Premature pubarche is characterized by pubic hair, adult type body odor, acne, and axillary hair before 8 yr of age in girls and 9.5 yr of age in boys. Causes of this premature virilization include premature adrenarche, mild errors of steroidogenesis, precocious puberty, and adrenal and gonadal tumors. To determine whether any clinical parameters are helpful in distinguishing which children should undergo further evaluation for mild congenital adrenal hyperplasia, we performed ACTH stimulation tests in 69 children with premature pubarche and 8 pubertal controls. Patients were categorized as having typical (pubic hair with or without axillary hair and body odor) or atypical (pubic hair and genital enlargement) premature pubarche. Blood samples, before and 30 min after iv bolus administration of synthetic ACTH, were obtained for progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, <em>androstenedione</em>, 11-deoxycortisol, and cortisol measurements. The patients were divided into <em>4</em> groups based on their individual responses to ACTH stimulation: premature adrenarche (no apparent defect in steroidogenesis), possible decreased 21-hydroxylase activity, possible decreased 3 beta-hydroxysteroid dehydrogenase activity, and indeterminate responses. Five of 11 (<em>4</em>5%) children with atypical premature pubarche and 7 of 58 (12%) children with typical premature pubarche were found to have evidence of mild defects in steroidogenesis. Similar to previous reports in postpubertal women, only responses to ACTH stimulation allowed accurate classification of these patients.

Twenty women using oral contraceptives and complaining of impaired sexual function were compared with twenty women without sexual problems, matched for age and oral contraceptive. Whilst the sexual behaviour differed in the two groups, the plasma testosterone, <em>androstenedione</em>, oestradiol and SHBG concentrations were very similar. The total androgen levels were low in both groups. Plasma testosterone and oestradiol concentrations were correlated with measures of sexual interest in the no-problem group, but not in the problem group. Administration of exogenous <em>androstenedione</em> to women in the problem group, using a double blind cross-over comparison with a placebo, failed to improve their sexual function except in one case. The majority of women showed a rise in androgen and oestradiol between day 2<em>4</em> of one pill cycle and day <em>4</em> of the next. The possible behavioural indications of this pattern are discussed.

Androgens produced by the primate corpus luteum (CL) serve as precursors for estrogen synthesis; moreover, detection of androgen receptors in luteal tissue suggests a regulatory role within the CL. To determine the cellular source(s) and agonist regulation of androgen production during the lifespan of the primate CL, luteal tissues were collected from rhesus monkeys in the early (days 3-5 post-LH surge), mid (days 7-8), mid-late (days 11-12), and late (days 1<em>4</em>-15) luteal phase of the menstrual cycle. Collagenase-dispersed cells (i.e., mixed cells) were analyzed by flow cytometry based on light scatter properties and sorted into populations of small (< or = 15 microns) and large >> 20 microns) luteal cells. Cells (n = <em>4</em> animals/stage) were incubated in Ham's F-10 and 0.1% BSA for 3 h at 37 C with or without hCG (100 ng/mL), PGE2 (1<em>4</em> mumol/L), or dibutyryl cAMP (dbcAMP; 5 mmol/L), and <em>androstenedione</em> (A<em>4</em>) and testosterone were measured. Basal A<em>4</em> production by large cells was markedly higher (P < 0.05) than that by small cells (e.g. mid-late luteal phase, 821 +/- 188 vs. 69 +/- 25 pg/mL.5 x 10(<em>4</em>) cells/3 h; mean +/- SEM), whereas that by mixed cells was intermediate (317 +/- 205 pg/mL). In the early luteal phase, hCG stimulated A<em>4</em> synthesis by mixed (1.6-fold; P < 0.05) and large (3.1-fold; P < 0.05) luteal cells, but not by small cells (1.3-fold). By the mid-late luteal phase, hCG did not increase A<em>4</em> production by any cell type, although hCG responsiveness returned to large cells (2.0-fold increase; P < 0.05) by the late luteal phase. PGE2 responsiveness by cell types was similar to that of hCG, except large cell responsiveness did not return in the late luteal phase. In all cell types, dbcAMP stimulated the largest increase in A<em>4</em> levels; in the mid-late luteal phase, small and large cells responded to dbcAMP with 8.2- and 3.0-fold increases (P < 0.05) in A<em>4</em> production, respectively. When luteal cells were incubated with the steroidogenic substrates, 17 alpha-hydroxyprogesterone or 17 alpha-hydroxypregnenolone (1 mumol/L), large cells produced much more (P < 0.05) A<em>4</em>, testosterone, estrone, and estradiol than small cells. Both substrates elicited similar patterns of androgen production, with A<em>4</em> synthesis predominant in all luteal cell types. Thus, cell subpopulations in the primate CL can be distinguished by their ability to produce androgen and estrogen. Changes in agonist-responsive androgen production may influence the local steroid milieu and function of the CL during the menstrual cycle.

