原田 信広

Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the I-Kr channel current in guinea pig ventricular myocytes and the human ether-a-go-go-related gene (hERG) channel currents in CHO-K1 cells regardless of estrogen receptor signaling and revealed that the partially suppressed hERG currents enhanced the sensitivity to the hERG blocker E-4031. To obtain in vivo proof-of-concept data to support the effects of estrogen on cardiac electrophysiology, we here employed an aromatase knockout mouse as an in vivo estrogen-null model and compared the acute effects of E-4031 on cardiac electrophysiological parameters with those in wild-type mice (C57/BL6J) by recording surface electrocardiogram (ECG). The ablation of circulating estrogens blunted the effects of E-4031 on heart rate and QT interval in mice under a denervation condition. Our result provides in vivo proof of principle and demonstrates that endogenous estrogens increase the sensitivity of E-4031 to cardiac electrophysiology.

The human CYP19 gene encodes aromatase, which converts androgens to estrogens. CYP19 mRNA variants are transcribed mainly from three promoters. Quantitative RT-PCR was used to measure the relative amounts of each of the three transcripts and determine the on/off state of the promoters. While some of the promoters were silent, CYP19 mRNA production differed among the other promoters, whose estimated transcription levels were 0.001% to 0.1% of that of the TUBB control gene. To investigate the structural aspects of chromatin that were responsible for this wide range of activity of the CYP19 promoters, we used a fractionation protocol, designated SEVENS, which sequentially separates densely packed nucleosomes from dispersed nucleosomes. The fractional distribution of each inactive promoter showed a similar pattern to that of the repressed reference loci; the inactive regions were distributed toward lower fractions, in which closed chromatin comprising packed nucleosomes was enriched. In contrast, active CYP19 promoters were raised toward upper fractions, including dispersed nucleosomes in open chromatin. Importantly, these active promoters were moderately enriched in the upper fractions as compared to active reference loci, such as the TUBB promoter; the proportion of open chromatin appeared to be positively correlated to the promoter strength. These results, together with ectopic transcription accompanied by an increase in the proportion of open chromatin in cells treated with an H3K27me inhibitor, indicate that CYP19 mRNA could be transcribed from a promoter in which chromatin is shifted toward an open state in the equilibrium between closed and open chromatin.

The post-translational regulation of aromatase has not been well characterized as compared with transcriptional regulation. Several studies of post-translational regulation have focused on decreases in catalytic activity following phosphorylation. We report here dual post-translational regulation of aromatase, at the catalytic activity and protein levels. Microsomal aromatase prepared from JEG-3 cells was rapidly inactivated and subsequently degraded in the presence of a cytosolic fraction with calcium, magnesium, and ATP. In a reconstituted system consisting of microsomal and cytosolic fractions, aromatase was protected from protein degradation by treatment with alkaline phosphatase, whereas degradation was enhanced by treatment with calcineurin inhibitors (FK506 and cyclosporin A). Furthermore, aromatase was protected from degradation by treatment with kinase inhibitors, especially the calcium/calmodulin kinase inhibitors KN62 and KN93. Similarly to the reconstituted system, aromatase in cultured JEG-3 cells was protected from degradation by KN93, whereas FK503 increased degradation in the presence of cycloheximide, although cellular aromatase mRNA levels were unchanged by these reagents. Knockdown of calcineurin and calcium/calmodulin kinase II (CaMKII) with small interfering RNAs resulted in a dose-dependent increase in aromatase degradation and protection from degradation, respectively. The cytosol fraction-dependent phosphorylation of microsomal aromatase was inhibited by calcineurin, KN62, and KN93, and promoted by CaMKII and FK506. These results indicate that aromatase is regulated acutely at the catalytic activity level and subsequently at the enzyme content level by CaMKII/calcineurin-dependent phosphorylation/dephosphorylation.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion and socio-sexual behaviours. Oestrogen (neuroestrogen) synthesized in the brain from androgen by aromatase regulates male socio-sexual behaviours. Here we show that GnIH directly activates aromatase and increases neuroestrogen synthesis in the preoptic area (POA) and inhibits socio-sexual behaviours of male quail. Aromatase activity and neuroestrogen concentration in the POA are low in the morning when the birds are active, but neuroestrogen synthesis gradually increases until the evening when the birds become inactive. Centrally administered GnIH in the morning increases neuroestrogen synthesis in the POA and decreases socio-sexual behaviours. Centrally administered 17 beta-oestradiol at higher doses also inhibits socio-sexual behaviours in the morning. These results suggest that GnIH inhibits male socio-sexual behaviours by increasing neuroestrogen synthesis beyond its optimum concentration for the expression of socio-sexual behaviours. This is the first demonstration of any hypothalamic neuropeptide that directly regulates neuroestrogen synthesis.

The expression pattern of aromatase (ARO), the enzyme converting androgens to estrogens, was analyzed in the olfactory bulb of adult male rats and was compared with the distribution of estrogen receptor beta (ER beta), the main estrogen receptor isoform expressed in this brain region. A strong ARO immunolabeling obtained with a specificity tested antibody was observed in juxtaglomerular neurons of the glomerular layer and a weaker immunoreaction was detected in the mitral cell layer of the main olfactory bulb, while the granule cell layer of the main olfactory bulb as well as all layers in the accessory olfactory bulb showed faint immunolabeling. Fluorescence double labeling experiments revealed that ARO detected in juxtaglomerular neurons of the main olfactory bulb colocalized with tyrosine hydroxylase (TH) and glutamic acid decarboxylase 67 (GAD67), while no colocalization between ARO and the calcium binding proteins calretinin (CR) and calbindin (CB) was observed. Furthermore, the TH immunoreactive neurons expressed metabotropic glutamate receptor 1 (mGluR1) too. ER beta immunoreactivity, in contrast to ARO, was detected in all layers of both the main and accessory olfactory bulb. In the glomerular layer of the main olfactory bulb it was expressed in TH and GAD67 containing juxtaglomerular neurons, and it colocalized with CR, CB and even with glial fibrillary acidic protein too. Our morphological findings suggest that ARO expression is a novel feature of dopaminergic/GABAergic juxtaglomerular neurons in the adult rat main olfactory bulb, and raise the possibility that ARO activity may change in function of olfactory input via mGluR1. In situ estrogen production in the olfactory bulb in turn may modulate interglomerular circuits through ER beta.

