原田 信広

The effect of a SNP in exon 10 of CYP19 on tumor mRNA levels and splice variants were studied and correlated with clinical parameters and risk of breast cancer. In the vast majority of breast cancers, the estrogen levels modulate the tumor growth and depend on the activity of CYP19, Patients (n=481) and controls (n=236) were genotyped by T-tracks in a single sequencing reaction (SSR), The frequency of TT genotypes was significantly higher in patients versus controls (P=0.007) particularly among those with stage III and IV disease (P=0.004) and with tumors larger than 5 cm (P=0.001), A significant association between presence of the T allele and the level of aromatase mRNA in the tumors was observed (P=0.018), as well as with a switch from adipose promoter to ovary promoter (P=0.004). Previously, we reported a rare polymorphic allele of CYP19 (repeat (TTTA)(12)) to be significantly more frequent in breast cancer patients than in controls, Here we describe another polymorphism, a C-T substitution in exon 10 of the CYP19 gene which is in strong linkage disequilibrium with the (TTTA)n polymorphism but with higher frequency of the variant allele, Our data suggest that the T-allele of the CYP19 gene is associated with a 'high activity' phenotype.

The present study investigates the expression patterns of 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isozymes in human fetal tissues to understand how estrogenic activity is regulated in the human fetus. Using enzyme assay, high 17 beta HSD activity was detected in the placenta and liver, and low levels of 17 beta HSD activity were also present in the gastrointestinal tract and kidney. After Northern blot analysis, we detected the messenger ribonucleic acid for 17 beta HSD type 1 (17 beta HSD1) only in the placenta, whereas that for 17 beta HSD type 2 (17 beta HSD2) was detected in the placenta, Liver, gastrointestinal tract, and urinary tract at 20 gestational weeks. In RT-PCR analysis of the messenger ribonucleic acid transcripts, 17 beta HSD 1 was predominantly expressed in the placenta, brain, heart, lung, and adrenal, whereas 17 beta HSD2 expression was predominantly detected in the liver, gastrointestinal tract, and kidney. In addition, we detected 17 beta HSD2 immunoreactive protein in surface epithelial cells of the stomach, absorptive epithelial cells of the small intestine and colon, hepatocytes of the liver, and interstitial cells surrounding the urinary tubules of the renal medulla. 17 beta HSD2 in these tissues may be functioning in the prevention of in utero exposure of the fetus to excessive estradiol from the maternal circulation and amniotic fluids.

The change in hepatic antioxidant defense system with the development of alpha-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). Liver injury with cholestasis did not occur 12 h after ANIT injection, but appeared at 24 h, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Liver lipid peroxide content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Liver superoxide dismutase and catalase activities decreased 24 and 48 h, respectively, after ANIT injection, although the catalase activity increased at 12 h, but these decreases were attenuated at 72 h. Liver Se-glutathione peroxidase activity remained unchanged 24, 48, and 72 h after ANIT injection, although the activity increased at 12 h. Liver reduced glutathione content increased 24 h after ANIT injection, but the increase was reduced time dependently thereafter. Liver ascorbic acid content increased 12 h after ANIT injection and further increased at 24 h, but the increase was reduced time dependently thereafter. These results indicate that the change in hepatic antioxidant defense system occurs before and with the development of ANIT-induced liver injury in rats, and suggest that the reduction of hepatic antioxidant defense system mediated by SOD and catalase could contribute to the liver injury development through an enhancement of hepatic lipid peroxidation. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

We identified a new point mutation in the CYP19 gene responsible for aromatase (P450(arom)) deficiency in a 46,XY male infant with unremarkable clinical findings at birth. This boy is homozygote for a l-bp (C) deletion in exon 5 of the aromatase gene causing a frame-shift mutation. The frame-shift results in a prematurely terminated protein that is inactive due to the absence of the functional regions of the enzyme. Aromatase deficiency was suspected prenatally because of the severe virilization of the mother during the early pregnancy, and the diagnosis was confirmed shortly after birth. Four weeks after birth, the baby boy showed extremely low levels of serum estrogens, but had a normal level of serum free testosterone; in comparison with the high serum concentration of androstenedione at birth, a striking decrease occurred by 4 weeks postnatally. We previously reported elevated basal and stimulated FSH levels in a female infant with aromatase deficiency in the first year of life. In contrast, in the male infant, basal FSH and peak FSH levels after standard GnRH stimulation tests were normal. This finding suggests that the contribution of estrogen to the hypothalamic-pituitary gonadotropin-gonadal feedback mechanism is different in boys and girls during infancy and early childhood. In normal girls, serum estradiol concentrations strongly correlate with circulating inhibin levels, and thus, low inhibin levels may contribute to the striking elevation of FSH in young girls with aromatase deficiency. In contrast, estradiol levels are physiologically about a 7-fold lower in boys than in girls, and serum inhibin levels remain elevated even though levels of FSH, LH, and testosterone are decreased.

A new cis-element, trophoblast-specific element 2 (TSE2) is located in the placenta-specific enhancer of the human aromatase gene that dictates its tissue-specific expression, In the minimum enhancer region, an element similar to the trophoblast-specific element (TSE), originally described for the human chorionic gonadotropin alpha-subunit gene, also exists (Yamada, K., Harada, N., Honda, S., and Takagi, Y. (1995) J. Biol. Chem, 270, 25064-25069). The co-presence of TSE and TSE2 is required to direct trophoblast-specific expression driven by a heterologous thymidine kinase promoter. A 2562-base pair cDNA clone encoding a 436-amino acid protein that binds to TSE2 was isolated from a human placental cDNA library using a yeast one-hybrid system with the TSE2 as a reporter sequence, The protein was revealed to be identical to hGCMa, a mammalian homologue of the Drosophila GCM (glia cells missing) protein. Expression of hGCMa is restricted to the placenta, The protein also binds to PLE1 in the leptin promoter among other cis-elements reported to confer placenta-specific expression, suggesting that hGCMa is a placenta-specific transcription regulator, possibly involved in the expression of multiple placenta-specific genes.

The sulfatase pathway has been thought to be a primary means of local production of estrone in human breast cancer tissue. We measured steroid sulfatase (STS) mRNA levels in 97 breast cancers and evaluated its association with disease-free survival. High levels of STS mRNA proved to be a significant predictor of reduced relapse-free survival, both as a continuous variable (log STS mRNA P = 0.028) and as a dichotomous variable with an optimized cutoff point (P = 0.002). In multivariate analysis a high level of STS mRNA was an independent factor for predicting relapse-free survival. These results suggest a putative role of STS in breast cancer growth and metastasis, and administration of sulfatase inhibitors to breast cancer patients with high levels of STS mRNA might be an additional treatment option.

