原田 信広

Background: Estrogens are suspected to play a role in the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer. In this study, the expression of aromatase messenger ribonucleic acid (mRNA) was determined, and these levels were quantitated, in human prostatic tissues to evaluate the role of estrogens in the pathogenesis of BPH and prostate cancer. Methods: Prostatic tissues were obtained either by retropubic prostatectomy, radical prostatectomy, or radical cystectomy from patients with BPH, prostate cancer, and bladder cancer. The expression of aromatase mRNA in the prostatic tissues was studied by Southern blot analysis of the reverse transcription and polymerase chain reaction technique (RT-PCR) products. Aromatase mRNA levels were measured in human prostatic tissues by the RT-PCR using a fluorescent primer. Results: Aromatase mRNA was identified in all specimens by Southern blot analysis of the RT-PCR products. The concentrations of aromatase mRNA (mean ± SD) which were measured by fluorometric quantitation in 16 of 19 patients with BPH and in 3 of 4 patients with prostate cancer, were 1.81 ± 3.02, and 0.84 ± 0.27, x 10-3 attomoles/μg of total RNA, respectively. Conclusions: These results demonstrate local formation of estrogen in the prostates of patients with BPH and prostate cancer. Controlled studies will be necessary to determine whether this may be a factor in the development of BPH and prostate cancer.

Treatment of castrated quail with testosterone (T) reliably activates male copulatory behavior and, at the same time, increases the aromatase activity (AA), the number of aromatase-immunoreactive (ARO-ir) cells and the concentration of aromatase mRNA as measured by RT-PCR in the brain. All these effects can be mimicked by estrogens. The behavioral effects of T can be blocked by a variety of aromatase inhibitors and, in parallel, the AA is strongly inhibited in the preoptic area (POA). We showed recently that the steroidal inhibitor, 4-OH-androstenedione (OHA) markedly decreases the immunostaining density of brain ARO-ir cells while the non-steroidal inhibitor, R76713 (racemic Vorozole; VOR) unexpectedly increased the density of this staining, despite the fact that the enzyme activity was completely inhibited. To generalize these findings and try to identify the underlying mechanism, we compared here the effects of two steroidal (OHA and androstatrienedione [ATD]) and two non-steroidal (VOR and Fadrozole [FAD]) aromatase inhibitors on the aromatase immunostaining and aromatase mRNA concentration in the brain of castrated quail concurrently treated with T. The 4 inhibitors significantly blocked the activation by T of male copulation. The two steroidal inhibitors decreased the immunostaining of brain ARO-ir cells but both VOR and FAD markedly enhanced the density of this staining. In parallel, OHA and ATD completely blocked the T-induced increase in aromatase mRNA concentration, while VOR and FAD had no effect on these RNA concentrations in the POA-anterior hypothalamus and they decreased them only slightly in the posterior hypothalamus. Taken together these results suggest that the inhibition of AA by ATD or OHA and the subsequent removal of locally produced estrogens blocks the synthesis of aromatase presumably at the transcriptional level. By contrast, the two non-steroidal inhibitors tested here block AA but in parallel increase the aromatase immunostaining. This effect does not result from an enhanced transcription and it is therefore speculated that these compounds increase either the translation of the aromatase mRNA or the half-life of the protein itself.

The aromatase (cytochrome P-450(AROM)) gene contains multiple untranslated exons I that are differentially transcribed in a tissue-specific manner. DNA sequences within the initial -301 upstream of placenta-specific exon I (exon Ia) are sufficient for placenta-specific expression of aromatase. In gel mobility shift assay, three separate domains in this region form specific binding complexes with proteins extracted from choriocarcinoma JEG-3 nuclei. A fragment containing these domains activates transcription driven by a heterologous promoter in a cell type-specific manner. Two of the binding domains that form major complexes in gel shift assay compete with each other and with a DNA fragment containing the trophoblast-specific element (TSE), which is derived from the enhancer region of the human chorionic gonadotropin alpha-subunit gene and is believed to confer placenta-specific expression of the gene. The core sequence RNCCTNNRG is sufficient for recognition of the TSE-binding protein, which is detected only in nuclear extracts prepared from placenta and choriocarcinoma. A mutation introduced in the distal TSE core in aromatase promoter resulted in marked reduction of transcriptional activity, although TSE region by itself did not show enhancer activity as that in human chorionic gonadotropin alpha-subunit gene.