Estrogens have been found to improve memory and reduce risk of dementia, although conflicting results such as failure of estrogen replacement therapy for treatment of Alzheimer's disease (AD) also has been reported. Only recently, our published human brain studies showed a depletion of brain estrogen in women with AD, while other studies have demonstrated cognitive impairment believed to be caused by inhibition of endogenous estrogen synthesis in females. To investigate whether the shortage of brain estrogen alters the sensitivity of response to estrogen replacement therapy, we have used genetic and surgical animal models to examine the response of estrogen treatment in AD neuropa-thology. Our studies have shown that early treatment with 17β-estradiol (E2) or genistein could reduce brain amyloid levels by increasing Aβ clearance in both APP23 mice with genetic deficiency of aromatase (APP/Ar+/-), in which the brains contain nondetectable levels of estrogen, and in APP23 mice with an ovariectomy (APP/OVX), in which the brains still contain certain levels of estrogen. However, only APP/Ar +/- mice showed a great reduction in brain amyloid plaque formation after E2 or genistein treatment along with downregulation of β-secretase (BACE1) mRNA and protein expression. Our results suggest that early and long-term usage of E2 and/or genistein may prevent AD pathologies in a dependent manner on endogenous brain estrogen levels in aged females. © Springer Science+Business Media New York 2012.

The enzyme aromatase is important for regulating sexual and aggressive behaviors during the reproductive season, including many aspects of courtship. In birds, aromatase is expressed at high levels in a number of different brain regions. Although this expression does vary among species, the extent to which the distribution of aromatase positive cells reflects species differences in courtship and other behaviors is not well established. Here, we examine the distribution of aromatase immunoreactive (ARO) neurons in the brain of a species with a unique courtship display, the ruffed grouse (Bonasa umbellus). Unlike most other galliforms, male ruffed grouse do not vocalize as part of their courtship and instead use their wings to create a non-vocal auditory signal to attract females. Because aromatase is involved in courtship behaviors in several bird species, including other galliforms, we hypothesized that aromatase distribution in the ruffed grouse would differ from that of other galliforms. We used an antibody raised against quail aromatase to examine aromatase immunoreactivity in the ruffed grouse, the closely related spruce grouse (Falcipennis canadensis) and the Japanese quail (Coturnix japonica). In all three species, ARO neurons were identified in the medial preoptic nucleus, the bed nucleus of the stria terminalis and the nucleus ventromedialis hypothalami. Both grouse species had ARO neurons in two regions of the telencephalon, the hyperpallium, and entopallium, and the ruffed grouse also in field L. ARO neurons were only found in one region in the telencephalon of the Japanese quail, the septum. In general, breeding male ruffed grouse had significantly more ARO neurons and those neurons were larger than that of both the non-breeding male and female ruffed grouse. Aromatase expression in the telencephalon of the ruffed grouse suggests that steroid hormones might modulate responses to visual and acoustic stimuli, but how this relates to species differences in courtship displays and co-expression with estrogenic receptors is yet to be determined. © 2012 Elsevier B.V.

Neurosteroidal oestrogen has been proposed to play important roles in a variety of reproductive behaviours. Aromatase, a key enzyme in oestrogen synthesis, is localised in neural nuclei of specific brain regions and is developmentally regulated, with a transient expression peak at the perinatal period. The brain-specific promoter of the aromatase gene was analysed aiming to determine the transcriptional control mechanisms that could help explain the spatiotemporal expression. We previously reported that a 202-bp sequence, which is upstream from the transcriptional initiation site, is essential for the basal transcriptional activity. The 202-bp upstream region of brain-specific exon 1 comprises at least three types of cis-acting elements: aro-AI (Arom-A alpha), aro-AII (Arom-A beta) and aro-B (Arom-B). To identify the binding proteins for the cis-acting elements, a yeast one-hybrid screen was performed with these cis-element sequences using a mouse foetal cDNA library. Lhx2, a LIM-homeodomain protein, was identified as one of the aro-B binding proteins. The identification was further confirmed using the gel shift assay, which demonstrated binding competition of nuclear proteins to the aro-B element with a typical Lhx2-binding element. In addition, a chromatin immunoprecipitation assay with an anti-Lhx2 antibody demonstrated that Lhx2 bound to the aro-B site in vivo. A reporter assay of the brain-specific promoter demonstrated increased Lhx2-dependent promoter activity. Furthermore, the time-dependent increase in aromatase mRNA in primary cultured foetal neurones was suppressed by an small-interfering RNA-mediated knockdown of Lhx2 expression. These results show that Lhx2 is involved in the transcriptional regulation of aromatase in the rodent brain.

In the present study we examined the protective effect of Brazilian propolis against hepatic oxidative damage in rats with water-immersion restraint stress (WIRS) in comparison with that of vitamin E (VE). Fasted rats orally received Brazilian green propolis ethanol extract (BPEE; 10, 50 or 100 mg/kg), VE (250 mg/kg) or vehicle at 30 min before the onset of WIRS. Exposure of vehicle-treated rats to 6 h of WIRS caused liver cell damage, judging from the levels of serum alanine aminotransferase and aspartate aminotransferease, increased hepatic lipid peroxide, NOx contents and myeloperoxidase activity, and decreased hepatic non-protein SH, ascorbic acid contents and superoxide dismutase activity. Preadministration of BPEE (50 or 100 mg/kg) or VE to the stressed rats protected against the hepatic damage and attenuated the increased hepatic lipid peroxide and NOx contents and myeloperoxidase activity and the decreased hepatic non-protein SH and ascorbic acid contents and superoxide dismutase activity. These protective effects of BPEE (50 mg/kg) were greater than those of BPEE (100 mg/kg) and were almost equal to those of VE. These results indicate that BPEE protects against hepatic oxidative damage in rats exposed to WIRS possibly through its antioxidant and antiinflammatory properties such as VE. Copyright (c) 2012 John Wiley & Sons, Ltd.

Neurosteroids are synthesized de novo from cholesterol in the brain. In rodents, the Purkinje cell actively produces several kinds of neurosteroids including estradiol during neonatal life, when cerebellar neuronal circuit formation occurs. Estradiol may be involved in cerebellar neuronal circuit formation through promoting neuronal growth and synaptic contact, because the Purkinje cell expresses estrogen receptor-beta. To test this hypothesis, in this study we examined the effect of estradiol on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using neonatal wild-type (WT) mice or cytochrome P450 aromatase knock-out (ArKO) mice. Administration of estradiol to neonatal WT or ArKO mice increased dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell. In contrast, WT mice treated with tamoxifen, an ER antagonist, or ArKO mice exhibited decreased Purkinje dendritic growth, spinogenesis, and synaptogenesis at the same neonatal period. Estrogen administration to neonatal WT or ArKO mice increased the expression of brain-derived neurotrophic factor (BDNF) in the cerebellum, whereas tamoxifen decreased the BDNF level in WT mice similar to ArKO mice. BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth. These results indicate that estradiol induces dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell via BDNF action during neonatal life.

