Stimulation Of Aromatase Activity Research Articles

Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF)

The effects of fibroblast growth factor (FGF) on testicular aromatase activity has been studied using primary cultures of porcine Leydig cells. After culture for 3 days in the absence or presence of FGF, the ability of the cells to produce estrogen was examined in a 4h-test period in which either (a) hCG (10 −9 M) or (b) androstenedione (3×10 −6 M) was added to the medium. FGF produced a 3- to 20-fold increase in estrogen formation from endogenous or exogenous substrate during the test period, in spite of a marked decrease (⋍ 60%) in [ 125I]-hCG binding and no significant change in testosterone concentration. Stimulation of estrogen secretion by FGF was dose-(ED 50⋍2 ng/ml) and time-dependent, the first and maximal effects were observed after 12h and 48h, respectively. Preliminary tests with several other factors (insulin, EGF, TGF-β, FSH and hCG) showed that hCG alone directly stimulated aromatase activity. From these findings a role is suggested for FGF as a paracrine/autocrine agent in the control of estrogen secretion by Leydig cells.

The Effects of Experimental Hyperinsulinemia on Steroid Secretion, Ovarian [125I]Insulin Binding, and Ovarian [125I]Insulin-Like Growth-Factor I Binding in the Rat*

We randomized 32 cycling female Sprague-Dawley rats (82 days old) into experimental and control groups (16 animals/group). Hyperinsulinemia was induced and maintained for 22 days in the experimental group with NPH human insulin (Novolin, Squibb-Novo, Princeton, NJ) as previously described. Controls received an identical volume of vehicle. Fifteen minutes before death, each rat received a sc injection of 100 ng synthetic GnRH (Factrel, Ayerst Laboratories, New York, NY). The mean serum insulin level was significantly higher in the insulin-treated group than in the control group (165 +/- 57 vs. 49 +/- 9 microU/ml; P less than 0.05). The mean final weight also was significantly higher in the insulin-treated group (283 +/- 4 vs. 242 +/- 7 g; P less than 0.001). There were no significant differences in mean final serum levels of testosterone, estradiol, estrone, or androstenedione or in GnRH-stimulated serum levels of LH or FSH. The androstenedione to estrone ratio, however, was significantly lower in the insulin-treated group (2.5 +/- 0.3 vs. 3.4 +/- 0.2; P less than 0.01), suggesting that aromatase activity increased with hyperinsulinemia. Specific [125I]insulin binding to ovarian tissue homogenates was lower in the insulin-treated group (1.7 +/- 0.1% vs. 2.6 +/- 0.6%/0.2 mg protein; P greater than 0.05), suggesting that ovarian insulin receptors tended to down-regulate with hyperinsulinemia. Specific [125I]insulin-like growth factor I [( 125I]IGF-I) binding to ovarian tissue homogenates, in contrast, was significantly higher in the insulin-treated group (13.3 +/- 1.4% vs. 7.2 +/- 0.6%/0.2 mg protein; P less than 0.05), suggesting that ovarian IGF receptors up-regulated with hyperinsulinemia. The affinity of neither [125I]insulin binding nor that of [125I]IGF-I binding changed significantly, with the 50% inhibition point remaining between 2.0 and 5.0 ng/ml for each peptide in both groups. We conclude that hyperinsulinemia increases ovarian [125I]IGF-I binding and stimulates aromatase activity in the rat. These phenomena, if also true in women, could be important factors contributing to the ovarian hyperstimulation observed in various hyperinsulinemic states.

