Feminization of hepatic steroid metabolism in male rats following electrothermic lesion of the hypothalamus.

The metabolism of [4-14C]androst-4-ene-3,17-dione, [4-14C]5alpha-androstane-3alpha,17beta-diol and [1,2-3H]5alpha-androstane-3alpha,17beta-diol, 3,17-disulfate in the 105,000 X g supernatant and microsomal fractions of liver was studied in male and female rats after electrothermic lesion of the hypothalamus including the median eminence. Following electrothermic lesion, hepatic steroid metabolism in male rats was generally "feminized" (increased 5alpha-reduction and decreased 6beta- and 16alpha-hydroxylation of 4-androstene-3,17-dione, decreased 2alpha-, 2beta-, 18- and 7beta-hydroxylation of 5alpha-androstane-3alpha, 17beta-diol and induced 15beta-hydroxylation of 5alpha-androstane-3alpha,17beta-diol,3,17-disulfate), whereas hepatic metabolism in female rats remained essentially unchanged. Previous investigations have pointed to the occurrence of a sex-specific secretion of "feminizing factor" from the female pituitary that is responsible for the "feminization" of the basically "masculine" type of metabolism characterizing the rat liver. Taken together with these findings, the present results indicate that the release of the pituitary "feminizing factor" is controlled by means of a release-inhibiting factor from the hypothalamus. This factor is not secreted in female rats; it is suggested that its secretion in male rats is turned on as a result of neonatal imprinting by testicular androgens.     

Estrogen formation in the central nervous system and characteristics of aromatase of rat hypothalamus

Estrogen formation from androst-4-ene-3,17-dione and its kinetics were studied using microsomes from rat hypothalamus. [4-14C] androst-4-ene-3,17-dione and a homogenate of rat hypothalamus were incubated in the presence of NADPH at 37 degrees C for 3 hrs. The estrogen fraction was extracted from the incubation mixture with ethyl acetate, purified by column chromatography on Sephadex LH-20 and Bond Elut C18, and separated into estrone and estradiol fractions by HPLC. In analysis of the trimethylsilyl (TMS) derivatives of each fraction by gas chromatography-mass spectrometry (GC-MS), the molecular ion peak of the estrone fraction appeared at m/z 344, within 2 amu of that for the TMS derivative of natural estrone. The retention time of the estrone fraction derivative was 11.6 min, the same as that of natural estrone. 14C-estrone was thus concluded to be biosynthesized from [4-14C]-androst-4-ene-3,17-dione in rat hypothalamus. The kinetics of the aromatase of rat hypothalamic tissue was studied by measuring 3H2O released from [1 beta-3H]-androst-4-ene-3,17-dione and estrone as the estrogen product by measured gas chromatography selected ion monitoring (GC-SIM). High correlation was found between 3H2O release and estrone measured by GC-SIM (r = 0.97). Aromatase activity was linear with respect to incubation time and quantity of tissue. Km and Vmax were 30.3 nM and 7.98 fmol estrogen/h/mg of wet tissue, respectively. 4-hydroxyandrostenedione (4-OH-A) suppressed the activity of aromatase in both rat hypothalamic and human placental tissue in a concentration-dependent manner. Polyclonal IgG to human placental aromatase also suppressed aromatase activity of human placental tissue, but only slightly suppressed that of rat hypothalamus. The molecular structure of aromatase in rat hypothalamus was thus concluded to differ from that in human placenta.     

Metabolism of (4-14C)progesterone and (7alpha-3H)pregnenolone by human ovaries perfused in vitro.

Nine human ovaries were perfused in vitro with [4-14C]progesterone and in addition one ovary with [7-3H]pregnenolone. From the perfusate unchanged progesterone and five different metabolites were isolated: 20alpha-dihydroprogesterone, 17alpha-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 5alpha-pregnane-3,20-dione and 4-androstene-3,17-dione. In the ovarian tissue saturated pregnane derivatives were the main metabolites. When [3H]pregnenolone and [14C]progesterone were perfused simultaneously a stimulation of delta4-5-isomerase and 3beta-dehydrogenase activity by HCG was demonstrated.     