Besides their slow genomic actions, estrogens also induce rapid physiological responses. To be functionally relevant, these effects must be associated with rapid changes in local concentrations of estrogens. Rapid changes in aromatase activity (AA) controlled by calcium-dependent phosphorylations of the enzyme can alter in a rapid manner local estrogen concentrations, but so far this mechanism was identified only in the avian (quail) brain. We show here that AA is also rapidly down-regulated by phosphorylating conditions in quail ovary homogenates and in various cell lines transfected with human aromatase (HEK 293, Neuro2A, and C6). Enzymatic activity was also rapidly inhibited after depolarization of aromatase-expressing HEK 293 cells with 100 mM KCl, and activity was fully restored when cells returned to control conditions. Western blot analysis demonstrated that the reduction of enzymatic activity is not due to protein degradation. We next investigated by site-directed mutagenesis the potential implication in the control of AA of specific aromatase residues identified by bioinformatic analysis. Mutation of the amino acids S118, S247, S267, T462, T493, or S497 to alanine, alone or in combination, did not block the rapid inhibition of enzymatic activity induced by phosphorylating conditions, but basal AA was markedly decreased in the S118A mutant. Altogether, these results demonstrate that the rapid inhibition of AA is a widespread and fully reversible process and that phosphorylation of specific residues modulate AA. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues. (Endocrinology 152: 4199-4210, 2011)

Background: 1-(2,4-Dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylpheny)Propane (DP) was reported as a novel tyrosinase inhibitor by Nesterov et al. In previous study, we showed that DP is an antioxidant and accelerates the fading of UVB-induced tan in human skin but details of inhibiting mechanism of DP in melanogenesis remain incomplete. Objective: To clarify additional mechanisms of DP inhibition of melanogenesis, we studied the effect of DP on tyrosinase processing and degradation. Methods: Tyrosinase inhibition was assessed using mushroom and human tyrosinase. The effect of DP on mRNA and protein levels as well as glycosylation and degradation of tyrosinase was examined using normal human epidermal melanocytes (NHEM). Results: DP was 200 times more potent than that of kojic acid in inhibiting mushroom tyrosinase activity. In contrast, DP (IC(50) = 200 mu M) was significantly less effective at inhibiting tyrosinase from NHEM. DP decreased melanin content in cultured NHEM after 7th day (IC(50) = 10 mu M). The IC(50) for DP against human tyrosinase activity was found to be at least 20 times higher than that of melanin synthesis. At a non-cytotoxic concentration DP did not decrease tyrosinase mRNA however protein level decreased by 46% after 48 h treatment. DP did not alter the ratio of mature and immature tyrosinase assayed by endo H cleavage. Tyrosinase degradation assays revealed that DP accelerated tyrosinase degradation in NHEM. Conclusions: We found that DP acts through dual mechanisms to reduce melanin synthesis; by inhibition of tyrosinase activity via an anti-oxidant effect, and, more importantly, by the acceleration of tyrosinase degradation. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Aromatization of testosterone into estradiol in the preoptic area plays a critical role in the activation of male copulation in quail and in many other vertebrate species. Aromatase expression in quail and in other birds is higher than in rodents and other mammals, which has facilitated the study of the controls and functions of this enzyme. Over relatively long time periods (days to months), brain aromatase activity (AA), and transcription are markedly (four-to sixfold) increased by genomic actions of sex steroids. Initial work indicated that the preoptic AA is higher in males than in females and it was hypothesized that this differ-ential production of estrogen could be a critical factor responsible for the lack of behavioral activation in females. Subsequent studies revealed, however, that this enzymatic sex dif-ference might contribute but is not sufficient to explain the sex difference in behavior. Studies of AA, immunoreactivity, and mRNA concentrations revealed that sex differences observed when measuring enzymatic activity are not necessarily observed when one mea-sures mRNA concentrations. Discrepancies potentially reflect post-translational controls of the enzymatic activity. AA in quail brain homogenates is rapidly inhibited by phosphoryla-tion processes. Similar rapid inhibitions occur in hypothalamic explants maintained in vitro and exposed to agents affecting intracellular calcium concentrations or to glutamate ago-nists. Rapid changes in AA have also been observed in vivo following sexual interactions or exposure to short-term restraint stress and these rapid changes in estrogen production modulate expression of male sexual behaviors. These data suggest that brain estrogens display most if not all characteristics of neuromodulators if not neurotransmitters. Many questions remain however concerning the mechanisms controlling these rapid changes in estrogen production and their behavioral significance. © 2011 Balthazart, Charlier, Cornil, Dickens, Harada, Konkle, VoigtandBall.

Aromatase in the mouse brain is expressed only in the nerve cells of specific brain regions with a transient peak during the neonatal period when sexual behaviors become orga-nized.The aromatase-knockout (ArKO) mouse, generated to shed light on the physiological functions of estrogen in the brain, exhibited various abnormal behaviors, concomitant with undetectable estrogen and increased androgen in the blood.To further elucidate the effects of neurosteroidal estrogens on behavioral phenotypes, we first prepared an brain-specific aromatase transgenic (bsArTG) mouse by introduction of a human aromatase transgene controlled under a -6.5kb upstream region of the brain-specific promoter of the mouse aromatase gene into fertilized mouse eggs, because the -6.5kb promoter region was previously shown to contain the minimal essential element responsible for brain-specific spatiotemporal expression. Then, an ArKO mouse expressing the human aromatase only in the brain was generated by crossing the bsArTG mouse with the ArKO mouse. The resulting mice (ArKO/bsArTG mice) nearly recovered from abnormal sexual, aggressive, and locomotive (exploratory) behaviors, in spite of having almost the same serum levels of estrogen and androgen as the adult ArKO mouse.These results suggest that estrogens locally synthesized in the specific neurons of the perinatal mouse brain directly act on the neurons and play crucial roles in the organization of neuronal networks participating in the control of sexual, aggressive, and locomotive (exploratory) behaviors. © 2011 Honda.