The effect of progestin on rabbit endometrial aromatase activity

The ability of rabbit endometriuna to synthesize estrogen was investigated. Aromatase activity was measured by incubating the viable tissue fragments with [ 3H]testosterone at 37°C and quantitated the [ 3H]estrogens at the end of 6 h incubation. The activity was not detectable in endometrium and myometrium of non-pregnant rabbits. In pregnant rabbit, the activity increased from 2 to 12 fmol/h/g of tissue in endometrium obtained from both gravid and nongravid uterine horns 4–8 days after mating. Aromatase activity in endometrium at implanted sites on the 8th day after mating reached up to 550 fmol/h/g of tissue. Hormonal regulation of the aromatase activity was studied directly in cultured rabbit endometrial stromal cells. Both progesterone and medroxyprogesterone acetate stimulated the activity with 2- to 25-fold increase over the control values (0.2–1 fmol/h/mg protein). The stimulation of aromatase activity by progestin was found to be both time- and dose-dependent. Estrogen, Bu 2cAMP, forskolin, HCG or relaxin had no effect on the activity. Aromatase activity in glandular epithelial cells was neither detectable nor affected by progestin. These results indicate that aromatase activity in rabbit endometrium is concentrated in stromal cells and progestin stimulates the activity. However, estrogen, forskolin and relaxin which enhanced the stimulation of the activity by progestin in human endometrium had no effect on aromatase in rabbit endometrium. The increase of aromatase activity observed after the onset of conception may be physiologically important to increase the local estrogen content for decidualization of the endometrium.

The effect of insulin on aromatase activity in isolated human endometrial glands and stroma

Hyperinsulinemic states have been associated with an increased incidence of estrogen-dependent endometrial neoplasia. To study the effect of insulin on the ability of endometrium to aromatize androgens to estrogens, late proliferative endometrium was obtained from normally cycling women at the time of indicated surgery, separated into component glands and stroma, and grown to confluence. Separated gland and stromal cultures were incubated in triplicate with increasing insulin concentrations and epidermal growth factor. Aromatase activity was assayed by the production of tritiated water from tritium-labeled androstenedione. The activity was noted to increase proportionally with increasing concentrations of insulin greater than 10 U/ml, and the effect was specific. These data suggest the following conclusions: (1) Insulin stimulates aromatase activity in both endometrial glands and stroma; (2) hyperinsulinemia may predispose to endometrial neoplasia by enhancing endogenous endometrial estrogen production.

Plasma concentrations of ovarian steroids in the freshwater European silver eel (Anguilla anguilla L.): effects of hypophysectomy and transfer to sea water.

Intact and hypophysectomized freshwater (FW) silver eels were transferred to tanks of FW or artificial sea water (SW; salinity = 0.60 osmol/l) which were simultaneously renewed twice a week. Fish were killed 2 months after transfer and plasma was assayed for ovarian steroids. In all fish, 5 alpha-androstane-3 beta,17 beta-diol was present, while 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol were undetectable. In intact FW eels, plasma levels of testosterone, 5 alpha-androstane-3 beta,17 beta-diol and oestradiol-17 beta were approximately 0.15 nmol/l. In intact SW eels, no change in plasma levels of testosterone and 5 alpha-androstane-3 beta,17 beta-diol was found, whereas the concentration of oestradiol-17 beta was increased significantly (P less than 0.01), indicating stimulation of aromatase activity. In hypophysectomized compared with intact FW fish, plasma levels of testosterone and 5 alpha-androstane-3 beta,17 beta-diol were decreased (P less than 0.05) and there was a slight but significant (P less than 0.01) augmentation of the plasma concentration of oestradiol-17 beta which may have involved the removal of pituitary-dependent inhibition of aromatase activity, possibly by 5 alpha-reduced compounds. In hypophysectomized compared with intact SW fish, plasma levels of testosterone, 5 alpha-androstane-3 beta,17 beta-diol and oestradiol-17 beta were decreased (P less than 0.05); in the case of oestradiol-17 beta, this may have reflected the diminished ovarian synthesis of testosterone, its precursor. The plasma level of oestradiol-17 beta was, however, higher in SW than in FW fish, even in hypophysectomized eels. This suggests that extra-pituitary mechanisms mediate, at least partly, the effects of transfer to SW on aromatase activity.

Effect of relaxin on aromatase activity in human endometrial stromal cells.

