Showing papers on "Aromatase published in 1989"

Anti-Müllerian hormone produces endocrine sex reversal of fetal ovaries.

Abstract: We have previously reported that anti-Mullerian hormone (AMH), also known as Mullerian-inhibiting substance, the testicular glycoprotein involved in regression of the Mullerian ducts of the male fetus, induces the formation of seminiferous cord-like structures in fetal ovaries exposed to it in organ culture We have now investigated the effect of bovine AMH, purified to homogeneity, on ovarian endocrine differentiation Ovine fetal ovaries exposed to AMH release testosterone instead of estradiol, an endocrine sex reversal due to suppression of aromatase activity AMH dramatically decreases the conversion rate of testosterone to estradiol and also decreases total aromatase activity, as measured by the tritiated water technique AMH acts by decreasing aromatase biosynthesis rather than by blocking enzyme activity, as suggested by the relatively long period of AMH exposure required to produce an effect In the rabbit fetal ovary, aromatase activity is AMH-responsive during the whole gestational period The basal steroidogenic activity of rat fetal ovaries is extremely low but can be markedly increased by cAMP AMH completely blocks the effect of cAMP Taken together, our results suggest that AMH plays a pivotal role in both morphological and endocrine gonadal sex differentiation

Regulation of Estrogen Biosynthesis by Human Adipose Cells

Abstract: Introduction THE BIOSYNTHESIS of estrogens is catalyzed by an enzyme complex termed aromatase which is localized in the endoplasmic reticulum of cells in which it is expressed. The aromatase enzyme complex consists of two components (1): The first is a form of cytochrome P-450 known as aromatase cytochrome P-450 (P-450AROM). This heme protein is responsible for binding the C19 steroid substrate and catalyzing the concerted series of reactions leading to the formation of the phenolic A ring. The second is a flavoprotein, NADPH-cytochrome P-450 reductase, which is an essentially ubiquitous protein in the endoplasmic reticulum of most cell types and is responsible for transferring reducing equivalents from NADPH to cytochrome P-450. Since there is only one gene encoding the reductase, this enzyme must be capable of transferring reducing equivalents to any form of microsomal cytochrome P-450 that it encounters (2). The aromatase reaction apparently utilizes 3 moles oxygen and 3 moles NADPH for every mole of C...

Localization of aromatase in synaptosomal and microsomal subfractions of quail (Coturnix coturnix japonica) brain.

Abstract: The subcellular distribution patterns of aromatase, 5α- and 5β-reductase in the hypothalamus/preoptic area of Japanese quail were studied using standard methods of centrifugation, and fractional const

Aromatase Activity in Quail Brain: Correlation with Aggressiveness*

Abstract: Testosterone (T) triggers aggressive behavior in males of many vertebrate species; however, the neural and hormonal basis of individual differences in the frequency or intensity of aggressive behavior is still debated. Using the Japanese quail (Coturnix coturnix japonica), a species in which individuals exhibit a wide range of aggressiveness in nature and the laboratory, together with a newly devised test procedure for quantifying aggressiveness, we recently demonstrated that aggression is estrogen dependent. Here we extend these studies by testing the hypothesis that aromatization in brain is a rate-limiting step in the expression of individual differences in aggressiveness. Using procedures previously validated for this species, aromatase and 5 alpha- and 5 beta-reductase activities were estimated in selected brain regions of reproductively active male quail by measuring conversion of [3H]androstenedione to [3H]estrone, [3H]5 alpha-androstanedione, and [3H]5 beta-androstanedione, respectively. In Exp 1, behaviorally inexperienced test birds were killed 90 sec after a single behavioral test. Aggressiveness of individuals in this group, as determined by pecking and locomotor activity in response to visualization of a conspecific, ranged 3- to 4-fold from high to low. Aromatase activity in the posterior hypothalamus (PHYP) was significantly higher in males rated high for aggressiveness than in animals rated low (1.04 vs. 0.59 pmol/h.mg protein; P less than 0.02). Similar differences were observed in the anterior hypothalamus/preoptic area (AHPOA) but were not significant. In Exp 2, sexually mature males were behaviorally tested eight times over 22 days and killed 24 h after the final test. Aggressiveness varied 5-fold from high to low, although the rating in a given bird remained constant with time and repeat testing. Aromatase activity in the AHPOA was significantly greater in birds rated high for aggressiveness than in low aggressiveness birds (3.77 vs. 2.80 pmol/h.mg protein; P less than 0.02). In addition, when AHPOA aromatase in all birds was plotted against behavioral intensity, there was a 2-fold variation and a significant positive correlation (r = 0.556; P less than 0.02). Similar differences were observed in PHYP, but these were of borderline significance. By contrast, aromatase levels outside the AHPOA and PHYP were unrelated to behavior. Moreover, in both Exp 1 and 2, 5 alpha- and 5 beta-reductase activities in AHPOA, PHYP, and other brain regions; plasma T, 5 alpha-dihydrotestosterone, and total estrogens; and relative testicular weights were not consistently related to aggression.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of aromatase with CGS 16949A in postmenopausal women.