The effects of two steroidal (4-hydroxyandrostenedione and atamestane) and three non-steroidal (fadrozole, vorozole, and pentrozole) aromatase inhibitors on the levels of aromatase mRNA and protein were examined in vitro and in vivo. Immunocytochemistry revealed increased quantities of immunoreactive aromatase in human choriocarcinoma-derived JEG-3 cells in response to pretreatment with the non-steroidal inhibitors. To elucidate this effect in detail, aromatase protein in JEG-3 cells after treatment with various inhibitors was quantified using an enzyme-linked immunosorbent assay (ELISA). A time-dependent increase in aromatase protein in the cells was observed with all the aromatase inhibitors except 4-hydroxyandrostenedione, whereas aromatase mRNA levels in the cells remained unchanged during the inhibitor treatment. The three non-steroidal agents caused an approximately fourfold increase in aromatase protein in the cells 24 h after the treatment, as compared with untreated controls. The increase in aromatase protein in the cells was not blocked by treatment with cycloheximide, an inhibitor of protein synthesis. The inhibitors also appeared to block the rapid degradation observed in JEG-3 cells after induction by forskolin. In vivo, daily injection of the inhibitors into adult female mice caused increases in levels of both aromatase mRNA and protein in the ovary. The increase in aromatase mRNA in this in vivo study could be explained by an increase in gonadotropin concentrations in response to decreased plasma concentrations of estrogens. In conclusion, we suggest that aromatase inhibitors increase aromatase protein through stabilization and reduced protein turnover.

The atheroprotective effects of estrogen are well established and the presence of an estrogen receptor in vascular tissues has recently been reported. Therefore, we investigated the localization of the estrogen-producing enzyme aromatase in vascular tissues to assess the possible contribution of endocrine, paracrine, and autocrine modes of action. Aromatase was found in human vascular smooth muscle cells (SMCs) but not in endothelial cells on in situ hybridization. These observations were further supported by quantitative analysis of aromatase mRNA and the activity in 15 human vascular specimens. Only trace levels of expression we:re detected in the 3 infants examined, whereas 0.0088 to 0.0806 amol/mu g RNA of aromatase mRNA and 12.9 to 122.3 fmol.h(-1).mg(-1) protein of the activity were detected in 12 of the adult individuals. The switching of tissue-specific exon 1 of the human aromatase gene was also observed in some cases. Aromatase was found to be expressed only in cultured SMCs and not in cultured endothelial cells of human aorta and pulmonary artery and to be regulated through dexamethasone and the signaling pathways of protein kinase A and C. Study results revealed the localized expression of aromatase in vascular SMCs, which indicated a possible direct action of locally produced estrogen in an autocrine or paracrine manner, with possible cross talk between smooth muscle and endothelial cells.

Among sex steroids, especially estrogen metabolism has been considered to play a role in the function and pathology of human veins. We investigated the expression and activity of the estrogen-producing enzyme aromatase and estrogen receptor (ER) in human vena cava to assess possible in situ biosynthesis of estrogens and their modes of action. We first examined aromatase expression by immunohistochemistry in human inferior vena cava obtained from 29 autopsy cases (11 males, 18 females, 63.6+/-3.0 years old). We then semiquantitated the level of aromatase mRNA by reverse transcriptase-polymerase chain reaction in 24 cases and aromatase activity by H-3-water assay in 15 cases to examine whether or not and in which cell types aromatase was expressed. We also studied alternative use of multiple exon Is of its gene and immunolocalization of 17 beta-hydroxysteroid dehydrogenase type I (17 beta-HSD I), which converts estrone produced by aromatase to estradiol, a biologically active estrogen and ER. Aromatase and 17 beta-HSD I immunoreactivity were both detected in smooth muscle cells (SMC) of the media in all the cases and in endothelial cells (EC) in 20 and 22 cases, respectively. ER immunoreactivity was detected in SMC of vena cava in 21 cases. The amount of aromatase mRNA was significantly greater in the cases utilizing 1c (I.3) or Id (P.II) of exon 1 (9 cases, 191.1+/-26.3 attomol/ng total RNA) than those utilizing 1b (I.4) as the promoter (14 cases, 50.6+/-13.0 attomol/ng total RNA) (p < 0.01). Significant correlation (p < 0.05) was observed between the amount of aromatase mRNA and aromatase activity in 15 cases examined. No significant correlation was detected between the amount of aromatase mRNA or aromatase labeling index and the ER status. These results suggest that estrone and estradiol are produced in the human vena cava and that their production is mediated by aromatase and 17 beta-HSD I, respectively but not all of these locally synthesized estrogens may not work directly in situ.

Among multiple exons 1 of the mouse aromatase gene, brain-specific exon 1 is only utilized in the hypothalamus and amygdala regions. In this study, identification of the promoter region necessary for basal transcription of the aromatase gene in the brain was undertaken. Deletions of various lengths were introduced into the overall promoter region, which was fused to the chloramphenicol acetyltransferase gene. The resulting reporters were transfected into cultured neurons from the diencephala of fetal mouse brains on embryonic day 13 and then their CAT mRNA levels were determined. The reporter plasmid containing the promoter region 202 bp upstream from the transcriptional initiation site gave the greatest expression. Then binding of trans-acting factors in a nuclear extract of the diencephala to the - 202 bp promoter region was investigated by DNase I footprint analysis, multiple protected areas, referred to as Arom-A alpha, A beta, A gamma, B and C, being found. Gel shift assays, performed with oligonucleotides corresponding to the protected areas, showed that nuclear DNA binding factors form specific complexes exhibiting different mobilities. Substitution in the Arom-A alpha or -B sequence in the promoter region in the CAT reporters decreased the CAT mRNA expression levels to about one-fifth the wild type one. These results suggest that multiple nuclear factors bound to the core promoter region participate in the expression of the aromatase gene in mouse brain neurons. (C) 1999 Elsevier Science B.V. All rights reserved.

An association between lipid peroxidation and alpha-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). The severity of liver injury was estimated 12, 24, 48, and 72 h after ANIT injection. Liver injury appeared 24 h after ANIT injection, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Serum lipid peroxide (LPO) concentration increased 24 h after ANIT injection and further increased at 48 h, but this increase was attenuated at 72 h. In contrast, liver LPO content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Similarly, myeloperoxidase (MPO) activity, an index of neutrophil infiltration, in the liver tissue increased 12 h after ANIT injection and further increased at 24 and 48 h, but this increase was attenuated at 72 h. Either serum LPO concentration or liver LPO content was significantly correlated with liver MPO activity (r = 0.661 for serum LPO concentration; r = 0.585 for liver LPO content). These results suggest that lipid peroxidation might be associated with ANIT-induced liver injury in rats and that this lipid peroxidation might occur via oxygen radicals derived from neutrophils infiltrated into the liver tissue of ANIT-intoxicated rats. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

About 28% of patients with the Crow-Fukase syndrome exhibit glucose intolerance which may be induced by low serum levels of dehydroepiandrosterone (DHEA). We report a patient with the Crow-Fukase syndrome who exhibited non-insulin dependent diabetes mellitus (NIDDM) worsened prior to admission. He received the DHEA sulfate (DHEA-S) infusion test to evaluate aromatase activity. This patient exhibited an increase in aromatase activity measured by the conversion of the intravenously loaded DHEA-S to estrogen, and low serum levels of DHEA and DHEA-S, These abnormalities returned to nearly normal during the administration of prednisolone, 60 mg per day. No adverse effect on his diabetes was observed during the corticosteroid treatment. Five control patients with diabetes but without the Crow-Fukase syndrome showed no increase in the conversion of DHEA-S to estrogen, which suggests that aromatase activity is normal in diabetes. The increase in aromatase activity in our patient may have led to a low serum concentration of DHEA that in turn caused glucose intolerance and a deterioration of the diabetes prior to admission. Glucocorticoid therapy may be beneficial in Crow-Fukase syndrome to improve the distorted metabolism of DHEA with no adverse effect on the diabetes.