Previous studies have shown that the aromatase activity in human endometrial stromal cells was stimulated by progestin and enhanced by estrogen and forskolin (Fk), an agent that stimulates the accumulation of intracellular cAMP. Present study was undertaken to investigate whether any peptide hormone would affect endometrial aromatase activity. Stromal cells were isolated from normal proliferative and secretory endometria and cultured in nutrient medium. Porcine relaxin (RLX) was added to culture medium individually or in combination with medroxyprogesterone acetate (MPA) and estradiol (E2). Cells treated with RLX alone did not affect the aromatase activity. RLX, however, exerted a synergistic effect on aromatase activity in the presence of MPA or MPA plus E2. On the other hand, human CG, epidermal growth factor, human PRL, and insulin did not increase the aromatase activity in the presence or absence of MPA and E2 studied in a limited number of specimens. The progestin-dependent effect of RLX on aromatase activity was dose dependent indicating that the biological effect of RLX is mediated through a saturable mechanism. When RLX was added to MPA-pretreated cells, additional increase of aromatase activity was seen after 24 h incubation indicating that the action of RLX on stromal cells is not an acute effect. Antiprogestin, RU486, inhibited the stimulation of aromatase activity in both MPA and MPA plus RLX treated cells. RLX has either no effect or a moderate increase (up to 2-fold over the control) on intracellular cAMP content. On the other hand, Fk increased the intracellular cAMP level and enhanced the aromatase activity in the presence of progestin. Also RLX did not replace the effect of Fk since additional increase of aromatase activity was noted when stromal cells were incubated with MPA plus RLX plus Fk in comparison to MPA plus RLX or MPA plus Fk. These results suggest that the action of RLX on stromal cells may be mediated through an intracellular messenger independent of cAMP. Present studies provide evidence that RLX exerts a synergistic effect on aromatase activity in the presence of progestin in human endometrial stromal cells. It is evident that human endometrium is a target organ of RLX.

Regulation of aromatase in estrogen-producing cells

Human adipose stromal cells in monolayer culture aromatize androstenedione to estrone. The rate of aromatization is stimulated 20- to 30-fold by glucocorticoids when fetal calf serum is present in the culture medium and by dibutyryl cyclic AMP in the absence of serum. The action of dibutyryl cyclic AMP to stimulate aromatase activity is potentiated markedly by phorbol esters and inhibited by growth factors, such as EOF. In order to investigate the mechanisms underlying this multifactorial regulation, we have prepared polyclonal and monoclonal antibodies specific for aromatase cytochrome P-450. By use of these antibodies it was demonstrated that the action of these various factors to regulate aromatase activity was caused by alterations in the rate of synthesis of aromatase cytochrome P-450, whereas the synthesis of the reductase component of the aromatase enzyme complex was relatively unaffected. The changes in the rate of synthesis of aromatase cytochrome P-450 were, in turn, reflective of changes in the levels of translatable mRNA specific for this protein. In order to analyze the levels of aromatase cytochrome P-450 mRNA directly, we have isolated a cloned cDNA insert complementary to the mRNA encoding aromatase cytochrome P-450, by screening a λgt 11 human placental cDNA library utilizing the polyclonal anti-aromatase P-450 IgG. Use of this cDNA probe in Northern analysis of RNA extracted from human adipose stromal cells revealed that the changes in translatable mRNA resulting from incubation of the cells with the various regulatory factors were due to changes in the absolute levels of mRNA encoding this protein.

Time-dependent biphasic response of aromatase to dexamethasone in cultured human skin fibroblasts.