The transformation of testosterone into estradiol in the brain plays a key role in several behavioral and physiological processes, but it has been so far impossible to localize precisely the cells of the mammalian brain containing the relevant enzyme, viz., aromatase. We have recently established an immunohistochemical technique that allows the visualization of aromatase-immunoreactive cells in the quail brain. In this species, a marked increase in the optical density of aromatase-immunoreactive cells is observed in subjects that have been treated with the aromatase inhibitor, R76713 or racemic Vorozole. This increased immunoreactivity, associated with a total blockade of aromatase activity, has been used as a tool in the present study in which the distribution of aromatase-immunoreactive material has been reassessed in the brain of mice pretreated with R76713. As expected, the aromatase inhibitor increases the density of the immunoreactive signal in mice. Strongly immunoreactive cells are found in the lateral septal region, the bed nucleus of the stria terminalis, the central amygdala, and the dorso-lateral hypothalamus. A less dense signal is also present in the medial preoptic area, the nucleus accumbens, several hypothalamic nuclei (e.g., paraventricular and ventromedial nuclei), all divisions of the amygdala, and several regions of the cortex, especially the cortex piriformis. These data demonstrate that, contrary to previous claims, aromatase-immunoreactive cells are present in all brain regions that have been shown previously to contain high aromatase activity.

Mouse and quail aromatase cDNAs were isolated from libraries of mouse ovary and quail brain by using a human aromatase cDNA fragment (hA-24) as a probe. These three cDNAs were inserted into plasmid vectors and expressed in Escherichia coli. Antisera against these purified recombinant proteins were raised in rabbit and purified by ammonium sulfate fractionation and affinity chromatography. The three antibodies directed against recombinant human, mouse and quail proteins were used to visualize aromatase-immunoreactive cells in the quail brain. They were compared with the antibody raised against human placental aromatase used in previous experiments and with another antibody recently developed by similar methods. The signal obtained with all antibodies was completely abolished by preadsorption with the homologous recombinant antigens and the signal produced by the two antibodies raised against placental aromatase was similarly abolished by a preadsorption with recombinant quail aromatase. The antibodies raised against recombinant proteins identified the major groups of aromatase cells previously described in the quail brain. The antibodies directed against the mouse and quail antigen identified more positive cells and stained them more densely than the antibodies raised against human recombinant antigen or purified placental aromatase. The new cell groups identified by the antibody raised against quail recombinant aromatase were located in an area ventral to the fasciculus prosencephali lateralis, the nucleus accumbens, the paleostriatum ventrale, the nucleus taeniae, the area around the nucleus ovoidalis, the caudal tuber and the mesencephalic central gray. A critical re-examination of the distribution and nomenclature of the aromatase-positive cells is proposed based on these new findings.

Immunoreactive aromatase enzyme (AROM-IR) was studied in the preoptic and septal areas of the male Japanese quail brain relative to the age-related decline in endocrine and behavioral components of reproduction. Additional analyses were conducted to determine if the co-localization of AROM-IR and estrogen receptor immunoreactivity (ER-IR) in the medial preoptic area change during aging. Young, sexually active, male quail (6 months of age) were compared to aged sexually active or inactive, male quail (36 months of age). Testis size decreased in old, sexually inactive males, similar to our previous observations. The numbers of AROM-IR neurons in the medial preoptic area (POM) and the lateral septum (LS) decreased significantly with aging and sexual activity. The number of cells that co-localized both AROM-IR and ER-IR did not differ with age. As a consequence of the age-related change in AROM-IR cells, the relative percentage of dual labelled (AROM-IR and ER-IR) and single labelled cells (AROM-IR) increased in aged males. These data provide histochemical evidence that alterations in the aromatase enzyme system in the medial preoptic area may underlie behavioral and endocrine events associated with reproductive aging.

In the hamster liver, 2,5,2',5'-tetrachlorobiphenyl (TCB) is metabolized to 3-hydroxy- and 4-hydroxy-2,5,2',5'-TCB to a similar extent, and formation of the former metabolite is stimulated by phenobarbital pretreatment of the animals, while that of the latter metabolite is stimulated by 3-methylcholanthrene pretreatment. In the present study, we identified a new isoform (designated P450HPB-1) of cytochrome P450 which proved to be phenobarbital-inducible and responsible for 3-hydroxylation of this TCB isomer. This isoform was purified from liver microsomes of phenobarbital-treated hamsters and characterized. P450HPB-1 has a molecular mass of 50 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the absorption maxima of the oxidized form at 417 nm and of the reduced CO-complex form at 450 nm. The sequence of 28 amino acids of P450HPB-1 at the aminoterminal has a 68% similarity with those of rat P450 2B1 and mouse P450 2b10, 57% similarity with that of guinea pig P450GP-1, and 54% similarity with those of rabbit P450 2B4 and dog P450 2B11. P450HPB-1 in the reconstituted system catalyzed the 3- but not 4-hydroxylation of 2,5,2',5'-TCB, at a rate of 19.0 pmol/min/nmol P450. The isoform also has high catalytic activity for 17-oxidation of testosterone but low activity for the N-demethylation of benzphetamine and 16 alpha- and 18 beta-hydroxylations of testosterone. In microsomal metabolism of 2,5,2',5'-TCB, rabbit antiserum against P450HPB-1 almost completely inhibited 3- but not 4-hydroxylation. Immunoblot analysis of hamster liver microsomes revealed that P450HPB-1 was constitutive and phenobarbital-inducible but was decreased by pretreatment with 3-methylcholanthrene or 3,4,5,3/,4'-pentachlorobiphenyl. These results suggest that P450HPB-1 belongs in the P450 2B subfamily and apparently plays a major role in the 3-hydroxylation of 2,5,2',5'-TCB, in hamster liver. (C) 1995 Academic Press, Inc.