Abstract: Potent, specific, and nontoxic inhibitors of aromatase would be useful for experimental studies and for use in the treatment of breast cancer and other disorders. We evaluated the effects of CGS 16949A, a nonsteroidal inhibitor of aromatase activity, in 12 postmenopausal women with breast cancer by measuring plasma and/or urinary androgens and estrogens after oral administration of CGS 16949A at doses ranging from 0.6-16 mg daily; each dose was given for 2 weeks. The 0.6-mg daily dose partially lowered estrogen levels, and maximum reduction occurred at doses of 2-16 mg daily. The fall in plasma and urinary estrogens without a concomitant fall in plasma androgens confirmed the blockade of aromatase activity. The degree of estrogen reduction was greatest for urinary estrone [to 27 +/- 3% (+/- SE) of basal], followed in order by plasma estrone sulfate (30 +/- 4%), plasma estrone (32 +/- 6%), urine estradiol (45 +/- 5%), and plasma estradiol (65 +/- 5%). Use of gas liquid chromatography-mass spectrometry techniques revealed similar patterns of reduction in catechol estrogens, estriol, and total urinary estrogens, suggesting that CGS 16949A does not alter the pathways of estrogen metabolism. The degree of estrogen reduction was remarkably similar to that caused with aminoglutethimide. At doses of 4-16 mg daily, CGS 16949A inhibited the C21-hydroxylase enzyme as well, based on concomitant rises in plasma androstenedione, testosterone, and 17 alpha-hydroxyprogesterone. This effect was insufficient to lower urinary cortisol excretion during the study. However, a statistically significant blunting of plasma cortisol responses to ACTH occurred with the 16-mg daily dose. No changes in plasma dehydroepiandrosterone sulfate levels or in thyroid, hematological, liver, or renal parameters were found. No significant side-effects of the medication were encountered. CGS 16949A appears to be a specific inhibitor of aromatase at doses below 4 mg daily and to lack apparent side-effects or toxic actions at doses up to 16 mg daily. This agent shows promise as a potent aromatase inhibitor for physiological and clinical studies.

Ovine placental steroid 17α-hydroxylase/C-17,20-lyase, aromatase and sulphatase in dexamethasone-induced and natural parturition

