Diagnostic Value of Autoantibodies against Steroidogenic Enzymes and Hormones in Infertile Women with Premature Ovarian Insufficiency.

The objective of the study was to evaluate the profile and diagnostic significance of serum autoantibodies in infertile patients with premature ovarian insufficiency (POI). The pilot study included 26 patients of reproductive age with POI and diminished ovarian reserve who received complex treatment using new surgical technologies (Group 1) and 18 patients without POI (Group 2). The profile of serum autoantibodies, including anti-ovarian antibodies, antibodies against thyroid peroxidase (TPO), steroidogenic enzymes, and steroid and gonadotropic hormones, was studied using modified ELISAs and human recombinant steroidogenic enzymes (CYP11A1, CYP19A1, CYP21A2). Patients in Group 1 had higher levels of IgG autoantibodies against steroidogenic enzymes, estradiol, progesterone, and TPO than those in Group 2. Tests for IgG antibodies against CYP11A1, CYP19A1, and CYP21A2 exhibited high sensitivity (65.4-76.9%), specificity (83.3-89.9%), and AUC values (0.842-0.910) for POI, the highest in the first test. Three-antibodies panel screening showed higher diagnostic accuracy (84.1% versus 75-79.6%). The levels of these antibodies correlated with menstrual irregularities and a decrease in the antral follicle count. Thus, antibodies against CYP11A1, CYP19A1, and CYP21A2 have a high diagnostic value for POI. Three-antibody panel screening may improve the accuracy of POI diagnosis and be useful for identifying high-risk groups, early stages of the disease, and predicting POI progression.

Regulation of aromatase in cancer.

The regulation of aromatase, an enzyme involved in the biosynthesis of estrogen in normal and cancer cells, has been associated with growth factor signaling and immune response modulation. The tissue-specific regulatory roles of these factors are of particular importance as local aromatase expression is strongly linked to cancer development/progression and disease outcomes in patients. Therefore, aromatase has become a chemotherapeutic target and aromatase inhibitors (AIs) are used in the clinic for treating hormone-dependent cancers. Although AIs have shown promising results in the treatment of cancers, the emerging increase in AI-resistance necessitates the development of new and improved targeted therapies. This review discusses the role of tumor and stromal-derived growth factors and immune cell modulators in regulating aromatase. Current single-agent and combination therapies with or without AIs targeting growth factors and immune checkpoints are also discussed. This review highlights recent studies that show new connections between growth factors, mediators of immune response, and aromatase regulation.

Aromatase: Contributions to Physiology and Disease in Women and Men.

Aromatase (estrogen synthetase; EC 1.14.14.1) catalyzes the demethylation of androgens' carbon 19, producing phenolic 18-carbon estrogens. Aromatase is most widely known for its roles in reproduction and reproductive system diseases, and as a target for inhibitor therapy in estrogen-sensitive diseases including cancer, endometriosis, and leiomyoma (141, 143). However, all tissues contain estrogen receptor-expressing cells, the majority of genes have a complete or partial estrogen response element that regulates their expression (61), and there are plentiful nonreceptor effects of estrogens (79); therefore, the effect of aromatase through the provision of estrogen is almost universal in terms of health and disease. This review will provide a brief but comprehensive overview of the enzyme, its role in steroidogenesis, the problems that arise with its functional mutations and mishaps, the roles in human physiology of aromatase and its product estrogens, its current clinical roles, and the effects of aromatase inhibitors. While much of the story is that of the consequences of the formation of its product estrogens, we also will address alternative enzymatic roles of aromatase as a demethylase or nonenzymatic actions of this versatile molecule. Although this short review is meant to be thorough, it is by no means exhaustive; rather, it is meant to reflect the cutting-edge, exciting properties and possibilities of this ancient enzyme and its products.

[Metformin does not suppress the aromatase expression in breast cancer tissue of patients with concurrent type 2 diabetes].