Steroid sulfatase (STS) hydrolyzes several sulfated steroids such as estrone sulfate, dehydroepiandrosterone sulfate, and cholesterol sulfate. Zn the present study, we have measured STS mRNA levels in 97 breast cancers by reverse transcription-PCR using a fluorescent primer in the presence of an internal standard RNA and evaluated its association with disease-free and overall survival. The median value was 728.0 amol/ng RNA (range, 0-11,778 amol/ng RNA), Levels were significantly higher in tumors demonstrating lymph node metastasis than in those without nodal involvement (P = 0.033) and in patients who experienced a recurrence during the follow-up period (mean, 40.8 months; median, 39 months) compared with those with no evidence of further disease (mean, 49.2 months; median, 48 months; P = 0.029), No significant associations were found between STS mRNA expression and age, menopausal status, tumor size, histological grade, estrogen receptor status, or postoperative adjuvant therapy. High levels of STS mRNA proved to be a significant predictor of reduced relapse-free survival as a continuous variable (log STS mRNA; P = 0.028). As a dichotomous variable with an optimized cutoff point of 1,240 amol/ng RNA, expression was also associated with a significantly shorter relapse-free survival rate (P = 0.002), but no significant correlation was found between the STS mRNA level and overall survival, Expression was found to be an independent factor for predicting relapse-free survival on multivariate analysis. The results thus support a putative role of STS in breast cancer growth and metastasis.

Estrogen is a major mitogenic stimulus to established breast cancer. Estrogen sources include ovarian, extraglandular sites and breast tissue. Which source primarily maintains benign and breast cancer tissue estrogen concentrations remains unclear. While macrophages may comprise up to 50% of the mass of breast carcinomas, previous studies neglected to study them as possible sources of estrogen. We present evidence that breast macrophages constitute an in situ source of estradiol and that the amount produced is sufficient to mediate cellular proliferation. We utilized immunohistochemistry and RT-PCR to study cell-specific aromatase expression in (i) 29 breast biopsies, (ii) human monocytes/macrophages and (iii) a myeloid cell, line (THP-1) capable of differentiating into macrophages. Use of a breast cancer cell line (MCF-7) provided biologic confirmation of the role of aromatization in cell proliferation. We demonstrated considerable amounts of immunoreactive-aromatase (irARO) in breast tissue macrophages and a positive correlation between the proportion of irARO present in macrophages and lesion severity. Using in vitro techniques, we demonstrated that monocytes and THP-1 cells require differentiation into macrophages to produce aromatase in amounts approaching placental levels. The amount of estrogen produced by THP-1 cells stimulated MCF-7 cells to proliferate, an effect blocked by aromatase inhibitors. Estrogen production by macrophages in breast tissue appears sufficient to stimulate the proliferation of adjacent epithelial cells and to autoregulate cytokine production. These findings represent a new dimension of cellular regulation in breast tissue with major biologic implications, amenable to pharmacological manipulation. (C) 1999 Elsevier Science Ltd. All rights reserved.

Aromatase is known to be involved in the control of morphological, neuroendocrine, and behavioral sex differences in the brain. To study the detailed functions of aromatase in mouse brain, we generated aromatase-deficient mice that lack exons 1 and 2 and the proximal promoter region of the cyp19 gene by homologous recombination. The genotypes of offspring from heterozygous matings conformed to the Mendelian rule. The aromatase knockout (ArKO) male and female mice displayed no significant external morphological abnormalities, although the uteri of adult ArKO female mice were remarkably underdeveloped. Male-typical sexual behavior directed toward females was, however, strongly modified in the ArKO male mice. The ArKO male mice showed significantly prolonged latencies to mount and decreased numbers of mounts in response to receptive stimulus females. These results suggest an important participation of the aromatase gene in the reproductive behaviors. (C) 1998 Academic Press.

Ad4BP/SF-1 is a transcription factor essential for the development of the adrenal gland and the gonads as well as for the maintenance of their functions through regulating tissue-specific gene transcription. In the whole body, hypothalamo-pituitary-gonadal and -adrenal axes are known to play prominent roles in mediating the function of the gonads and adrenal, In this study, the effects of the tropic peptide hormones secreted by the pituitary on the regulation of the rat Ftz-F1 (rFtz-F1) gene encoding Ad4BP/SF-1 were investigated, Immunochemical studies revealed that Ad4BP/SF-1 was expressed even in the adrenal cortex of hypophysectomized rats, Such persistent expression of Ad4BP/SF-1 was also observed in the testes and ovaries of the hypophysectomized animals. In contrast to Ad4BP/F-1, the expressions of steroidogenic P450s were reduced significantly. The transcriptional activities of the endogenous and transfected rFtz-F1 genes were examined with Y-1 and I-10 cells derived from mouse adrenocortical and testicular Leydig cell tumors, respectively, Neither gene appeared to be activated significantly by cAMP, whereas both endogenous and exogenous CYP11A genes encoding P450(SCC) were activated. Taken together, these observations indicate that the expression of the rFtz-F1 gene is mainly regulated by a mechanism independent of the neuroendocrine axes.

The anatomical distribution and seasonal variations in aromatase activity and in the number of aromatase-immunoreactive cells were studied in the brain of free-living male pied flycatchers (Ficedula hypoleuca). A high aromatase activity was detected in the telencephalon and diencephalon but low to negligible levels were present in the optic lobes, cerebellum, and brain stem. In the diencephalon, most aromatase-immunoreactive cells were confined to three nuclei implicated in the control of reproductive behaviors: the medial preoptic nucleus, the nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus. In the telencephalon, the immunopositive cells were clustered in the medial part of the neostriatum and in the hippocampus as previously described in another songbird species, the zebra finch. No immunoreactive cells could be observed in the song control nuclei. A marked drop in aromatase activity was detected in the anterior and posterior diencephalon in the early summer when the behavior of the birds had switched from defending a territory to helping the female in feeding the nestlings. This enzymatic change is presumably controlled by the drop in plasma testosterone levels observed at that stage of the reproductive cycle. No change in enzyme activity, however, was seen at that time in other brain areas. The number of aromatase-immunoreactive cells also decreased at that time in the caudal part of the medial preoptic nucleus but not in the ventromedial nucleus of the hypothalamus (an increase was even observed), suggesting that differential mechanisms control the enzyme concentration and enzyme activity in the hypothalamus. Taken together, these data suggest that changes in diencephalic aromatase activity contribute to the control of seasonal variations in reproductive behavior of male pied flycatchers but the role of the telencephalic aromatase in the control of behavior remains unclear at present. (C) Academic Press.