Concern over the potential negative ecological effects of steroid hormones from human- and animal-derived wastes has resulted in an increased interest regarding the mobility and persistence of these compounds in the environment. Batch experiments were conducted to examine the simultaneous sorption and dissipation of three reproductive hormones (testosterone, 17beta-estradiol, and 17alpha-ethynyl estradiol) in four midwestern U.S. soils and one freshwater sediment. Sorption isotherms were generated by measuring aqueous concentrations and by extracting the sorbed parent chemical or transformation products (e.g., estrone, <em>androstenedione</em>). Apparent sorption equilibrium is reached within a few hours. Measured sorption isotherms for the three parent chemicals and their principal transformation products were generally linear. Average organic carbon normalized sorption coefficients (K(oc)) resulted in standard deviations of less than 0.2 log units and were consistent with reported aqueous solubilites and octanol-water partition coefficients, indicating hydrophobic partitioning as the dominant sorption mechanism. Large log K(oc) values (approximately 3-<em>4</em>) suggest that leaching from soils will be limited, runoff of soil- and land-applied biosolids are the most likely inputs into surface waters, and that a significant fraction of these compounds will be associated with sediments. Half-lives for hormone dissipation in the aerobic soil and sediment slurries estimated assuming pseudo first-order processes ranged from a few hours to a few days with testosterone having the shortest half-life.

Using gas chromatography/mass spectrometry we have developed a method for the simultaneous determination of six plasma steroids: testosterone, <em>4</em>-<em>androstenedione</em>, 17 alpha-hydroxyprogesterone, 5 alpha-androstane-3 alpha,17 beta-diol, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone. For each analyte, a deuterium-labeled internal standard was used for quantification. Due to the high isotopic purity of our standards, no complex corrections for isotope contributions were necessary. The procedure provides a sensitive and specific technique with good accuracy and precision.

In this study, we investigated follicular dynamics and ovarian steroid secretion during the follicular and early luteal phases of the estrous cycle in sheep. Six Finn-Merino ewes with ovarian autotransplanted ovaries were monitored for 10 days during the follicular phase and subsequent early luteal phase after luteal regression was induced with cloprostenol (a potent analogue of prostaglandin F2alpha). Over this period, follicular diameter was measured by serial ultrasound scans, and the concentration of gonadotropins and steroids in ovarian venous blood was measured at intervals of 6-12 h. All animals had an LH surge (Day 0) 59 +/- <em>4</em>.7 h after injection of cloprostenol. The ovulatory follicles were derived mainly from large antral follicles present at the time of injection of cloprostenol (5.1 +/- 0.<em>4</em> mm; mean +/- SEM, n = 6), although in some animals recruitment of additional small follicles was observed after luteolysis. The concentration of FSH decreased during the follicular phase and peaked synchronously with the LH surge, while estradiol and <em>androstenedione</em> concentrations in ovarian venous plasma increased progressively from luteal regression to a maximum at the LH surge. The rise in concentration of FSH on Day 1 was followed by the growth of a new cohort of follicles. Follicular size and ovarian steroid secretion increased in a linear fashion from Day 1 to Day 3, with ovarian steroid secretion reaching a maximum when the first wave of luteal phase follicles achieved a diameter of 5 mm or more. On Day <em>4</em>, steroid secretion began to decline without significant changes in follicular diameter, and a second wave of follicles emerged. We conclude that 1) the preovulatory follicles are usually derived from the large follicle population present at the time of luteal regression, but the sheep has the ability to promote smaller follicles if required; and 2) the second peak of FSH stimulates the development of large estrogenic follicles during the early luteal phase, but the period of functional dominance is shorter than the period of morphological dominance.

OBJECTIVE

To assess the association of genetic polymorphisms related to cardiovascular disease (CVD) risk with anthropometric parameters and indices of androgenicity in healthy postmenopausal women.

METHODS

Cross-sectional study in a University Menopause Clinic.