The aromatase enzyme (estrogen synthetase) catalyzes the conversion of testosterone to estrogen in peripheral and central nervous tissue. Light and electron microscopic immunocytochemistry was used to study the localization of this enzyme in the septal area of adult male and female albino rats. Aromatase-immunoreactivity was found restricted to neuronal somata and dendritic arbors, and no sex differences were detected in its distribution or intensity. Most aromatase-immunoreactive neurons formed two oblique bands in the lateral and the medial zones of the lateral septum; in addition, labeled cells were present in the septohippocampal nucleus and the laterodorsal portion of the bed nucleus of the stria terminalis. Electron microscopy revealed that the majority of aromatase-positive neurons in the lateral septum exhibit somatic spines, a characteristic marker of a neuron population that is known to contribute to local and extraseptal projections. The presence of aromatase in lateral septal somatospiny neurons suggests that estrogen formed by these neurons may be critically involved in the septal control of steroid-dependent behaviors.

CASTRATED quail were injected with testosterone or with the synthetic hormones diethylstilbestrol (DES) or methyltrienolone (R1881)to analyse the steroid specificity in the induction of brain aromatase. R1881 produced a moderate (generally non-significant) increase in the number of aromatase-immunoreactive cells. DES significantly increased the number of positive cells in most brain areas. A clear synergism between DES and R1881 was observed in all brain regions: more immunoreactive cells were found in birds receiving both compounds than in those injected with DES or R1881 alone. DES and R1881 are highly specific ligands for oestrogen and androgen receptors respectively. It appears likely that both androgens and oestrogens directly modulate brain aromatase, presumably at the transcription level.

The volume and cytoarchitectonic organization of the sexually dimorphic medial preoptic nucleus (POM) of the quail are sensitive to plasma levels of testosterone (T). We previously showed that, in castrated quail, T or its estrogenic metabolite, estradiol (E(2)), increases the size of the large neurons located in the lateral part of POM. Embryonic treatments with estrogens are also known to affect permanently the size of these large neurons. Since the lateral POM also contains a dense population of aromatase-immunoreactive (ARO-ir) cells, and these are known to be a target for steroids, we hypothesized that the effects of steroids identified in previous experiments were primarily directed to these ARO-ir cells. This idea was tested in two experiments in which the size of these cells was measured in male quail under various endocrine conditions. In experiment 1, a detailed analysis of ARO-ir and of non-immunoreactive cells in the POM of adult, sexually mature males revealed that the immunoreactive perikarya are larger than the non-immunoreactive cells and that they constitute the vast majority of the large cells(area > 50 mu m(2)) in the POM. In experiment 2, it was shown that T and E(2) actually increase the size of ARO-ir cells in the POM while the androgenic metabolite of T, dihydrotestosterone has no effect at this level. Taken together, these data suggest that the sex differences and the steroid-induced changes in cell size previously described in the study of POM sections stained for Nissl material largely concern aromatase-containing cells. Since aromatization of T plays a limiting role in the activation of male copulatory behavior, these changes may represent the morphological signature of the mechanisms causally involved in the control of this behavior.

Castrated quail were treated with Silastic implants filled with testosterone (T) in association with injections of the aromatase inhibitors, R76713 (racemic vorozole; 1 mg/kg twice a day) or 4-hydroxyandrostenedione (OHA; 5 mg/bird twice a day). Control birds received no treatment (CX group) or were implanted with T capsules only (CX + T group). Both R76713 and OHA strongly inhibited the T-activated male copulatory behavior. This inhibition had the same magnitude in both groups. The growth of the cloacal gland, a strictly androgen-dependent process was not affected by these compounds. The treatments significantly affected the number of aromatase-immunoreactive (ARO-ir) cells in each of the six brain areas that were studied: the anterior and posterior parts of the sexually dimorphic medial preoptic nucleus (POM), the septal region, the bed nucleus of the stria terminalis (BNST) and the anterior and posterior parts of the tuber. This number was significantly increased in all areas by T. In agreement with our previous study, R76713 significantly inhibited this effect of T in the tuberal hypothalamus but not in the anterior POM nor in the BNST. By contrast the effect of T on the number of ARO-ir cells was completely blocked by OHA in all brain nuclei. The two inhibitors had statistically different effects in all brain regions. Like in a previous study, R76713 increased the intensity of the staining of all ARO-ir cells. This effect took several days to develop suggesting a progressive build-up of the enzyme concentration. This was also suggested by the fact that a rebound in aromatase activity was observed 16 to 24 h after a single injection of R76713. The increased immunoreactivity was not observed in OHA-treated birds. The denser immunoreactivity in R76713-treated birds and the better tissue preservation due to the aldehyde fixative that had been used provided here a clearer picture of the cellular and subcellular localization of ARO-ir material. This allowed to identify new groups of immunoreactive cells, namely in the nucleus accumbens, in the area of the paleostriatum ventrale, in the nucleus taeniae, in the medial and caudal hypothalamus and in the medial part of the mesencephalon and of the pens. Most of the immunoreactive material was located in perikarya but some of these cells were also surrounded by dense networks of ARO-ir fibers often associated with immunopositive punctate structures. These fibers and punctate structures were seen also in areas that were quite distant from the closest ARO-ir cells. They were detected in periventricular position throughout the preoptic area and hypothalamus.