Aromatase, a key enzyme of estrogen biosynthesis, is transcriptionally regulated by many growth factors. IGF-I enhances aromatase activity in a variety of cells, but the mechanism of action has not been determined. We herein report our finding of a novel mechanism of action for IGF-I. IGF-I enhanced the dexamethasone (DEX)-induced aromatase activity by 30% in serum-starved THP-1 cells. The increase was associated with a corresponding increase in the level of aromatase protein but not with any change in the mRNA level. Metabolic labeling experiments revealed that IGF-I inhibited the degradation of aromatase. We identified pepstatin A as the most effective inhibitor of aromatase degradation by in vitro assay. Using a nontoxic concentration of pepstatin A, we examined IGF-I's action on aromatase distribution in microsomes and lysosomes. In the presence of pepstatin A, DEX caused an increase in the amount of aromatase in both microsomes and lysosomes, and IGF-I attenuated the DEX-induced accumulation of aromatase in lysosomes and, conversely, enhanced its accumulation in the microsomes. The addition of serum abolished the IGF-I-induced changes. The transport from microsome to lysosome was fluorescently traced in cells using a recombinant aromatase. IGF-I selectively reduced the aromatase signal in the lysosomes. Finally, we observed that IGF-I enhanced the aromatase activity by 50% as early as 1 h after treatment; furthermore, rapamycin, an enhancer of autophagy, completely negated the effect of IGF-I on the enzyme. These results indicate that IGF-I enhances aromatase by the inhibition of autophagy. (Endocrinology 151: 4949-4958, 2010)

The biochemical properties, neuroanatomical location, and function of aromatase (ARO), the enzyme that converts testosterone to 17 beta-estradiol, have been studied extensively in the adult quail brain. Conversely, very little is known about ARO in quail embryos. This study investigated the distribution of ARO in quail prosencephalon at embryonic days (E) 9, 11, and 15 by immunocytochemistry. ARO-immunoreactive cells were observed within the walls of the cerebral ventricles, the ventral striatum, medial preoptic nucleus (POM), medial part of the bed nucleus of the stria terminalis (BSTM), lateral part of the BST, and in the tuberal region. The BSTM and to a lesser extent the POM showed transient, female-biased sex-differences. In the BSTM, the number of the ARO-immunoreactive cells, the fractional area covered by ARO-immunoreactive structures, and the overall extension of ARO-immunoreactivity were greater in females at E9 and E11, but these differences largely disappeared at E15 and post-hatch day 1. The sex differences were confirmed at the transcriptional level by in situ hybridization. In the lateral part of the POM, females showed slightly more ARO-immunoreactivity than males at E11. Treatment of E9 male embryos with estradiol completely feminized ARO-immunoreactivity at E11. The origins and the functional significance of these sex differences remain unknown. (c) 2010 Elsevier B.V. All rights reserved.

Aromatase is the enzyme that catalyzes the biosynthesis of estrogens. It is implicated in neuroprotection. The present study investigated aromatase expression in the cerebellum of dogs infected with canine distemper virus (CDV), a disease characterized by demyelination in the white matter of the cerebellum. The presence of CDV infection was confirmed on the basis of histopathology and immunohistochemical localization of CDV antigen in glial cells of the white matter. The number of aromatase immunoreactive astrocytes were significantly (p < 0.05) higher in CDV-infected dogs compared to control dogs. The results suggest that astrocytes respond to invasion and persistence of CDV by means of increased estrogen production. The results also suggest that the high level of estrogen expression is maintained similarly throughout all stages of the disease since the number of aromatase immunoreactive astrocytes did not vary during the different stages of CDV infection.

As brain testosterone plays both androgenic and estrogenic actions due to its conversion into estrogen via aromatase naturally, it is unclear that the age-related reduction of testosterone increased risk of Alzheimer's disease (AD) in men is mediated through androgen alone or both androgen and estrogen mechanisms. Our previous studies using a gene-based approach in mouse model to block the conversion of testosterone into estrogen (aromatase gene knock-out, ArKO), found a depletion of estrogen and increase in testosterone endogenously in males. Here, we use crossing the ArKO mice with APP23 transgenic mice, a mouse model of AD, to produce APP23/Ar+/- mice to study the estrogen-independent effect of testosterone on AD. We found a significant reduction in brain plaque formation, improved cognitive function and increase NEP activity in male APP23/Ar+/- mice compared with age-matched male APP23 controls. In addition, we found, for the first time, a reduction of beta-secretase (BACE1) enzyme activity, mRNA level and protein expression in the male APP23/Ar+/- mice, suggesting that endogenous testosterone, independent from estrogen, may protect against AD in males via two major mechanisms, downregulation of BACE1 activities at transcriptional level to reduce beta amyloid production and upregulation of NEP activities to enhance bate amyloid degradation.

Testosterone and estrogen are essential for male behaviors in vertebrates. How these two signaling pathways interact to control masculinization of the brain and behavior remains to be established. Circulating testosterone activates the androgen receptor (AR) and also serves as the source of estrogen in the brain. We have used a genetic strategy to delete AR specifically in the mouse nervous system. This approach permits us to determine the function of AR in sexually dimorphic behaviors in males while maintaining circulating testosterone levels within the normal range. We find that AR mutant males exhibit masculine sexual and territorial displays, but they have striking deficits in specific components of these behaviors. Taken together with the surprisingly limited expression of AR in the developing brain, our findings indicate that testosterone acts as a precursor to estrogen to masculinize the brain and behavior, and signals via AR to control the levels of male behavioral displays.

Sex hormones are essential for neural circuit development and sex-specific behaviors. Male behaviors require both testosterone and estrogen, but it is unclear how the two hormonal pathways intersect. Circulating testosterone activates the androgen receptor (AR) and is also converted into estrogen in the brain via aromatase. We demonstrate extensive sexual dimorphism in the number and projections of aromatase-expressing neurons. The masculinization of these cells is independent of AR but can be induced in females by either testosterone or estrogen, indicating a role for aromatase in sexual differentiation of these neurons. We provide evidence suggesting that aromatase is also important in activating male-specific aggression and urine marking because these behaviors can be elicited by testosterone in males mutant for AR and in females subjected to neonatal estrogen exposure. Our results suggest that aromatization of testosterone into estrogen is important for the development and activation of neural circuits that control male territorial behaviors.