Although there is data suggesting the in vitro inhibition of aromatase in cell lines by antidiabetic biguanide metformin (MF), there is no data on the intratumoral breast cancer (BC) aromatase expression in patients already receiving therapy for type II diabetes. Paraffinized tumor samples obtained from 57 BC pts aged 48-77 yrs, >80% of pts had stage T1-2N0-3M0 BC. Thirteen of the pts didn't have diabetes, 44 pts were previously diagnosed type II diabetes and reseaved the following therapy for at least 1 year: diet only (n=14), sulphonylurea (SU, n=14), metformin (MF, n=9) or MF with SU (n=7). Tumor samples were deparaffinized in xylene and treated with the monoclonal aromatase antibody 677. The rate and intensity of tissue staining was then analyzed by semi-quantitative method using conventional scores. Negative controls were processed with 0.01 M PBS instead of the specific antibody. For positive control paraffin-embedded human placenta samples were used. By conventional scores method the following values were obtained: 1.31 (pts without diabetes), 1.47 (all diabetic patients), 2.22 (MF), 1.50 (SU), 1.29 (MF+SU), 1.81 (MF and MF+SU), 1.07 (diet). Allred scores for progesterone receptor (PR) were the highest in the samples from pts treated with MF or MF+SU and the lowest in the samples obtained from SU-treated pts. Thus, in contrast to previous findings suggesting the suppressive effect of MF on aromatase in vitro, no such trend was discovered for aromatase expression in tumor samples from diabetic patients treated with MF. Although the investigated patients population is still small, this data combined with clinical data (higher PR levels) may suggest the better responses to hormonal therapy in MF-treated diabetic patients.

Transplacental transfer and metabolism of buprenorphine in preterm human placenta.

We sought to determine whether gestational age affects the transplacental transfer and metabolism of buprenorphine (BUP). Transfer of BUP (10 ng/mL) and its [ (3)H]-isotope was determined across placentas of 30 to 34 weeks of gestation utilizing the technique of dual perfusion of placental lobule. Concentration of the drug in trophoblast tissue and in maternal and fetal circuits was determined by liquid scintillation spectrometry. Microsomes prepared from placentas of 17 to 37 weeks of gestation were divided into three groups: late second, early third, and late third trimesters. Antibodies raised against human cytochrome P450 (CYP) isoforms were utilized to identify the enzyme(s) catalyzing BUP biotransformation by preterm placental microsomes. The amount of norbuprenorphine formed was determined by liquid chromatography-mass spectrometry (LC-MS). BUP transfer across the placentas of 30 to 34 weeks of gestation was similar to those at term. CYP19 antibodies caused 60% inhibition of BUP metabolism by microsomes of late second and early third trimesters and 85% by microsomes of late third trimester. The developmental changes occurring in human placenta between 30 weeks of gestation through term do not affect the transfer of BUP across human placenta. CYP19 is the major enzyme responsible for biotransformation of BUP beginning at 17 weeks of gestation until term.

Plasma membrane localization of CYP4Z1 and CYP19A1 and the detection of anti-CYP19A1 autoantibodies in humans.

It is thought that autoantibody (aAb) production can be caused by (aberrant) protein targeting to the plasma surface of cells. We recently demonstrated the presence of the human cytochrome P450 enzyme CYP4Z1 on the plasma membrane of MCF-7 breast cancer cells and the detection of high titers of anti-CYP4Z1 aAbs in breast cancer patients, but not in healthy controls. In the present study we show that cells of the normal breast cell line MCF-10A do not display CYP4Z1 on their surface. By contrast, we detected CYP19A1 (aromatase) on the plasma membrane of both cell lines. Interestingly, the presence of CYPs on the cell surface did not correlate with their relative expression levels in these cell lines. Indirect ELISA experiments demonstrated the presence of anti-CYP19A1 aAbs in female breast cancer patient sera as well as in male and female controls, respectively; aAb titers in all three groups varied considerably and overall, the results obtained for each group were not significantly different from those of either of the other two groups. Based on these data we propose the hypothesis that CYP translocation to the plasma membrane, but not the intracellular expression level, is the crucial precondition for the generation of anti-CYP aAbs.