METHODS

The following polymorphisms were assessed in 8<em>4</em> healthy postmenopausal women: glycoprotein IIIa Leu33Pro, apolipoprotein E2/E3/E<em>4</em>, methylenetetrahydrofolate reductase (MTHFR) Ala222Val, apolipoprotein B Arg3500Gln, paraoxonase 1 Gln192Arg, plasminogen activator inhibitor 1 <em>4</em>G/5G, cholesterol-7 alpha-hydroxylase A-20<em>4</em>C, and cholesterol ester transfer protein (TaqIB) B1/B2. Hormonal assays included FSH, LH, 17-beta-estradiol, testosterone, sex hormone-binding globulin (SHBG), DHEA sulfate, Delta-<em>4</em>-<em>androstenedione</em> (Delta<em>4</em>A), free androgen index (FAI), free estrogen index (FEI), and homocysteine (Hcy). The anthropometric components were body mass index (BMI) and waist-to-hip ratio (WHR).

RESULTS

MTHFR Ala222Val polymorphism was positively associated with testosterone, FAI, and FEI (P=0.001, P=0.000<em>4</em>, and P=0.01<em>4</em> respectively) and negatively with SHBG (P=0.0<em>4</em>7). Furthermore, women bearing this polymorphism had higher BMI and WHR compared with women with the wild-type variant (P=0.027 and P=0.0<em>4</em><em>4</em> respectively).

CONCLUSIONS

MTHFR Ala222Val polymorphism is associated with increased androgenicity and elevated BMI and WHR in healthy postmenopausal women. The significance of this association with respect to the CVD risk of postmenopausal women remains to be elucidated in future studies.

To investigate the enzymatic basis for abnormal steroid metabolism in subjects with male pseudohermaphroditism due to 5 alpha-reductase deficiency, the ring A reduced urinary 5 beta and 5 alpha metabolites of testosterone, <em>androstenedione</em>, 11 beta-hydroxy<em>androstenedione</em>, cortisol and corticosterone were measured by gas chromatography. Assays of the four pairs of urinary 5 beta and 5 alpha steroid metabolites revealed decreased conversion of the parent steroids to 5 alpha-reduced urinary metabolites, with increased 5 beta to 5 alpha urinary steroid metabolite ratios. These studies establish that increased urinary 5 beta/5 alpha ratios are distinctive for this disorder, and represent the most reliable method for confirming the diagnosis of primary inherited 5 alpha-reductase deficiency. These data also suggest that the conversion on the many delta <em>4</em>-3 ketosteroids to 5 alpha-reduced steroids may be due to a single enzyme with broad specificity, or multiple enzyme reductases with a common regulator.

Mammalian 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3beta-HSD type 1 (3beta-HSD_1) with an NAD cofactor and <em>androstenedione</em> product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose <em>4</em>-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD_1) with an NADP cofactor and <em>androstenedione</em> (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3beta-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose <em>4</em>,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3beta-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.

OBJECTIVE

Pioglitazone is an insulin sensitizer used for the management of type 2 diabetes mellitus (T2DM). It has been shown to reduce testosterone level in patients with polycystic ovarian syndrome. However, its effect on testosterone in men has not been studied.

METHODS

A randomized, double-blind, placebo-controlled trial with 6 months follow-up. Fifty (25 in each group) eugonadal men (well virilized and total testosterone ≥ 12 nm) with T2DM, aged 30-55 year and HbA1c of ≤ 7.5% were randomly assigned to receive pioglitazone 30 mg per day or placebo along with existing glimepiride and metformin therapy.

RESULTS

As compared to placebo, 6 months of pioglitazone therapy in patients with T2DM resulted in significant reduction in mean total testosterone level (16.1 to 1<em>4</em>.9 vs 17.1 to 17.0 nm; P = 0.031), calculated free testosterone (P = 0.001) and bioavailable testosterone (P = 0.000) despite significant increase in sex hormone-binding globulin (P = 0.000). Plasma <em>androstenedione</em> (∆(<em>4</em>) ) level increased (1.5 to 1.9 vs 1.7 to 1.7 ng/ml; P = 0.051) following pioglitazone therapy. The decrease in testosterone was independent of change in body weight, body fat and HbA1c.

CONCLUSIONS

Pioglitazone therapy significantly decreases total, free and bioavailable testosterone in eugonadal men with T2DM. The effects of these alterations need to be determined by further long-term studies.

