A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17β-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen both in men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we use steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in 3 different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11β-hydroxy-17β-estradiol binds and activates estrogen receptors α and β and that 11β-hydroxy-17β-estradiol and the classic androgen pathway-derived active estrogen, 17β-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data show that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.

Brain regulation of glucose homeostasis is sexually dimorphic; however, the impact sex hormones have on specific neuronal populations within the ventromedial hypothalamic nucleus (VMN), a metabolically sensitive brain region, has yet to be fully characterized. Glucose-excited (GE) and -inhibited (GI) neurons are located throughout the VMN and may play a critical role in glucose and energy homeostasis. Within the ventrolateral portion of the VMN (VL-VMN), glucose sensing neurons and estrogen receptor (ER) distributions overlap. We therefore tested the hypothesis that VL-VMN glucose sensing neurons were sexually dimorphic and regulated by 17β-estradiol (17βE).

Menopause is characterized by depletion of ovarian follicles, a reduction of ovarian hormones to castrate levels and elevated levels of serum gonadotropins from the anterior pituitary gland. Although this process has significant repercussions throughout the body and affects a large proportion of our society, the neuroendocrine control mechanisms that accompany menopause are poorly understood. This review aims to examine rigorously the most accredited literature to provide an update about our current understanding of the role of the hypothalamic-pituitary axis in the onset of and transition into female reproductive senescence, focusing on the role of some specific neuropeptides in regulating the HPG axis and on their effects on several menopausal symptoms, especially referring to the cardiovascular risk, to open up new horizons for new therapeutic strategies.

Fluorination preventing metabolic hydroxylation of 17β-estradiol (E2) was applied to investigate the mechanisms underlying estrogen-induced carcinogenesis. Either 2-fluoro-17β-estradiol (2-FE2) or 4-fluoro-17β-estradiol (4-FE2) was administered subcutaneously for 52 weeks to August Copenhagen Irish (ACI) rats, the preferred animal model for human breast cancer. 4-FE2 induced frequent mammary tumors whereas 2-FE2 did not. The cumulative incidence of mammary tumors in rats treated with 4-FE2 was comparable to that observed with E2. The carcinogenic results were supported by histological examination of mammary glands of fluorinated estrogen-treated ACI rats. To evaluate the estrogenic potential of the fluorinated estrogens, 2-FE2 or 4-FE2 was administrated subcutaneously to ovariectomized rats. Both 4-FE2 and 2-FE2 showed high uterotrophic potency. Our results indicate that estrogenic potential may not be the sole factor driving mammary tumorigenesis. Since fluorination inhibits metabolic hydroxylation of E2 at the substituted position, the carcinogenic effect may occur through the metabolic activation of 2-hydroxylated E2, in combination with the compound's estrogenic potency.

It is well documented that steroid hormones modulate cytokine gene expression. In some tissues estrogens are known to suppress cytokine production while in other tissue types, cytokine expression is enhanced by the hormone. This study was conducted to investigate the regulatory mechanisms which underlie the modulation of the interleukin-1beta (IL-1beta) gene at the transcription level. To accomplish this, the macrophage cell line RAW264.7, which appeared insensitive to 17beta-estradiol (E2) treatment, was stably transfected with the human estrogen receptor (ER) and an IL-1beta promoter-CAT reporter construct. E2 markedly enhanced LPS-induced IL-1beta promoter-driven CAT activity in an E2 dose dependent manner. This responsiveness was estrogen specific since no synergism was observed between LPS and the sex steroids testosterone or progesterone while the estrogen analogue 17alpha-estradiol stimulated only at 10 to 100 times the amount required for 17beta-E2. Several antiestrogens, H1285, ICI 182 780, and tamoxifen inhibited the estrogen stimulated enhancement of IL-1beta promoter activity in a dose-dependent manner, indicating that this effect was indeed mediated through the ER in a ligand dependent manner. The estrogenic effect appeared to be indirect and time dependent since the addition of E2 was required hours prior to LPS stimulation; addition of E2 and LPS at the same time resulted in a greatly reduced estrogenic effect. The estrogen metabolites 17-epiestriol and 16-keto-17beta-E2 displayed an estrogenic response virtually indistinguishable from E2. 4-Hydroxyestradiol displayed activity only at 100-fold the concentration of E2 while 2-hydroxyestrone showed no activity at any of the concentrations tested. Overall the results demonstrate that E2 and some metabolites of E2 synergize with LPS to markedly enhance IL-1beta promoter activity through ER mediated processes.