Intratumoral aromatase in human breast, endometrial, and ovarian malignancies.

In human estrogen-dependent neoplasms such as breast, endometrioid endometrial, and surface epithelial-stromal ovarian carcinomas, intratumoral aromatase is considered to play important roles in converting circulating androgens derived from adrenal cortex and/or ovary to estrogens, possibly in association with 17 beta-HSD type 1 and estrogen sulfatase. Analysis of intratumoral aromatase in these estrogen-dependent neoplasms is important not only in understanding the development and biological behavior of these tumors, but also in the clinical management of these patients, because suppression of intratumoral aromatase by newly developed aromatase inhibitors may provide new potentials in endocrine therapy of these patients.

Aromatase and estrogen receptor immunoreactivity in the coronary arteries of monkeys and human subjects.

OBJECTIVE: The objective of this study was to determine whether estrogen could be formed locally in the coronary arteries. DESIGN: Coronary arteries were examined from monkeys (Macaca fascicularis, one male and one female) and human subjects (one premenopausal woman, one postmenopausal woman, and one man) by immunocytochemistry, using purified antisera against human placental estrogen synthetase (aromatase) and ER α. The arteries were graded for the amount of atherosclerosis. RESULTS: There was clear immunopositivity for both aromatase and estrogen receptors in all arteries studied. Although all endothelial cells (CD31 positive) stained for both antigens, the staining in macrophages, fibroblasts, and smooth muscle cells was irregular. CONCLUSION: The present results provide the first evidence for the local formation of estrogen in the coronary arteries. In addition to complementing the evidence of a cardioprotective effect of estrogen on the coronary circulation, our results highlight the potential importance of local regulation of estrogen formation and the role of available precursor androgens in maintaining the cardiovascular system.

Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between aromatase expression and aromatase inhibitors are warranted.

Aromatase in zebrafish: a potential target for endocrine disrupting chemicals.

In zebrafish, two isoforms of the aromatase gene exist, namely cyp19a1 and cyp19a2, expressed predominantly in the gonads and brain, respectively. In this study, we focus on characterizing the specificity of antibodies against the aromatase isoforms, and on (xeno)estrogen-induced changes of individual cyp19a2 mRNA concentrations in the brains of adult male zebrafish. Among three polyclonal antibodies studied, the one against CYP19A2 was found to be specific in Western blots and immunohistochemistry. Real-time RT-PCR analyses revealed strong interindividual variation of cyp19a2 levels in the brains of adult male zebrafish. After a three-week-exposure to (xeno)estrogens, mean values of cyp19a2 mRNA levels tended to increase, with significant induction at 200 ng 17beta-estradiol/L, but interindividual variation of cyp19a2 expression was maintained.

Multiple forms of aromatase and response of breast cancer aromatase to antiplacental aromatase II antibodies.

Two distinct aromatase-active protein complexes are solubilized by use of deoxycholate and separated by diethylamino-ethyl-cellulose chromatography from lyophilized powder of 900 X g precipitate fraction of human term placenta. Aromatase activity to produce estriol, the major estrogen of human pregnancy, was designated to be aromatase I activity and measured by estriol formation from 16 alpha-hydroxytestosterone. Aromatases II activity was the designation for that which produces estrone plus estradiol and was measured by androstenedione aromatization. Aromatases II and I are eluted with 0.25 M and 0.5 M Tris buffer, respectively, from diethylaminoethyl-cellulose column in an Mr 2 million soluble complex. Each has a minimum active Mr 135,000 subunit, which is isolated by Bio-Gel filtration in the presence of detergents, and consists of a reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (Mr 83,000) and a cytochrome P-450 (Mr 52,000). Aromatase II was found to be the major aromatase, containing approximately five times more aromatase activity, reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity, cytochrome P-450, and protein than did aromatase I. Antibodies raised in rabbits against aromatase II and its reductase suppressed aromatase II activity of breast cancer tissues, as well as of adult male lung tissue, placental microsomes, and solubilized aromatase. The breast carcinoma specimens responded to the antibodies in different degrees, but there was no response to antibodies against rat liver cytochrome P-450. The results indicate similar antigenic structures for breast cancer and placental aromatase but not for rat liver cytochrome P-450.