Aspiration of bovine follicles 12-36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High <em>androstenedione</em> (A<em>4</em>;>><em>4</em>0 ng/ml; mean = 102) and another with Low A<em>4</em> (<20 ng/ml; mean = 9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A<em>4</em> cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A<em>4</em> (158-fold) and testosterone (31-fold) in the dominant follicle of High A<em>4</em> cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A<em>4</em> cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A<em>4</em> cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A<em>4</em> cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07). There was a tendency for reduction in stage <em>4</em> follicles in ovarian cortex samples from High A<em>4</em> cows suggesting that progression to antral stages were impaired. High A<em>4</em> cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A<em>4</em> cows suggesting reduced fertility in the High A<em>4</em> population. These data suggest that the dominant follicle environment of High A<em>4</em> cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.

Postmenopausal women convert circulatory C19 steroids to estrogen. In order to study the possible role of such estrogen in endometrial cancer, the determination of the fractional conversion of circulation delta-<em>4</em>-androstene-3,17-dione to estrone was attempted. However, in the course of this work it became apparent that the mathematical model upon which this determination is based does not adequately represent the true physiological conditions. The reasons for the inadequacy of the model are not apparent, although they seem to bear some relationship to obesity. The direction and magnitude of the deviation of the values; determined from the true values are unknown. The values for the apparent fractional conversion of <em>androstenedione</em> to estrone in 9 postmenopausal women with endometrial cancer are strongly correlated with values indices of obesity. These values also correlate well with values for the apparent rate of production of estrone in our 9 patients and also in 5 postomenopausal patients with uterine bleeding reported in the literature, but no such correlation is evident for postmenopausal women without endometrial abnormality reported in the literature. The range of values for both the apparent fractional conversion and the apparent rate of production of estrone are similar to those reported by other workers using the same model. However, the data suggest that women with endometrial cancer may produce estriol by a pathway not involving circulating estrone.

OBJECTIVE

Aromatase inhibitors are known to prevent the conversion of androgens to estrogens and play a significant role in the treatment of estrogen dependent diseases such as breast cancer. Some flavonoids have been reported as potent aromatase inhibitors; therefore, in an effort to develop novel anti breast cancer agents, B ring substituted flavanones with a 7-methoxy group on A ring were synthesized and tested to assess their ability to inhibit aromatase activity and to determine the optimal B ring substitution pattern.

METHODS

A series of flavanones was prepared by cyclisation of 2'-hydroxychalcones previously obtained by Claisen-Schmidt condensation and the aromatase inhibitory activity of these compounds was investigated using human placental microsomes and radiolabeled [1,2,6,7-(3)H]-androstenedione as substrate.

RESULTS

Almost all flavanones exhibited inhibitory effect on the aromatase activity but their potency was dependent on their B ring substitution pattern. Hydroxylation at position 3' and/or 4' enhanced the anti-aromatase activity; thus, 3',4'-dihydroxy-7-methoxyflavanone was found to be twice more potent than aminoglutethimide, the first aromatase inhibitor clinically used.

CONCLUSIONS

These results indicated that these flavanones could be considered as potential anti breast cancer agents through the inhibition of aromatase activity and allowed us to select some of these compounds as skeleton for the development of flavonoid structurally-related aromatase inhibitors.

Plasma levels of androstane-3 alpha, 17 beta-diol glucuronide (3 alpha-diol-G) and androsterone glucuronide (ADT-G) have been found to be effective markers of C-19 steroid metabolism in periphery in man. The present study has been performed in order to study in castrated patients the effect of antiandrogen administered alone or in combination with aminoglutethimide (AG) on the metabolism of adrenal C-19 steroid. Ten castrated patients with prostatic cancer received flutamide (FLU) alone for 2 months and, afterwards, the combined therapy of FLU and AG for 2 months. Antiandrogen treatment alone reduces the levels of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone glucuronide (DHEA-G) and <em>androstenedione</em> (<em>4</em>-ene-dione) by <em>4</em>3, 3<em>4</em> and 38% (P less than or equal to 0.01) respectively while dehydroepiandrosterone (DHEA), androst-5-ene-3 beta,17 beta-diol (5-ene-diol) and androst-5-ene-3 beta,17 beta-diol-glucuronide (5-ene-diol-G) levels show a nonsignificant inhibition. In these patients, plasma 3 alpha-diol-G and ADT-G concentrations are nonsignificantly stimulated to 122 and 1<em>4</em>3%. Moreover, when patients were receiving the combined administration of FLU and AG, adrenal C-19 steroids were further inhibited while both 3 alpha-diol-G and ADT-G show a small but nonsignificant decrease. Our data indicate that the antiandrogen increases the formation and/or the metabolism of adrenal C-19 steroids into steroid glucuronides.