Human genital skin fibroblasts grown in cell culture possess aromatase activity and, therefore, provide a model to investigate the molecular mechanisms that control aromatase in extraglandular tissues. Following the observation by other investigators that glucocorticoids stimulated aromatase activity in cultured stromal-vascular cells from adipose tissue, we examined the influence of dexamethasone (DEX) on aromatase in cultured skin fibroblasts. Preincubation of skin fibroblasts with DEX stimulated aromatase expression in all cell strains. In time-course studies, aromatase activity showed a biphasic curve, with peak levels at 12 h and a return to baseline levels by 72 h. When DEX was removed after 12 h, aromatase activity could be completely restimulated by DEX only after a period of 60-72 h. The DEX stimulation appeared to involve glucocorticoid receptor function, since the concentration of DEX required for half-maximal stimulation of aromatase activity (4.2 nM) was similar to the dissociation constant (Kd, 4.3 nM) of the receptor (for DEX). Actinomycin D and cycloheximide (CHX) inhibited DEX stimulation of aromatase when they were present in the preincubation and assay media. When cells were preincubated with DEX and CHX and then washed free of CHX and DEX before the assay, superinduction of aromatase activity occurred. Our data concerning the time course and superinduction of aromatase activity by DEX are in contrast to the findings reported by others for adipose tissue stromal-vascular cells and suggest that the mechanisms for the control of aromatase in extraglandular tissue may vary significantly in different tissues.

Synergism between granulosa and theca-interstitial cells in estrogen biosynthesis by gonadotropin-treated rat ovaries: studies on the two-cell, two-gonadotropin hypothesis using steroid antisera.

In mammalian ovaries, luteinizing hormone (LH) induces androgen biosynthesis by theca interna cells, whereas both follicle-stimulating hormone (FSH) and LH stimulate aromatase activity by granulosa cells. The joint action of these two types of cell and pituitary hormones forms the basis of a 2-cell, 2-gonadotropin hypothesis for biosynthesis of estrogen. We tested the synergism between these cell types using antisera against the estrogen precursors progesterone and testosterone. Ovaries were obtained from immature rats two days after a single injection of pregnant mare serum gonadotropin (PMSG). Granulosa cells were obtained by puncturing the follicles, and ovarian remnants were dissociated with collagenase to derive cells from the theca interstitium. Granulosa and theca interstitial cells were cultured alone or in combination for 20 h. Granulosa cells secreted negligible amounts of estrogen and testosterone, but contained high aromatase activity. Treatment with both FSH and human chorionic gonadotropin (hCG) increased production of progesterone in granulosa cells. In contrast, theca interstitial cells had negligible aromatase activity. Treatment with hCG, but not FSH, increased androgen production by theca interstitium; the hCG action was further augmented by the inclusion of exogenous progesterone. Furthermore, co-culture of gonadotropin-treated granulosa and theca interstitial cells resulted in substantial increases in estrogen production, indicating synergism between the two types of cell. The increases in estrogen production in the co-cultures were accompanied by decreases in progesterone content. To test the possibility that progesterone released by granulosa cells may serve as a substrate for production of androgen in theca cells, specific antiserum was used to adsorb progesterone present in the medium. Addition of the progesterone antibody inhibited gonadotropin-stimulated production of testosterone (39% decrease) and estrogen (64% decrease) by the combined cell cultures. The inhibitory effect on estrogen production could be reversed by the addition of progesterone (10(-6) M) or testosterone (10(-6) M) but not by the addition of a synthetic progestin, R5020. Since testosterone released from theca cells may serve as the substrate for aromatases in granulosa cells, specific testosterone antiserum was also used. Production of estrogens by the combined cell cultures was inhibited (78%) by the testosterone antiserum, but the inhibitory effect was completely reversed by exogenous testosterone or progesterone. Thus, synergistic interactions between granulosa and theca interstitial cells are important in effecting maximal estrogen biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of aromatase activity by dihydrotestosterone in human skin fibroblasts

In order to study the regulation of aromatase activity by androgens in cultured fibroblasts derived from genital skin of normal prepubertal boys, aromatase activity was evaluated in the presence of various concentrations of non-aromatizable androgen DHT(5α-dihydrotestosterone). The estrogen formation was assayed by an enzymatic method, after 24 h incubation of the cells with 10 −6 M androstenedione. Aromatase activity was stimulated 3- to 20-fold by DHT at concentrations 10 −10 and 10 −9 M. It was necessary to preincubate the cells with DHT for 48 h in order to bring about this stimulation. The stimulatory effect was not significant after preincubation for only 24 h. The basal value of aromatase activity was in the range of 8 ± 1.2 pmol/mg protein/day (mean ± SEM), while the maximal stimulation 1043 ± 46 pmol/mg protein/day was obtained at the concentration of 10 −8 MDHT. This stimulation was partially blocked with cyproterone acetate at level of 20 ± 4 pmol/mg protein/day; stimulation of aromatase activity by DHT could thus be mediated by the androgen receptor. This stimulatory effect was prevented by incubation of the cells with cycloheximide or actinomycin D, suggesting that DHT acts to increase aromatase activity in cultured fibroblasts by inducing the synthesis of new proteinaceous material. In vitro regulation of aromatase activity by androgens could contribute to a new approach to the extraglandular formation of estrogen.

