Identification of aromatase in the reptilian brain

The present study tests the hypothesis that brain aromatase is an "ancient" property of nervous tissue and may be identified in homologues of the limbic system in a non-mammalian vertebrate, the turtle Chrysemys picta. Tissue homogenates (180 mg wet weight/2 ml) were incubated with [7 alpha-3H]androstenedione and cofactors for 60 min at 37 C. Estrone (E1) was isolated and characterized by thin layer chromatography, methylation and recrystallization to constant specific activity. No estradiol-17 beta was detected. Aromatase was found only in the forebrain but was diffusely distributed throughout this major brain division. No other neural or non-neural tissues, including mid- and hindbrain structures, testis, and ovary, synthesized detectable quantities of E1 in our system. The strio-amygdaloid complex of both sexes synthesized more E1 per unit weight than the preoptic-hypothalamic area (POA-HTH) or other forebrain structures. It is possible that the conversion of androgen to estrogen has biological significance in this species since the reaction occurred throughout the physiological temperature range experienced during activity in nature and sex differences in brain aromatase activity during the breeding season were apparent. These experiments in Chrysemys demonstrate that the synthesis of estrogen from androgen by the brain is not limited to mammals, but also occurs at a more primitive level of phylogenetic development. Restriction of aromatase to forebrain structures of the turtle is consistent with the neuroanatomic distribution of enzyme activity in the limbic system of mammals. Estrogen yield from adult turtle brain incubates (3.2-22.6 pmol/g) is more like that reported for fetal (2.7-33 pmol/g) than for adult (0.1-1.9 pmol/g) mammals. We suggest that the in situ synthesis of estrogen by the central nervous system has its orgins early in vertebrate evolution and may be a primitive characteristic of brain-steroid interactions that regulate physiological and behavioral sex in vertebrates.     

Brain formation of oestrogen in the mouse: sex dimorphism in aromatase development

Steroid sex hormones have an organizational role in gender-specific brain development. Aromatase, converting testosterone (T) to oestradiol-17 beta (E2), is a key enzyme in the brain and the regulation of this enzyme is likely to determine availability of E2 effective for neural differentiation. In rodents, oestrogens are formed very actively during male perinatal brain development. This paper reviews work on the sexual differentiation of the brain aromatase system in vitro. Embryonic day 15 mouse hypothalamic culture aromatase activity (AA: mean Vmax = 0.9 pmol/h/mg protein) is several times greater than in the adult, whereas apparent Km is similar for both (approximately 30-40 nM). Using microdissected brain areas and cultured cells of the mouse, sex differences in hypothalamic AA during both early embryonic and later perinatal development can be demonstrated, with higher E2 formation in the male than in the female. The sex differences are brain region-specific, since no differences between male and female are detectable in cultured cortical cells. AA quantitation and immunoreactive staining with an aromatase polyclonal antibody both identify neuronal rather than astroglial localizations of the enzyme. Kainic acid eliminates the gender difference in hypothalamic oestrogen formation indicating, furthermore, that this sex dimorphism is neuronal. Gender-specific aromatase regulation is regional in the brain. Oestrogen formation is specifically induced in cultured hypothalamic neurones of either sex by T, since androgen has no effect on cortical cells. Androgen is clearly involved in the growth of hypothalamic neurones containing aromatase. It appears that differentiation of the brain involves maturation of a gender-specific network of oestrogen-forming neurones.     