The validation of new aromatase monoclonal antibodies for immunohistochemistry--a correlation with biochemical activities in 46 cases of breast cancer.

Intratumoral aromatase is a therapeutic target for the treatment of post-menopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. Immunohistochemistry (IHC) is considered one of the most suitable methods in this regard. A multi-centre collaborative group has been established to generate and validate new aromatase monoclonal antibodies. We have selected two monoclonal antibodies, #677 against native aromatase protein and F2 against formalin-fixed protein for this purpose. With these two monoclonal antibodies 43 cases of invasive ductal carcinoma, which had been previously assayed for aromatase activity by product isolation methodology, were immunostained in three laboratories in UK, USA and Japan and independently evaluated by three pathologists (H.S., T.A. and S.G.S.). Staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments of the tumors were assessed by estimating the proportion of positive staining cells and the relative intensity of staining in this fashion. Immunoreactivity could be detected in each component of the tissue specimens but a significant positive correlation with biochemical activity was detected only in malignant epithelium stained with 677 not in other components with #677 and not in any of the components. Staining using F2 as a primary antibody did not produce a positive correlation in any components with aromatase activity. These results suggest that we now have a monoclonal antibody against aromatase (#677) which may be used to stain archival materials. A methodology and scoring system is recommended whereby staining significantly correlates with aromatase activity of the resected tissue specimens of breast cancer.

Structure of an activity suppressing Fab fragment to cytochrome P450 aromatase: insights into the antibody-antigen interactions.

The crystal structure of a Fab fragment (Fab3-2C2) of a monoclonal antibody raised against aromatase cytochrome P450 P450arom) has been determined at 3.0 A resolution. P450arom is a membrane bound enzyme responsible for the catalysis of indrogens to estrogens, the process of aromatization, and hence has been implicated in hormone-dependent breast cancer. The Fab fragment of MAb3-2C2 IgG suppresses P450arom activity in a dose dependent manner. The Fab3-2C2 molecule crystallizes n the space group P2(1)2(1)2(1) with a unit cell of a= 154.89 A, b = 73.51 A, and c= 36.90 A. The crystal structure consists of a light and a heavy chain in the asymmetric unit, each characterized by the greek-key antiparallel beta barrel folding seen in all Fab structures. The average elbow angle between the two domains is 143 degrees. Modeling of the interactions between the variable domains of the antibody and a known model of P450arom maps the epitope to a region of the enzyme that is consistent with the available biochemical data and the activity-suppressing function of the antibody. The epitope mapping result is further supported by the inability of MAb3-2C2 IgG to suppress the activity of, or to interact with placental porcine P450arom, which is 81% identical (86% similar) to human P450arom but has a few key substitutions in the putative epitope region.

An enzyme-linked immunosorbent assay for quantitation of aromatase cytochrome P-450.