Estrogen formation in endometrial and cervix cancer cell lines: Involvement of aromatase, steroid sulfatase and 17beta-hydroxysteroid dehydrogenases (types 1, 5, 7 and 12)

The involvement of aromatase, steroid sulfatase (STS) and reductive 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in the production of estrogens was determined in four cell lines of endometrial cancer (Ishikawa, HEC-1A, HEC-1B and RL-95) and one cell line of cervix cancer (Hela) in culture. After incubation with 4-androstene-3,17-dione (4-dione), there are no estrogens, estrone (E1) and estradiol (E2), detected suggesting that the pathway of aromatase is not important in these cell lines. In whole cells, the results show low percentages of transformation of estrone sulfate (E1S) into E1 suggesting that the entrance of E1S is difficult. However, in homogenized cells the STS activity was much higher and fully blocked by an inhibitor. Using selective inhibitors for each reductive 17beta-HSD (types 1, 5, 7 and 12), alone or in combination, we did not succeed in completely blocking the conversion of E1 into E2, suggesting that another 17beta-HSD (known or unknown) is involved in the formation of E2 from E1.     

Aromatase overexpression enhances the stimulatory effects of adrenal androgens on MCF7 breast cancer cells

The intratumoral conversion of adrenal androgens into estrogens by the aromatase enzyme complex may be an important mechanism of autocrine stimulation in hormone-dependent breast tumor. The effects of estrogens on tumor development are mediated by the activity of estrogen receptor alpha that induces gene expression and cell proliferation. Thus, estrogen biosynthesis 'in situ' and/or estrogen receptor action are the main targets of endocrine treatment in endocrine-dependent breast carcinoma. In the present study we demonstrate that three major adrenal androgens, dehydroepiandrosterone, 5-androstene-3beta, 17beta-diol and 4-androstene 3,17-dione, all acquire an estradiol-like biological efficacy in aromatase transfected MCF7 breast cancer cells. Our results suggest that in postmenopausal women aromatase inhibitors might be considered as an adjuvant approach to the treatment of hormone-dependent breast tumors that overexpress aromatase.     

Feminizing Leydig cell tumor: endocrine and incubation studies

A 32-year-old patient with a history of surgery for left gynecomastia four years previously presented with right gynecomastia; a tumor in the left testis proved to be a Leydig cell tumor. Preoperative investigations showed elevated but variable levels of plasma estradiol (E2) and estrone (E1), and reduced serum LH and FSH and plasma testosterone (T). After hCG stimulation, E2 response was increased and abnormally prolonged; T reached normal values, which has predictive value for a return to normal of post-operative T level. After left orchiectomy, gynecomastia regressed within a few days, gonadotropins increased by day 2, estrogens dropped by day 2 and were normal at day 7, T and 5 alpha-dihydrotestosterone dropped at day 2 but reached normal levels at day 16. Pathophysiology of these hormonal data are discussed. An incubation procedure with 3H testosterone showed an aromatase activity 21 times greater in the tumor than in normal peritumoral tissue, while the percentage of the volume occupied by Leydig cells was 34 times higher. This suggests that the aromatase activity of a single tumor cell is very similar to that of a normal Leydig cell. Furthermore, evidence of juxtatumoral Leydig cell hyperplasia in areas where the tumor was well encapsulated suggests the existence of a factor stimulating Leydig cell multiplication.     

Aromatase activity in purified Leydig cells from adult rat. Comparative effects of insulin, IGF-I, and hCG

The comparative effects of insulin and IGF-I on aromatization in adult rat purified Leydig cells were examined to elucidate the mechanism of action of the peptides in testicular steroidogenesis. Aromatase activity was measured in short-time incubations, using the tritiated water release method with [1 beta-3H] and androstenedione as substrate. In the presence of varying concentrations of substrate, the apparent Km for androstenedione was 0.945 mol/l; treatment of cells with insulin, IGF-I and hCG markedly increased the apparent maximal velocity, without modifying Km; peptides were more potent in aromatase stimulation than hCG alone or in combination with either peptides. When related to time (0-4 h) and expressed as percent of control values, aromatase activity in the presence of insulin, IGF-I and/or hCG exhibited a significant and transient increase at 15-30 min. In order to clarify the nature of this early stimulation, the effects of dibutyryl cAMP, various antibiotics, and cytochalasin B on treated Leydig cells were analysed. Results indicated that insulin and IGF-I action on aromatization was not cAMP-dependent; peptides could intervene by increasing RNA and protein, but not DNA, synthesis; they were also effective in glucose transport. These data suggest that insulin and IGF-I are able to modulate aromatization in Leydig cells.     