Human estrogens prescribed for hormone replacement therapy (HRT) are known to be potent carcinogens. To find safer estrogens, several chlorinated estrogens were synthesized and their carcinogenic potential were determined. A pellet containing either 2-chloro-17β-estradiol (2-ClE2) or 4-chloro-17β-estradiol (4-ClE2) was implanted subcutaneously for 52 weeks into August Copenhagen Irish (ACI) rats, a preferred animal model for human breast cancer. 17β-Estradiol (E2) frequently induced mammary tumors while both 2-ClE2 and 4-ClE2 did not. Their 17α-ethinyl forms, thought to be orally active estrogens, were also synthesized. Neither 2-chloro-17α-ethinylestradiol (2-ClEE2) nor 4-chloro-17α-ethinylestradiol (4-ClEE2) induced tumors. The less carcinogenic effects were supported by histological examination of mammary glands of ACI rats treated with the chlorinated estrogens. A chlorine atom positioned at the 2- or 4-position of E2 may prevent the metabolic activation, resulting in reducing the carcinogenicity. 2-ClE2 and 4-ClE2 administered subcutaneously and 2-ClEE2 and 4-ClEE2 given orally to ovariectomized rats all showed uterotrophic potency, albeit slightly weaker than that of E2. Our results indicate that less carcinogenic chlorinated estrogens retaining estrogenic potential could be safer alternatives to the carcinogenic estrogens now in use for HRT.

The roots of licorice (Glycyrrhiza glabra) are a rich source of flavonoids, in particular, prenylated flavonoids, such as the isoflavan glabridin and the isoflavene glabrene. Fractionation of an ethyl acetate extract from licorice root by centrifugal partitioning chromatography yielded 51 fractions, which were characterized by liquid chromatography-mass spectrometry and screened for activity in yeast estrogen bioassays. One third of the fractions displayed estrogenic activity towards either one or both estrogen receptors (ERs; ERα and ERβ). Glabrene-rich fractions displayed an estrogenic response, predominantly to the ERα. Surprisingly, glabridin did not exert agonistic activity to both ER subtypes. Several fractions displayed higher responses than the maximum response obtained with the reference compound, the natural hormone 17β-estradiol (E(2)). The estrogenic activities of all fractions, including this so-called superinduction, were clearly ER-mediated, as the estrogenic response was inhibited by 20-60% by known ER antagonists, and no activity was found in yeast cells that did not express the ERα or ERβ subtype. Prolonged exposure of the yeast to the estrogenic fractions that showed superinduction did, contrary to E(2), not result in a decrease of the fluorescent response. Therefore, the superinduction was most likely the result of stabilization of the ER, yeast-enhanced green fluorescent protein, or a combination of both. Most fractions displaying superinduction were rich in flavonoids with single prenylation. Glabridin displayed ERα-selective antagonism, similar to the ERα-selective antagonist RU 58668. Whereas glabridin was able to reduce the estrogenic response of E(2) by approximately 80% at 6 × 10(-6) M, glabrene-rich fractions only exhibited agonistic responses, preferentially on ERα.

Metabolic reprogramming remains largely understudied in relation to hormones in estrogen receptor (ER) and progesterone receptor (PR) positive breast cancer. In this study, we investigated how estrogens, progestins, or the combination, impact metabolism in three ER and PR positive breast cancer cell lines. We measured metabolites in the treated cells using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). Top metabolic processes upregulated with each treatment involved glucose metabolism, including Warburg effect/glycolysis, gluconeogenesis, and the pentose phosphate pathway. RNA-sequencing and pathway analysis on two of the cell lines treated with the same hormones, found estrogens target oncogenes, such as MYC and PI3K/AKT/mTOR that control tumor metabolism, while progestins increased genes associated with fatty acid metabolism, and the estrogen/progestin combination additionally increased glycolysis. Phenotypic analysis of cell energy metabolism found that glycolysis was the primary hormonal target, particularly for the progestin and estrogen-progestin combination. Transmission electron microscopy found that, compared to vehicle, estrogens elongated mitochondria, which was reversed by co-treatment with progestins. Progestins promoted lipid storage both alone and in combination with estrogen. These findings highlight the shift in breast cancer cell metabolism to a more glycolytic and lipogenic phenotype in response to combination hormone treatment, which may contribute to a more metabolically adaptive state for cell survival.