Neuroblastoma and Alzheimer's disease brain cells contain aromatase activity

Human brain steroidogenic mechanisms, particularly aromatase, have been investigated in healthy and diseased conditions. Aromatase activity was measured in differentiated and undifferentiated neuroblastoma cell lines from mouse (TMN) and human (5H SY5Y) and in human post mortem brain samples. Neuroblastomas show much higher aromatase activity than human brain samples. Homogenates of adult human male and female cortex and frontal and temporal areas of both Alzheimer's and control patients all show considerably lower activity. The temporal area has significantly higher aromatase activity than the frontal. Aromatisation activity in differentiated neuroblastoma cells of both species is lower than in undifferentiated cells. These results are consistent with an inverse relationship between brain estrogen formation and stage of neuronal differentiation and the hypothesis that aromatase may be involved in the early stages of neuronal growth. Significant but variable activities of other androgen-metabolising enzymes, such as 5 alpha-reductase, 3 alpha/beta-hydroxysteroid dehydrogenases, and 17 beta-hydroxysteroid dehydrogenase, which generate a spectrum of regulatory molecules, are also found.     

Aromatase expression and its localization in human breast cancer

Aromatization or in situ estrogen production by aromatase has been considered to play an important role in the development of human breast carcinoma. In the human breast, aromatase overexpression is observed in the stromal or interstitial cells of the carcinoma, especially at the sites of frank invasion and/or adipose tissue. Transplantation experiments in the nude mouse employing MCF-7 and/or SF-TY human fibroblast cell lines revealed that aromatase activity and expression were much higher in the tumour with MCF-7 and SF-TY than that with MCF-7 alone. Aromatase overexpression in human breast carcinoma tissue is considered to occur as a result of carcinoma-stromal cell interactions, i.e. paracrine communication between stromal and carcinoma cells. Aromatase overexpression is correlated with the malignant phenotype in the human breast, but not with stage, age, clinical stages, clinical course, or proliferative activity of breast carcinoma. Aromatase overexpression may be correlated with development, rather than the biological behaviour of breast malignancy. Aromatase overexpression is not necessarily correlated with expression of 17beta-hydroxysteroid dehydrogenase type 1, which converts estrone to estradiol and estrogen receptor. Different mechanisms may be involved in the regulation of expression of these two important estrogen-metabolizing enzymes and estrogen receptor in human breast cancer. Aromatase overexpression in intratumoral stromal cells was much more frequently detected in male breast cancer than in female counterparts, which confers a growth advantage on cancer cells in a male hormonal environment with low serum estrogen levels.     

Pharmacological analysis of the haemodynamic effects of 5-HT1B/D receptor agonists in the normotensive rat

Our previous findings in female rats suggest that the potent effects of sex steroids on mood and mental state may be mediated, in part, by the effect of estrogen on the 5-hydroxytryptamine2A receptor (5-HT2AR) in brain. The aim of the present study was to determine the effect of acute (approximately 32h) sex steroid manipulation on central 5-HT2AR in the adult male Wistar rat. Castration (under halothane anesthesia) decreased while testosterone or estrogen, but not 5alpha-dihydrotestosterone (5alpha-DHT), increased significantly the 5-HT2AR mRNA content in dorsal raphe nucleus and the density of 5-HT2AR binding sites in frontal, cingulate and primary olfactory cortex and nucleus accumbens. The lack of effect of 5alpha-DHT, a potent androgen which cannot be converted to estrogen, suggests that the action of testosterone depends upon its conversion to estrogen by aromatase. This may also explain why estrogen, but not testosterone or 5alpha-DHT, increased the density of 5-HT2AR binding sites in the caudate-putamen, a brain region where aromatase is scarce. These findings are discussed in relation to the possible role of the 5-HT2AR in depression, schizophrenia and Alzheimer's Disease.     

Immunolocalization of aromatase in human minor salivary glands of the lower lip with primary Sj?gren's syndrome