Abstract: Parturition in the sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to oestrogen production. This change is believed to be a consequence of the preparatum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17) alpha), steroid C-17,20-lyase, and possibly aromatase and steroid sulphatase. The activity levels have been determined of steroid 17 alpha-hydroxylase, aromatase and steroid sulphatase in placental microsomes in late pregnancy, dexamethasone-induced labour and in natural labour at term. Over the gestational period of 118-140 days, basal levels of placental aromatase were relatively constant (mean value (+/- S.E.M.) of 5.6 +/- 0.5 pmol/min per mg microsomal protein (n = 10]. Pregnenolone and progesterone 17 alpha-hydroxylase activities were undetectable (less than 0.5 pmol/min per mg microsomal protein (n = 7]. In six animals in labour induced with infusion of dexamethasone into the fetus, placental aromatase activity increased to a value of 14.0 +/- 1.0 pmol/min per mg protein; placental pregnenolone 17 alpha-hydroxylase, measured in four of the animals, also increased to 453 +/- 77 pmol/min per mg microsomal protein. In five animals in natural spontaneous labour with vaginal delivery, aromatase activity was 26.7 +/- 5.2 pmol/min per mg microsomal protein and pregnenolone 17 alpha-hydroxylase activity was 141 +/- 14 pmol/min per mg microsomal protein. Steroid sulphatase activity was barely detectable (less than 1.5 pmol/min per mg microsomal protein) during late pregnancy, dexamethasone-induced labour or natural parturition.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Abstract: 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 μl/ 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 wash...

Testosterone regulates aromatase activity in discrete brain areas of male rhesus macaques.

Abstract: The nonhuman primate brain contains two divergent pathways for testosterone (T) metabolism. Estradiol is biosynthesized from T by aromatization through the first pathway, whereas dihydrotestosterone is produced by the action of Sct-reductase through the second pathway. Previously, we mapped the distribution of these enzyme activities within specific microdissected brain area and determined that aromazase activity (AA), but not 5a-reductase activity (SaRA), was reduced in certain brain areas after castration. In the present study, we measured M and SaRA in thirteen brain nuclei and subregions from five castrated and five T-treated castrated male rhesus monkeys to determine whether exogenous androgen treatment could reverse the effects of castration on brain AA. We found that T, administered in a dose that maintained serum levels at 14.2 ± 1.6 (SEM) ng/ml, suppressed circulating luteinizing hormone (Castrates = 491.9 ± 86 ng/ml vs. Ttreated castrates = 1.8 ± 0.2 ng/ml), and stimulated AA in specific nuclei including the suprachiasmatic nucleus (n.), periventricular area, ventromedial n., and lateral hypothalamus. T treatment had no sign #{231}ficant effect on AA in nine other nuclei or on SaRA in any brain areas that we studied. These data indicate that AA in diencephalic and limbic structures of the nonhuman primate brain is distributed heterogeneously into androgen-dependent and androgen-independent regions. This distribution is similar to that found in rodents. SaRA, on the other hand, is more homogeneously distributed than AA in these same brain regions and is not controlled by androgens.

Lack of Evidence for Aromatase in Human Prostatic Tissues: Effects of 4-Hydroxyandrostenedione and Other Inhibitors on Androgen Metabolism

Abstract: The effects of 4-hydroxyandrostenedione (4-OHA) and other aromatase inhibitors, 10-propargylestr-4-ene-3,17-dione and imidazo[1,5-α]-3,4,5,6-tetrahydropyrin-6-yl-(4-benzonitrile), as well as 5α-reductase inhibitors N,N-diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxyamide and 4-methyl-3-oxo-4-aza-androsta-5-ene-17-ol were investigated in prostatic tissue from six patients with benign prostatic hypertrophy and seven patients with prostatic cancer, and from normal men at autopsy. We attempted to measure aromatase activity in the tissue incubations by quantitating 3H2O released from androstenedione or testosterone labeled at the C-1 position. High performance liquid chromatography and thin layer chromatography were used to isolate steroid products. Although the amount of 3H2O released was at least twice that of the heat-inactivated tissue samples, no estrone or estradiol was detected on high performance liquid chromatography. The 3H2O release was significantly inhibited by 4-OHA and N,N-diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxyamide, but not by the other aromatase inhibitors. 4-OHA also inhibited 5α-reductase in both benign prostatic hypertrophy and cancer tissue, although to a lesser extent than N,N-diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxyamide. The other aromatase inhibitors were without effect on 5α-reductase. Our results indicate that 3H2O released from [1β-3H]androstenedione and [1,2,6,7-3H]androstenedione does not correlate with estrogen formation and may be the result of other metabolic reactions. Although it appears that the prostate lacks aromatase, 4-OHA may be of benefit in patients with benign prostatic hypertrophy or prostatic cancer by inhibiting this enzyme in peripheral tissue.