A number of studies have been devoted to the analysis of the anatomical distribution, control by steroids and functional significance of aromatase (the enzyme metabolizing testosterone into 17 beta-estradiol) in the quail brain. In particular, the sexually dimorphic nucleus preopticus medialis has been the main focus of investigation because testosterone aromatization in this structure mediates the activation of male sexual behavior and aromatase activity is itself testosterone-dependent in this nucleus. No information on the anatomical distribution of aromatase gene expression is, however, available so far in this avian species. In the present study we applied a non-radioactive in situ hybridization technique to describe the distribution of aromatase mRNA containing neurons in the quail prosencephalon. We also analyzed, at a neuronal level of resolution, the induction by testosterone of this mRNA in the medial preoptic nucleus. Dense clusters of aromatase gene expressing neurons were observed within the medial preoptic nucleus, the nucleus of the stria terminalis, the ventro-medial hypothalamus and the tuberal region. Scattered neurons expressing lower levels of aromatase mRNA were also found in the dorsal thalamic area and central gray. The specificity of the staining was confirmed by demonstrating the absence of signal in sections that had been hybridized with a sense probe. Moreover, the distribution of the aromatase mRNA containing cells completely overlapped with the distribution of the aromatase-immunoreactive cells. Aromatase-mRNA expression was controlled by testosterone (or its metabolites) in the entire medial preoptic nucleus. Castration resulted in a decrease in the number of aromatase mRNA-containing cells and this effect was totally reversed by testosterone treatment. These data further support the idea that testosterone regulates the rate of its own aromatization by modulating the expression of aromatase rather than by acting at a post transcriptional lever. (C) 1998 Elsevier Science B.V. All rights reserved.

OBJECTIVE Oestrogen produced locally by aromatase is thought to participate in numerous biological functions in the adult central nervous system (CNS), However, little is known about aromatase expression in the human CNS, DESIGN We examined aromatase expression in human brain regions, (4 men, 2 women) obtained from autopsy, by reverse transcriptase (RT)-polymerase chain reaction (PCR) and also studied alternative use of multiple exons 1 of its gene, which is involved in tissue specific expression of aromatase in human. RESULTS The amount of aromatase mRNA determine by RT-PCR assay in 6 cases tended to be highest in pens, thalamus, hypothalamus and hippocampus. Analysis of multiple exons 1 revealed that If, considered specific for brain, as well as Ib (fibrolast type) and Id (gonadal type), were expressed, Id and If tended to be utilized in hypothalamus, thalamus and amygdala, The amount of overall mRNA expression was also higher in hypothalamus, thalamus and amygdala than in other regions of the brain, There were no differences of utilization of exons 1 and mRNA expression of aromatase between female and male brain, CONCLUSIONS These results demonstrate that aromatase is expressed widely in various regions of human brain tissues in both men and women.

The activation of male sexual behavior in Japanese quail (Coturnix japonica) requires the transformation of testosterone to 17 beta-estradiol by the enzyme aromatase (estrogen synthetase). There are prominent sex differences in aromatase activity that may be regulated in part by sex differences in catecholaminergic activity. In this study, we investigate, with double-label immunocytochemistry methods, the anatomical relationship between the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) and aromatase (ARO) in the quail brain. The immunoreactivity observed for each antigen generally matched the previously described distribution. One exception is the observation that cells weakly labeled for aromatase were found widely distributed throughout the telencephalon. The presence of telencephalic aromatase was confirmed independently by radioenzymatic assays. There was an extensive overlap between the distribution of the two antigens in many brain areas. In all densely labeled aromatase-immunoreactive (ARO-ir) cell groups, including the preoptic medial nucleus, nucleus of the stria terminalis, mediobasal hypothalamus, and paleostriatum ventrale, ARO-ir cells were found in close association with TH-ir fibers. These TH-ir fibers often converged on an ARO-ir cell, and one or more TH-ir punctate structure(s) were found in close contact with nearly every densely labeled ARO-ir cell. In the telencephalon (mostly the neostriatum), all TH-ir fibers were found to be part of fiber groups that surrounded weakly immunoreactive aromatase cells. The few cells exhibiting an intracellular colocalization were detected in the anteroventral periventricular nucleus. These results are consistent with the hypothesis that catecholaminergic inputs regulate brain aromatase. (C) 1998 Wiley-Liss, Inc.

The present study was designed to demonstrate localized aberrant expression of aromatase in primary and metastatic malignant liver tumors. Immunocytochemistry revealed the presence of locally increased aromatase protein in regions around tumors in all specimens from seven primary and seven metastatic liver tumors. This observation was further confirmed by Western blotting analysis and assay of aromatase activity in tumorous, proximal, and distal regions. Western blots showed most intensely immunoreactive bands at the position corresponding to aromatase in proximal tissues where aromatase activities also were higher (2.75 +/- 1.59 pmol/mg.h) than in tumors (0.137 +/- 0.115 pmol/mg.h) and distal tissues (1.90 +/- 1.47 pmol/mg.h), in spite of a gradient decline of NADPH-cytochrome P-450 reductase activity from the distal regions to the tumors. RT-PCR analysis indicated that the aberrant increase in aromatase protein and enzyme activity in the regions proximal to tumors is caused by locally elevated aromatase messenger RNA.

Aromatase (estrogen synthetase) catalyzes a key step in estrogen biosynthesis and plays an important role in reproductive processes in the ovary and placenta. In this context, aromatase is an enzyme producing estrogen as an endocrine female sex hormone. Recently, it has reported that aromatase is also present in various extra-gonadal tissues and is tissue- specifically regulated by various factors. This tissue-specific regulation of human aromatase gene is realized by alternative utilization of multiple exon 1's exons 1a, 1b, 1c, 1d, 1e, and 1f that are specific for expression in the placenta, skin fibroblasts/fetal liver, ovary, ovary/prostate/testis placenta, and brain, respectively. Each of the tissue-specific exon 1's is flanked by a unique promoter containing basic and regulatory elements. The facts that (i) aromatase is distributed in various gonadal and extra-gonadal tissues and (ii) regulated tissue-specifically by various factors, (iii) estrogen participates in specific physiological functions of various tissues, and (iv) estrogen receptor is also distributed in various tissues strongly indicate that estrogen locally produced by aromatase acts in various tissues as a multi-functional paracrine or autocrine hormone. This idea was discussed in connection with the Kd value for the estrogen receptor and serum concentration of estrogen.