In situ synthesis of estrogens by breast cancer tissue provides a potential explanation for the high concentrations of estradiol in mammary neoplasms in postmenopausal women. A major metabolic pathway for estrogen biosynthesis is the conversion of androstenedione to estrone via the enzyme aromatase. Biochemical studies have demonstrated aromatase in tumor tissue, but at relatively low and not clearly biologically significant levels. The present study tested the hypothesis that tumor levels of aromatase, albeit low, could be biologically important if present in high concentrations in focal clusters of specific cell types. A pilot study used an immunohistochemical method in frozen sections of fresh breast tumors as an optimal means to detect aromatase. Twelve of 18 tumors contained aromatase-positive cells, some with highly intense staining. A follow-up study then attempted to precisely define the types of cells containing aromatase and correlate the immunohistochemical findings with biochemical aromatase activity. A modified H-score (histological scoring system) was used to semiquantitate the amount of aromatase staining in tumor epithelial, stromal spindle, stromal inflammatory, and normal breast epithelial cells. We found that immunohistochemical staining for aromatase predominated in stromal spindle cells with a median H-score of 13, whereas tumor epithelial, stromal inflammatory, and normal breast elements contained lesser amounts (median H-scores of 4.8, 0.03, and 0.5, respectively). The H-score for stromal spindle cells, but not those for other cell types, correlated highly with the biochemical aromatase assay (P < 0.01). Using a cut-off parameter estimated by a sensitivity/specificity (receiver operating curve) analysis, 62% of tumors were classified as aromatase positive based on stromal spindle cell staining. A similar number were also positive by biochemical assay, with concordance between the two methods of 77%. These observations provide substantial evidence for the presence of aromatase in human breast tumors, particularly in stromal spindle cells, and support the biological importance of aromatase for in situ production of estradiol.

The expression of aromatase mRNA in cultured mouse brain cells was measured by a quantitative reverse transcription-PCR method using an internal standard. Aromatase mRNA was expressed in the cultured neurons prepared from diencephalon at 0.037 +/- 0.005 attomol/mu g total RNA. However, the mRNA was detected in neither the neurons from cerebral cortex nor astrocytes. These results demonstrate that expression of aromatase mRNA is regulated in cell type- and region-specific manners in cultured brain cells. The aromatase mRNA levels in neurons obtained from diencephalon were not affected by administration of testosterone, estradiol, dexamethasone, forskolin, or 12-O-tetradecanoyl 13-acetate. The results are in apparent disagreement with previous reports concerning regulation by androgens of brain aromatase activity in vivo and may suggest that aromatase expression in brain neurons is not directly induced by androgens. Androgen induction of brain aromatase may be mediated by several steps including cell-cell (neuron-neuron and/or neuron-glia) interaction.

An efficient expression system for a cDNA clone of human placental aromatase has been developed using the baculovirus expression system in TN5 (Tricoplusia ni) cells. The protein was expressed at high levels, with specific aromatase activity and specific P450 content comparable to that found in human placental microsomes. To achieve these high levels of activity, hemin had to be added to the cultures of infected cells and NADPH-cytochrome P450 reductase had to be included in the assay buffer. The spectral properties of ligand bound forms of the baculovirus expressed aromatase were very similar to those exhibited by the same ligand bound forms of the enzyme purified from placental microsomes. This expression system appears to be a suitable source for the purification of milligram quantifies of recombinant aromatase. (C) 1994 Academic Press, Inc.