Screening of aromatase-containing neurons in rat forebrain: an immunohistochemical study with antibody against human placental antigen X-P2 (hPAX-P2).

Abstract: Aromatase-containing neurons were immunohistochemically examined in rat brains by using a polyclonal antibody against human placental antigen. The antibody recognizes cytochrome P-450 portion of aromatase, an enzyme converting androgen to estrogen. A large group of strongly immunoreactive cells was identified in the ventral pallidum, which extends caudally from the area surrounding the islands of Calleja. Other strongly or moderately stained cell groups were observed in the cerebral cortex, the amygdaloid area, the nucleus of the diagonal band, and the area anterior to the posterior commissure. Only a few stained cells were present in the medial preoptic region. These findings cast doubt upon the previous assumption, based on biochemical analysis of tissue samples, that the center of the aromatizing system is in the medial preoptic region. They indicate instead that most aromatase-containing neurons of rats lie within the ventral pallidum ventromedially adjacent to the preoptic area.

Aromatase cytochrome P450 and cholesterol side-chain cleavage cytochrome P450 in corpora lutea of pregnant rats: diverse regulation by peptide and steroid hormones

Abstract: In previous studies we have shown that aromatase cytochrome P450 (P450arom) mRNA and protein increase markedly in luteal tissue between days 10-19 of gestation, whereas cholesterol side-chain cleavage cytochrome P450 (P450scc) appears to be constitutively maintained regardless of hormonal changes occurring during pregnancy. To identify pituitary and placental hormones that regulate these two P450 enzymes in the rat corpus luteum, serum LH activity and pituitary PRL release were selectively inhibited by administration of LH antiserum (LH-Ab) or CB-154, respectively. Placental hormones were removed by hysterectomy. Hormonal activities were replaced by the administration of hCG, PRL, testosterone (T), or estradiol (E), given individually or in combination. Induction of aromatase mRNA transcripts (3.3, 2.6, and 1.9 kilobases) and protein (54,000 mol wt) between days 10-15 of gestation was blocked by either surgical hysterectomy or LH-Ab treatment. Hysterectomy on day 10 combined with CB-154 abolished not only aromatase mRNA, but also markedly reduced P450scc mRNA (2.0 kilobases) by day 12. Induction of aromatase was partially restored in the day 10-15 hysterectomized rats by treatment with PRL plus E (most effective), PRL plus T, or PRL alone, but not by either T or E alone. Similar results were observed 2 days after hysterectomy (day 12), except that hysterectomy alone caused a transient 3.5-fold increase in P450arom mRNA and protein, most likely due to a transient release of pituitary LH. Aromatase mRNA and protein were also increased in intact pregnant rats treated with hCG between days 10-12. However, no effect of hCG was observed before (days 8-10) or after (days 13-19) midgestation. Likewise, LH-Ab had no effect if given after day 13. Despite hormone-specific regulation of the content of aromatase protein, E biosynthesis in vitro was not strictly related to aromatase enzyme content. We conclude that aromatase mRNA and protein are maintained by PRL at a low level of expression in the first half of pregnancy, can be modulated by LH at midgestation, and are subsequently induced to high levels in the second half of gestation by placental factors (rat placental lactogen-1 and T) and the conversion of T to E in the corpus luteum. P450scc appears to be constitutively maintained. Thus, two P450 genes known to be regulated by LH/cAMP in the rat follicle are controlled by diverse peptide and steroid signal transduction mechanisms in the corpus luteum.