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic'' porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

Sex differences in song behavior and in the neural system controlling song in songbirds are well documented but relatively little is known about sex differences in hearing. We recently demonstrated the existence of sex differences in auditory brainstem responses in a songbird species, the zebra finch (Taeniopygia guttata). Many sex differences are regulated by sex steroid hormone action either during ontogeny or in adulthood. As a first step to test the possible implication of sex steroids in the control of sex differences in the zebra finch auditory system, we evaluated via immunocytochemistry whether estrogens are produced and act in the zebra finch inner ear. Specifically we examined the distribution of aromatase, the enzyme converting testosterone into an estrogen, and of estrogen receptors of the alpha subtype (ER alpha) in adult zebra finch inner ears. The anatomy of the basilar papillae was visualized by fluorescein-phalloidin, which delineated the actin structure of hair cells and supporting cells at their apical surface. Whole mount preparations of basilar papillae stained by immunocytochemistry revealed in both males and females an abundant aromatase distribution in the cytoplasm of hair cells, while ER alpha was identified in the nuclei of hair cells and of underlying supporting cells. Double-labeled preparations confirmed the extensive co-localization of aromatase and ER alpha in the vast majority of the hair cells. These results are consistent with studies on non-avian species, suggesting a role for estrogens in auditory function. These findings are also consistent with the notion that estrogens may contribute to a sex difference in hearing. To our knowledge, this is the first demonstration of the presence of aromatase and of the co-localization of aromatase and ER alpha in the sensory epithelium of the inner ear in any animal model. (C) 2009 Elsevier B.V. All rights reserved.

Exposure to estrogens during critical developmental periods and in adulthood affects sex differences in the brain. We examined the roles of estradiol (E2) and phytoestrogens. and their interactions, on potential sex differences in brain. We used aromatase knockout (ArKO) mice, which cannot produce endogenous estrogens, along with wild type (WT) littermates. Mice were gestated, raised and maintained on a diet either rich in phytoestrogens or a diet virtually void of soy-derived phytoestrogens. Adult males and females were gonadectomized and received implants filled with 17-beta-estradiol to induce progestin receptors (PR), while controls received empty implants. Mice were sacrificed five days later and brain sections containing the posterodorsal medial amygdala (MePD) were processed for PR immunoreactivity. Activation of sex differences in PR required adult E2 treatment. A diet high in phytoestrogens was required for expression of sex differences in PR after E2 treatment. Our data underscore the important contribution of dietary phytoestrogens for the development of sex differences in PR-it in the adult mouse medial amygdala. We hypothesize that both aromatization of androgens to estrogens and dietary sources of additional estrogens are part of the normal requirement for sex differences in the rodent brain. (c) 2009 Elsevier Inc. All rights reserved

The brain aromatase (oestrogen synthase) hypothesis predicts that oestrogen plays important roles in both sexual behaviours and brain sexual differentiation. To elucidate the functions of oestrogen in the brain, we generated aromatase knockout (ArKO) mice, which showed undetectable oestrogen and enhanced androgen levels in blood. These ArKO mice exhibited an enhanced appetite and disorders in sexual motivation, sexual partnership preference, sexual performance, aggressive behaviour, parental behaviour, infanticide behaviour and exploratory (anxiety) behaviour. We characterised the brain-specific promoter of the mouse aromatase gene, and identified several crucial cis-acting elements and the minimal essential promoter region for brain-specific expression. Next, we introduced a transgene of human aromatase, controlled by the minimal promoter region, into the ArKO mouse. The resulting mouse (ArKO/hArom), whose preoptic area, hypothalamus and amygdala were exposed to oestrogens only in the perinatal period, and then to enhanced androgens and no oestrogens in adulthood, showed near recovery from behavioural disorders. These results suggest that local oestrogens acting in specific brain regions are involved in the organisation of sex-specific neural networks during the perinatal period. Finally, we examined effects of oestrogens on gene expression within specific brain regions in mice during the perinatal period using DNA microarray analysis. This assay revealed both up-regulated and down-regulated brain-specific genes, including those related to neuronal function. Specifically, genes involved in energy metabolism, cell proliferation/apoptosis and secretory/transport system were altered in ArKO mice compared to wild mice. These results suggest that brain oestrogens participate in the sexual differentiation of the brain by influencing gene expression.

Introduction Peripherally localized aromatase, which converts circulating androgens into estrogens, is important in the pathogenesis of postmenopausal breast carcinomas. We have previously shown that aromatase mRNA levels are higher in elderly breast carcinomas (EldCa) than breast carcinomas of the control group (ContCa) or normal breast tissues. Aromatase expression has been reported to be regulated through the alternative use of multiple exons 1 (exons 1a-1f and so on); however, the preferential usage of exons 1 in elderly breast tissue has never been systematically examined. In order to properly treat and protect against EldCa, the regulation mechanism of aromatase expression in elderly breast tissues should be elucidated. The aim of the present study is to elucidate whether there are any specific patterns in use of multiple exons 1 in elderly breast tissue. Methods Usage of multiple exons 1 of the aromatase gene and mRNA levels of aromatase were examined by reverse transcription-polymerase chain reaction analysis in breast tissues of 38 elderly patients with breast cancer (age 80-99), and the results were compared with those in 35 patients of the control group (age 37-70). One-factor analysis of variance and the Scheffe test were used for the comparison of aromatase mRNA levels. Patterns of preferential utilization of multiple exons 1 of the aromatase gene were compared by chi(2) test for independence or Fisher exact test for independence using a contingency table. Results Exon 1d was utilized much more frequently in elderly tissue than in the control group irrespective of cancerous or normal tissue (EldCa, 36/38, 95% versus ContCa, 7/35, 20%, P < 0.0001; normal tissue of the elderly, EldNorm, 30/34, 88% versus normal tissue of controls, ContNorm, 2/29, 7%, P < 0.0001). Twenty EldCa (53%) and 12 EldNorm (35%) used both exons 1c and 1d; however, their dominance was reversed (EldCa, all 1d > 1c; EldNorm, all 1c > 1d). Conclusions Elderly breast tissues exhibited specific patterns in use of multiple exons 1, which at least partly explained the higher aromatase levels in EldCa. The mechanisms of how these specific patterns occur during aging and carcinogenesis should be further examined.

Human aromatase (CYP19) responsible for the conversion of androgens to estrogens is expressed not only in gonads and adrenals but also in many other tissues, including normal lungs and lung cancers. To investigate the involvement of CYP19 in lung cancer development, purified CYP19 protein and antibody are required. In this study, we have developed an efficient expression method of human aromatase in E. coli (> 1000 nmol/L culture). The protein purified from E. coli was used to raise an antibody against the human CYP19 in rabbits. The resulting antibody showed a high titer judged by ELISA, which allowed us to determine the expression of CYP19 in non-small cell lung cancer (NSCLC). Of 78 NSCLC specimens from Japanese patients, 50 (64%) NSCLC aberrantly expressed CYP19. This CYP19 expression in NSCLC was independent of any clinical and pathological parameters as well as the expression of other P450s, except tumor stage. The results suggest that the aromatase inhibitors might be useful for the management of non-small cell lung cancer in postmenopausal women.