We previously demonstrated that testosterone (T) increases aromatase activity (AA) and that AA is sexually dimorphic (males > females) in the quail preoptic area (POA). The precise anatomical localization of these effects is, however, impossible to obtain by biochemical assays even when samples are dissected by the Palkovits punch technique. We were recently able to set up an immunocytochemical (ICC) procedure that permits visualization of aromatase-immunoreactive (ARO-ir) cells in the quail brain. This showed that the ARO-ir cells of the quail POA actually outline the sexually dimorphic medial preoptic nucleus (POM). This ICC technique was used here to analyze the sex dimorphism of the quail preoptic aromatase and the localization of T effects on ARO-ir cells. In Experiment 1, the number of ARO-ir cells was counted in one section every 100 mu m throughout the rostral to caudal extent of the POM of castrated birds that had been treated with increasing doses of T (5, 10, or 20 mm long Silastic implants). These T-treatments produced a dose-related increase in the sexual behavior of the birds and they increased the number of ARO-ir cells in POM, in the septal regions, and in the bed nucleus of the stria terminalis (BNST). The effect had a particularly large amplitude in the part of the POM located under the anterior commissure (AC). In Experiment 2, the same procedure was used to reanalyze the sex difference of the preoptic aromatase system. This showed that the POM of adult males contains more stained cells than the POM of females but only in a restricted region located just under and rostral to the AC. No significant sex difference was observed in the septum or in the BNST. In Experiment 3, the number of ARO-ir cells was determined in the POM of males and females that had been gonadectomized and treated with a same dose of T (40 mm implants). No sex difference in the number of ARO-ir cells could be detected in these conditions. This suggests that the sex difference in AA that had been previously observed in T-treated birds results either from a difference in aromatase concentration or activity in a similar number of positive cells or from a difference in the number of ARO-ir cells that is very discrete from the anatomical point of view. These experiments also indicate that ARO-ir cells in the region of POM located just under and rostral to AC are sexually dimorphic and T-sensitive, which suggests their involvement in the control of male sexual behavior. This conclusion is also supported but our recent studies based on electrolytic lesion and stereotaxic implantation of T in POM.

In the female musk shrew (Suncus murinus) neural aromatization of testosterone to estradiol is critical for the expression of sexual behavior. To localize the brain regions capable of aromatization, we used immunocytochemistry to map the distribution of aromatase enzyme. Aromatase immunoreactivity (AROM-ir) has a discrete distribution primarily limited to the lateral septum (LS), central nuclei of the amygdala (Ce) and the bed nucleus of the stria terminalis (BST). In these nuclei the intensity of immunoreactivity varies with hormonal status. Ovariectomy (OVX) significantly reduces the optical density of AROM-ir neurons in all nuclei as compared with brains of normal females. Combined OVX and adrenalectomy (ADX) further reduces optical density readings in AROM-ir cells in the LS and BST, as compared with readings from the brains of OVX animals. Normal and ovariectomized females implanted with testosterone had qualitatively equivalent AROM-ir. High levels of aromatase activity have been measured in the preoptic area and hypothalamus in a number of mammals, including the musk shrew. However, in this experiment AROM-ir was absent in these areas. We present several hypotheses to account for this discrepancy between previously reported biochemical data and these histological data. In summary, these data suggest that limbic nuclei may play a role in the expression of sexual behavior in female musk shrews.

We isolated overlapping cDNA clones encoding human histidase (histidine ammonia-lyase) from a human lambdagt10 library. The cDNA predicted a 657 amino acid protein of 72 651 Da. The human histidase amino acid sequence was 93% conserved with both rat and mouse histidase sequences, including four N-glycosylation consensus sites.

It was found that cytochrome P-450(arom) purified from human placenta microsomes is glycosylated, and the sugar chain was cleaved with endoglycosidase H (Endo H). The core glycosylation of P-450(arom) was examined with two heterologous expression systems, cultured insect cells and in vitro translation system. The P-450(arom) protein expressed in the insect cells was glycosylated, and the sugar chain was sensitive to Endo H. It was also glycosylated when translated with the wheat germ cell-free system in the presence of rough microsomal membrane, and the sugar chain could be removed by Endo H treatment. Since the P-450(arom) molecule has two potential glycosylation sites (Asn-12 and Asn-180), we replaced each of the 2 asparagine residues with alanine by site-directed mutagenesis and examined the glycosylation of the two mutant proteins in the cell-free system. The core glycosylation did not occur when the Asn-12 residue was mutated, whereas the mutant protein with modified Asn-180 residue was glycosylated. These results demonstrated that the potential glycosylation site (Asn-12) in the N-terminal portion of P-450(arom) is the site of glycosylation. We conclude that the N terminus of P-450(arom) is translocated across the endoplasmic reticulum membrane to be glycosylated at the luminal side.