The new aromatase inhibitor CGS-16949A suppresses aldosterone and cortisol production by human adrenal cells in vitro.

Abstract: CGS-16949A is a new orally active nonsteroidal aromatase inhibitor which is more than 100-fold more potent than aminoglutethimide. This compound is an imidazole derivative, and therefore, its possible effect on cytochrome P-450-dependent enzyme activities in the adrenal gland was evaluated. In vitro investigations with dispersed normal and hyperplastic human adrenocortical cells showed that CGS-16949A at 10(-7)-10(-6) M is a potent 11 beta-hydroxylase inhibitor, which inhibits ACTH-stimulated cortisol release to a similar extent as an equimolar concentration of metyrapone (IC50 for both compounds, 10(-7)-5 X 10(-7) M). Etomidate was a more potent 11 beta-hydroxylase inhibitor (IC50, approximately 10(-8) M), while 10(-7)-10(-6) M ketoconazole caused (via 17 alpha-hydroxylase inhibition) a similar inhibition of cortisol release as 10(-7) M CGS-16949A (IC50, 10(-7)-5 X 10(-7) M). The 11 beta-hydroxylase inhibition by CGS-16949A was accompanied by a dose-dependent increase in the release of precursor steroids by the adrenocortical cells in vitro, including deoxycortisol, 17-hydroxyprogesterone, and androstenedione. Aldosterone release was suppressed 50% by 10(-9) M CGS-16949A, while the IC50 for cortisol in the same cells was 10(-7) M. Aldosterone release by the dispersed adenoma cells obtained from a patient with primary aldosteronism was also significantly suppressed by CGS-16949A. We concluded that 1) the new nonsteroidal aromatase inhibitor CGS 16949A is an inhibitor of 11 beta-hydroxylase which is equipotent to metyrapone. At present it is unclear whether the compound at the dose that causes complete aromatase inhibition in vivo also affects stress-induced cortisol release in man. 2) CGS-16949A exerts a very potent inhibitory effect on normal aldosterone release (IC50, 10(-9) M) and on tumorous aldosterone secretion. CGS-16949A might, therefore, be a drug that can be used in the treatment of primary hyperaldosteronism.

Aromatase, 5α- and 5β-reductase in brain, pituitary and skin of the sex-role reversed Wilson's phalarope

Abstract: While intrasexual competition for mates is generally considered to be an androgen-dependent characteristic of reproductively active males, in the Wilson's phalarope (Phalaropus tricolor) it is the female that acquires the brighter nuptial plumage and aggressively competes for access to the less aggressive males. Despite this pronounced sex-role reversal, circulating sex steroid hormones of breeding phalaropes are similar to those of avian species displaying traditional male-female reproductive roles. To investigate whether these behavioural and morphological steroid-dependent differences may be due to differences in target organ metabolism of circulating androgen, [3H]androstenedione in the presence of an NADPH-generating system was incubated with homogenates of brain, pituitary and skin of male and female Wilson's phalaropes collected from a naturally breeding population. Oestrone, 5 alpha-androstanedione and 5 beta-androstanedione were measured as endpoints of aromatization, 5 alpha-reduction and 5 beta-reduction respectively. Aromatase activity in the anterior hypothalamus/preoptic area (AHPOA) and posterior hypothalamus was greater in breeding males with high circulating concentrations of testosterone than in females, and activity in the AHPOA was greater in breeding than in non-breeding males (with low circulating testosterone). Aromatase levels did not differ in septum, archistriatum, hyperstriatum or pituitary. 5 alpha- and 5 beta-reductase were detected in all neuroendocrine tissues sampled and although there were no significant male-female differences, 5 alpha-reductase was greater in the AHPOA of breeding than of nonbreeding males.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Human Placental Aromatase in Saccharomyces cerevisiae

Abstract: A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3′-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 μg a...

Estrogen receptors in quail brain: a functional relationship to aromatase and aggressiveness.