Apocrine carcinoma of the breast, which frequently expresses oestrogen receptor-beta (ER-beta) in the absence of ER-alpha and only infrequently is treated endocrinologically, gives an opportunity to investigate the clinicopathological role of ER-beta in breast cancer independent of ER-alpha expression or tamoxifen treatment. Several isotypes of ER-beta, ER-beta 1-5 etc., have been identified thus far; however, the clinicopathological importance of each ER-beta isotype in breast cancer is still uncertain. Here we aimed to clarify the clinicopathological importance of ER-beta 1 and ER-beta cx (ER-beta 2) in apocrine carcinomas, immunohistochemically examining expressions of ER-beta 1 and ER-beta cx in 47 apocrine carcinomas. Positivity for ER-beta 1 and ER-beta cx was observed in 41 (87%) and 18 (38%) of 47 cases, respectively. ER-beta 1 positivity was related to smaller tumor size (P=0.0359), lower histological grade (P=0.0322), and higher disease-free survival (P<0.0001), whereas ER-beta cx status was related to none of these parameters. ER-beta 1 positivity was also associated with favorable clinical outcome in 24 so-called triple-negative (ER-alpha-negative/PR-negative/HER2-negative) apocrine carcinomas. ER-beta 1 itself, independent of ER-alpha expression and tamoxifen treatment, seems to have a tumor-suppressive effect, at least in apocrine carcinomas. Further study of ER-beta 1 is desired to optimize breast cancer treatment.

Female gender is a risk factor for drug-induced arrhythmias associated with QT prolongation, which results mostly from blockade of the human ether-a-go-go-related gene (hERG) channel. Some clinical evidence suggests that oestrogen is a determinant of the gender-differences in drug-induced QT prolongation and baseline QT(C) intervals. Although the chronic effects of oestrogen have been studied, it remains unclear whether the gender differences are due entirely to transcriptional regulations through oestrogen receptors. We therefore investigated acute effects of the most bioactive oestrogen, 17 beta-oestradiol (E2) at its physiological concentrations on cardiac repolarization and drug-sensitivity of the hERG (I-Kr) channel in Langendorff-perfused guinea pig hearts, patch-clamped guinea pig cardiomyocytes and culture cells over-expressing hERG. We found that physiological concentrations of E2 partially suppressed J(Kr) in a receptor-independent manner. E2-induced modification of voltage-dependence causes partial suppression of hERG currents. Mutagenesis studies showed that a common drug-binding residue at the inner pore cavity was critical for the effects of E2 on the hERG channel. Furthermore, E2 enhanced both hERG suppression and QT(C) prolongation by its blocker, E4031. The lack of effects of testosterone at its physiological concentrations on both of hERG currents and E4031-sensitivity of the hERG channel implicates the critical role of aromatic centroid present in E2 but not in testosterone. Our data indicate that E2 acutely affects the hERG channel gating and the E4031-induced QT(C) prolongation, and may provide a novel mechanism for the higher susceptibility to drug-induced arrhythmia in women.

The final step in the physiological synthesis of 17 estradiol (E-2) is aromatization of precursor testosterone by a CYP19 gene product, cytochrome P450 estrogen aromatase in the C19 steroid metabolic pathway. Within the central nervous system (CNS) the presence, distribution, and activity of aromatase have been well characterized. Developmental stage and injury are known modulators of brain enzyme activity, where both neurons and glial cells reportedly have the capability to synthesize this key estrogenic enzyme. The gonadal steroid E2 is a critical survival, neurotrophic and neuroprotective factor for dopaminergic neurons of the substantia nigra pars compacta (SNpc), the cells that degenerate in Parkinson's disease (PD). In previous studies we underlined a crucial role for the estrogenic status at the time of injury in dictating vulnerability to the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our ongoing studies address the contribution of brain aromatase and extragonadal E-2 as vulnerability factors for PD pathology in female brain, by exposing aromatase knockout (ArKO, -/-) female mice which are unable to synthesize estrogens to MPTP. Our initial results indicate that aromatase deficiency from early embryonic life significantly impairs the functional integrity of SNpc tyrosine hydroxylase-positive neurons and dopamine transporter innervation of the caudate-putamen in adulthood. in addition, ArKO females exhibited a far greater vulnerability to MPTP-induced nigrostriatal damage as compared to their Wt type gonadally intact and gonadectomized counterparts. Characterization of this novel implication of P450 aromatase as determining factor for PD vulnerability may unravel new avenues for the understanding and development of novel therapeutic approaches for Parkinson's disease. (c) 2007 Elsevier B.V. All rights reserved.

The roles of extragonadal estrogen in the skin are poorly understood, due to the lack of proper animal models. We examined the skin phenotypes of aromatase-knockout hairless (ArKO) mice and wild-type hairless (WT) mice, both of which were obtained through crossbreeding of Ar mice and hairless mice. Differences in the skins of ArKO and WT mice were compared with those of ovariectomized (OVX) and control (Sham) mice. A difference was observed in the skin tone of ArKO mice, which is pale white and differs from the pinkish tone of all other mice. However, both ArKO and OVX mice similarly exhibited deteriorations of skin properties as compared to their respective controls. Furthermore, all the deteriorations were similarly amplified by chronic UVB irradiation in both ArKO and OVX mice as compared to their respective controls. The unique skin phenotype of ArKO mice was observed in sunburn reactions. Specifically, skins of ArKO mice showed no reaction after an acute UVB irradiation at dose intensities caused sunburn in others. However, follow-up observation found delayed reactions associated with brownish skin color and swelling only in ArKO mice, thereby suggesting that the role of extragonadal estrogen may be connected with the protective reactions of skin. (c) 2007 Elsevier Ltd. All rights reserved.