Recently, in situ formation of active sex steroids at the sites of their actions from biologically inactive precursors in the circulation have been demonstrated to play very important roles in sex steroid-dependent neoplasms. These tissues in which the conversion occurs are designated as intracrine tissues and their mechanisms of actions can be designated as intracrinology in contrast to endocrinology. Aromarase, which converts serum androgens to estrone, and 17β-hydroxysteroid dehydrogenase I, which is involved primarily in the conversion of estrone to estradiol, are two major enzymes which function in the in situ formation of biologically active estrogens from circulatory androgens. In human estrogen-dependent neoplasms, including breast, endometrioid endometrial, and common epithelial ovarian carcinoma, we recently demonstrated overexpression of aromatase, especially in stromal cells at sites of frank invasion possibly under a new promoter usage and that of 17β-hydroxysteroid dehydrogenase I in these carcinoma cells. These estrogen-dependent carcinomas are considered to have a common characteristic in estrogen metabolism (i.e., the expression of aromatase in the stromal cells and of 17β-hydroxysteroid dehydrogenase I in the epithelial cells). With these in situ mechanisms of generating biologically active estrogens from circulating androgens, that is, 'intracrine manner,' these estrogen- dependent neoplasms can exert estrogenic actions on carcinoma cells despite low circulating serum estrogen levels, as observed in postmenopausal women. Evaluation of intracrine mechanisms can provide new insights into various estrogen-related biological phenomena in humans.

Peripheral aromatization of androgens exert estrogenic actions in many tissues, Recently in situ production of estrogens by aromatase,vas detected in human bone and cultured osteoblasts and has been proposed to participate in the maintenance of bone mass. We examined aromatase expression by immunohistochemistry and mRNA in situ hybridization in 16 cases of tibia (female 2 male, 14 female, 62 +/- 5.2 years old) and quantified the level of aromatase mRNA in 28 cases of rib, femur, and lumbar vertebrae (16 male, 12 female, 58.0 +/- 11.3 years old) by reverse transcriptase-polymerase chain reaction (RT-PCR) in order to study whether or not and in which cell types aromatase was expressed in human bone tissues. We also studied alternative use of multiple exons 1 of its gene and immunolocalization of type I 17 beta-hydroxysteroid dehydrogenase (HSD), which converts estrone produced by aromatase to estradiol. Strong aromatase immunoreactivity and mRNA hybridization as well as type I 17 beta-HSD immunoreactivity were detected in lining cells, osteoblasts, chondrocytes of articular cartilage, and adipocytes adjacent to bone trabeculae in all the cases examined. Amounts of aromatase mRNA varied greatly among the subjects (11.25 +/- 9.77, 0.61 +/- 42.84 attomol/ng of total RNA). The amount of aromatase expression was not correlated with age or gender of the subjects but positively correlated with the degree of osteroporotic changes evaluated by radiological findings of lumbar vertebrae. Analysis of multiple exons 1 revealed that 1b or fibroblast type was predominantly (23/26) utilized as a promoter of aromatase gene expression. These results demonstrated that aromatase is expressed widely in human bone tissue and may play important roles in maintenance of human bone tissue.

The expression and activity of aromatase was evaluated in 19 individuals with benign prostatic hyperplasia (BPH) and 26 prostatic carcinoma (PC) patients to elucidate the possible biological significance of in situ estrogen production in the development of human prostatic disorders. Marked aromatase immunoreactivity was observed in proliferative stromal cells, especially those around hyperplastic glands in 18 (95%) BPH patients and in stromal cells surrounding carcinomatous glands in 18 (69%) PC patient specimens. The percentage of aromatase-positive stromal cells did not differ between BPH and PC. No significant correlation was apparent between the percentage of aromatase-positive cells and either the extent of carcinoma differentiation or surgical stage in the PC patients. Quantitation of aromatase activity by the [H-3] water assay yielded values of 27.23 +/- 6.87 and 26.52 +/- 9.12 fmol/hr/mg of protein for BPH (nine patients) and PC (nine patients), respectively. Reverse transcriptase and polymerase chain reaction analysis revealed that the mean aromatase mRNA content was 1.671 +/- 0.82 and 1.11 +/- 0.51 attomole/ng of total RNA (tRNA) for BPH (seven patients) and PC (four patients), respectively. There were no significant differences in aromatase activity or aromatase mRNA concentration between PC and BPH. The alternative use of multiple exons 1 of the aromatase gene was also examined. Predominant aromatase gene transcripts contained exon 1b in three of four of PC specimens and two of three BPH specimens examined, in contrast to the use of exon 1d previously described in normal prostate. Unlike breast and endometrium, therefore, aromatase expression in human prostate was not associated with malignancy. However, overexpression of aromatase, possibly attributable to abnormal gene regulation, may result in estrogen production in situ and play a role in the induction or development of human prostatic disorders. (C) 1997 Wiley-Liss, Inc.

The expression of aromatase is tissue-specifically regulated through the alternative use of multiple exons 1 and promoters. We analysed expression levels of aromatase mRNA, preferential utilization of multiple exon I of the human aromatase gene, and transcriptional regulation of their multiple promoters in breast cancer tissues by newly developed fluorometric methods. The expression levels of aromatase mRNA in breast cancer tissues were significantly higher than those in regions distal to tumours or in non-malignant breast tissues. Aromatase mRNA in these non-malignant tissues was transcribed from skin fibroblast/fetal liver-specific exon 1 (exon 1b) of the aromatase gene. However, in half the cases of breast cancer patients, the utilization of multiple exons I in the aromatase mRNA changed from exon 1b to ovary-specific exon 1 (exon 1c) in their breast tissues. Aromatase mRNA in HepG2 cells as well as in non-malignant breast tissues was also transcribed from exon Ib. Then, the promoter region responsible for the exon 1b-specific utilization in HepG2 cells was examined by fluorometric promoter assay using a new reporter containing four major alternative exons 1 and promoters. The results suggested that transcriptional elements determining preferential utilization of exon Ib in the cells was located on the promoter region of exon 1b from -255 to -1145. To investigate further the cause of the elevation of aromatase mRNA and the switching from exon 1b to exon 1c in the transcription of the aromatase gene, the effects of various factors on the expression levels and preference of alternative exons 1 were examined in cultured adipose stromal cells from breast tissues. Aromatase mRNA was transcribed from exon 1b in the stromal cells, cultured in the presence of calf serum. However, removal of the serum or the addition of forskolin or phorbol ester (TPA) induced a rapid elevation of aromatase mRNA and the switching of aromatase transcripts to exon 1c in the cells, whereas TGF beta almost abolished the expression of aromatase mRNA. Because co-culture of cancer cells such as MCF-7 increased aromatase mRNA of the cells cultured in the serum-containing medium, it is possible that cancer cells secret stimulatory factors acting like forskolin or TPA, or consume serum inhibitory factors acting like TGF beta, consequently causing levels of aromatase mRNA to increase. (C) 1997 Elsevier Science Ltd.