The effect of spironolactone on aromatase activity

Spironolactone, an aldosterone antagonist, causes decreased plasma testosterone (T) levels and gynecomastia in men and is clinically useful in the treatment of hirsutism in women. Many mechanisms of action for spironolactone have been proposed. It has been suggested that one cause for low plasma T levels may result from an increased metabolic clearance rate of T due to increased extraglandular aromatization to 17 beta-estradiol. The purpose of this investigation was to determine the effect of spironolactone on the rate of activity of aromatase in human fetal liver (hFL) cells. The activity of aromatase was assayed by determining the rate of incorporation of [1-3H]androstenedione into [3H]water in hFL cells exposed to spironolactone (10(-10) to 10(-4) M) for 24 or 72 hours. The aromatase activity in control hFL cells remained constant during the study. Dibutyryl cyclic adenosine monophosphate significantly stimulated aromatase activity from 63 to 257 pmol X mg-1 protein X 2 hours-1 after 24 hours and 72 hours exposure, respectively. In contrast, when hFL cells were exposed to spironolactone (10(-10) to 10(-5) M) for up to 72 hours, the activity of aromatase did not differ significantly from that in control hFL cells. It is concluded that spironolactone in therapeutic concentrations does not stimulate aromatase activity in hFL cells maintained in vitro.

Modulation of aromatase activity in human endometrial stromal cells by steroids, tamoxifen and RU 486.

The regulation of aromatase activity (AA) in human endometrial stromal cells by various steroids was studied in primary cell culture. Various progestins, but not androgens or glucocorticoids, stimulated AA. Medroxyprogesterone acetate (MPA) was the most potent progestin. Estrogen (E) alone did not change the activity but it potentiated the stimulation of AA by progestin. Biphasic regulation of AA by progestin was noted in both time- and dose-dependent manners. Endometrial AA was stimulated by MPA and reached the maximum rate between 2-5 days of incubation with subsequent decline of AA in prolonged culture. When stromal cells were treated with MPA (0.03 to 30 microM) for 3 days, AA was increased over the control at all the concentrations tested. The maximum was found at doses between 0.1-1 microM. The activities reduced steadily from the maximum stimulation to less than 50% when the concentration of MPA increased from 1-30 microM. In addition, initial treatment of stroma cells with MPA (1-3 days) resulted in further increase of activity after progestin withdrawal. The enhancement of the induction of AA by E did not alter the biphasic pattern regulated by progestin alone, i.e. E enhanced both the stimulation and the decay of AA. The time study of the effect of E showed that enhancement of AA required at least 10 h of incubation of E with MPA conditioned cells. The effect of E is dose dependent between 0.04-40 nM and shows the greatest effect in the presence of MPA between 0.01-1 microM. The optimal concentrations of E and progestin that stimulate AA in culture are similar to the plasma concentrations after pregnancy, suggesting that the physiological function of the endometrial aromatase is at the time of decidualization. The effects of antiprogestin, Ru 486, and antiestrogen, tamoxifen (TAM), on AA were studied. Ru 486 or TAM alone did not alter AA. Ru 486 inhibited the MPA stimulated AA in a dose-dependent manner suggesting that the effect of progestin may be mediated through a receptor mechanism. Enhancement, but no inhibitory effect, was observed when cells were treated with TAM + MPA and TAM + MPA + E. The effectiveness of Ru 486 to inhibit the induction of AA in endometrial cells may be of primary importance for contraception.