Traditionally, aromatase has been quantified as aromatase activity according to its ability to produce estrogen from androgen. We have developed a quantitative assay based on the protein mass of catalytically active aromatase cytochrome P-450. A solid phase sandwich enzyme-linked immunosorbent assay for aromatase cytochrome P-450 has been devised using mouse monoclonal antibody (MAb3-2C2) and rabbit polyclonal antiserum (PAb R-8-2). Two rabbit antisera (PAb R-8-1 and R-8-2) were raised by immunization against human placental aromatase cytochrome P-450 which had been isolated by immunoaffinity chromatography of MAb3-2C2-coupled to Sepharose 4B resin. Both antisera were capable of suppressing human placental aromatase activity with IC50 values of 0.6 and 0.8 microliter/ml incubate, respectively, and showed monospecific to aromatase cytochrome P-450 in the Western blot analyses. Solubilized human placental microsomal samples were incubated in microtiter wells precoated with MAb3-2C2. The unbound proteins were washed out, and the aromatase cytochrome P-450 bound with the MAb3-2C2 in the wells was then reacted with PAb R-8-2, the binding of which was subsequently probed with goat antirabbit immunoglobulin G antibody alkaline phosphatase conjugate. Immunoaffinity-purified aromatase cytochrome P-450 of human placental microsomes was used for the standard, with the current assay detection limit at 1 ng/ml. There was a positive correlation between aromatase activity and the immunoreactive aromatase cytochrome P-450 level in solubilized microsomal samples after preincubation at 22 and 37 C, indicating that the enzyme-linked immunosorbent assay measures the level of aromatase cytochrome P-450 that has catalytic activity. The mean level of aromatase cytochrome P-450 in solubilized human term placental microsomes was 16.4 +/- 10.3 (+/- SD) micrograms/ml, corresponding to 0.38 +/- 0.19% of the original microsomes. The mean specific activity of aromatization of the solubilized samples was 0.650 +/- 0.163 nmol estrogen formed/min.mg protein. These results indicate that aromatase in the solubilized placental microsomal fraction has catalytic ability of 5.3 +/- 1.6 min-1 based on the immunoassayable cytochrome P-450.

Diverse functions of aromatase: O-deethylation of 7-ethoxycoumarin.

In studying the diverse functions of aromatase we found that purified and reconstituted aromatase also catalyzes O-deethylation of 7-ethoxycoumarin. Aromatase cytochrome P450 was purified from human term placentas by monoclonal antiaromatase P450 antibody-Sepharose 4B column chromatography. Kinetic analysis of the O-deethylation of 7-ethoxycoumarin by reconstituted aromatase showed Km of 200 microM, Vmax of 12.5 nmol.min-1.mg-1, and turnover rate of 1.06 min-1. 7-Ethoxycoumarin competitively inhibited androstenedione aromatization, the Ki was 180 microM. Fadrozole (CGS16949A), a specific competitive aromatase inhibitor, and MAb3-2C2, an antiaromatase P450 monoclonal antibody, inhibited both aromatase and 7-ethoxycoumarin O-deethylase activities dose responsively. The IC50 of Fadrozole was 33 nM for aromatase and 67 nM for 7-ethoxycoumarin O-deethylase. The IC50 of MAb3-2C2 was 1.1 micrograms IgG for aromatase and 4.0 micrograms IgG for 7-ethoxycoumarin O-deethylase. These results indicate that the two enzyme activities are catalyzed by the same active site of the cytochrome P450. Contrary to the previous postulate on the mechanism-based inactivation of microsomal aromatase by 4-androstene-3,6,17-trione, we found that with purified aromatase, both the initial 19-hydroxylase and the after lyase reactions are simultaneously inactivated by the steroid suicide inhibitor.

Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies.