A transient increase in aromatase activity is known to occur in the hypothalamus of rodents in pre- and postnatal periods. The mechanisms regulating such a developmental increase of brain aromatase was studied in fetal mouse diencephalic cells, by measuring aromatase mRNA levels by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. When slices of diencephalon were cultured on embryonic day (E) 12, E13 and E15, the level of aromatase mRNA continued to increase for the first 2 to 3 days. A time-dependent increase of mRNA was also shown for 3 days in E13 neuronal cells dissociated with papain and cultured in chemically defined medium. However, no significant increase was observed in E10 or E11 brain cells cultured by either method. Aromatase mRNA was detected in neither cerebral cortex neurons nor astrocytes. An cll-selective adrenergic agonist, phenylephrine, increased aromatase in the E13 diencephalic neurons in culture, whereas prazosin, an alpha(1)-antagonist, suppressed the mRNA level. Ligands for alpha(2)- or beta-adrenergic receptors did not alter the mRNA level. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide as well as phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP all increased the mRNA level. We concluded that: (a) the developmental increase of aromatase mRNA in diencephalic neurons is an autonomous event and is perhaps genetically regulated after E12; (b) aromatase mRNA is expressed in a cell type-and region-specific manner; and (c) protein kinases C and G activated via receptors of the specific neurotransmitters may be involved in modulation of the developmental expression of aromatase mRNA. (C) 1997 Elsevier Science Ltd.

Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by alpha(1)-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the alpha(1)-agonist phenylephrine than in control neurons, whereas prazosin, an alpha(1)-antagonist, suppressed this increase, and ligands for alpha(2)- or beta-adrenergic receptors did not exert any influence. The profile of alpha(1)-adrenergic receptor subtypes during actual development in vivo suggested that the alpha(1B) subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyrylcyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via alpha(1) (possibly alpha(1B))-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expres sion of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. beta-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.

The in situ formation of estradiol plays an important role in the development and biological behavior of human breast cancer Aromatase and 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD type 1) are two principal enzymes involved in in situ estradiol production. We evaluated the expression of aromatase and 17 beta-HSD type 1 by immunohistochemistry in 41 cases of invasive breast carcinoma (19 lobular and 22 ductal). We then examined the correlation among the expression of these enzymes, estrogen (ER) and progesterone (PR) receptor status, Ki67 labeling index of carcinoma cells, age, and the clinical stage of the patients. Marked aromatase immunoreactivity was observed in stromal cells around carcinomatous glands in 32 of 41 cases (78%), and 17 beta-HSD type 1 immunoreactivity was detected in carcinoma cells in 23 of 41 cases (56%). There was a significant correlation observed between expression of 17 beta-HSD type 1 and aromatase in invasive lobular carcinoma (P = 0.0119), but not in invasive ductal carcinoma. There was an inverse correlation between aromatase and ER status in invasive ductal carcinoma (P = 0.0213), but not in invasive lobular carcinoma. No other correlations were observed among 17 beta-HSD type 1, aromatase, PR, ER, clinical stage, age, and Ki67 labeling indexes. Aromatase and 17 beta-HSD are not always expressed simultaneously in human breast carcinoma, but their simultaneous expression is more frequent in invasive lobular carcinoma than invasive ductal carcinoma. Consequently, different mechanisms may be involved in the regulation of expression of these two enzymes in human breast carcinoma.

Cells immunoreactive for the enzyme aromatase were localized in the forebrain of male zebra finches with the use of an immunocytochemistry procedure. Two polyclonal antibodies, one directed against human placental aromatase and the other directed against quail recombinant aromatase, revealed a heterogeneous distribution of the enzyme in the telencephalon, diencephalon, and mesencephalon. Staining was enhanced in some birds by the administration of the nonsteroidal aromatase inhibitor, R76713 (racemic Vorozole) prior to the perfusion of the birds as previously described in Japanese quail, Large numbers of cells immunoreactive for aromatase were found in nuclei in the preoptic region and in the tuberal hypothalamus. A nucleus was identified in the preoptic region based on the high density of aromatase immunoreactive cells within its boundaries that appears to be homologous to the preoptic medial nucleus (POM) described previously in Japanese quail, In several birds alternate sections were stained for immunoreactive vasotocin, a marker of the paraventricular nucleus (PVN), This information facilitated the clear separation of the POM in zebra finches from nuclei that are adjacent to the POM in the preoptic area-hypothalamus, such as the PVN and the ventromedial nucleus of the hypothalamus, Positively staining cells were also detected widely throughout the telencephalon, Cells were discerned in the medial parts of the ventral hyperstriatum and neostriatum near the lateral ventricle and in dorsal and medial parts of the hippocampus. They were most abundant in the caudal neostriatum where they clustered in the dorsomedial neostriatum, and as a band of cells coursing along the dorsal edge of the lamina archistriatalis dorsalis, They were also present in high numbers in the ventrolateral aspect of the neostriatum and in the nucleus taeniae, None of the telencephalic vocal control nuclei had appreciable numbers of cells immunoreactive for aromatase within their boundaries, with the possible exception of a group of cells that may correspond to the medial part of the magnocellular nucleus of the neostriatum. The distribution of immunoreactive aromatase cells in the zebra finch brain is in excellent agreement with the distribution of cells expressing the mRNA for aromatase recently described in the finch telencephalon, This widespread telencephalic distribution of cells immunoreactive for aromatase has not been described in non-songbird species such as the Japanese quail, the ring dove, and the domestic fowl. (C) 1996 John Wiley & Sons, Inc.

A female infant with partial androgen insensitivity (PAIS) was first seen at 4 months of age with slight virilization of the genitalia and externally palpable testes. Sex chromosome was 46,XY. She received left orchidectomy and exploratory laparotomy at 2 yr of age. At exploratory laparotomy, neither a uterus nor fallopian tubes were found. The right testis was preserved by fixing it at the external inguinal ring expecting spontaneous pubertal maturation. After discharge, serum levels of LH, FSH, testosterone (T) and estradiol (E(2)) were measured annually, and the steroid responses to hCG stimulation were examined every two yr. At the age of 10 yr, she developed breasts and a very feminine body habitus. At 12 yr, she received a clitoroplasty and right orchidectomy. The fibroblast cultures were made from the genital skin whereby androgen receptor (AR) binding was assessed by radioreceptor assay using H-3-DHT as the ligand, and thermoinstability of AR was noted despite normal maximum binding (B-max) and dissociation constant (Kd) at 22 degrees C. But another binding experiment with H-3-Mibolerone resulted in the lack of receptor binding. AR gene analysis with direct sequencing of coding exons of the gene revealed no abnormality of the AR gene. 5 alpha-reductase activity was normal. Aromatase activity appeared to be enhanced in the genital skin fibroblast (GSF) cells as well as in the testicular tissue. The results of these-studies indicated that the patient had PAIS with impaired AR functions and increased aromatase activity. After the discharge, the patient has maintained feminine phenotype, receiving estrogen therapy with mestranol 0.02 mg/day po.