Growth factors suppress and phorbol esters potentiate the action of dibutyryl adenosine 3',5'-monophosphate to stimulate aromatase activity of human adipose stromal cells.

In previous studies, we observed that the stimulatory effect of (Bu)2cAMP on aromatase activity of human adipose stromal cells was markedly attenuated when fetal calf serum was present in the culture medium. To determine whether growth factors may be the inhibitors of (Bu)2cAMP-stimulated aromatase activity in serum, the effects of growth factors and phorbol esters on aromatase activity of human adipose stromal cells in monolayer culture were investigated. Epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) were all without effect on aromatase activity when added by themselves, but markedly inhibited aromatase activity stimulated by (Bu)2cAMP. On the other hand, nerve growth factor, multiplication-stimulating activity, relaxin, and insulin had no effect on aromatase activity, either by themselves or in the presence of (Bu)2cAMP. Thus, EGF, PDGF, and FGF can mimic the inhibitory action of fetal calf serum on (Bu)2cAMP-stimulated aromatase activity of these cells. By contrast, none of these substances was capable of mimicking the effect of serum to facilitate the stimulatory action of dexamethasone on aromatase activity of these cells. The phorbol esters phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate were also capable of facilitating the action of (Bu)2cAMP to stimulate aromatase activity, but had little or no action on dexamethasone-stimulated aromatase activity or when added by themselves. It is concluded that aromatase is under multifactorial regulation in human adipose stromal cells. The activity is induced by glucocorticoids and by agents that stimulate cAMP-dependent protein kinase; the latter effect is potentiated by factors that stimulate protein kinase C, but is suppressed by growth factors such as EGF, FGF, and PDGF, whose actions are believed to be mediated by receptor-linked tyrosine kinase activity.

A gonadotropin-like thermostable peptide, prepared from bovine anterior pituitary, inducing spermiation in the frog Rana esculenta (L.) and binding to a rat ovarian FSH receptor

A thermostable peptide, inducing sperm release in Rana esculenta L. and having immunological properties which resemble those of the gonadotropins but nonidentical with any one of the gonadotropins or their subunits, has been prepared from pars distalis of bovine anterior pituitaries by means of acetic acid extraction, heating, ethanol precipitation, and reverse-phase high-performance liquid chromatography (HPLC). During the purification, the biological activity was regularly followed by means of the potency of collected fractions to induce sperm release in frogs. Using HPLC (5 μm C 18 silica), the activity was eluted with 38% of acetonitrile:water:trifluoroacetic acid (80:19.5:0.5) and 62% of 0.5% trifluoroacetic acid, pH 3.3. The active principle was also found to bind to a rat ovarian follitropin (FSH) receptor. In contrast, bovine FSH and LH treated identically to the peptide lost all such affinity, indicating that the peptide is not created from either of these during the purification procedure. Further, the peptide was, like FSH, also shown to stimulate aromatase activity in intact Sertoli cells from immature male rats in vitro.

Factors affecting the conversion of androstenedione to estrogens by human fetal hepatocytes in monolayer culture.

The purpose of the present investigation was to characterize and determine what hormones affect the activity of aromatase in human fetal hepatocytes maintained in primary monolayer culture. The major product of aromatization of androstenedione was estrone sulfate. Optimal conditions for assay of aromatase activity in fetal liver cells were determined. The apparent Km for androstenedione was 50 nM. Aromatase activity was stimulated by glucocorticoids in the presence of fetal calf serum. The concentration of dexamethasone required for half-maximal stimulation was 10(-8) M, similar to the concentration required for half-maximal binding to glucocorticoid receptors. This action of dexamethasone was inhibited by cortisol 21-mesylate, a glucocorticoid antagonist. Aromatase activity was also stimulated by (Bu)2cAMP and cholera toxin, and was inhibited by fetal calf serum. This effect of fetal calf serum was mimicked by epidermal growth factor. However, epidermal growth factor did not mimic the permissive action of serum to stimulate aromatase activity by dexamethasone. In these respects, the regulation of aromatase activity of human fetal hepatocytes is similar to that of human adipose stromal cells. A polycyclic hydrocarbon, benzo(a)pyrene, which causes induction of aryl hydrocarbon hydroxylase activity in fetal hepatocytes, inhibited the stimulation of aromatase activity by dexamethasone. Of a number of hormones tested, including glucagon, insulin, angiotensin II, ACTH, hCG, GH, PRL, and T3, only glucocorticoids were effective in stimulating aromatase activity of human fetal hepatocytes. These results emphasize the complex and multiparameter nature of the regulation of aromatase activity in this as in other tissues.