Steroid hormones act on developing neural circuits that regulate the hypothalamic-pituitary-gonadal axis and are involved in hormone-sensitive behaviours. To test the hypothesis that developmental exposure to oestradiol (E-2) organises the quantity of adult oestrogen receptors (ER alpha and ER beta), we used male mice with a targeted mutation of the aromatase enzyme gene (ArKO) and their wild-type (WT) littermates. These mice are unable to aromatise testosterone to E-2, but still express both ER alpha and beta. To evaluate adult responsiveness to E-2, gonadectomised males were implanted with Silastic capsules containing E-2, or an empty implant, 5 days prior to sacrifice. Immunoreactivity for ER alpha and ER beta was quantified in the caudal ventromedial nucleus (VMN) and the medial preoptic area (POA). Regardless of genotype, adult treatment with E-2 reduced ER alpha-immunoreactive (ir) and ER beta-ir cell numbers in the POA, as well as ER beta-ir, but not ER alpha-ir, cell numbers in the VMN. Genotype, and thus endogenous exposure to E-2, produced opposite effects on ER expression in the two brain areas. In the VMN, ArKO males had more ER alpha-ir and ER beta-ir cells than did WT males. In the POA, ArKO males had fewer ER alpha-ir and ER beta-ir cells than did WT males. Thus, numbers of immunoreactive neurones containing both ERs in the adult ArKO male were enhanced in the POA, but decreased in the VMN, and most likely these patterns were established during the developmental critical period. Furthermore, although both ER alpha and beta-ir cell numbers are altered by the disruption of the aromatase gene, ER beta is altered in a more robust and region-specific manner.

Neurosteroids are synthesized de novo from cholesterol in the brain. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently, we identified the Purkinje cell as an active neurosteroidogenic cell. In rodents, this neuron actively produces several neurosteroids including estradiol during neonatal life, when cerebellar neuronal circuit formation occurs. Estradiol may be involved in cerebellar neuronal circuit formation through promoting neuronal growth and neuronal synaptic contact, because the Purkinje cell expresses estrogen receptor-beta (ER beta). To test this hypothesis, in this study we examined the effects of estradiol on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using neonatal wild-type (WT) mice or cytochrome P450 aromatase knock-out (ArKO) mice. Administration of estradiol to neonatal WT or ArKO mice increased dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell. In contrast, WT mice treated with tamoxifen, an ER antagonist, or ArKO mice exhibited decreased Purkinje dendritic growth, spinogenesis, and synaptogenesis at the same neonatal period. To elucidate the mode of action of estradiol, we further examined the expression of brain-derived neurotrophic factor ( BDNF) in response to estrogen actions in the neonate. Estrogen administration to neonatal WT or ArKO mice increased the BDNF level in the cerebellum, whereas tamoxifen decreased the BDNF level in WT mice similar to ArKO mice. BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth. These results indicate that estradiol induces dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell via BDNF action during neonatal life.

Objective: To assess the effect of aromatase inhibitors with GnRH agonist for a severe symptomatic adenomyosis that is refractory to GnRH agonist and danazol with GnRH agonist. Design: Case report. Setting: Clinical practice in university hospital. Patient(s): A 34-year-old woman with a complaint of severe dysmenorrheal, symptomatic anemia, and a desire to retain fertility. Intervention(s): Aromatase inhibitor anastrozole given orally (1.0 mg or 2.0 mg daily) for 16 weeks and GnRH agonist given monthly (injected SC, 1.8 mg) for 4 months. Main Outcome Measure(s): Measurements of uterine volume and levels of serum E 2, estrone, A, dehydroepiandrosterone sulfate, LH, FSH, and CA125. Result(s): Uterine volume was reduced. The reduction rate of uterine volume estimated by magnetic resonance imaging and ultrasonography was 60% after 8 weeks of treatment. Conclusion(s): Aromatase inhibitor with GnRH agonist therapy was useful for the management of a severely adenomyotic woman whose desire was for conservative treatment. (Fertil Steril (R) 2007;87:1468.e9-12. (C) 2007 by American Society for Reproductive Medicine.)

Aims: Apocrine carcinoma of the breast seldom expresses oestrogen receptors (ER) or progesterone receptors (PR), but frequently expresses androgen receptors (AR). Because of this unusual hormone receptor status, it has been suggested that oestrogens have a less important role in the pathogenesis of apocrine carcinoma. The ER status of apocrine carcinoma has been studied for one kind of ER, the classic receptor now named ER-alpha; however, the status of ER-beta, a secondary oestrogen receptor, has not been examined systematically in apocrine carcinoma. The aim was to study ER-beta status in apocrine carcinoma. Methods and results: The expression of ER-beta was examined immunohistochemically in 48 apocrine carcinomas and compared with clinicopathological factors and ER-alpha, PR and AR status. ER-beta positivity was observed in 35 cases (73%), regardless of any clinicopathological factors or the status of other receptors. The results of ER-beta mRNA analysis supported the immunohistochemical results. Conclusions: The significance of oestrogens in apocrine carcinoma should not be dismissed at present when the role of ER-beta remains to be determined. Studying the action of oestrogen or antioestrogen in apocrine carcinoma may reveal a role for ER-beta independent of ER-alpha and raise the potential of hormonal therapy for these tumours.

High doses of estradiol (E-2) can impair spatial learning in the Morris water maze, in ovariectomized mice, but the same dose has no effect on adult castrated males. Here, we test the hypothesis that this sex difference is caused by neonatal actions of E-2. In Experiment 1, C57BL/6J pups were given daily estradiol benzoate (EB) or oil injections from the day of birth until postnatal Day 3. Adults were gonadectomized and received EB (s.c.) or oil 28 h before the first day of training, and 4 h before each of four daily training sessions on the Morris water maze. Females given oil as neonates, and EB prior to training displayed the poorest performance. Females that received EB as neonates and EB prior to training were insensitive to the deleterious effects of adult EB and performed better than males given the same hormone treatments. We conducted a second experiment using aromatase enzyme knockout (ArKO) mice. Adult male and female ArKO and wild-type (WT) littermates were gonadectomized and received either injections of oil or EB prior to and during water maze training (as described above). Hormone treatment failed to affect performance, yet, female but not male ArKO mice showed impaired learning compared to WT littermates. Thus, exposure to estradiol during neonatal development can counteract the deleterious effects of EB on adult spatial learning. (c) 2006 Elsevier Inc. All rights reserved.

Steroid receptors such as the androgen and estrogen receptors require the presence of several proteins, known as coactivators, to enhance the transcription of target genes. The first goal of the present study was to define the role of SRC-1 on the steroid-dependent expression of the aromatase protein and its activity in male Japanese quail. The second goal was to analyze the rapid plasticity of the POM following antisense treatment interruption. We confirm here that the inhibition of SRC-I expression by daily intracerebroventricular injections of locked nucleic acid antisense oligonucleotides in the third ventricle at the level of the preoptic area-hypothalamus (HPOA) significantly reduces testosterone-dependent male sexual behavior. In the first experiment, aromatase protein expression in HPOA was inhibited in SRC-1-depleted males but the enzymatic activity remained at the level measured in controls. We observed in the second experiment a recovery of the behavioral response to testosterone treatment after interruption of the antisense injection. However, several morphological characteristics of the POM were not different between the control group, the antisense-treated birds and antisense-treated birds in which treatment had been discontinued 3 days earlier. Antisense was also less effective in knocking-down SRC-1 in the present experiments as compared to our previous study. An analysis of this variation in the degree of knock-down of SRC-1 expression suggests dissociation among different aspects of steroid action on brain and behavior presumably resulting from the differential sensitivity of behavioral and neurochemical responses to the activation by testosterone and/or its estrogenic metabolites. (c) 2006 Elsevier B.V. All rights reserved.