It is established that testosterone (T) increases aromatase activity (AA) in the quail brain and that this induction of AA represents a limiting factor in the activation of male copulatory behavior. This action of T presumably results from an induction of aromatase synthesis since the number of aromatase-immunoreactive (ARO-ir) cells increases and, in parallel, there is an increase in aromatase mRNA as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) technology. The specific role of androgenic and estrogenic metabolites of T in this induction is not yet clear but product-formation assays suggest that both types of compounds synergize to increase AA. The exact role of androgens and estrogens in the induction of aromatase was examined by studying both the aromatase protein by immunocytochemistry and the aromatase mRNA by RT-PCR in castrated quail that had been treated with T or its androgenic metabolite, 5alpha-dihydrotestosterone (DHT) or its estrogenic metabolite, estradiol-17beta (E2) or both DHT and E2 simultaneously. A specific quantitative PCR technique using a modified aromatase as internal standard was developed for this purpose. T increased the number of ARO-ir cells in all brain areas and increased the concentration of ARO mRNA in the preoptic area-anterior hypothalamus (POA-aHYP) and in the posterior hypothalamus (pHYP). E2-treated birds had more ARO-ir cells than castrates in the posterior part of the medial preoptic nucleus (POM), in the bed nucleus stria terminalis (BNST) and tuber. Their aromatase mRNA concentration was significantly increased in the POA-aHYP but this effect did not reach significance in the pHYP. DHT by itself had no effect on either the number of ARO-ir cells (all brain regions considered) or the concentration of aromatase mRNA. DHT, however, synergized with E2, both in inducing ARO-ir neurons and in increasing aromatase mRNA concentration. This synergism was shown to be statistically significant in several brain areas. These data demonstrate that both androgens and estrogens regulate aromatase at the pretranslational level. Because the percentage increase in the number of ARO-ir cells was in general very similar to the increase in aromatase mRNA concentration, these data also suggest that these steroids regulate aromatase mostly by changing its mRNA synthesis or catabolism.

Placental aromatase deficiency, which was characterized by maternal and fetal virilization and by a low level of estrogen excretion into urine during pregnancy, was studied by biochemical and molecular genetical techniques. Among enzymes participating in the electron transport system of the patient's placental microsomes, only aromatase activity was observed to be reduced (<3% of normal levels). Northern and Western blotting analyses showed that the transcription of the aromatase gene and the translation of its mRNA seemed to proceed normally in the patient's tissue. However, the aromatase cDNA isolated from the patient was found to contain an extra DNA fragment of 87 base pairs (bp) which encoded 29 amino acids in frame but no termination codon. The insertion was located at the splicing point between exon 6 and intron 6 of the normal aromatase gene. The extra DNA fragment represented the first part of intron 6 except that its initial GT was altered to GC. These findings indicated that, in the patient's aromatase gene, the splicing between exon 6 and intron 6 did not occur at the normal position. This reflected the presence of one point mutation in its consensus sequence which caused the next cryptic consensus sequence 87 bp downstream, to be used according to the canonical GT/AG rule. The protein molecule thus translated contained an extra 29 amino acids. Furthermore, the patient's aromatase cDNA was observed to produce a protein molecule with a trace of activity in the transient expression system of COS-7 cells and in the high level expression system of baculovirus-insect cells. Direct DNA sequencing of aromatase genes from the patient and parents confirmed that this deficiency is a hereditary disease with an autosomal recessive inheritance pattern. The patient and parents are homozygote and heterozygotes, respectively, for this mutation.

The aromatase (estrogen synthetase) enzyme catalyzes the conversion of androgens to estrogens in peripheral tissues, as well as in the brain. Our study aimed at comparing the brain distribution of aromatase-immunoreactive neurons in male and female, normal and gonadectomized rats. Light microscopic immunostaining was employed using a purified polyclonal antiserum raised against human placental aromatase. Two anatomically separate aromatase-immunoreactive neuronal systems were detected in the rat brain: A ''limbic telencephalic'' aromatase system was composed by a large population of labeled neurons in the lateral septal area, and by a continuous ''ring'' of neurons of the laterodorsal division of the bed nucleus of stria terminalis, central amygdaloid nucleus, stria terminalis, and the substantia inominata-ventral pallidum-fundus striati region. The other, ''hypothalamic'' aromatase system consisted of neurons scattered in a dorsolateral hypothalamic area including the paraventricular, lateral and dorsomedial hypothalamic nuclei, the subincertal nucleus as well as the zona incerta. In addition, a few axon-like processes (unresponsive to gonadectomy) were present in the preoptic-anterior hypothalamic complex, the ventral striatum, and midline thalamic regions. No sexual dimorphism was observed in the distribution or intensity of aromatase-immunostaining. However, 3 days, 2, 3, 8, 16, or 32 weeks after gonadectomy, aromatase-immunoreactive neurons disappeared from the hypothalamus, whereas they were still present in the limbic areas of both sexes. The results indicate the existence of two distinct estrogen-producing neuron systems in the rat brain: (1) a ''limbic ring'' of aromatase-labeled neurons of the lateral septum-bed nucleus-amygdala complex unresponsive to gonadectomy; and (2) a sex hormone-sensitive ''hypothalamic'' aromatase neuron system.