The enzyme aromatase is involved in the conversion of androgens to estrogens and in the modulation of various androgenic and estrogenic actions. Abnormalities of estrogen metabolism have been postulated to play roles in the development and/or pathophysiology of Sj?gren's syndrome. In the present study, aromatase was immunolocalized in 75 cases of inflammatory disorders of human minor salivary glands of the lower lip. These included cases of primary Sj?gren's syndrome (19 cases), of chronic sialadenitis (34 cases) and of mucous extravasation cysts (22 cases), in order to clarify the possible involvement of in situ estrogen production in primary Sj?gren's syndrome. Aromatase immunoreactivity was detected in myoepithelial cells of acini and in interstitial cells adjacent to acini and ducts in 13/19 (68%) cases of primary Sj?gren's syndrome. In contrast, aromatase expression was detected in only six of 34 (18%) cases of chronic sialadenitis and in seven of 22 (32%) cases of mucous extravasation cyst. These results suggest that increased aromatase expression in minor salivary glands with primary Sj?gren's syndrome in premenopausal women may be involved in the biological features of primary Sj?gren's syndrome through the production of estrogens in situ and possibly through the aggravation of the inflammatory reaction.     

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.     

Photodynamic therapy for rheumatoid arthritis?

Songbirds have emerged as extremely important animals for investigating sex steroid hormone effects on the central nervous system. The masculinizing effects of exogenous estrogen on the neural circuits controlling song in female zebra finches are well documented. There is evidence that estrogens are necessary for the full activation of singing behavior in several species. These kinds of studies have led us and others to investigate the mechanisms whereby estrogens are made available to the brains of songbirds during development and adulthood. In this article, I review results of some of these studies examining the estrogen synthetic enzyme aromatase and its expression and activity in brain and in other tissues of songbirds. I will discuss some results and thoughts we have about the interactions of aromatase with the two remaining androgen-metabolizing enzymes in the avian brain, 5alpha-reductase, the enzyme that converts T into the active androgen 5alpha-dihydrotestosterone (DHT); and 5beta-reductase, the enzyme that converts T into the inactive 5beta-DHT. Finally, I will consider some ideas raised by these studies concerning potential sources of the androgen substrate for brain aromatization as well as some possible new functions that aromatase might be playing in the songbird telencephalon.     

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and the sexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.     

An analysis of automatic teller machine usage by older adults: a structured interview approach

Sex differences in the activity of aromatase cytochrome P450 (CYP19) in the rat brain have been reported during pre- and postnatal development. It is unclear, however, whether these differences are reflected by corresponding differences in specific mRNA levels. To address this question, we have examined aromatase mRNA levels in specific regions of male and female rat brains by means of in situ hybridization (ISH). At prenatal stages of development, i.e. at gestational day 18 (GD18) and GD20, aromatase mRNA was detected in several preoptic, hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal sex differences in any of these regions. In contrast, clear-cut sex differences were determined at postnatal day (PN) 2; male animals expressed significantly more aromatase mRNA in the bed nucleus of stria terminalis (BST) and the sexually dimorphic nucleus of the preoptic area (SDN). Smaller but still significant differences (females > males) were obtained in the medial preoptic area (MPO). At PN6, sex differences of aromatase mRNA signals (males > females) were still present in the BST, but were no longer detectable in the SDN and the MPO. At PN15 and in adult animals, aromatase mRNA levels were similar in BST and medical amygdaloid nucleus of male and female rats. Since aromatase mRNA expression decreases during postnatal development, no ISH signals could be detected anymore in MPO, SDN and ventromedial hypothalamic nucleus. Our results are consistent with the concept that differential regulation of aromatase mRNA expression might be important for the establishment of different neuronal circuitry in male and female animals.     