The incidence of rheumatoid arthritis (RA) increases at the same time as menopause when estrogen level decreases. Estrogen treatment is known to reduce the IgG pathogenicity by increasing the sialylation grade on the terminal glycan chain of the Fc domain, inhibiting the binding ability to the Fc gamma receptor. Therefore, treatment with estrogen may be beneficial in pre-RA patients who have autoantibodies and are prone to get an autoimmune disease. However, estrogen treatment is associated with negative side effects, therefore selective estrogen receptor modulators (SERMs) have been developed that have estrogenic protective effects with minimal side effects. In the present study, we investigated the impact of the SERM bazedoxifene on IgG sialylation as well as on total serum protein sialylation. C57BL6 mice were ovariectomized to simulate postmenopausal status, followed by ovalbumin immunization, and then treated with estrogen (estradiol), bazedoxifene, or vehicle. We found that estrogen treatment enhanced IgG levels and had a limited effect on IgG sialylation. Treatment with bazedoxifene increased the sialic acids in plasma cells in a similar manner to E2 but did not reach statistical significance. However, we did not detect any alteration in IgG-sialylation with bazedoxifene treatment. Neither estrogen nor bazedoxifene showed any significant alteration in serum protein sialylation but had a minor effect on mRNA expression of glycosyltransferase in the bone marrow, gonadal fat, and liver.

Mammalian sperm must undergo a series of controlled molecular processes in the female reproductive tract called capacitation before they are capable of penetrating and fertilizing the egg. Capacitation, as a complex biological process, is influenced by many molecular factors, among which steroidal hormone estrogens play their role. Estrogens, present in a high concentration in the female reproductive tract are generally considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive parameters. The purpose of this study is to investigate the effect of three natural estrogens such as estrone (E1), 17beta-estradiol (E2) and estriol (E3) as well as the synthetical one, 17 alpha-ethynylestradiol (EE2) on boar sperm capacitation in vitro.

Estrone, estradiol, ethynylestradiol and estrone 3-methyl ether underwent a biotransformation process in the submerged culture of Isaria fumosorosea KCh J2. Estrone was transformed into seven metabolites, four of which were glycosylated. Estradiol was selectively glycosylated at C-3 and then transformed to D-ring lactone. Ethynylestradiol was coupled with methylglucoside and 6β-hydroxyderivative was obtained. Estrone 3-methyl ether was not transformed indicating that a free hydroxyl group at C-3 is necessary for glycosylation. Baeyer-Villiger oxidation combined with hydroxylation and glycosylation was observed. All glycosides obtained in this study are 3-O-β-methylglucosides.

The effect of estrogens, including estrone (E1), estradiol-17beta (E2), estriol (E3) and 2-hydroxyestradiol (2-OH-E2), on the oxidative damage induced by ferrylmyoglobin (ferrylMb) was investigated. These estrogens inhibited lipid peroxidation induced by ferrylMb. The ability of 2-OH-E2 to inhibit lipid peroxidation was much greater than the other estrogens. Furthermore, 2-OH-E2 trapped 2,2'-azobis-(2-amidinopropane)-dihydrochloride peroxyl radicals more rapidly, and among these estrogens only 2-OH-E2 reacted with 2,2-diphenyl-1-picrylhydrazyl. These results suggest that the ability of 2-OH-E2 to inhibit lipid peroxidation is because it scavenges lipid peroxyl and carbon-centered radicals. Estrogens, except for 2-OH-E2, partially prevented the inactivation of alcohol dehydrogenase (ADH) induced by ferrylMb. Of interest, however, the exposure of sulfhydryl (SH) enzymes to ferrylMb in the presence of 2-OH-E2 dramatically increased the inhibition of the enzyme activity. Ascorbic acid (ASA) and reduced glutathione (GSH) strongly inhibited the inactivation of ADH induced by ferrylMb in the presence of 2-OH-E2. During the reaction of ferrylMb with ASA or GSH in the presence of 2-OH-E2, large amounts of oxymyoglobin were formed, suggesting the involvement of the semiquinone from 2-OH-E2 in the reduction of metmyoglobin. Presumably, the semiquinone formed from 2-OH-E2 oxidizes the SH group of enzymes to facilitate the rapid inactivation of the SH enzymes induced by ferrylMb.