A sensitive method was developed for the determination of trace amounts of aromatase mRNA in various tissues. Aromatase mRNA was quantitated by subjecting it to reverse transcription in the presence of an internal standard RNA and then amplifying the resulting cDNA by polymerase chain reaction with a fluorescent primer. The tissue distribution of aromatase mRNA in mice was examined by this polymerase chain reaction method. Results showed that aromatase mRNA is expressed in the brain, testis, and ovary, but scarcely at all in other tissues, including the placenta. In mouse brain, aromatase is mainly located in the forebrain, especially the diencephalon. In adult male and female diencephala, aromatase mRNA levels were 0.022 +/- 0.004 and 0.014 +/- 0.003 attomoles/mug total RNA, respectively. Aromatase in the diencephalon is known to participate in brain differentiation and sexual behavior, so changes in its mRNA levels in the brain during development were examined. Aromatase mRNA was first detected in the 12-day-old fetus and was found to increase rapidly during the fetal and neonatal periods. Its mRNA levels in male and female brains reached maxima of 0.068 +/- 0.008 and 0.059 +/- 0.006 attomoles/mug total RNA, respectively, 3-4 days after birth and then gradually decreased to adult levels. These observations are consistent with previous data indicating that the marginal period of neonatal imprinting of sexual differences is within a week after birth and suggest that brain aromatase may be important in sexual differentiation and behavior.

The aromatase cytochrome P-450 (P-450AROM) cDNA, which was identified by homologies in the DNA and in the deduced amino acid sequences with human P-450AROM cDNA, was isolated from a brain cDNA library of Japanese quail, demonstrating the presence of RNA transcripts of P-450AROM in the quail brain. To determine trace amounts of P-450AROM mRNA in the brain and to examine the effects of testosterone on its expression, a quantitative PCR method of RNA transcripts was developed. Brain total RNA was subjected to reverse transcription reaction and then quantitated by PCR from cDNA with a fluorescent dye-labeled primer. The quantity of P-450AROM mRNA was calculated by using an internal standard of modified P-450AROM (m-P-450AROM) RNA. The brain P-450AROM was primarily transcribed in the hypothalamus area (1.15 +/- 0.14 amol/mu-g of RNA) and traces of transcripts only were detected in the cerebellum (0.038 +/- 0.005 amol/mu-g of RNA). The P-450AROM mRNA in the hypothalamus of castrated quail was low (0.270 +/- 0.078 amol/mu-g of RNA) and increased 4- to 5-fold following treatment with testosterone. These results demonstrate, for the first time, that the increase in P-450AROM activity that is observed in the brain following treatment with testosterone results from a pretranslational regulation of the P-450AROM by androgens.

Biochemical and molecular genetic studies were made on a case of placental aromatase (P-450AROM) deficiency. Of the enzymes participating in the electron transport system of placental microsomes, only aromatase activity was decreased specifically in the patient, being less than 0.3% of the normal activity. Northern and Western blotting analyses showed that the transcription of the aromatase gene and the translation of its mRNA proceeded normally in the placenta of this patient. However, aromatase cDNA isolated from a placental cDNA library of the patient was found to have an insert of 87 base pairs, encoding 29 amino acids in frame with no termination codon. The insert was located at the splicing point between exon 6 and intron 6 of the normal aromatase gene, and the extra DNA fragment was the first part of intron 6, except that its initial GT was altered to GC. These findings indicated that in this patient with aromatase deficiency, splicing between exon 6 and intron 6 did not occur at the normal position because of a point mutation in its consensus sequence and was forwarded to GT in the next cryptic consensus sequence 87 base pairs downstream according to the canonical GT/AG rule, resulting in translation of an abnormal protein molecule with 29 extra amino acids. During the transient expression in COS-7 cells, the aromatase cDNA of the patient was found to produce a protein with a trace of activity. This is the first report of a genetic defect for aromatase deficiency.

Testosterone (T) increases brain aromatase activity (AA) in quail and other avian and mammalian species. It was shown both in quail and in rat that this enzymatic induction results from a synergistic action of androgens and estrogens. These studies provide little information on possible anatomical or cellular specificity of the effect. Using a polyclonal antiserum against human placental aromatase, we have previously identified aromatase-immunoreactive (ARO-ir) neurons in the quail brain and demonstrated that T increases the number of ARO-ir cells in the quail preoptic area (POA) supporting previous evidence that T increases AA in the brain. However, which T metabolites are involved, the actual mechanism of regulation and the possibility of anatomical specificity for these effects are not yet clear. In the present study, we disassociated the effects of androgens and estrogens in aromatase induction by comparing ARO-ir neurons of quail treated with T alone or T in the presence of a potent aromatase inhibitor (R76713), which has been shown to depress AA levels and to suppress T-activated copulatory behavior. T increased the number of ARO-ir cells in POA, bed nucleus striae terminalis (BNST) and tuberal hypothalamus (Tu). The T effect was inhibited by concurrent treatment with aromatase inhibitor in Tu, but not in POA and BNST. This differential effect of the aromatase inhibitor fits in very well with our previous studies of the co-localization of aromatase and estrogen receptors. The T effect was blocked by R76713 in areas where ARO-ir and estrogen receptor-ir are generally co-localized (Tu) and was not affected in areas with mainly ARO-ir positive, estrogen receptor-ir negative cells (POA, BNST). This suggests anatomical differences in the expression or clearance of aromatase which may be differentially sensitive to androgens and estrogens and dependent upon the presence of sex steroid receptors.