Anastrozole: a new selective nonsteroidal aromatase inhibitor

Aromatase (estrogen synthetase) is the enzyme complex responsible for the final step in estrogen synthesis--the conversion of androstenedione and testosterone to estrone and estradiol, respectively. Inhibitors of this enzyme have been shown to be clinically effective in the treatment of advanced breast cancer in postmenopausal women, in whom the major source of estrogen production derives from aromatization of adrenal androgens in peripheral tissues, such as muscle, liver, and fat. The most widely used aromatase inhibitor has been aminoglutethimide; however, it is nonselective and also inhibits adrenocorticosteroid synthesis, necessitating hydrocortisone supplementation. Aminoglutethimide is also associated with frequent and troublesome side effects. Formestane, the first selective aromatase inhibitor to be developed, has an improved safety profile and selectivity, but its use has been limited somewhat by its inconvenient administration via intramuscular injection. In this article, the preclinical and clinical data published to date on the new third-generation aromatase inhibitor anastrozole (Arimidex) are presented in the context of current endocrine therapies. Future applications of aromatase inhibitors, both as monotherapy and in combination with other endocrine therapies, are discussed. The use of aromatase inhibitors in advanced disease, the adjuvant setting, and as possible chemopreventive agents are examined.     

New insights into the regulation and function of brain estrogen synthase (aromatase)

In the brain, conversion of androgens into estrogens by the enzyme aromatase (estrogen synthase) is a key mechanism by which testosterone regulates many physiological and behavioral processes, including the activation of male sexual behavior, brain sexual differentiation and negative feedback effects of steroid hormones on gonadotropin secretion. Studies on the distribution and regulation of brain aromatase have led to a new perspective on the control and function of this enzyme. A growing body of evidence indicates that the estrogen regulation of aromatase is, at least in part, trans-synaptic. Afferent catecholamine pathways appear to regulate aromatase activity in some brain areas and thereby provide a way for environmental cues to modulate this enzyme. The localization of aromatase in pre-synaptic boutons suggests possible roles for estrogens at the synapse.     

A reappraisal of the hormonal regulation of larval fat body histolysis in female Drosophila melanogaster

The histolysis of larval fat body cells in adult female Drosophila melanogaster was examined in wild type and mutant animals. The fat body cells of wild type (Canton-S), apterous56f homozygotes, apterous78jts homozygotes and heterozygotes, apterous4/+, ecdysoneless1 homozygotes and heterozygotes all underwent histolysis normally during the 72 h following adult eclosion. Only in the case of ap4/ap4 adults did the cells fail to histolyze normally. The fat body cells of both diapausing and non-diapausing wild type females underwent histolysis at the same rate. Attempts to demonstrate histolysis in vitro were unsuccessful, even in the presence of juvenile hormones (JHs), larval ring glands, or adult ovaries. In all strains other than the ap4 homozygotes, a significant proportion of larval fat body cells were dead at any time while the ap4/ap4 animals, almost all cells remained viable. It is postulated that fat body cell lysis following eclosion is not a JH-mediated event, but is elicited by an as yet unidentified factor(s), possibly originating in the ovary.     

Further evidence of increased aromatase activity in granulosa luteal cells from polycystic ovary

This study evaluated the effect of atamestane (a competitive inhibitor of P-450 aromatase) on granulosa luteal cells from polycystic and normal ovaries. Treatment with atamestane (10 micromol/l) determined a strong inhibition of basal aromatase activity in both types of cells; however, its effect was markedly more pronounced in granulosa cells from normal ovary than in granulosa cells from polycystic ovaries (PCO; P < 0.01). Concomitant treatment with insulin (25 microg/ml) and increasing doses of atamestane (0.01-10 micromol/l) caused a dose-dependent inhibition of insulin-stimulated aromatase activity, but again with marked differences between the two types of cells. In granulosa cells from PCO, the minimal effective dose of atamestane was 1 micromol/l and it had an EC50 of 2.23 +/- 0.4 micromol/l and a maximal inhibitory effect of 75%; in granulosa cells from normal ovary, the minimal effective dose of atamestane was 0.01 micromol/l, the EC50 was 0.4 +/- 0.07 micromol/l, and the maximal inhibitory effect was 94%. Significant differences were observed between the different cells at all the studied dose points. Reversibility studies showed that resumption of aromatase activity in granulosa cells from PCO is basally greater and more inducible with insulin treatment. This study provides further evidence of an increased in-vitro function of the aromatase complex in granulosa cells from PCO, that could be induced by an altered cellular autoregulation.