Estrogen formation is catalyzed by the aromatase cytochrome P450 (P450AROM) enzyme. Aromatase activity has been detected in several regions in the rat brain. In the present study, we used peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of the rat P450AROM protein (as deduced from the nucleic acid sequence of the rat P450AROM complementary DNA), to determine the location of this enzyme in rat brain sections. Immunoreactive antisera were titered by means of an enzyme-linked immunosorbent assay and purified by diethylaminoethyl-Affigel Blue chromatography. Specific immunoreactivity was confirmed by Western blot analysis using known aromatase-containing tissue (rat ovary homogenates and microsomal fractions). Evaluation of the distribution of P450AROM immunoreactivity in brain sections of male and female rats (30 and 60 days of age) was performed using the avidin biotin peroxidase immunocytochemical technique and light microscopy. P450AROM immunoreactivity appeared to be localized to neurons, and was present in brain regions and nuclei where enzymatic activity has been reported. For example, intense immunoreactivity was observed in the amygdaloid structures and supraoptic nucleus, whereas moderate to light immunoreactivity was evident in the paraventricular and arcuate nuclei and hippocampus. Surprisingly, neurons in the bed nucleus stria terminalis, medial basal hypothalamic, and preoptic areas displayed little aromatase immunoreactivity. However, P450AROM immunoreactivity was detected in specific brain regions not previously recognized to contain the enzyme (i.e. intense staining was seen in the reticular thalamic nucleus, olfactory tract and piriform cortex, as well as other brain structures). The pattern, distribution, and intensity of P450AROM immunoreactivity was similar regardless of sex or age. In this study, microsomal preparations derived from a new brain area (i.e. the reticular thalamic nucleus; Rt) displaying P450AROM immunoreactivity were observed to contain detectable levels of aromatase enzymatic activity, as determined by the 3H2O-release assay. The activity in the Rt was inhibited by a known aromatase inhibitor, 4-hydroxyandrostenedione. These results confirm histologically the localization of P450AROM to brain regions where aromatase enzymatic activity has been detected and extend the knowledge of its location to areas previously unknown as sites of aromatase activity, which may be involved in the modulation of neuroendocrine function and reproductive behavior.

Expression of aromatase cytochrome P-450 in premenopausal and postmenopausal human ovaries: an immunocytochemical study.

The cellular distribution of the aromatase cytochrome P-450 enzyme in human ovaries has been investigated immunocytochemically, using an aromatase-specific monoclonal antibody. Ovaries of females ranging in age from prepubertal infant girl through to postmenopausal adulthood were obtained from immediate autopsy or after surgery. The results have revealed temporal and spatial changes in expression of aromatase at different stages of development. No immunoreactive aromatase was detected in the ovary of the 2.5 month infant. In premenopausal ovaries, aromatase was absent from the stromal compartment, but in follicles, a consistent pattern in expression of aromatase was observed, related to their size and developmental stage. Aromatase was not expressed in primordial, primary, or small secondary follicles less than 250 microns diameter. In slightly larger follicles (250-700 microns diameter) aromatase was first detected in a few thecal cells (TC). In more developed secondary through to large preovulatory follicles (greater than 1 cm) TC aromatase immunostain increased in intensity and number of positive cells, and the reaction was localized to a band of theca interna (TI) cells at the TI/theca externa interface. In granulosa cells (GC), aromatase was first detected in follicles in the initial stages of antrum formation (greater than 700 microns), and staining intensified as follicle diameter and antral cavity increased, being maximal in preovulatory follicles. GC aromatase was always found in the presence of TI immunostain. These two cell populations were separated by an unstained layer of TI cells giving the follicle walls a banded appearance. Immunostain was most intense in mural GC, was weaker in antral GC cells and was absent from the cumulus GC. Immunoreactive aromatase was also detected in functional corpora lutea (CL) but was absent from involuting CL's and corpora albicans. Our findings indicate that the immunostained cells of the CL are comprised of the former GC and possibly a subpopulation of former TI cells. In perimenopausal ovaries there was no evidence of any follicular or stromal aromatase immunostain. In postmenopausal ovaries no follicles were observed, but individual cells and clusters of cells in the stromal compartment of 3/7 specimens were found to have an aromatase immunostain reaction. In all cases, the aromatase immunostain reaction was cytoplasmic. The results provide the first direct evidence of the existence of TC aromatase, and of stromal cell aromatase in postmenopausal women.

Distribution and regulation of telencephalic aromatase expression in the zebra finch revealed with a specific antibody.