We used gene expression profiling to investigate whether the molecular effects induced by estrogens of different provenance are intrinsically similar. In this article we show that the physiologic estrogen 17-beta-estradiol, the phytoestrogen genistein, and the synthetic estrogen diethylstilbestrol alter the expression of the same 179 genes in the intact immature mouse uterus under conditions where each chemical has produced an equivalent gravimetric and histologic uterotrophic effect, using the standard 3-day assay protocol. Data are also presented indicating the limitations associated with comparison of gene expression profiles for different chemicals at times before the uterotrophic effects are fully realized. We conclude that the case has yet to be made for regarding synthetic estrogens as presenting a unique human hazard compared with phytoestrogens and physiologic estrogens. Key words: diethylstilbestrol, estrogen, gene expression, genistein, microarray, phytoestrogen, toxicogenomics, uterus.

In this study mouse Leydig cell (MA-10) were treated with G-protein coupled membrane estrogen receptor antagonist (G-15; 10 nM). Cells were analyzed by Western blotting for expression of estrogen-related receptors (ERRα, β and γ), steroidogenic markers (lutropin receptor; LHR and 3β-hydroxysteroid dehydrogenase; 3β-HSD) and lipid droplet markers (perilipin; PLIN and microtubule-associated protein 1 A/1B-light chain 3; LC3). Concomitantly, microscopic analyses by light microscope (immunofluorescent staining for lipid droplets, PLIN and LC3) as well as by electron microscope (for lipid droplet ultrastructure) were utilized. For analysis of cholesterol content, cAMP level and progesterone secretion, G-15, estrogen receptor (ER) antagonist (ICI 182,780; 10 μM), 17β-estradiol (10 mM) and, bisphenol A (BPA; 10 nM) were used alone or in combinations. We revealed no changes in ERRs expression but alterations in ERRβ and γ localization in G-15-treated cells when compared to control. Partial translocation of ERRβ and γ from the cell nucleus to cytoplasm was observed. Decreased expression of LHR, 3β-HSD, PLIN and LC3 was detected. Moreover, in treated cells large lipid droplets and differences in their distribution were found. Very strong signal of co-localization for PLIN and LC3 was found in treated cells when compared to control. In ultrastructure of treated cells, degenerating lipid droplets and double membrane indicating on presence of lipophagosome were observed. We found, that only (i) BPA and G-15 did not effect on cholesterol content, (ii) BPA, G-15 and ICI did not effect on cAMP level and (iii) BPA, ICI alone and in combination, and BPA with G-15 did not modulate progesterone secretion. These findings showed complex and diverse estrogen effects on mouse Leydig cells at various steps of steroid hormone production (cholesterol storage, release and processing). Lipid homeostasis and metabolism in these cells were affected by endogenous and exogenous estrogen, interactions of receptors (GPER, ER and ERR) and GPER and ER antagonists.

Sexually dimorphic brain volumes (sexually dimorphic nucleus of the preoptic area (SDN-POA) and anteroventral periventricular (AVPV) nucleus) are influenced by estrogens. Phytoestrogens, derived from plants (especially soy products), are molecules structurally and functionally similar to estradiol. The purpose of this study was to examine: the consumption of phytoestrogen (using a phytoestrogen-rich (Phyto-600) versus a phytoestrogen-free (Phyto-free)) diets from conception to adulthood (or changing the diets during adulthood) and characterizing (a) circulating plasma phytoestrogen levels, (b) testosterone levels in males, (c) sexually dimorphic brain volumes (i.e. the SDN-POA and AVPV) and (d) the presence of apoptotic cells in these brain structures in Long-Evans rats. Phyto-600 fed animals displayed total serum phytoestrogens levels 37-fold higher compared to Phyto-free values. Circulating testosterone levels were not significantly altered by the diets. Female SDN-POA volumes were not altered by the diets. Whereas, males fed a Phyto-free diet displayed decreased SDN-POA volumes compared to male Phyto-600 values. Females fed the Phyto-600 diet displayed larger AVPV volumes compared to males on the same diet or females on the Phyto-free diet. Males fed the Phyto-free diet had the largest AVPV values compared to Phyto-600 fed males. When the SDN-POA region was examined in lifelong Phyto-free fed males, apoptotic cells were present versus males fed the Phyto-600 diet and in the AVPV region the opposite results were obtained. In summary, consumption of dietary phytoestrogens (estrogen mimics) can alter hormone-sensitive hypothalamic brain volumes in rodents during adulthood.

