dependent on the activity of local aromatase, the final enzyme in estrogen synthesis. In view of the abundant gonadotropin-releasing hormone receptor (GnRH-R) messenger RNA expression in the hippocampus and the direct effect of GnRH on estradiol (E2) synthesis in gonadal cells, we asked whether GnRH serves as a regulator of hippocampal E2 synthesis. In hippocampal cultures, E2 synthesis, spine synapse density, and immunoreactivity of spinophilin, a reliable spine marker, are consistently up-regulated in a dose-dependent manner at low doses of GnRH but decrease at higher doses. GnRH is ineffective in the presence of GnRH antagonists or aromatase inhibitors. Conversely, GnRH-R expression increases after inhibition of hippocampal aromatase. As we found estrus cyclicity of spine density in the hippocampus but not in the neo-cortex and GnRH-R expression to be fivefold higher in the hippocampus compared with the neocortex, our data strongly suggest that estrus cycle-dependent synaptogenesis in the female hippocampus results from cyclic release of GnRH.

Estradiol-17[beta] ([E.sub.2]) causes cell proliferation in the uterine epithelium of mice and humans by signaling through its transcription factor receptor [beta] (ER[alpha]). In this work we show that this signaling is mediated by the insulin-like growth factor 1 receptor (IGF1R) expressed in the epithelium, whose activation leads to the stimulation of the phosphoinositide 3-kinase/protein kinase B pathway leading to cyclin D1 nuclear accumulation and engagement with the canonical cell cycle machinery. This cyclin D1 nuclear accumulation results from the inhibition of glycogen synthase kinase 3[beta] (GSK3[beta]) activity caused by an inhibitory phosphorylation by protein kinase B. Once the IGF1 pathway is activated, inhibition of ER signaling demonstrates that it is independent of ER. Inhibition of GSK3[beta] in the absence of [E.sub.2] is sufficient to induce uterine epithelial cell proliferation, and GSK3[beta] is epistatic to IGF1 signaling, indicating a linear pathway from [E.sub.2] to cyclin D1. Exposure to [E.sub.2] is the major risk factor for endometrial cancer, suggesting that downstream activation of this IGF1-mediated pathway by mutation could be causal in the progression to ER-independent tumors. cell cycle | cyclin D | estrogen | glycogen synthase kinase 3[beta](GSK[beta]) | estrogen receptor

Estrogen is a key regulator of vascular responses and angioadaptation in multiple organs and tissues, including brain. However, the consequences of a loss of ovarian steroid hormone secretion on the status of microvascular networks in brain and meninges are largely unknown. Here, using the perfused dura mater model coupled with high-resolution digital epifluorescence and laser scanning confocal microscopy and computer-assisted morphometric analysis, we demonstrate that cessation of ovarian hormone production causes dramatic vascular remodeling in meningeal microvascular networks characterized by a threefold decrease in microvessel density and capillary rarefaction and an almost fourfold increase in vascular permeability. These changes were accompanied by a significant decrease in angiopoietin-1 (Ang-l) expression and Ang-1/Tie-2 ratio (1.4-fold, P < 0.01, and 1.5-fold, P < 0.05, respectively) in ovariectomized animals compared with intact females, but no changes were detected in the expression of estrogen receptors (ER)-[alpha] and -[beta]. We conclude that estrogen-dependent control of Ang-1 expression plays an important role in stabilizing meningeal microvessel and maintaining healthy microvascular networks. hormones; microcirculation; imaging

Intravaginal impedance (IVI) fluctuates during the goat estrous cycle. To understand which ovarian steroids are responsible for IVI changes and whether IVI variations are associated with precopulatory and copulatory behaviors, 8 ovariectomized females were assigned to 4 treatments in a 4 x 4 Latin square replicated over four 8-d periods. The treatments were as follows: progesterone plus estradiol-17[beta] ([P.sub.4] + [E.sub.2]), oil plus estradiol-17[beta] ([E.sub.2]), progesterone plus oil (P.sub.4]), or oil (OIL). Daily IVI measurements at the vaginocervical junction were taken at 1 and 70 KHz. Progesterone was given on d 2 and 3. Estradiol was given in the evening of d 5. On d 1 to 8, goats were group-exposed to a sexually experienced male and observed for the expression of sexual behaviors. On d 6 and 7, IVI was less when goats received [P.sub.4] + [E.sub.2] or [E.sub.2] compared with goats given [P.sub.4] or OIL (P < 0.05). Impedance measured at 1 kHz tended to remain lower on d 8 in [P.sub.4] + [E.sub.2] treated females compared with those given [P.sub.4] or OIL (P < 0.055). Like previous results, [P.sub.4] + [E.sub.2] or [E.sub.2] treatment induced behavioral estrus; 5 of 8 [P.sub.4] + [E.sub.2]-treated and 5 of 8 [E.sub.2]-treated females were sexually receptive on d 6. On d 7, although IVI remained low and 2 of 8 [P.sub.4] + [E.sub.2]-treated goats and 4 of 8 [E.sub.2]-treated goats remained sexually receptive, no additional females were in estrus. No IVI decreases and no estrous behavior were observed in goats given [P.sub.4] or OIL. This experiment demonstrated that E2 initiates the periestrous drop in IVI, and [P.sub.4] may delay baseline return. Key words: estrogen, estrus, goat, impedance, progesterone, vagina

Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for [beta]-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by [beta]-catenin. Additionally, [beta]-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH-and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of [beta]-catenin. The stimulatory effect of [beta]-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that [beta]-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for [beta]-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation. ovary | steroidogenic factor-1 | granulosa cells | steroidogenesis | estrogen