Abstract: Estradiol (E2) mediates many of the activational effects of testosterone (T) on masculine reproductive and aggressive behaviors. Using Japanese quail (Coturnix coturnix japonica) as an animal model, together with a newly devised procedure for quantifying aggressiveness, we recently showed that aggression is E2 -dependent and that individual differences in behavioral intensity are correlated with aromatase in the hypothalamus/preoptic area (HPOA). In thisstudywe characterized estrogen receptors (ER) in quail brain and tested the hypothesis that aromatase in brain regulates T-induced behavioral responsiveness by regulating the quantity of E2 available for receptor binding. Based on standard binding assays and Sephadex LH-20 chromatography, quail brain ER was shown to be estrogen-specific, of high affinity (Kd = 0.88 nM), and of limited capacity with highest concentrations in limbic brain areas (Bmax 23-27fmoles/gm HPOA). In addition, this ER adhered to DNAcellulose under activating conditions. The quantitative relationship between aromatization, ER, and aggressiveness was tested in reproductively inactive (nonaggressive) males by treatment with T ± the aromatase inhibitor 4-hydroxyandrostenedione (OHA). After 5 days, T markedly stimulated aggressiveness, and elevated aromatase and nuclear (occupied) ER in HPOA. Simultaneous treatment with OHA blocked effects on aggressiveness and aromatase, and lowered nuclear ER, but increased cyrosolic (empty) ER. Total ER (nuclear plus cytosolic) was higher after T treatment whether or not OHA was administered, suggesting that androgen per se induces ER in quail HPOA. Taken together, these data provide good evidence that aromatization is a rate-limiting determinant of ER occupancy and that T serves not only as a substrate for aroma tase but also up-regulates the enzyme and total ER abundance in the HPOA, a behaviorally important area.

Estrogen biosynthesis in human uterine adenomyosis

Abstract: Estrogen biosynthesis (aromatase activity) was investigated in human adenomyosis tissue and compared with that of the normal myometrium, endometrium, and endometrial cancer tissues. Homogenates were incubated with [1,2,6,7-3H]androstenedione and NADPH at 37 degrees C for 1 h. After stopping the enzymatic reaction with ethyl acetate, [4-14C]estrone and [4-14C]estradiol-17 beta were added to the incubated sample. Estrone and estradiol were purified and identified by Bio-Rad AG1-X2 column chromatography, thin-layer chromatography and co-crystallization. Estrogen formed in the incubated sample was calculated from the 3H/14C ratio of the final crystal. The value for estrone formed from androstenedione was 52-132 fmol.h-1.g-1 wet weight. Aromatase activity in the adenomyosis tissues was higher than that in normal endometrial or myometrial tissues, but lower than that found in myometrial or endometrial tumour tissue. Furthermore, we investigated the effect of danazol, progesterone, and medroxyprogesterone acetate on adenomyosis cells in primary cultures. Aromatase activity in adenomyosis was blocked by danazol, but stimulated by progesterone and MPA. These results indicate that aromatase activity in adenomyosis may contribute to the growth of the ectopic endometrial tissue which occurs in this disease.

Further immunocytochemical study on the localization of aromatase in the ovary of rats and mice.

Abstract: The precise localization of estrogen biosynthesis in the ovary of rats and mice were immunocytochemically studied using new antisera against aromatase cytochrome P-450. The positive reaction for aromatase was detected mainly on the granulosa cells of large, apparently preovulatory follicles. In addition, the cells of some corpora lutea showed very weak positive reaction but most corpora lutea were negative to the staining. Those cells such as the granulosa cells of smaller follicles, the theca interna cells, the interstitial gland cells, oocytes, peritoneal epithelial cells were entirely negative. These results indicate that in the ovary of rats and mice, the granulosa cells of preovulatory follicles are the main site for synthesis of estrogen from androgen which is provided by the theca interna cell and the interstitial gland cell.

Immunocytochemical studies of aromatase in early and full-term human placental tissues: comparison with biochemical assays.