Metabolism of androsterone and 5 alpha-androstane-3 alpha,17 beta-diol in human lung tissue and in pulmonary endothelial cells in culture

The metabolism of [3H]androsterone and [3H] 5 alpha-androstane-3 alpha,17 beta-diol ( [3H] 3 alpha-diol) was studied in slices of human lung tissue and cultures of human pulmonary artery endothelial cells. Lung tissue metabolized [3H]androsterone (0.25 microM) to 5 alpha-androstane-3,17-dione (30.3 pmol 100 mg-1 tissue h-1), isoandrosterone (0.7 pmol 100 mg-1 tissue h-1), 5 alpha-dihydrotestosterone (5 alpha-DHT; 0.1 pmol 100 mg-1 tissue h-1), 3 alpha-diol (0.1 pmol 100 mg-1 tissue h-1), and two polar metabolites. Pulmonary arterial endothelial cells produced the same metabolites of [3H]androsterone (0.083 microM), with the exception of the polar compounds [5 alpha-androstane-3,17-dione (1.3 pmol mg-1 protein h-1), isoandrosterone (0.1 pmol mg-1 protein h-1), 5 alpha-DHT (0.2 pmol mg-1 protein h-1), and 3 alpha-diol (0.2 pmol mg-1 protein h-1)]. Thus, the principal metabolite of [3H]androsterone in both lung tissue and endothelial cells was 5 alpha-androstane-3,17-dione. Human lung tissue metabolized [3H]3 alpha-diol (0.28 microM) to 5 alpha-DHT (8.8 pmol 100 mg-1 tissue h-1), androsterone (2.2 pmol 100 mg-1 tissue h-1), 5 alpha-androstane-3,17-dione (0.8 pmol 100 mg-1 tissue h-1), isoandrosterone (0.1 pmol 100 mg-1 tissue h-1), and four polar metabolites (0.2 pmol 100 mg-1 tissue h-1). 5 alpha-DHT was the principal metabolite of [3H]3 alpha-diol within the first hour of incubation, but the concentration of this androgen declined thereafter to 3.6 pmol 100 mg-1 tissue after 4 h of incubation. This decline was correlated with increased 5 alpha-androstane-3,17-dione synthesis (6.7 pmol 100 mg-1 tissue 4 h-1). Androsterone formation from [3H]3 alpha-diol, however, was linear with time of incubation for 4 h (8.9 pmol 100 mg-1 tissue 4 h-1). The formation of these products demonstrates that the principal 5 alpha-reduced-C19-steroid-metabolizing enzymes in human lung are 3 alpha-hydroxysteroid oxidoreductase.     

Estradiol formation from testosterone by continuously cultured human breast cancer cells.

Two human breast cancer cell lines (MCF-7 and MDA-MB-231) and one cell line derived from normal human breast (HBL-100) were examined for the presence of aromatase activity by determining the amounts of [3H]estradiol ([3H]E2) formed by cell cultures incubated with [3H]testosterone. Aromatase activity was demonstrable in both breast cancer cell lines, but estradiol synthesis was not observed in HBL-100 cultures. The [3H]E2 content of MCF-7 cultures rose as a function of incubation time and substrate concentration. Furthermore, [3H]E2 formation by this cell line was suppressed by several known inhibitors of human placental aromatase. These observations represent the first evidence that some lines of continuously cultured human breast cancer cells, like some human breast tumors, are capable of forming estrogen from an extracellular precursor steroid. Cultured breast cells may provide model systems for investigating the relative importance of intracellular estrogen formation in the regulation of human breast cancer cell growth.     