No definitive information is yet available on the steroidogenic capacity of the two morphologically distinct cell types forming the bovine trophoblast, the uninucleated trophoblast cells (UTCs) and the trophoblast giant cells (TGCs). Hence, in order to localise 17 alpha-hydroxylase-C17,20-lyase (P450c17) on a cellular level and to monitor its expression as a function of gestational age, placentomes from pregnant (days 80-284; n = 19), prepartal (days 273-282; 24-36h prior to the onset of labour; n = 3) and parturient cows (n = 5) were immunostained for P450c17 using an antiserum against the recombinant bovine enzyme. At all stages investigated, P450c17 was exclusively found in the UTCs of chorionic villi (CV), where staining was ubiquitous between days 80 and 160, but was largely restricted to primary CV and the branching sites of secondary CV between days 160 and 240. Thereafter, a distinct ubiquitous staining reoccurred in the UTCs of all CV in late pregnant, prepartal and parturient animals. Using an antiserum against human aromatase cytochrome P450 (P450arom), specific cytoplasmic staining was observed in TGCs. In placentomes from pregnant cows, staining intensity was higher in mature compared with immature TGCs and was more pronounced in the trophoblast covering big stem villi compared with the trophoblast at other sites of the villous tree. In placentomes of a parturient cow, specific staining was only found in mature TGCs that survived the normal, but substantial, prepartal decline in TGC numbers. These results clearly showed that bovine UTCs and TGCs exhibit different steroidogenic capacities, constituting a 'two-cell' organisation for oestrogen synthesis. P450c17 expression appears to be quickly down-regulated and P450arom is up-regulated when UTCs enter the TGC differentiation pathway.

Context: Aromatase, steroid sulfatase, and 17 beta-hydroxysteroid dehydrogenase type 1 (HSD-1) peripherally up-regulate, whereas estrogen sulfotransferase ( EST) and HSD-2 down-regulate, the synthesis of active and more potent estrogens. These estrogen-metabolizing enzymes (EMEs) are important in postmenopausal breast cancers, but have never been systematically examined in breast cancers of the elderly. Objective and Design: mRNA levels of EMEs in cancerous and normal breast tissues from 39 elderly patients (age, 80-99 yr) were compared with those from 39 controls (age, 37-70 yr) or compared according to estrogen (ER)/progesterone (PR) receptor status. Results: Aromatase levels were higher in cancers of the elderly (EldCa) than in normal tissue of the elderly (P = 0.0008) or cancers of controls (P = 0.0033). In contrast, levels of steroid sulfatase and EST were higher in cancers of controls than normal tissue of controls (P = 0.0046 and P < 0.0001, respectively) or EldCa (P < 0.0001 and P < 0.0001, respectively). Levels of HSD-1 and HSD-2 did not differ significantly between any two of the categories. Among EldCa, HSD-1 levels were higher in ER/PR-positive than in ER/PR-negative carcinomas, whereas EST and HSD-2 exhibited opposite results. Conclusions: The importance of aromatase is relatively increased in EldCa. ER/ PR-positive EldCa exhibited a pattern of EMEs more beneficial to the production of estrogen than did ER/PR-negative EldCa. The specific pattern exhibited in EldCa may elucidate the role of EMEs in the absence of ovarian estrogens in the pathogenesis of breast cancer.

Much evidence indicates that women have a higher risk of developing Alzheimer's disease (AD) than do men. The reason for this gender difference is unclear. We hypothesize that estrogen deficiency in the brains of women with AD may be a key risk factor. In rapidly acquired postmortem brains from women with AD, we found greatly reduced estrogen levels compared with those from age- and gender-matched normal control subjects; AD and control subjects had comparably low levels of serum estrogen. We examined the onset and severity of AD pathology associated with estrogen depletion by using a gene-based approach, by crossing the estrogen-synthesizing enzyme aromatase gene knockout mice with APP23 transgenic mice, a mouse model of AD, to produce estrogen-deficient APP23 mice. Compared with APP23 transgenic control mice, estrogen-deficient APP23 mice exhibited greatly reduced brain estrogen and early-onset and increased beta amyloid peptide (A beta) deposition. These mice also exhibited increased A beta production, and microglia cultures prepared from the brains of these mice were impaired in A beta clearance/degradation. In contrast, ovariectomized APP23 mice exhibited plaque pathology similar to that observed in the APP23 transgenic control mice. Our results indicate that estrogen depletion in the brain may be a significant risk factor for developing AD neuropathology.

We found that both benzyl isothiocyanate (ITC) and phenyl ITC inhibited respiration in the mitochondria in an electrophilic reaction-dependent manner. ITG induced mitochondrial swelling and cytochrome c release were prevented by cyclosporin A, indicating that they are mediated through the ITC moiety-dependent reaction to critical thiol groups for the opening of membrane permeability transition-dependent pores.

Dioxins exert a variety of adverse effects on organisms, including teratogenesis, immunosuppression, tumor promotion, and estrogenic action. Studies using aryl hydrocarbon receptor (AhR)-deficient mice suggest that the majority of these toxic effects are mediated by the AhR. In spite of the adverse effects mediated by this receptor, the AhR gene is conserved among a number of animal species, ranging from invertebrates to vertebrates. This high degree of conservation strongly suggests that AhR possesses an important physiologic function, and a critical function is also supported by the reduced fertility observed with AhR-nuil female mice. We demonstrate that AhR plays a crucial role in female reproduction by regulating the expression of ovarian P450 aromatase (Cyp19), a key enzyme in estrogen synthesis. As revealed by in vitro reporter gene assay and in vivo chromatin immunoprecipitation assay, AhR cooperates with an orphan nuclear receptor, Ad4BP/SF-1, to activate Cyp19 gene transcription in ovarian granulosa cells. Administration to female mice of an AhR ligand, DMBA (9,10-dimethyl-1,2-benzanthracene), induced ovarian Cyp19 gene expression, irrespective of the intrinsic phase of the estrus cycle. In addition to elucidating a physiological function for AhR, our studies also suggest a possible mechanism for the toxic effects of exogenous AhR ligands as endocrine disruptors.