19 Aromatase assay by bioluminescence: Stimulation of aromatase activity by androgens in human skin fibroblasts

19 Aromatase assay by bioluminescence: Stimulation of aromatase activity by androgens in human skin fibroblasts

Gonadotropic regulation of aromatase activity in the adult rat testis.

Aromatase activity during gonadotropin action in vivo and in vitro was examined in purified Leydig cells to further define the early effects of LH and for elucidation of the enzymatic processes involved in the development of late lesions of androgen biosynthetic pathway. Aromatase was measured by the tritiated water release method using [1 beta-3H]testosterone as substrate. The enzyme activity was proportional to the amount of cells (0.1-1.0 X 10(6] incubated, increased with the incubation time (2-60 min), was inhibited by androstatriendione (ED50, 5.0 microM), and showed a Km for testosterone of 1.69 microM. Aromatase activity was stimulated (10-20%; P less than 0.05) 1 h after treatment of rats with a single sc dose of 5 micrograms hCG. This activation preceded the late steroidogenic lesion at the site of 17 alpha-hydroxylase and 17,20-desmolase activity by 2-5 h. A RIA of improved sensitivity (0.5 pg) was developed to detect the very low cellular and secretory levels of estradiol. The testicular contents of testosterone and estradiol showed small increases (P less than 0.05) within 40 and 60 min after hCG treatment, respectively. Testicular testosterone levels reached a peak by 1 h after injection and preceded the peak level of estradiol formation by 2 h. After in vitro treatment of cultured Leydig cells with 100 ng hCG, the aromatase activity was significantly increased within 30 min (P less than 0.05) and then returned to control levels for up to 16 h of culture. A similar temporal pattern for enzyme activation was observed after treatment of cultures with 8-bromo-cAMP or forskolin (23-27% above control; P less than 0.05), while cholera toxin stimulated aromatase activity at 2 h. Net testosterone accumulation in the incubation medium increased 30 min after the hCG treatment and reached a plateau by 4 h. A small but significant increase in estradiol levels (P less than 0.05) was also observed at 30 min, remaining constant until 120 min, which was followed by a sharp rise parallel to that of testosterone. These results suggest that the estradiol-mediated desensitization of the Leydig cell observed after hCG administration is consistent with an early cAMP-dependent activation of aromatase and a further rise in estradiol formation due to increased substrate availability.

Regulation of aromatase activity of cultured adipose stromal cells by catecholamines and adrenocorticotropin

Adipose tissue is the major site of estrogen formation in postmenopausal women. We have previously reported (Simpson E.R., Ackerman G.E., Smith M.E. and Mendelson C.R. (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78, 5690–5694; Mendelson C.R., Cleland W.H., Smith M.E. and Simpson E.R. (1982) Endocrinology 111, 1077–1085) that aromatase activity of human adipose stromal cells in culture is stimulated by glucocorticoids and by dibutyryl cyclic AMP (Bt 2-cAMP). In order to establish which physiological factors might stimulate aromatase activity of these cells by activation of adenylate cyclase, we have investigated the roles of adrenocorticotropin (ACTH) and isoproterenol to increase cyclic AMP levels and stimulate the aromatization of androstenedione. In the presence of methylisobutylxanthine (MIX), ACTH stimulated cyclic AMP formation and aromatase activity in a time- and concentration-dependent manner. The concentration of ACTH required for half-maximal stimulation was ~ 10 −8 M. Isoproterenol, in the presence of MIX, stimulated cyclic AMP formation in a time- and concentration-dependent fashion, and also stimulated aromatase activity. These effects of isoproterenol appeared to be mediated by binding of the agonist to a population of β-adrenergic receptors. On the basis of these and our previous studies, we suggest that ACTH may play an important role in stimulating estrogen formation by human adipose tissue, both directly, and by stimulating the adrenal cortex to produce both substrate, androstenedione, and inducing agent, namely cortisol.