19-Hydroxylation of androgens by rat granulosa cells

The transformation of androgens by rat granulosa cells was examined employing [19-C3H3]-, [1 beta-3H]-, and [1,2,6,7-3H]androgens as substrates. Rat granulosa cell homogenates incubated with [19-C3H3]androstenedione generated [3H] water and [3H]formic acid in a ratio of 8-9, indicating considerable 19-hydroxylation which was not followed by aromatization. This ratio remained relatively constant regardless of the time in the estrous cycle when the ovaries were removed, although there were large differences in the extent of the reactions. Parallel incubations with [1 beta-3H]]androstenedione showed that the aromatization of [19-C3H3]androstenedione in this tissue proceeds with a negative isotope effect of approximately 3, similar to that in human placenta. Incubation of the same substrates with granulosa cell cultures produced [3H]water and [3H]formic acid in ratios of 4-5 and showed a smaller negative isotope effect in the aromatization of [19-C3H3]androstenedione. FSH stimulation of the cell cultures had no influence on the ratio of 19-hydroxylation to aromatization with respect of either the duration of stimulation or the concentration of the pituitary hormone. Incubation of the cell cultures with [1,2,6,7-3H]androstenedione yielded tritium-labeled 19-hydroxy- and 19-oxoandrostendiones and estrogens in relative quantities corresponding to those expected from the [3H]water and [3H]formic acid formation. Virtually all of the products were found in the medium, with only trace quantities located intracellularly. Similarly, incubation of granulosa cell homogenates with [14C]androstenedione yielded [14C]19-oxygenated androgens in excess of [14C] estrogens. These results indicate that rat granulosa cells effect C-19-hydroxylation of androgens greater than that linked to aromatization and that the rat ovaries produce 19-oxygenated androgens in quantities exceeding those of estrogens. The excess 19-hydroxylation is synchronous with aromatization, but it is not known whether it is catalyzed by the same or a different enzyme. The formation of 19-oxygenated androgens in cell cultures indicates that they are distinct metabolites of androgens in the rat ovary and are not merely trapped transient aromatization intermediates.     

Oestrogen secreting Leydig cell tumour and GnRH agonist in-vivo and in-vitro studies

OBJECTIVE: The purposes of our study concerning two patients with oestrogen secreting Leydig cell tumour were to determine whether endogenous LH levels are involved in testicular tumour steroidogenesis and whether aromatase activity of oestrogen secreting Leydig cell tumours is directly or indirectly dependent on LH levels. MEASUREMENTS: E2 and T were evaluated after hCG injection (5000 IU) during 96 hours. Bio and immuno LH, T, E2, were determined at the basal state and after administration of D-Trp-6-GnRH agonist (3.75 mg) every 3 weeks. The abnormal testis was removed after the third injection and testicular venous blood was collected during the operation. Testicular tumour was incubated with 4-14C-T. RESULTS: Oestradiol (E2) response to hCG injection (5000 IU) was prolonged and exaggerated while that of testosterone (T) was similar to that of the controls. The aromatase index (E2/T) remained elevated even 96 hours after hCG. Intramuscular injection of the GnRH agonist, D-Trp-6-GnRH (3.75 mg) resulted in a reduction of immunoreactive and bioactive LH. T was decreased to about 10% of baseline levels and E2 fell from 240 to 36 pmol/l. In the blood of the spermatic veins collected in the course of surgery, E2 levels were found to be lower in comparison with the controls. E2 was found to be twofold higher in the spermatic vein draining the tumoral side than in that of the contralateral testis. Incubation of the testicular tumours with 4-14C-T, displayed a reduced aromatase activity (conversion of T to E2: 0.3 and 0.1% in patients 1 and 2 respectively). CONCLUSIONS: The kinetics of E2 response to hCG administration would suggest a modification of the regulation of the aromatase activity in this type of oestrogen secreting tumour. A certain endogenous LH level may be necessary to supply a sufficient quantity of T substrate, and to maintain aromatase activity of such Leydig cell tumours secreting oestrogens. These tumours seem to be responsive to endogenous LH levels.     

Cellular localization of rat testicular aromatase activity during development

The aromatase activity from purified testicular sources (Leydig and Sertoli cells) of immature (5- and 15-day-old) and adult rats (60-day-old) was investigated by the tritiated water release method in isolated Leydig and Sertoli cells that were morphologically and functionally characterized. Electron micrographs of Sertoli cell preparations from different ages showed no marked changes, except that tight junctions between Sertoli cells normally present in 60-day-old rats were not observed in 5-day-old and rarely found in 15-day-old animals. Leydig cells underwent ultrastructural changes along with development, such as the appearance of thicker nuclear heterochromatin and laminar-like mitochondria. The 15-day-old rat interstitial tissue possessed less than 10% of Leydig cells morphologically similar to those present in the adult, whereas the rest were probably transition cells, since they did not show typical Leydig cell structure but were able to bind [125I]iodo-hCG, as evaluated by autoradiography. The number of LH/hCG-binding sites increased with age in Leydig cells, but was not detectable in Sertoli cells. The highest number of FSH-binding sites in Sertoli cells was observed in the 15-day-old animals. Minor FSH binding was found in Leydig cell preparations, which was consistent with the known LH contamination of the human FSH tracer preparation. cAMP production increased significantly in Leydig cells only after hCG treatment and in Sertoli cells after FSH stimulation. Both types of cells were shown to have the capacity for aromatization. The aromatase activity increased in the Leydig cell but decreased in the Sertoli cell during testicular development. The highest aromatase activity was found in adult rat Leydig cells, and the enzyme activity was significantly higher (2-fold) in purified Leydig cells than in crude interstitial cell preparations. Estradiol production in response to hCG stimulation in vitro was not different from the basal value in 5-day-old rat Leydig cells, but increased significantly in 60-day-old rat Leydig cells. In conclusion, 1) Leydig cells are the major site of estrogen synthesis in adult rat testis; and 2) the low aromatase activity observed in immature rat Leydig cells could partially explain the differential response of the mature and immature rat testis to hCG-induced desensitization.     