The distribution of aromatase-immunoreactive cells was studied in the medial preoptic nucleus of male and female quail that were sexually mature and gonadally intact, or gonadectomized, or gonadectomized and treated with testosterone. The study first confirmed the existence of a significant difference in the number of aromatase-immunoreactive cells between males and females (males > females) and the marked effect of castration and testosterone treatment which, respectively, decrease and restore the number of these cells. An analysis of the distribution in space of this neurochemically defined cell population was also carried out. This study revealed that castration does not uniformly decrease the density of aromatase-immunoreactive cells, but local increases are observed in an area directly adjacent to the third ventricle. A number of new sex differences in the organization of the medial preoptic nucleus and its population of aromatase cells have, in addition,been identified. The density of aromatase-immunoreactive cells is not higher in males than in females throughout the nucleus, but a higher density of immunoreactive cells is present in the ventromedial part of the nucleus in females as compared to males. In addition, the cross-sectional area of the nucleus as defined by the population of aromatase-immunoreactive cells is larger in males than in females in its rostral part and its shape is more elongated in the dorso-ventral direction in females than in males. Some of these differences (e.g. higher density of ARO-ir cells in the ventromedial part of the female POM, shape of the nucleus) appear to be organizational in nature, because they are still present in birds exposed to the same endocrine conditions during adult life (e.g. gonadectomized and treated with a same dose of testosterone). This conclusion should now be tested by experiments manipulating the endocrine environment of quail embryos. The anatomical heterogeneity of the medial preoptic nucleus revealed by this study also suggests a functional heterogeneity and the specific roles of the medial and lateral parts of the nucleus should also be investigated.

The expression of aromatase (estrogen synthetase) is tissue specifically regulated through the alternative use of multiple exons 1 and promoters. We have determined the amounts of aromatase messenger ribonucleic acid (mRNA) and which type of multiple exons 1 of the human aromatase gene is used in breast tissues of 49 patients with breast cancer by reverse transcription-PCR analysis. The aromatase mRNA levels in these breast cancer tissues (4.53 +/- 0.66 x 10(-3) attomoles/mu g RNA) were significantly (P < 0.01) higher than those in 16 nonmalignant breast tissues (1.73 +/- 0.40 x 10(-3) amol/mu g RNA). Aromatase mRNA in all nonmalignant breast tissues was transcribed from skin fibroblast/fetal liver-specific exon 1 (exon 1b) of the gene. In 23 breast cancer tissues, the utilization of multiple exons 1 in the aromatase mRNA was the same as that in nonmalignant breast tissues, whereas in the other 26 cases, it changed from exon 1b to ovary-specific exon 1 (exon 1c). Such switching of tissue-specific exons 1 may affect strict regulation of the tissue-specific expression of aromatase, leading to abnormal expression of the aromatase. The consequent overproduction of local estrogen might promote carcinogenesis or the proliferation of breast cancers.

Aromatase cDNA clones were isolated from cDNA libraries of mouse hypothalamus, amygdala and ovary. Analysis of the nucleotide sequences of the 5' regions of the obtained cDNAs suggested that the mouse aromatase gene is tissue-specifically regulated by alternative exons 1. There were obvious differences between the 5' regions of the brain and ovary aromatase cDNAs, but no difference was found between the sequences of the hypothalamus and amygdala ones. We further isolated a mouse genomic DNA clone containing brain- and ovary-specific exons 1. The brain specific exons 1 and their promoters were highly homologous in the human and mouse aromatase genes. In contrast there were several differences in the sequences among the promoter regions of the ovary-specific exons 1 of the mouse, human and rat aromatase genes, significant homology between their sequences was also observed. The present results demonstrate that expression of the mouse aromatase gene is also tissue-specifically regulated through the use of alternative exons 1 and promoters, as reported for man.

Aromatase enzyme is essential for the expression of normal sexual behavior in many mammals and birds. Here we report that vorozole (R83842), a non-steroidal aromatase inhibitor, blocks sexual behavior in the female musk shrew. In addition, vorozole treatment lowers aromatase activity in male and female preoptic area, and reduces plasma estradiol concentrations in females. Our findings confirm and extend results demonstrated in other species, conducted with the active enantiomer (R83842), or the racemic mixture (R76713, racemic vorozole). We also report that vorozole treatment affects the immunocytochemical distribution of aromatase immunoreactivity (AROM-ir) in musk shrew brain. The histological identification of neurons that contain this enzyme has been difficult in mammals. Several aromatase enzyme antisera have been developed and used in brain, and each gives a different pattern of immunoreactivity. Moreover, despite the fact that aromatase activity is very high in the bed nucleus of the stria terminalis, several amygdala nuclei, the preoptic area and hypothalamus, AROM-ir in these regions has been very limited. The distribution of AROM-ir in female musk shrew brain tissues is modified by treatment with vorozole prior to sacrifice. Female musk shrew brains contain aromatase immunoreactive cell bodies, as reported previously, in the central amygdala, lateral septum and to a limited extent in the bed nucleus of the stria terminalis (BST). Brains of females treated with vorozole show additional immunoreactivity in the preoptic area, hypothalamus, and medial amygdala, and have a broad distribution of AROM-ir in several subdivisions of the BST. Several sexual dimorphisms are apparent in musk shrews brains after treatment with vorozole. We have quantified this sexual dimorphism in the medial preoptic area (MPO) by counting immunoreactive cells. In both the rostral and caudal portions of the MPO, female brains contain significantly fewer AROM-ir cell bodies than males. These data are in complete agreement with sex differences in biochemical analyses of aromatase activity in the MPO. At this time we do not know if these dimorphisms are the result of differences in circulating levels of steroids in males and females, and/or if the AROM-ir nuclei regulate sexually dimorphic behaviors.

The expression of aromatase cytochrome P450 (P450(arom)) in the adrenal glands, testes, and placentas of fetal and newborn pigs was investigated. Western immunoblot analysis detected a single 48-50-kDa protein band in these tissues as well as in other porcine tissues known to express P450(arom) including Day 12 tubular conceptuses, theca interna, and granulosa. Slight differences in migration suggested that the P450 protein expressed in the testis was larger than that in the adrenal gland, which was, in turn, larger than that in placenta, theca, and granulosa. Consistent with P450(arom) expression in these tissues, a cDNA encoding porcine P450(arom) hybridized to a 2.3-kb transcript in Northern analyses of porcine blastocysts, placentas, and fetal and newborn adrenal glands and testes, as well as in theca and granulosa tissues from preovulatory follicles. No differences in transcript size were detectable among tissues. The identity of P450(arom) transcripts was confirmed by sequence analysis of partial cDNA clones amplified from porcine fetal adrenal glands, testes, and placentas according to the RACE procedure. The sequences of the adrenal and testis clones were identical but differed from the placental sequence, which represented the first 85 amino acids of porcine P450(arom). Specifically, the adrenal and testis clones expressed transcripts that resembled the ovarian isoform of porcine P450(arom) rather than the porcine placental isoform, predicting a two-amino acid deletion and 12 predicted amino acid substitutions. P450(arom) activity was examined to further define expression in these tissues. Activity in adrenal, testis, and placental homogenates was inhibited by 4-hydroxyandrostenedione (4OH-A4) whereas inhibition by etomidate was demonstrated in the adrenal and testis homogenates but not in the placental homogenates. The sensitivity of activity in the newborn porcine adrenal glands and testes to inhibition by etomidate was similar to that of ovarian P450(arom) activity. The level of P450(arom) activity was highest in the placenta and lowest in the adrenal gland, and no effect of fetal sex was noted in either tissue. Immunocytochemical studies localized the expression of P450(arom) in the adrenal gland of newborns to cells at the corticomedullary junction and, with greater intensity, to cells around the developing medullary lobules. The same cells expressed cytochrome P450 17 alpha-hydroxylase, the expression of which also extended throughout the zona fasciculata. The interstitial cells were the site of P450(arom) expression in the testis, but no expression could be detected in any cells within the spermatic tubules. These data demonstrate that fetal and newborn porcine adrenal glands and testes express an active P450(arom) that resembles the isoform expressed in the ovary. The localization of adrenal expression suggests a possible role in medullary maturation and function in the fetal and newborn pig.