Abstract: An unmunocytochemical method for visualizing the aromatase P450 enzyme with a specific monoclonal anti- body has been developed for use with wIxed, frozen tissue sections. We compared both monoclonal and poly- clonal aromatase-specific antibodies andfound that placental aromatase was consistently and exclusively located in the syncytiotrophoblast layer of chorionic viii. The monoclonal antibody had the highest #{128}�1nity, with negli- gible associated background stain. Fixation wasfound to impair stain reaction. Erainination offlrst trimester and term placentae revealed identical immunostaining patterns of similar intensity in 9 of 10 samples. The immunostain reactions offirst trimester and term placentae were compared with their respective micro- somal aromatase activity, determined simultaneously by both indirect radioneetric tritiated water (3H20) assay, and direct product isolation by IIPLC, using (1, 2, 6, 7-3HJandrostenedione as substrate. The two assays were found to be comparable for enzyme activity estimates of term placental specimens. However, when first trimester specimens were analyzed, the direct-product measurements were significantly larger than the corresponding �I12O assay results. Nonetheless, biochemical aromatase activity was found to correlate positively with immunos- fain reaction. Although 17�-hydroxysteroid dehydrogenase activity was not directly measured, differences in the estradiol :estrone product ratio (2.49 ± 0.68 first trimester vs. 0.89 ± 0.15 term) suggest djfferential control of this enzyme at the two stages of pregnancy. One first trimester specimen with an atypical, patchy invnunostain distri- bution also had extremely low arontatase activity. The results indicate that both antibodies recognize functi onal aromatase enzyme and suggest that immunocytochenucal detection is a sensitive, qualitative technique for inves- tigating this important steroidogenic enzyme.

5α-Reductase, Aromatase, and Androgen Receptor Levels in the Monkey Brain during Fetal Development*

Abstract: To elucidate the metabolic fate and possible role of androgens and their derivatives during primate fetal development, aromatase (AROM), 5 alpha-reductase (5 alpha R), and androgen receptor (AR; cytosolic) levels were assessed in the brain, heart (HRT), lung (LNG), and skeletal muscle (MUS) of fetal rhesus monkeys. Analyses were performed on tissues taken on days 100 and 160 postconception. Five male and four or five female fetuses were examined at each stage. Brain tissues analyzed included medial basal hypothalamus (MBH), amygdala (AMG), cerebellum (CB), corpus callosum (CAL; splenial region), cerebral cortex (CTX), and cingulate cortex (CNG). In the following, enzyme activities are reported as picomoles per mg protein/h, while receptor levels are femtomoles per mg protein. 5 alpha R activity was measurable in all tissues. Analysis of variance revealed significant tissue differences [P less than 0.001, combined stages and sexes; CAL (2.05) greater than MBH (1.08) greater than AMG (0.63) greater than CB (0.4)-CNG-CTX-LNG-HRT-MUS (0.02); -indicates not significantly different]. A significant age x tissue interaction (P less than 0.001) was noted which could be explained by higher MBH and CAL levels in older vs. younger fetuses and higher AMG levels in younger vs. older fetuses. There was also a significant sex x tissue interaction which was attributed to higher female values in the MBH and CAL. AROM activity was detected in all tissues. Levels varied significantly among tissues [P less than 0.001, combined stages and sexes; MBH (0.80)-AMG (0.76) greater than CAL (0.4)-CNG-CB-CTX-LNG-HRT-MUS (0.07)]. Significant age (P less than 0.001) and age x tissue (P less than 0.001) effects were noted, which were due to higher MBH and AMG levels in younger vs. older fetuses. No sex difference in AROM levels was evident in any tissue. AR was measurable in all cases. Although stage and sex differences were not significant, tissue levels varied significantly [P less than 0.001; LNG (2.8)-MUS (2.6)-MBH (2.2) greater than HRT-AMG-CB-CTX-CAL-CNG (0.9)]. These findings indicate that neural and nonneural fetal primate tissues have the potential for transforming androgens to products that could have greater or lesser biological activity. AR were also noted through which dihydrotestosterone or testosterone could effect a genomic response. Since stage, tissue, and sex differences were evident in neural tissues, metabolic and receptor activities may be important for the normal differentiation of sexually dimorphic behavioral systems in monkeys as well as for potential teratogenic changes under abnormal metabolic or physiological conditions.