Aromatase inhibitors prevent granulosa cell differentiation: an obligatory role for estrogens in luteinizing hormone receptor expression

To determine the role of newly synthesized estrogens in LH receptor expression, granulosa cells from diethylstilbestrol-implanted immature rats were cultured with FSH plus aromatase inhibitors. When present throughout the 48-h culture period, 4-hydroxy-4-androstene-3,17-dione (4-OHA; greater than or equal to 100 microM) and 1,4,6-androstatriene-3,17-dione (greater than or equal to 5 microM) inhibited FSH-induced LH receptor formation by 40% and 90%, respectively. Both aromatase inhibitors caused relatively greater inhibition of LH receptor formation when added from 20-48 h of culture, the period during which FSH-stimulated estrogen synthesis occurs (85% maximal inhibition with 4-OHA and 95% with 1,4,6-androstatriene-3,17-dione). Addition of estradiol, but not androstenedione, reversed the reduction of LH receptor formation by 4-OHA, indicating that the effects of the aromatase inhibitors were specifically related to their blockade of estradiol synthesis. The stimulation of estrogen production by FSH alone (8-fold) or with androstenedione (80-fold) during the 48-h culture period was prevented by 4-OHA. FSH-stimulated cAMP production was initially enhanced by 4-OHA from 0-20 h of culture, but was reduced from 20-48 h. Lower concentrations of 4-OHA (less than or equal to 50 microM) amplified FSH-stimulated cAMP production and LH receptor formation. However, these responses were blocked by the antiestrogen keoxifene or the antiandrogen flutamide, indicating that 4-OHA or a metabolite may have partial estrogenic or androgenic properties. The inhibitory effects of higher concentrations of 4-OHA on LH receptor expression were potentiated by keoxifene or flutamide. These results indicate that estrogen production and action are necessary for LH receptor expression in the granulosa cell.     

In situ subcellular distribution and metabolism of progesterone, estradiol and androstenedione in the vascularly separated and isolated hypothalamus of the female rhesus monkey

A neurosurgical procedure has been developed for the vascular isolation of the hypothalamus-thalamus region of the rhesus monkey brain. The circulation to the left and right halves of the hypothalamus was also isolated and each half of the hypothalamus was perfused simultaneously, but separately, with a dextran-blood solution which contained radioactive gonadal steroids. The hypothalamus in situ efficiently converted [3H]androstenedione to [3H]estrone and this aromatization was inhibited by the presence of androsta-1,4,6-triene-3,17-dione (ATD) in the perfusate. [3H]Progesterone was metabolized predominantly to 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-OHP). Subcellular fractionation of the hypothalamus after the in situ perfusion with [3H]-progestin or [3H]estradiol to the hypothalamus of estrogen-treated ovariectomized monkeys or oil-treated ovariectomized monkeys, respectively, indicated that the retention of [3H]estradiol in the nucleus was a saturable, limited-capacity phenomenon. No saturable subcellular distribution of [3H]progesterone or [3H]R 5020 was observed. This latter observation might be attributable to the presence of a progesterone receptor in too small a concentration to be detected by the methods used.     