Intraneuronal production of estradiol from testosterone has been shown to play a pivotal role in gender-specific brain development of most vertebrates, and to participate in numerous functions of the adult central nervous system. Previous biochemical and morphological approaches demonstrated that estrogen synthetase (aromatase) is present in specific limbic and hypothalamic structures. On the other hand, less attention has been paid to revealing its subcellular distribution. The possibility of aromatase presence in axonal processes has been indicated by recent biochemical and morphological observations suggesting new insights for the role of aromatase in neural functions. The objective of the present study was to provide morphological evidence for the subcellular location of aromatase in neurons of different vertebrate species including Japanese quail, rat, monkey, and human. Immunocytochemistry using a purified polyclonal antiserum against human placental aromatase localized immunoreactivity to hypothalamic and limbic cell groups in all of these species. Light and electron microscopic examination of vibratome sections revealed the presence of aromatase immunoreactivity throughout the neuronal perikarya, including dendrites and axonal processes. In each species there were numerous boutons which contained labeled small clear synaptic vesicles. Many of these axon terminals formed synapses with immune-negative and immune-positive dendrites and perikarya, This study furnishes the first immunolocalization of aromatase in the brains of two primate species, humans and monkeys. The provision of further evidence for estrogen synthesis in axons and axon terminals may help resolve apparent differences between the measurement of aromatase activity and the lack of aromatase-immunopositive cell bodies in previous studies. The present findings may be coupled with recent evidence regarding the molecular biology and the diversity of functional properties of P450 aromatase to indicate previously unexpected effects of brain aromatase at the synaptic level.

This study was designed to (1) examine the ability of fetal diencephalic neurons cultured in vitro to express aromatase mRNA and (2) evaluate the involvement of several environmental factors which may regulate the development and differentiation of the neurons in the central nervous system. Brain cells from fetal mice at various developmental stages were cultured as tissue slices and as primary monolayer cells, and the expression levels of aromatase mRNA in the cultured cells were measured by a quantitative reverse transcription-polymerase chain reaction method using an internal standard. On cultured slices of the diencephalic region from fetal mice on embryonic day 12 (E12), E13, or E15 on collagen-coated membranes, the expression level of the mRNA continued to increase for the initial 2-3 days as that in vivo. Time-dependent increase of aromatase mRNA was also observed for 3 days in E13 neuronal cells dissociated with papain and cultured on poly L-Lys-coated dishes in serum-free medium. However, no significant time-dependent increase of the aromatase mRNA level was observed in E10 or E11 brain cells cultured by either method. These findings suggest that the developmental increase of aromatase mRNA in diencephalic neurons is an endogenous characteristic and probably genetically determined after E12.

The enzyme aromatase converts testosterone (T) into 17 beta-estradiol and plays a pivotal role in the control of reproduction. In particular, the aromatase activity (AA) located in the preoptic area (POA) of male Japanese quail is a limiting step in the activation by T of copulatory behavior. Aromatase-immunoreactive (ARO-ir) cells of the POA are specifically localized within the cytoarchitectonic boundaries of the medial preoptic nucleus (POM), a sexually dimorphic and steroid-sensitive structure that is a necessary and sufficient site of steroid action in the activation of behavior. Stereotaxic implantation of aromatase inhibitors in but not around the POM strongly decreases the behavioral effects of a systemic treatment with T of castrated males. AA is decreased by castration and increased by aromatizable androgens and by estrogens. These changes have been independently documented at three levels of analysis: the enzymatic activity measured by radioenzymatic assays in vitro, the enzyme concentration evaluated semi-quantitatively by immunocytochemistry and the concentration of its messenger RNA quantified by reverse transcription-polymerase chain reaction (RT-PCR). These studies demonstrate that T acting mostly through its estrogenic metabolites regulates brain aromatase by acting essentially at the transcriptional level. Estrogens produced by central aromatization of T therefore have two independent roles: they activate male copulatory behavior and they regulate the synthesis of aromatase. Double label immunocytochemical studies demonstrate that estrogen receptors (ER) are found in all brain areas containing ARO-ir cells but the extent to which these markers are colocalized varies from one brain region to the other. More than 70% of ARO-ir cells contain detectable ER in the tuberal hypothalamus but less than 20% of the cells display this colocalization in the POA. This absence of ER in ARO-ir cells is also observed in the POA of the rat brain. This suggests that locally formed estrogens cannot control the behavior and the aromatase synthesis in an autocrine fashion in the cells where they were formed. Multi-neuronal networks need therefore to be considered. The behavioral activation could result from the action of estrogens in ER-positive cells located in the vicinity of the ARO-ir cells where they were produced (paracrine action). Alternatively, actions that do not involve the nuclear ER could be important. Immunocytochemical studies at the electron microscope level and biochemical assays of AA in purified synaptosomes indicate the presence of aromatase in presynaptic boutons. Estrogens formed at this level could directly affect the pre- and post-synaptic membrane or could directly modulate neurotransmission namely through their metabolization into catecholestrogens (CE) which are known to be powerful inhibitors of the catechol-O-methyl transferase (COMT). The inhibition of COMT should increase the catecholaminergic transmission. It is significant to note, in this respect, that high levels of 2-hydroxylase activity, the enzyme that catalyzes the transformation of estrogens in CE, are found in all brain areas that contain aromatase. On the other hand, the synthesis of aromatase should also be controlled by estrogens in an indirect, transynaptic manner very reminiscent of the way in which steroids indirectly control the production of LHRH. Fibers that are immunoreactive for tyrosine hydroxylase (synthesis of dopamine), dopamine beta-hydroxylase (synthesis of norepinephrine) or vasotocine have been identified in the close vicinity of ARO-ir cells in the POM and retrograde tracing has identified the origin of the dopaminergic and noradrenergic innervation of these areas. A few preliminary physiological experiments suggest that these catecholaminergic inputs regulate AA and presumably synthesis.