Effect of sex, intrauterine position and androgen manipulation on the development of brain aromatase activity in fetal ferrets.

Abstract: Experiments were conducted to explore the possible relationship between testicular androgen secretion and the development of brain aromatase activity in fetal ferrets. Aromatase activity in the preoptic+mediobasal hypothalamus and temporal lobe was similar in fetuses of both sexes between embryonic Days 26 and 36 even though whole body androgen content was invariably higher in males than females. Whole body androgen content was significantly higher in females located caudally (downstream) from two or more as opposed to zero or one males in the same uterine horn; nevertheless their brain aromatase activity was similar. Finally, maternal treatment with either the androgen receptor antagonist Flutamide or 5alpha-dihydrotestosterone propionate beginning on gestational Day 24 did not affect brain aromatase activity in fetal offspring of either sex, delivered on embryonic Day 34. Previous studies suggest that the biosynthesis of estrogen in the fetal ferret brain is normally greater in males than females. The present results suggest that this sex difference results primarily from increased androgenic substrate being available to non-saturated aromatizing enzymes and not from an androgen-dependent activation of aromatase.

Granulosa cell aromatase bioassay for follicle-stimulating hormone.

Abstract: Publisher Summary Follicle-stimulating hormone (FSH) is required for the maturation of ovarian follicles and testicular tubules during pubertal development. This chapter describes the granulosa cell aromatase (estrogen synthetase) bioassay (GAB) and its applications to the measurement of FSH bioactivities. The extreme sensitivity of the present in vitro bioassay allows for the measurement of circulating or urinary levels of bioactive FSH. Measurement of serum and urine levels of bioactive FSH should provide insight regarding the role of FSH in various physiological, pharmacological, and pathophysiological conditions. Because rat granulosa cells respond to FSH preparations from different species, this in vitro assay also provides valuable information on FSH levels in diverse animal species, including those that lack a specific radioimmunoassay. Because of its low sensitivity, the classic Steelman–Pohley FSH bioassay cannot be used to measure serum FSH levels. For urine samples, up to 1 liter of urine is extracted for the in vivo bioassay.

Selective inhibition of androstenedione-induced prostate growth in intact beagle dogs by a combined treatment with the antiandrogen cyproterone acetate and the aromatase inhibitor 1-methyl-androsta-1,4-diene-3,17-dione (1-methyl-ADD).

Abstract: Interference with estrogenic and androgenic actions might result in an inhibitory effect of benign prostatic hyperplasia (BPH). In the present study the effects of the treatment of intact, adult beagle dogs with the antiandrogen cyproterone acetate (CPA) and the aromatase inhibitor 1-methyl-ADD either alone or in combination on androstenedione-induced prostate growth and on testes, epididymides, and the pituitary was investigated. 1-Methyl-ADD induced a marked counterregulatory increase in the serum testosterone and dihydrotestosterone (DHT) concentrations leading to hyperplasia of the glandular part of the prostate. However, the aromatase inhibitor antagonized the androstenedione-induced (estrogen-related) stimulation of the fibromuscular stroma of the prostate. CPA caused a complete atrophy of the prostate that was also present after treatment with both the aromatase inhibitor and CPA in spite of a striking elevation of the serum testosterone and DHT levels and in spite of the antagonization of the inhibition of testes and epididymal weight induced by androstenedione plus CPA. This indicates a selective inhibition of the prostate of intact beagle dogs treated with CPA and 1-methyl-ADD.