In songbirds, aromatase (estrogen synthase) activity and mRNA are readily detectable in the brain. This neural aromatization presumably provides estrogen to steroid-sensitive targets via autocrine, paracrine, and synaptic mechanisms. The location of immunoreactive protein, however, has been difficult to describe completely, particularly in distal dendrites, axons, and terminals of the forebrain. Here we describe the neuroanatomical distribution of aromatase in the zebra finch by using a novel antibody raised specifically against zebra finch aromatase. The distribution of aromatase-positive somata in the zebra finch brain is in excellent agreement with previous reports. Additionally, this antibody reveals elaborate, spinous dendritic arbors, fine-beaded axons, and punctate terminals of telencephalic neurons that may synthesize estrogen. Some of these axon-like fibers extend into the high vocal center (HVC) and the robust nucleus of the archistriatum (RA) in males and females, suggesting a role for presynaptic aromatization in cellular processes within these loci. Adult males have more aromatase-positive fibers in the caudomedial neostriatum (NCM) and the preoptic area (POA) compared to females, despite the lack of detectable sex differences in the number of immunoreactive somata at these loci. Thus, the compartmentalization of aromatase in dendrites and axons may serve a sexually dimorphic function in the songbird. Finally, in adult males, aromatase expression is down-regulated by circulating estradiol in the hippocampus, but not in the NCM or POA. The distribution of aromatase suggests a role for aromatization in the regulation of pre- and postsynaptic function in steroid sensitive areas of the songbird forebrain.

Aromatase is abundantly expressed by neonatal rat penis but downregulated in adulthood.

Although synthesis of estrogen by male gonads has been well documented for over half a century, it is only recently that the role of estrogen in male reproductive events has gained appreciation. We recently reported abundant expression of estrogen receptor (ER)-alpha and -beta in different cell types of the rat penis, whose levels diminished with advancing age. The present study, which builds on data from the ER study, was designed to determine whether the penis is capable of generating its own local estrogen by examining evidence of the expression of aromatase, a microsomal enzymatic complex which irreversibly converts androgens to estrogens, using immunohistochemistry, Western blotting, in situ hybridization and real-time PCR analyses. Secondly, the effects of sex steroid hormones on penile aromatase were examined. Discrete aromatase immunoreactive cells were localized in primordial corpus cavernosum, corpus spongiosus and os penis, blood vessels and sensory corpuscle of glans penis. In situ hybridization signals corresponded with immunohistochemical findings. Western blot, enzyme immunoassay and real-time PCR analyses of rat penile samples revealed an age-dependent expression of aromatase and estrogen, with levels at week 1 almost resembling those of the ovary, but they decreased sharply by week 8, and decreased further by week 35. This expression pattern was strikingly similar to that of ER-alpha reported previously. Testosterone and diethylstilbesterol administered prenatally upregulate levels of aromatase mRNA and protein, and estrogen postnatally. Dihydrotestosterone upregulated aromatase mRNA and protein, but not estrogen. We conclude that estrogen acts via ER in a paracrine and/or autocrine manner to regulate penile events, particularly during development, and that estrogen synthesis is regulated by estrogen and androgens.

Immunohistochemical localization of cytochrome P450 aromatase in equine gonads.

Estrogens are the major steroids produced by equine gonads. To identify the cells responsible for estrogen synthesis, an antiserum against purified equine testicular cytochrome P450 aromatase was produced in rabbits. The reactivity and specificity of the antiserum were assessed by ELISA, immunoblot analysis, and immunoneutralization studies. Immunofluorescence microscopy demonstrated that in the male gonad, cytochrome P450 aromatase (P450arom) was localized in the interstitial tissue, whereas, under the experimental conditions used, the Sertoli and germ cells did not show any specific staining. In the ovary, the granulosa cells of small follicles exhibited faint immunofluorescent staining for P450arom and the granulosa cells of large, viable more follicles showed a high degree of immunoreactivity. In the corpus luteum, all the luteinized cells showed immunoreactivity. No immunoreactivity was detected in other cells of small and large viable follicles. Immunolocalization of P450arom in the equine testicular Leydig cells and in ovarian granulosa and luteinized cells indicates that these cells have the ability to metabolize androgens to estrogens and possibly to catechol estrogens.