Although controversial, estrogens remain one of the few agents purported to influence the incidence of Alzheimer's disease and one of their postulated mechanisms of action is their effects on basal forebrain cholinergic neurons. However, it is unclear whether the responses of cholinergic neurons to estrogens are direct or mediated via the retrograde influences of neurotrophins, known to be induced by estrogens in the hippocampus and neocortex. In the present study, we explore the issue of the primary site of action of estrogens by studying the regulation of expression of genes that characterize mature cholinergic neurons, i.e., choline acetyltransferase, trkA, and p75(NTR) in the medial septum and the nucleus basalis complex. In parallel, we study the hippocampal expression of NGF, BDNF, and NT-3, i.e., neurotrophins with known trophic roles on cholinergic neurons. Gene expression is studied by RT-PCR in ovariectomized female rats with and without estrogen supplementation within the physiological estradiol range and in rats with complete fimbria-fornix transactions treated with estrogen or vehicle. To clarify mechanisms of estrogen transduction in cholinergic neurons, we study the effects of estrogen treatment on fimbria-fornix-lesioned mice with genetic ablations of ER subtypes alpha and beta. The results of the present study suggest that, while estrogens do regulate BDNF expression in the hippocampus and neocortex, they also exert stimulatory non-trophic effects on basal forebrain cholinergic neurons, primarily on ChAT expression. Cholinergic neurons retain their ability to respond to estrogens after their complete separation from the hippocampus. The elimination of ERalpha alters significantly the phenotypic responsiveness of cholinergic neurons to estrogens, whereas elimination of ERbeta appears to have no effect. Our findings support the idea that estrogens directly enhance cholinergic neuron function and that ERalpha plays a significant role in transducing these regulatory effects.

Estrogen deprivation is associated with delayed healing, while estrogen replacement therapy (ERT) accelerates acute wound healing and protects against development of chronic wounds. However, current estrogenic molecules have undesired systemic effects, thus the aim of our studies is to generate new molecules for topic administration that are devoid of systemic effects. Following a preliminary study, the new 17β-estradiol derivatives 1 were synthesized. The estrogenic activity of these novel compounds was evaluated in vitro using the cell line ERE-Luc B17 stably transfected with an ERE-Luc reporter. Among the 17β-estradiol derivatives synthesized, compounds 1e and 1f showed the highest transactivation potency and were therefore selected for the study of their systemic estrogenic activity. The study of these compounds in the ERE-Luc mouse model demonstrated that both compounds lack systemic effects when administered in the wound area. Furthermore, wound-healing experiments showed that 1e displays a significant regenerative and anti-inflammatory activity. It is therefore confirmed that this class of compounds are suitable for topical administration and have a clear beneficial effect on wound healing.

The liver is a vital organ that sustains multiple functions beneficial for the whole organism. It is sexually dimorphic, presenting sex-biased gene expression with implications for the phenotypic differences between males and females. Estrogens are involved in this sex dimorphism and their actions in the liver of several reptiles, fishes, amphibians, and birds are discussed. The liver participates in reproduction by producing vitellogenins (yolk proteins) and eggshell proteins under the control of estrogens that act via two types of receptors active either mainly in the cell nucleus (ESR) or the cell membrane (GPER1). Estrogens also control hepatic lipid and lipoprotein metabolisms, with a triglyceride carrier role for VLDL from the liver to the ovaries during oogenesis. Moreover, the activation of the vitellogenin genes is used as a robust biomarker for exposure to xenoestrogens. In the context of liver diseases, high plasma estrogen levels are observed in fatty liver hemorrhagic syndrome (FLHS) in chicken implicating estrogens in the disease progression. Fishes are also used to investigate liver diseases, including models generated by mutation and transgenesis. In conclusion, studies on the roles of estrogens in the non-mammalian oviparous vertebrate liver have contributed enormously to unveil hormone-dependent physiological and physiopathological processes.

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28°C as compared to 37°C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology.