OBJECTIVE

Aromatase inhibitors that block the synthesis of estrogen are proving to be superior to antiestrogens and may replace tamoxifen as first-line treatment for postmenopausal estrogen receptor (ER)-positive breast cancer patients. However, acquisition of resistance to all forms of treatments is inevitable and a major clinical concern. In this study, we have investigated the effects of long-term estrogen deprivation in the breast cancer xenograft model and whether sensitivity to antiestrogens can be restored in vivo. We also compared whether combining wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone.

METHODS

Long-term estrogen-deprived aromatase-transfected human ER-positive breast cancer cells (UMB-1Ca) were grown as tumors in ovariectomized athymic nude mice. Twelve weeks after inoculation, when tumors reached 300 mm(3), animals were grouped and injected with vehicle, Delta(<em>4</em>)A, letrozole, tamoxifen, fulvestrant, wortmannin, tamoxifen plus wortmannin, and wortmannin plus fulvestrant. Tumor volumes were measured weekly.

RESULTS

Tumors of UMB-1Ca cells grew equally well with and without androstenedione, indicating the ability of the cells to proliferate in the absence of estrogen. The combination of wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. The combination of wortmannin plus fulvestrant was the most effective treatment that maintained tumor regression for a prolonged time.

CONCLUSIONS

These results suggest that blocking both ER and growth factor receptor pathways could provide effective control over tumor growth of long-term estrogen-deprived human breast cancers.

We conducted studies to determine the magnitude and sources of variability in androgen assay results and to identify laboratories capable of performing such assays for large epidemiological studies. We studied androstanediol (ADIOL), androstanediol glucuronide (ADIOL G), <em>androstenedione</em> (ADION), androsterone glucuronide (ANDRO G), androsterone sulfate (ANDRO S), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA S), dihydrotestosterone (DHT), and testosterone (TESTO). A single sample of plasma was obtained from five postmenopausal women, five premenopausal women in the midfollicular phase of the menstrual cycle, and five women in the midluteal phase, divided into aliquots, and stored at -70 degrees. Four sets of two coded aliquots from each woman were then sent to participating labs for analysis at monthly intervals over <em>4</em> months. Using the logarithm of assay measurements, we estimated the components of variance and three measures of reproducibility. The usual coefficient of variation is a function of the components that are under the control of the laboratory. The intraclass correlation between measurements for a given individual is the proportion of the total variability that is associated with individuals. The minimum detectable relative difference is important to evaluate study feasibility. Results suggest that a single sample of ADIOL G, DHEA, DHEA S, and ANDRO G (with two lab replicates per sample) can be used to discriminate reliably among women in a given menstrual phase or menopausal status. The results for DHT, TESTO, ADION, and ANDRO S are more problematic and suggest that the present measurement techniques should be used with care, especially with midluteal phase women. The results for ADIOL suggest that this assay is not yet ready for use in epidemiological studies.

Estrogen, if it is produced in the gastrointestinal tract, may overflow into the systemic circulation in the case of increased portal-systemic shunting. This idea is in accord with a significant step-up in serum estradiol (E2) concentration in the portal vein of rats, compared with that in the artery. Gene expression of aromatase, estrogen synthetase, was demonstrated by RT-PCR in the gastric mucosa of male and female adult rats, equivalent to that in the ovary. Aromatase activity and production of E2 in the gastric mucosa were demonstrated by (3)H(2)O assay and gas chromatography-mass spectrometry, and they were inhibited by aromatase inhibitor, <em>4</em>-hydroxy<em>androstenedione</em>. Conversion of (1<em>4</em>)C-<em>androstenedione</em> to (1<em>4</em>)C-E2 through (1<em>4</em>)C-testosterone in cultured gastric mucosa was also demonstrated. Parietal cells exhibited strong signals for aromatase mRNA and immunoreactive protein by in situ hybridization histochemistry and immunohistochemistry. Estrogen receptor alpha mRNA and immunoreactive protein were demonstrated in hepatocytes by RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Total gastrectomy reduced portal venous E2 concentration, without changing systemic E2 concentration, together with down-regulation of estrogen receptor alpha mRNA level in the liver. These findings indicate that gastric parietal cells play a potent endocrine role in secreting estrogen that may function as a regulator of the gastro-hepatic axis.

This study investigated the effects of progesterone (P<em>4</em>) on the production and survival of neurons in the hippocampal dentate gyrus of adult male mice. The administration of P<em>4</em> (<em>4</em> mg/kg) for 3 consecutive days beginning on the 0-2nd day after the first BrdU-injection (BrdU-D(0-2)) produced an approximately twofold increase in the number of 28- and 56-day-old BrdU(+) cells in comparison to the controls, whereas it did not alter the number of 2<em>4</em>/<em>4</em>8-h-old BrdU(+) cells. P<em>4</em> preferentially promoted the survival of newborn neurons when administered at BrdU-D(5-7), but not at BrdU-D(10-12) and BrdU-D(15-17). <em>Androstenedione</em> (Ad), testosterone (TE), or estradiol (E2) at the same-dose of P<em>4</em>, when administered at BrdU-D(0-2), could not replicate the effect of P<em>4</em>, while the inhibition of 5alpha-reductase by finasteride did not affect the P<em>4</em>-action, indicating that the P<em>4</em>-effect is exerted by P<em>4</em> itself but not by its metabolites. On the other hand, the P<em>4</em>R antagonist RU<em>4</em>86 partially suppressed the P<em>4</em>-effect, while inhibitors for Src, MEK, or PI3K totally suppressed the P<em>4</em>-effect. Finally, the P<em>4</em>-enhanced survival of newborn neurons was accompanied by a potentiation of spatial learning and memory, which was P<em>4</em>R-dependent. These findings suggest that P<em>4</em> enhances the survival of newborn neurons through P<em>4</em>R and/or the Src-ERK and PI3K pathways independent of its influence on cell proliferation, which is well correlated with the potentiated spatial cognitive function of P<em>4</em>-treated animals.

The factors responsible for the marked gender differences in risk of coronary heart disease and atherosclerosis severity remain largely undetermined. While some clinical and experimental evidence supports a protective effect of endogenous estrogen on the initiation and progression of atherosclerosis and incidence of coronary heart disease, much of the epidemiological data do not support this conclusion. The possibility that endogenous androgens may have adverse effects on atherosclerosis progression and coronary risk has received little attention. We investigated the effects of experimentally induced hyperandrogenism in female cynomolgus monkeys with diet-induced atherosclerosis. Animals were assigned randomly to one of four treatment groups: (1) untreated controls, (2) ovariectomized (sex hormone-deficient) controls, (3) treated with <em>androstenedione</em> and estrone (mild hyperandrogenism), or (<em>4</em>) treated with testosterone (male plasma androgen pattern). At necropsy, coronary atherosclerosis was approximately twice as extensive (P < .05) in testosterone-treated animals relative to untreated controls, while treatment with <em>androstenedione</em> and estrone had no effect on atherosclerosis extent. Coronary plaque size was positively correlated with lumen size in intact and ovariectomized controls; however, there was no evidence of a similar relation between animals in either androgen treatment group. The atherogenic effects of testosterone were independent of variations in plasma lipoprotein and nonlipoprotein risk variables. Although chronic hyperandrogenism had adverse effects on atherosclerosis progression, it reversed (P < .03) atherosclerosis-related impairment of endothelium-dependent vasodilator responses. We conclude that an experimentally induced male plasma androgen pattern results in exacerbation of diet-induced atherosclerosis-related arterial remodeling in female monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----<em>4</em>-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and <em>androstenedione</em> from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = <em>4</em>1,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = <em>4</em>8.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.<em>4</em> microM, Vmax = <em>4</em>8.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 91<em>4</em>.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.<em>4</em> nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and <em>androstenedione</em>, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.<em>4</em> +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.

To define the hormonal criteria via genotypic proof for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency in the adrenals and gonads, we investigated the type II 3beta-HSD genotype in 55 patients with clinical and/or hormonal presentation suggesting compromised adrenal with or without gonadal 3beta-HSD activity. Fourteen patients (11 males and 3 females) had ambiguous genitalia with or without salt wasting and with or without premature pubarche. One female neonate had salt wasting only. Twenty-five children (<em>4</em> males and 21 females) had premature pubarche only. Fifteen adolescent and adult females had hirsutism with or without menstrual disorder. The type II 3beta-HSD gene, including the promoter region up to -1053 base, all exons I, II, III, IV, and exon and intron boundaries, was sequenced in all subjects. Eight patients had a proven or predictably deleterious mutation in both alleles of the type II 3beta-HSD gene, and <em>4</em>7 patients had no apparent mutation in the gene. ACTH-stimulated (1 h post iv bolus of 250 microg Cortrosyn) serum 17-hydroxypregnenolone (Delta5-17P) levels and basal and ACTH-stimulated ratios of Delta5-17P to cortisol (F) in the genotypic proven patients were unequivocally higher than those of age-matched or pubic hair stage matched genotype-normal patients or control subjects (n = 7-30 for each group). All other baseline and ACTH-stimulated hormone parameters, including dehydroepiandrosterone (DHEA) levels, ratios of Delta5-17P to 17-OHP and DHEA to <em>androstenedione</em> in the genotype-proven patients, overlapped with the genotype-normal patients or control subjects. The hormonal findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3beta-HSD deficiency congenital adrenal hyperplasia (numeric and graphic reference standards from infancy to adulthood are provided): ACTH-stimulated Delta5-17P levels in 1) neonatal infants with ambiguous genitalia at or greater than 378 nmol/liter equivalent to or greater than 5.3 SD above the control mean level [95 +/- 53 (SD) nmol/liter]; 2) Tanner I children with ambiguous genitalia at or greater than 165 nmol/liter equivalent to or greater than 35 SD above the control mean level [12 +/- <em>4</em>.3 (SD) nmol/liter]; 3) children with premature pubarche at or greater than 29<em>4</em> nmol/liter equivalent to or greater than 5<em>4</em> SD above Tanner II pubic hair stage matched control mean level [17 +/- 5 (SD) nmol/liter]; and <em>4</em>) adults with at or greater than 289 nmol/liter equivalent to or greater than 21 SD above the normal mean level [25 +/- 12 (SD) nmol/liter]. ACTH-stimulated ratio of Delta5-17P to F in 1) neonatal infants at or greater than <em>4</em>3<em>4</em> equivalent to or greater than 6.<em>4</em> SD above the control mean ratio [88 +/- 5<em>4</em> (SD)]; 2) Tanner I children at or greater than 216 equivalent to or greater than 23 SD above the control mean ratio [12 +/- 9 (SD)]; 3) children with premature pubarche at or greater than 363 equivalent to or greater than 38 SD above the control mean ratio [20 +/- 9 (SD)]; and <em>4</em>) adults at or greater than <em>4</em>010 equivalent to or greater than 221 SD above the normal mean ratio [29 +/- 18 (SD)]. Conversely, the hormonal data in the genotype-normal patients suggest the following hormonal criteria are not consistent with 3beta-HSD deficiency congenital adrenal hyperplasia: ACTH-stimulated Delta5-17P levels in children with premature pubarche up to 72 nmol/liter equivalent to up to 11 SD above the control mean level, and in hirsute females up to 150 nmol/liter equivalent to up to 12 SD above the normal female mean level [28 +/- 10 (SD) nmol/liter]; and ACTH-stimulated Delta5-17P to F ratio in children with premature pubarche up to 67 equivalent to up to 5 SD above the control mean ratio, and in hirsute females up to 151 equivalent to up to 10 SD above the normal mean ratio [32 +/- 12 (SD)]. These findings help define newly proposed hormonal criteria to accurately predict inherited 3beta-HSD deficiency.

To investigate the role of androgen receptors in the regulation of brain aromatase activity (AA) in adult rats, the levels of AA in discrete brain areas of androgen-insensitive testicular feminized (Tfm) rats were compared with those in their normal male littermates (NL). AA was measured in homogenates of brain tissue by using a radiometric assay that quantifies the production of 3H2O from [1 beta-3H]<em>androstenedione</em> as an index of estrogen formation. Initially, we assessed the capability of block-dissected tissues to aromatize androgens. We found that the AA in the amygdala and hypothalamus-preoptic area of Tfm rats was significantly lower (P less than 0.001) than the AA in NL despite the fact that circulating androgen concentrations in the Tfm were significantly higher. Kinetics studies demonstrated that the apparent Michaelis constant was equivalent for both groups (0.02-0.03 microM). Administration of testosterone propionate to castrated males produced 3 to <em>4</em>-fold elevations of AA in NL, but did not affect brain AA in Tfm rats. To pinpoint specific sites where AA is affected in Tfm rats, we measured AA in 10 hypothalamic and limbic nuclei that were dissected from 300-micron frozen brain sections. Compared to NL, Tfm rats exhibited significantly lower levels of AA in all micro-dissected brain regions studied, except for the medial and cortical amygdala. These data provide genetic evidence for both androgen-dependent and independent regulation of AA in the rat brain.

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 169<em>4</em>9A, 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 169<em>4</em>9A 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 +/- <em>4</em>%), plasma estrone (32 +/- 6%), urine estradiol (<em>4</em>5 +/- 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 169<em>4</em>9A does not alter the pathways of estrogen metabolism. The degree of estrogen reduction was remarkably similar to that caused with aminoglutethimide. At doses of <em>4</em>-16 mg daily, CGS 169<em>4</em>9A inhibited the C21-hydroxylase enzyme as well, based on concomitant rises in plasma <em>androstenedione</em>, 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 169<em>4</em>9A appears to be a specific inhibitor of aromatase at doses below <em>4</em> 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.

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-<em>4</em>50 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-<em>4</em>50 with approx. 600-fold increase of both specific activities, while other cytochrome P-<em>4</em>50 enzyme activities found in the microsomes were completely eliminated. The purified P-<em>4</em>50 showed M(r) of 55 kDa, specific heme content of 12.9 +/- 2.6 nmol.mg-1 (+/- SD, n = <em>4</em>), reconstituted aromatase activity of 111 +/- 19 nmol.min-1.mg-1 and estradiol 2-hydroxylase activity of 5.85 +/- 1.23 nmol.min-1.mg-1. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-<em>4</em>50 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH beta-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-<em>4</em>50 in 0.1 M phosphate buffer (pH 7.6) showed Km of 1.58 microM and Vmax of 8.9 nmol.min-1.mg-1. A significant shift of the optimum pH and Vmax, but not the Km, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and <em>androstenedione</em> competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and <em>androstenedione</em>. Estrone and estradiol showed Ki of <em>4</em>.8 and 7.3 microM, respectively, for testosterone aromatization, and 5.0 and 8.1 microM, respectively, for <em>androstenedione</em> aromatization. <em>Androstenedione</em> and testosterone showed Ki of 0.32 and 0.61 microM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-<em>4</em>50 functions as estrogen 2-hydroxylase as well as androgen 19-, 1 beta-, and 2 beta-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid substrates to face their beta-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.

The expression of hedgehog (Hh) genes, their receptor, and the co-receptor in mice, rat, and bovine ovaries were investigated. RT-PCR of ovarian transcripts in mice showed amplification of transcripts for Indian (Ihh) and desert (Dhh) Hh, patched 1 (Ptch1), and smoothened (Smo) genes. Semi-quantitative RT-PCR and northern blot analyses showed that whole ovarian Ihh and Dhh transcripts decreased <em>4</em>-2<em>4</em> h after hCG versus 0-<em>4</em>8 h after pregnant mares serum gonadotrophin treatment in mice, whereas mouse Ptch1 and Smo transcripts were expressed throughout the gonadotropin treatments. Quantitative real-time RT-PCR (qRT-PCR) revealed that the expression of the Hh-patched signaling system with Ihh mRNA abundance in granulosa cells was greater, whereas Smo and Ptch1 mRNA abundance was less in theca cells of small versus large follicles of cattle. In cultured rat and bovine theca-interstitial cells, qRT-PCR analyses revealed that the abundance of Gli1 and Ptch1 mRNAs were increased (P<0.05) with sonic hedgehog (SHH) treatment. Additional studies using cultured bovine theca cells indicated that SHH induces proliferation and <em>androstenedione</em> production. IGF1 decreased Ihh mRNA abundance in bovine granulosa cells. The expression and regulation of Ihh transcripts in granulosa cells and Ptch1 mRNA in theca cells suggest a potential paracrine role of this system in bovine follicular development. This study illustrates for the first time Hh activation of Gli1 transcriptional factor in theca cells and its stimulation of theca cell proliferation and androgen biosynthesis.

BACKGROUND

Conventional hydrocortisone dosing schedules do not mimic the normal circadian rhythm of cortisol, making it difficult to optimize treatment in congenital adrenal hyperplasia (CAH).

METHODS

We report a 14.5-year-old boy with CAH who had reduced bioavailability [42% (normal 80% orally and 100% by im route)] and increased clearance [half-life 50 min (normal range, 70-100 min)] of oral doses of hydrocortisone leading to ambient serum 17-hydroxyprogesterone concentrations of 400 nmol/liter (14.5 ng/ml) and androstenedione concentrations of 24.9 nmol/liter (7.1 ng/ml).

METHODS

Using a continuous but variable sc hydrocortisone infusion via an insulin pump, rapid control of his CAH was attained with a normal cortisol circadian profile. Average daily hydrocortisone dose was 17.4-18.6 mg/m(2), which produces on average 24-h serum cortisol and 17-hydroxyprogesterone concentrations of 316 nmol/liter (115 ng/ml) and 4.3 nmol/liter (1.4 ng/ml), respectively. Therapy has been maintained over 4 yr with suppression of normal adrenal androgen production and normal progression through puberty.

CONCLUSIONS

Continuous sc infusion of hydrocortisone may prove a valuable adjunct to therapy for CAH, particularly in patients requiring high doses of oral hydrocortisone and in those with abnormal hydrocortisone pharmacokinetics.

The normal response to a single 0.25-mg dose of ACTH-(1-2<em>4</em>) is not well established in infancy or childhood. We report the adrenal steroidogenic responses of 17-hydroxypregnenolone (17OH Preg), 17-hydroxyprogesterone (17OH Prog), 11-deoxycortisol, cortisol, deoxycorticosterone, dehydroepiandrosterone (DHEA), DHEA sulfate, <em>androstenedione</em> (A'dione), and testosterone in 102 healthy children who were divided into 5 groups: group 1 (less than 1 yr old; n = 22), group 2 (1-5 yr old; n = 22), group 3 (6-12 yr old; n = 15), group <em>4</em> (early-midpuberty; n = 21), and group 5 (late puberty; n = 22). Baseline and stimulated levels of 17OH Preg were significantly higher in group 1 infants than in group 2 children (P less than 0.01). Baseline levels of 17OH Prog increased in late puberty (P less than 0.01). Baseline and stimulated levels of DHEA rose in late puberty (group 5 vs. group 3, P less than 0.01). DHEA levels in late pubertal females were higher than those in their male counterparts (P less than 0.01). DHEA sulfate levels did not change after ACTH administration in any age group. Baseline and stimulated levels of A'dione rose significantly before the onset of puberty in female children (group 2 vs. group 3, P less than 0.01). The calculated ratio of 17OH Preg/17OH Prog in group 1 was significantly higher than that in other groups of children (P less than 0.01). The calculated, baseline DHEA/A'dione ratio was higher in group 1 than in older children (P less than 0.01). Stimulated ratios were higher in late pubertal females than in males (P less than 0.01). In both sexes baseline and stimulated ratios of 17OH Prog/deoxycorticosterone increased in puberty, such that late pubertal children had higher levels than prepubertal children (P less than 0.01). These data confirm the need for interpretation ACTH stimulation test data to be based upon age- and sex-specific norms.

We identified a new point mutation in the CYP19 gene responsible for aromatase (P<em>4</em>50arom) deficiency in a <em>4</em>6,XY male infant with unremarkable clinical findings at birth. This boy is homozygote for a 1-bp (C) deletion in exon 5 of the aromatase gene causing a frame-shift mutation. The frame-shift results in a prematurely terminated protein that is inactive due to the absence of the functional regions of the enzyme. Aromatase deficiency was suspected prenatally because of the severe virilization of the mother during the early pregnancy, and the diagnosis was confirmed shortly after birth. Four weeks after birth, the baby boy showed extremely low levels of serum estrogens, but had a normal level of serum free testosterone; in comparison with the high serum concentration of <em>androstenedione</em> at birth, a striking decrease occurred by <em>4</em> weeks postnatally. We previously reported elevated basal and stimulated FSH levels in a female infant with aromatase deficiency in the first year of life. In contrast, in the male infant, basal FSH and peak FSH levels after standard GnRH stimulation tests were normal. This finding suggests that the contribution of estrogen to the hypothalamic-pituitary gonadotropin-gonadal feedback mechanism is different in boys and girls during infancy and early childhood. In normal girls, serum estradiol concentrations strongly correlate with circulating inhibin levels, and thus, low inhibin levels may contribute to the striking elevation of FSH in young girls with aromatase deficiency. In contrast, estradiol levels are physiologically about a 7-fold lower in boys than in girls, and serum inhibin levels remain elevated even though levels of FSH, LH, and testosterone are decreased.

The effects of glucocorticoids on the steroidogenesis of ovarian granulosa cells were investigated. Cortisol and dexamethasone inhibited the increase in aromatase activity induced by FSH in cultured rat granulosa cells. In the same cultures progesterone production was stimulated to a maximum of 167% of the control level. This differential effect of glucocorticoids on estrogen and progesterone production by the granulosa cells indicates that glucocorticoids exert specific inhibition of the induction of aromatase by FSH and do not cause a general suppression of granulosa cell activity. In contrast to their inhibition of the FSH induction of aromatase enzymes, glucocorticoids did not interfere with the activity of pre-existing aromatase enzymes. In granulosa cells containing full aromatase activity, treatment with cortisol and dexamethasone did not inhibit aromatization of <em>androstenedione</em> to estrogens whereas two known aromatase inhibitors (dihydrotestosterone and <em>4</em>-androstene-3, 6, 17-trione) were effective. These results indicate that the glucocorticoids exert a selective inhibition of the FSH-induction of aromatase activity in rat granulosa cells by a mechanism other than directly interfering with the aromatization reaction.

Steroids synthesized de novo from cholesterol in the brain are generally called neurosteroids. We have recently demonstrated, using biochemical and molecular biological methods, that certain structures in the quail brain possess cytochrome P<em>4</em>50 side-chain cleavage enzyme (P<em>4</em>50scc) and 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(<em>4</em>)-isomerase (3beta-HSD) and produce pregnenolone, pregnenolone sulfate and progesterone. To clarify the biosynthetic pathway of neurosteroids in the avian brain, therefore, we examined the expression of messenger RNA (mRNA) encoding for the enzyme cytochrome P<em>4</em>50 17alpha-hydroxylase/c17,20-lyase (P<em>4</em>50(17alpha,lyase)), which converts pregnenolone to dehydroepiandrosterone via 17alpha-hydroxypregnenolone or progesterone to <em>androstenedione</em> via 17alpha-hydroxyprogesterone. RT-PCR analysis followed by Southern hybridization indicated the expression of P<em>4</em>50(17alpha,lyase) mRNA in the brain of sexually mature birds without a clear-cut sex difference. Employing biochemical techniques combined with HPLC analysis, the conversion of progesterone to 17alpha-hydroxyprogesterone was also found in brain slices of the mature male. P<em>4</em>50(17alpha,lyase) mRNA was detected in various brain regions, but there was a clear regional difference in the expression. The expressions of P<em>4</em>50(17alpha,lyase) mRNA in the diencephalon and mesencephalon were significantly higher than those in the cerebrum and cerebellum, unlike 3beta-HSD mRNA, which showed no regional difference in the expression. In situ hybridization revealed the cellular localization of P<em>4</em>50(17alpha,lyase) mRNA. The cells expressing P<em>4</em>50(17alpha,lyase) mRNA were detected several diencephalic and mesencephalic regions, such as the preoptic area, the anterior hypothalamus, the dorsolateral thalamus, the optic tectum and the ventral midbrain. The expression was also localized in the septum, the hyperstriatum accessorium, and the ventral portions of the archistriatum in the telencephalon. Cerebellar Purkinje cells also expressed P<em>4</em>50(17alpha,lyase) mRNA. These results suggest that the avian brain possesses P<em>4</em>50(17alpha,lyase) as well as P<em>4</em>50scc and 3beta-HSD in both sexes. The expression of P<em>4</em>50(17alpha,lyase) in the avian brain may be region-dependent.

OBJECTIVE

To prove that low-dose hCG alone can be clinically used to replace FSH-containing gonadotropins to complete controlled ovarian hyperstimulation (COH).

METHODS

Controlled, prospective, randomized study.

METHODS

Academic center.

METHODS

Infertile patients who are candidates for assisted reproduction.

METHODS

Patients received [1] recombinant FSH or hMG throughout COH (group A); [2] ovarian priming with recombinant FSH/hMG followed by low-dose hCG (200 IU/day) alone (group B).

METHODS

Medication consumption; daily serum and follicular fluid (FF) measurements of LH, FSH, hCG, E2, P, T, and androstenedione (A); number and size of follicles; intracytoplasmic sperm injection (ICSI) outcome.

RESULTS

In group B: [1] duration and dose of recombinant FSH/hMG administration were reduced; [2] preovulatory serum hCG, E2, and T were higher, whereas FSH was lower; [3] FF hCG, E2, T levels, and E2/T, E2/A, and E2/P ratios were higher, whereas A was lower; [4] small but not large preovulatory follicles were reduced; [5] fertilization rates were higher; and [6] serum and FF P levels, and ICSI outcome did not differ.

CONCLUSIONS

Low-dose hCG alone in the late COH stages: [1] reduced recombinant FSH/hMG consumption whereas ICSI outcome was comparable to traditional COH regimens; [2] stimulated follicle growth and maturation independent of FSH administration; [3] was associated with a reduced number of small preovulatory follicles; [4] did not cause premature luteinization; [5] resulted in a more estrogenic intrafollicular environment.

A phase I study was performed of CGS 20267, an oral nonsteroidal, highly potent, and selective aromatase inhibitor, in 21 postmenopausal patients with advanced breast cancer. The patients were recruited in 3 successive groups of 7, receiving 0.1, 0.5, and 2.5 mg p.o./day, respectively. All patients had received at least one prior endocrine treatment (range, 1-<em>4</em>), and six patients had received prior chemotherapy. The treatment was very well tolerated, and no toxicity was seen at any of the three doses. There was a statistically significant suppression of estradiol (E2) and estrone (E1) levels by 7<em>4</em>% and 79% from baseline levels, respectively (P < 0.0001). Suppression occurred in all three patient groups, with many patients having serum concentrations of estradiol and estrone, which were below the limit of detection of the assays (3 and 10 pM, respectively), which corresponds to a maximum measurable estrogen suppression of 86%. CGS 20267 had no significant effect on serum levels of follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, cortisol, 17 alpha-hydroxyprogesterone, <em>androstenedione</em>, and aldosterone. Seven (33%, 95% confidence interval, 15-57%) of the 21 patients have responded to treatment (one complete remission, 6 partial remissions according to criteria of the Union Internationale contre le Cancer), and 6 are still responding to CGS 20267 (duration of response; <em>4</em>+, 6+, 6+, 9+, 9, 12+, and 12+ months). Five have had stable disease for more than 3 months, and 9 had progressive disease. These results suggest that CGS 20267 is a very potent and specific aromatase inhibitor, and phase II studies are now required to confirm its clinical efficacy.

The influence of the aromatase inhibitor <em>4</em>-hydroxy<em>androstenedione</em> (<em>4</em>OHA) given intramuscularly on the peripheral aromatisation of <em>androstenedione</em> into oestrone was investigated in postmenopausal women with breast cancer and compared with the suppression of plasma oestradiol (E2). 7 patients were investigated before and during treatment on day 7, i.e. midway between two weekly injections. After an intravenous injection of [3H] <em>androstenedione</em> and [1<em>4</em>C] oestrone, urine was collected for 96 h and the isotope ratio determined in the urinary oestrogen metabolites after isolation with high performance liquid chromatography. At 250 mg, <em>4</em>OHA inhibited aromatisation to [mean (S.D.)] 15.2 (5)% of baseline (P < 0.002). There was significantly greater inhibition to 8.1 (2.7)% at <em>4</em>OHA 500 mg (P < 0.01). Plasma E2 was reduced to <em>4</em>1.2 (1<em>4</em>.1)% of baseline at <em>4</em>OHA 250 mg with a further reduction to 32.7 (19.8)% at 500 mg (P < 0.05). These results confirm the dose-response relation previously established with plasma oestrogen measurements alone.

The aim of this study was to determine 1) the time of onset and cellular localization of gene expression for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase/Delta(5),Delta(<em>4</em>) isomerase (3beta-HSD), and the cytochrome P<em>4</em>50 enzymes for cholesterol side-chain cleavage (P<em>4</em>50(scc)), 17alpha-hydroxylase (P<em>4</em>50(17alphaOH)), and aromatase (P<em>4</em>50(arom)) during gonadal development; and 2) the amount of progesterone, <em>androstenedione</em>, testosterone, and 17beta-estradiol present in the fetal sheep gonad. Fetuses were collected on Days 2<em>4</em>, 26, 28, 30, 32, 35, <em>4</em>0, 55, and 75 of gestation, and gene expression was determined by in situ hybridization. The steroid content of gonads collected on Days 30, 35, 55, and 75 of gestation was determined by RIA. Developing gonads collected from both male and female fetuses were steroidogenically active around the time of morphological sexual differentiation. In the female, the steroidogenic cells were initially located at the boundary of the cortex and medulla but become increasingly restricted to the mesonephric-derived cell streams. In the male, once tubules were identifiable, steroidogenesis was restricted to the interstitial regions. Interestingly, expression of both SF-1 and 3beta-HSD was observed prior to morphological sexual differentiation. In addition, expression of both of these genes was more widespread than the other genes in both males and females.

Male rhesus monkey fetuses have significantly more testosterone (T) in their circulation than females on days 35--50 of gestation (P less than 0.01; n = 6 males and 6 females). However, we found no sex differences for <em>androstenedione</em> (delta <em>4</em>). T concentrations remained significantly higher in male fetuses than in females later in gestation, e.g. days 79--8<em>4</em>, 100--133, and 1<em>4</em>0--160. Levels of delta <em>4</em> differed between the sexes only on days 79--8<em>4</em>, and dihydrotestosterone concentrations were significantly higher in male fetuses than in females on days 100--133 and 1<em>4</em>0--163. The fact that delta <em>4</em> concentrations were not different between the sexes at the earliest period studied (days 35--50) indicates that systemic concentrations of this hormone in the fetus probably are not important for sexual differentiation, especially of the central nervous system. Quantification of three steroids (T, delta <em>4</em>, and dihydrotestosterone) in umbilical arterial and venous plasma from five male and nine female fetuses (days 35--100) revealed significant arterial/venous differences only for T in males (arterial greater than venous). These data, which suggest that fetal testes secrete T during morphological differentiation, lend credence to the hypothesis that endogenous T partially regulates sexual differentiation.

As stated earlier, the mammalian ovary maintains the continuous development of follicles, but only a few are selected to ovulate and form corpora lutea. These processes are regulated primarily by the gonadotropins and involve specific, sequential changes in the function of theca cells and granulosa cells. Data from recent studies (summarized in Figure 3) show that specific genes are turned on or off at different stages of follicular growth in response to estradiol and different amounts of gonadotropins and cAMP. For example, mRNA for RII51 in granulosa cells and theca cells increases in association with small increased in cAMP but is markedly reduced by the LH surge and high cAMP. The content of mRNA for other kinase subunits, RI and C alpha, show little or no change during similar hormonal changes. In theca cells, mRNA for 17 alpha-hydroxylase increased and decreased in a manner similar to that for RII51. In contrast, levels of mRNA for P<em>4</em>50scc increased only gradually in follicles but were markedly increased by the LH surge and high concentrations of cAMP and then appeared to be constitutively expressed in rat corpora lutea in a cAMP-independent manner. PGS and t-PA appear to follow yet another pattern: rapid induction by the LH surge followed by a rapid decline in association with ovulation. One major task for reproductive endocrinologists and molecular biologists now is to determine how low and high concentrations of cAMP act to turn on and turn off the expression of these specific genes at specific times during follicular maturation. A working model of the molecular events occurring in theca and granulosa cells of PO follicles is shown in Figure <em>4</em>. LH acts on theca cells via cAMP ro regulate both P<em>4</em>50scc and P<em>4</em>50(17) alpha mRNA levels, leading to increased biosynthesis of <em>androstenedione</em>. The mechanisms by which cAMP acts in theca cells remain to be determined but appear to involve an increase in the content of RII51, P<em>4</em>50scc, and P<em>4</em>50(17) alpha. In granulosa cells, <em>androstenedione</em> is converted to estradiol by the aromatase P<em>4</em>50 enzyme system. Estradiol, in turn, binds to estradiol receptors present in these cells and may thereby regulate gene expression. However, despite the presence of estradiol and estradiol receptors, little or no effect of estradiol is observed unless FSH acts via the FSH receptor to increase intracellular concentrations of cAMP. In a manner not yet understood, cAMP appears to enhance the actions of estradiol.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

OBJECTIVE

To compare the effects of cyproterone acetate and desogestrel, as part of combined oral contraceptives, on lipid metabolism and hirsutism of adolescents with polycystic ovary syndrome (PCOS).

METHODS

Prospective randomized clinical trial.

METHODS

Outpatient gynecology clinic (referral center) of a university.

METHODS

Twenty-eight adolescent girls with clinical and biological hyperandrogenism and six or less menses during the past 12 months.

METHODS

Group A (n = 1<em>4</em>) received 0.15 mg of desogestrel plus 0.030 mg of ethinyl estradiol daily. Group B (n = 1<em>4</em>) received 2 mg of cyproterone acetate plus 0.035 mg of ethinyl estradiol daily. Treatment was given for 21 days followed by a 7-day rest for a period of 12 months.

METHODS

Hirsutism and lipid profile were evaluated before initiation and at 3, 6, 9, and 12 months of treatment. Androgen profile was evaluated before and at 12 months of treatment.

RESULTS

A significant decline of the Ferriman-Gallway hirsutism score was observed from the sixth month of therapy in both groups. During therapy, the levels of testosterone, free testosterone, Delta(<em>4</em>)-<em>androstenedione</em>, and 17OH-progesterone decreased significantly, whereas sex hormone-binding globulin (SHBG) increased significantly in both groups. The level of total cholesterol and low density lipoprotein (LDL) cholesterol increased significantly, whereas high density lipoprotein (HDL) cholesterol and apolipoprotein A-I increased significantly from the third month of therapy in both groups. Total cholesterol/HDL cholesterol and LDL cholesterol/HDL cholesterol ratios remained unchanged. The levels of triglycerides increased significantly in the cyproterone acetate-treated group after the third month.

CONCLUSIONS

Treatment of adolescent girls with PCOS with the two studied formulations is comparably effective in decreasing hirsutism and androgen levels. Both combined oral contraceptives are associated with an increase of total cholesterol, LDL cholesterol, and HDL cholesterol levels and no change of the total cholesterol/HDL cholesterol and LDL cholesterol/HDL cholesterol ratios. Treatment with the cyproterone acetate combined oral contraceptive is associated with a tendency toward increasing the levels of triglycerides.

Sex steroids play a crucial role in the development and differentiation of normal mammary gland as well as in the regulation of breast cancer growth. Local intracrine formation of sex steroids from inactive precursors secreted by the adrenals, namely, dehydroepiandrosterone and its sulfate, may regulate growth and function of peripheral target tissues, including the breast. Both endocrine and paracrine influences on the proliferation of human breast cancer cells are well recognized. Breast tumors harbor tumor-associated macrophages and tumor-infiltrating lymphocytes that secrete a wide spectrum of cytokines. These factors may also contribute to neoplastic cell activity. The present study was designed to investigate the action of cytokines on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity, which is an essential step in the biosynthesis of active estrogens and androgens in human breast cancer cell lines and in normal human mammary epithelial cells in primary culture. 3Beta-HSD activity was undetectable in ZR-75-1 and T-<em>4</em>7D estrogen receptor-positive (ER)+ cells under basal growth conditions. This activity was markedly induced after exposure to picomolar concentrations of interleukin (IL)-<em>4</em> or IL-13. The potent stimulatory effect of these cytokines on 3beta-HSD activity was also observed in the ER- MDA-MB-231 human breast cancer cell line and in normal human mammary epithelial cells (HMECs) in primary culture. The stimulation of 3beta-HSD activity by IL-<em>4</em> and IL-13 results from a rapid increase in 3beta-HSD type 1 mRNA levels as measured by RT-PCR and Northern blot analyses. Such an induction of the 3beta-HSD activity may modulate androgenic and estrogenic biological responses as demonstrated using ZR-75-1 cells transfected with androgen- or estrogen-sensitive reporter constructs and treated with the adrenal steroid 5-androstene-3beta,17beta-diol. The DNA-binding activity of Stat6, a member of the signal transducers and activators of transcription gene family, is activated 30 min after exposure to IL-<em>4</em> and IL-13 in human breast cancer cell lines as well as in HMECs in primary culture. In these cells, Stat6 activated by IL-<em>4</em> or IL-13 binds to two regions of the 3beta-HSD type 1 gene promoter, containing Stat6 consensus sequences. IL-<em>4</em> induction of 3beta-HSD mRNA and activity is sensitive to staurosporine. This protein kinase inhibitor also inhibits IL-<em>4</em>-induced Stat6 DNA-binding activity. Our data demonstrate for the first time that IL-<em>4</em> and IL-13 induce 3beta-HSD type 1 gene expression, thus suggesting their involvement in the fine control of sex steroid biosynthesis from adrenal steroid precursors in normal and tumoral human mammary cells. Furthermore, aromatase and/or 5alpha-reductase(s) are expressed in the mammary gland and in a large proportion of human breast tumors. An increase in the formation of their substrates, namely, <em>4</em>-<em>androstenedione</em> and testosterone, may well have a significant impact on the synthesis of active estrogens and androgens in these tissues.