Low molecular weight substance from rat ovary induces steroidogenesis in cultured granulosa cells

Ovaries from immature intact rats contain an apparently low molecular weight substance which mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induced cell-shape changes followed by intensive progestin production. Like FSH action, OS-induced steroidogenesis reversibly ceased upon washing the factor from the cultured cells, and could be blocked in the presence of cycloheximide or α-amanitin. Although OS stimulated aromatase activity in granulosa cells, it failed to elicit LH responsiveness in the cultured cells. Androstenedione synergistically augmented OS-induced progestin production and aromatase activity. OS itself synergistically augmented FSH-induced progestin but did not have any effect on FSH-induced aromatase activity. In contrast to FSH action which is mediated via cAMP formation, OS doses which evoked extensive synthesis of progestin products failed to stimulate significant increases in intracellular cAMP accumulation. These results suggest the existence of a putative intraovarian hormone-like substance which can mimic some effects of the gonadotropins on the follicular granulosa cell differentiation and may facilitate FSH action at yet unknown stages of the follicular development.

Aromatization of androgens by human adipose tissue in vitro

Stromal cells prepared from adipose tissue of women were maintained in monolayer culture to study the regulation of aromatase activity by hormones. Aromatase activity was stimulated 20–100-fold by dexamethasone. Half-maximal stimulation of aromatase activity was attained at a dexamethasone concentration of 2.5 nM. The stimulatory effect of dexamethasone was apparent after a preincubation time of 4 h, and stimulation was maximal after 24 h of preincubation. The stimulatory effect of dexamethasone was observed only when fetal calf serum also was present in the culture medium. Of the various steroids tested, dexamethasone was the most potent in stimulating aromatase activity. Cytosolic fractions prepared from stromal cells that had been maintained in monolayer culture were found to contain a homogeneous population of sites that specifically bound [ 3H]-dexamethasone with relatively high affinity ( K d = 2.9 nM) and low capacity (38 fmol/mg of protein). The stimulatory effect of dexamethasone on aromatase activity was prevented by simultaneous incubation with Cortisol 21-mesylate (0.1–10μM), a compound known to block the binding of glucocorticosteroids to cytoplasmic receptors. These findings are suggestive that glucocorticosteroids act to increase aromatase activity in stromal cells by binding to a glucocorticosteroid receptor. This presumably results in the induction of synthesis of specific proteins, perhaps the form of cytochrome P-450 involved in aromatization, or else NADPH-cytochrome P-450 reductase. Aromatase activity was also stimulated by dibutyryl cyclic AMP ((Bu) 2cAMP). In contrast to the stimulation by dexamethasone, the effect of (Bu) 2cAMP was diminished by 70% when fetal calf serum (15%) was present in the culture medium. Aromatase activity of cells incubated with (Bu) 2cAMP continued to increase over an 8 day period, at which time, enzyme activity was increased by 200-fold. Aromatase activity of stromal cells maintained in the presence or absence of serum was unaffected after incubation for 5 days with oFSH, hCG, bPRL, hGH or soterenol. On the other hand, incubation of cells for 5 days with cholera toxin (10μg/ml) or l-methyl-3-isobutylxanthine (0.1 mM) resulted in a marked increase in aromatase activity; the effect of these agents was diminished greatly when serum was present in the culture medium. The findings that serum inhibited the stimulation of aromatase activity by (Bu) 2cAMP and that the time courses of aromatase induction by (Bu) 2cAMP and dexamethasone were markedly different are suggestive that different molecular events mediate aromatase induction by glucocorticosteroids and cyclic AMP.