FSH induction of aromatase in cultured rat granulosa cells measured by a radiometric assay

The effects of FSH on the aromatase activity of rat granulosa cells in culture were studied by measuring the stereospecific transfer of 3H from [1,2,6,7-3H]testosterone or [1 beta-3H]testosterone into 3H2O. The use of 3H2O release as a specific assay for aromatization in granulosa cells was validated by various means. 2 days after plating, cultures of granulosa cells released only minimal amounts of 3H2O from [1 beta-3H]testosterone during a 3-h incubation, whereas cells which had been treated with FSH, or with (Bu)2cAMP plus 3-isobutyl-1-methylxanthine (MIX), from the time of plating released considerable amounts of 3H2O into the culture medium. The release of 3H2O from [1 beta-3H]testosterone by cultured cells was inhibited by the aromatizable androgens, 19-hydroxytestosterone and 19-hydroxyandrostenedione, indicating the specificity of the method for aromatization. In order to study the mechanism by which FSH enhanced the release of 3H2O, optimal conditions for aromatization by cell-free sonicates were determined. Optimal aromatase activity was achieved by incubating cell-free sonicates at 37 degrees C in the presence of O2 in 20 mM phosphate buffer (pH 7.4) containing 5 mM dithiothreitol, 20 mM MgCl2, 0.5 mM NADP+, 20 mM glucose 6-phosphate and 2 U/ml glucose-6-phosphate dehydrogenase. A concentration of 0.25 microM testosterone and 0.1 microCi tritium was used in the standard assay. Under these conditions the assay was linear for 1 h with up to 150 microgram protein from granulosa cells having maximal aromatase activity. When cells in culture were stimulated with purified FSH for 48 h from the time of plating, the aromatase activity in cell-free sonicates increased in a dose-dependent fashion. The ED50 for Sairam's FSH S-1528 C2 was 12 ng/ml. It was concluded from these studies that FSH increases estrogen levels primarily by inducing an active aromatase rather than by influencing secretion or availability of substrate or cofactor for the aromatization reaction.     

Vitamin E prolongs survival and function of porcine Leydig cells in culture

Vitamin E (alpha-tocopherol) is known to be required for testicular function but its action on specific testicular cells has not yet been studied. The present study used porcine Leydig cell cultures, in a hormone-supplemented medium, to study the effect of vitamin E (vit E) on Leydig cells. It was seen that the addition of vit E to the medium led to an increase in cell survival, lengthening the life span of the cultures from 3-4 days to more than a week. The Leydig cells maintained their LH/hCG receptors and responsiveness throughout this period as evidenced by an increase in testosterone (T) and prostaglandin secretion. The hCG stimulated T levels were synergistically increased in the presence of vitamin E, while basal levels of T secretion were not changed. Other secretory products of Leydig cells are prostaglandins E2 and F2 alpha. It was found that the addition of vit E inhibited both the basal prostaglandin levels and the stimulated levels by 90%. Maximal effects on all of these parameters were seen at 10 ng/ml vit E. It is obvious that vit E plays a critical role in maintaining porcine Leydig cells in primary cultures beyond the first 3 days. This vitamin seems to be involved both in steroidogenesis and in prostaglandin production in the Leydig cells. The exact mechanism of the action of vit E these two biosynthetic pathways remains to be determined.     

Use of granulosa-luteal cell culture to evaluate low and high clinical responses to menotropin stimulation.

The cause of a poor response to human menopausal gonadotropin (hMG) remains unexplained. To determine whether aromatase activity of cultured granulosa cells obtained from relatively low estradiol (E2) responders (serum E2 < 1000 pg/ml) to hMG therapy differed from that of good responders (E2 > or = 1000 pg/ml), we prospectively compared serum E2 on the day of human chorionic gonadotropin administration to in vitro aromatase activity following a 72-h culture. Granulosa cells were obtained from seven women undergoing hMG therapy and oocyte aspiration. Follicle stimulating hormone (FSH) was added to one-half of the cultures. Serum E2 was determined by radioimmunoassay, and aromatase activity was determined indirectly by measuring tritiated water formed by aromatization of 1-beta [3H] androstenedione to estrogen in 1 h. In this study, luteinized granulosa cells from patients with a relatively low serum E2 produced less estrogen in cultures when compared to cells from higher responders (p < 0.01). Aromatase activity was not significantly increased by FSH in the relatively high responders, whereas FSH stimulated a significant increase in aromatase activity in cells from lower responders (p < 0.001). Our results indicate that the clinical response to hMG is at least partly due to the "quality" of granulosa cell aromatase activity. A clinically relevant "block" to FSH action may be present in vivo in low responders which can be reversed in culture by addition of FSH.