We recently showed, using a new immunocytochemical technique, that aromatase-immunoreactive neurons are a specific marker for the sexually dimorphic medial preoptic nucleus (POM) in quail and that the number of these immunoreactive cells is markedly increased by a systemic treatment with testosterone (T). Since the POM is a key site for the activation of copulatory behavior by T and this androgen must be converted into estrogen by local aromatization within the POM before it can exert its behavioral effects, we used aromatase immunocytochemistry to map, at a cellular level of resolution, the areas that are destroyed by electrolytic lesions or that are stimulated by the stereotaxic implantation of T in the preoptic area (POA). These measures of the cellular action of T in the preoptic area were then correlated with the behavior of the animals to identify the parts of the POA that are critical in the activation of behavior. The electrolytic lesions of the POA disrupted the activation of male sexual behavior by T only if they destroyed a significant part of the POM. All lesions reduced the volume of the dimorphic nucleus and the absolute number of its aromatase-immunoreactive neurons, but the density of these cells in the remaining POM was not affected, suggesting that the volume change in the nucleus reflected a centripetal displacement of its boundaries rather than an overall shrinkage of the structure. Stereotaxic T implants in or close to POM activated male copulatory behavior and increased the volume of the POM and the number of its aromatase-immunoreactive cells. These neuroanatomical effects were more prominent on the side of the implant, but they were also detected on the contralateral side. Correlative analyses suggested that a part of the POM just rostral to the anterior commissure is critical for the activation of copulatory behavior. The best correlations between the behavioral deficits induced by electrolytic lesions and the size of the lesions were indeed observed in this area. In addition, high correlations were also observed between the behavior activated by T implants and the POM size or number of aromatase-immunoreactive cells that were induced by T in this area. Aromatase immunocytochemistry therefore appears as a useful tool to map the brain areas in which T action is presumably critical for the activation of male sexual behavior. It has allowed us to identify in the present studies a small part of the sexually dimorphic POM that is closely associated with behavior. Experimental studies involving the lesion, disconnection or specific stimulation by steroids of this area should now be undertaken to confirm the causal meaning of these correlations.

The relative distributions of aromatase and of estrogen receptors were studied in the brain of the Japanese quail by a double-label immunocytochemical technique. Aromatase immunoreactive cells (ARO-ir) were found in the medial preoptic nucleus, in the septal region, and in a large cell cluster extending from the dorso-lateral aspect of the ventromedial nucleus of the hypothalamus to the tuber at the level of the nucleus inferioris hypothalami. Immunoreactive estrogen receptors (ER) were also found in each of these brain areas but their distribution was much broader and included larger parts of the preoptic, septal, and tuberal regions. In the ventromedial and tuberal hypothalamus, the majority of the ARO-ir cells (over 75%) also contained immunoreactive ER. By contrast, very few of the ARO-ir cells were double-labeled in the preoptic area and in the septum. More than 80% of the aromatase-containing cells contained no ER in these regions. This suggests that the estrogens, which are formed centrally by aromatization of testosterone, might not exert their biological effects through binding with the classical nuclear ER. The fact that significant amounts of aromatase activity are found in synaptosomes purified by differential centrifugation and that aromatase immunoreactivity is observed at the electron microscope level in synaptic boutons suggests that aromatase might produce estrogens that act at the synaptic level as neurohormones or neuromodulators.

The distribution of aromatase-immunoreactive cells was studied by immunocytochemistry in the mouse forebrain using a purified polyclonal antibody raised against human placental aromatase. Labeled perikarya were found in the dorso-lateral parts of the medial and tuberal hypothalamus. Positive cells filled an area extending between the subincertal nucleus in the dorsal part, the ventromedial hypothalamic nucleus in the ventral part, and the internal capsule and the magnocellular nucleus of the lateral hypothalamus in the lateral part. The same distribution was seen in the two strains of mice that were studied (Jackson and Swiss), and the number of immunoreactive perikarya did not seem to be affected by castration or testosterone treatment. No immunoreactivity could be detected in the medial regions of the preoptic area and hypothalamus; these were expected to contain the enzyme based on assays of aromatase activity performed in rats and on indirect autoradiographic evidence in mice. Our data raise questions concerning the distribution of aromatase in the brain and the mode of action of the centrally produced estrogens.