Twelve postmenopausal women suffering from advanced breast cancer had plasma estrogens, androgens, cortisol, and gonadotropins determined before therapy and during treatment with megestrol acetate (MA) in oral doses escalated from <em>4</em>0 to 160 mg. The plasma clearance and production rate of estrone and estrone sulfate were determined before treatment and after <em>4</em> weeks of therapy with 160 mg MA. Treatment with MA suppressed plasma levels of dehydroepiandrosterone sulfate, <em>androstenedione</em>, and cortisol in a dose-dependent manner to <10% of pretreatment values. Plasma testosterone, estradiol, estrone, and estrone sulfate were suppressed to 18-29% of pretreatment values, whereas the gonadotropins were suppressed to 35-52%. The plasma clearance rates of estrone and estrone sulfate were increased by a mean value of 23.7% (P < 0.01) and 23.5% (P < 0.025), whereas the production rates were reduced by 76.7% (P < 0.0005) and 76.1% (P < 0.0005), respectively. Our findings indicate that MA causes profound suppression of adrenal steroid production but in addition suppresses ovarian secretion of androgens in postmenopausal breast cancer patients. The reduction in plasma estrogens is comparable to values obtained with commonly used aromatase inhibitors and may be responsible for its antitumor effects in breast cancer.

Fast-growing strain of Mycobacterium sp. VKM Ac-1815D is capable of effective oxidizing of sterols (phytosterol, cholesterol, ergosterol) to <em>androstenedione</em> and other valuable 3-oxo-steroids. To elucidate the role of cholesterol oxidase in sterol catabolism by the strain, the choD gene has been cloned and sequenced. The deduced gene product (M(r) 63.5kDa) showed homologies over its entire length to a large number of proteins belonging to the InterPro-family EPR006076, which includes various FAD dependent oxidoreductases. The expression of choD in Escherichia coli was shown to result in the synthesis of membrane associated cholesterol oxidase. In addition to cholesterol, the enzyme oxidized β-sitosterol, dehydroepiandrosterone, ergosterol, pregnenolone, and lithocholic acid. Knock-out of choD in Mycobacterium sp. VKM Ac-1815D strain was obtained by the gene replacement technique. The mutant strain transformed sitosterol forming exclusively 3-keto-<em>4</em>-ene steroids with <em>androstenedione</em> as a major product, thus evidencing that choD knock out did not abrogate sterol A-ring oxidation. The results indicated that ChoD is not a critical enzyme responsible for modification of 3β-hydroxy-5-ene- to 3-keto-<em>4</em>-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms.

A 31-yr-old hirsute female with oligoamenorrhea since menarche had markedly elevated peripheral plasma testosterone (T) concentrations of 250-255 ng/100 ml (normal 20-60 ng/100 ml), which lacked a diurnal rhythm, were not suppressed by dexamethasone, were decreased by ACTH, and were massively increased to 2,530 ng/100 ml by human chorionic gonadotropin (hCG). The binding capacity of T-binding globulin (TeBG) was 0.2 mug/100 ml (normal = 1.1-3.3 mug/100 ml). Plasma delta <em>4</em>-<em>androstenedione</em> (A) was elevated at 37<em>4</em>-681 ng/100 ml (normal = 90-135 ng/100 ml). Plasma estrone (E1) and estradiol (E2) were normal. The endometrium was proliferative. A T-secreting tumor was suspected because the plasma T levels were higher than those observed in polycystic ovarian disease. Exploratory surgery revealed bilateral polycystic ovaries and a pure thecoma in the right ovary which was not visible on surface examination. The thecoma did not contain granulosa cells. Plasma T in the right ovarian vein, draining the tumor, was 28,200 ng/100 ml and in the left ovarian vein was 2,600 ng/100 ml. Plasma A was elevated in both ovarian veins: 11,170 ng/100 ml on the left and 8,360 ng/100 ml on the right. The thecoma contained 1.35 mug/g of T and only 0.01<em>4</em> mug/g and 0.007 mug/g of E2 and E1, respectively. Plasma A and T after bilateral oophorectomy and removal of the thecoma were normal at 18<em>4</em> ng/100 ml and <em>4</em>0 ng/100 ml, respectively.

CONCLUSIONS

1) This pure thecoma produced primarily T rather than E1 OR E2 and was gonadotropin-responsive. 2) A very high plasma androgen level in a female is an important clue to the presence of a tumor. A T-secreting tumor should be ssupected when the peripheral plasma T is over 250 ng/100 ml and when plasma T increases to over 1,000 ng/100 ml following hCG stimulation. 3) Tumors cannot be classified as estrogenic or androgenic on the basis of the character of the endometrium.

Susceptibility to pubertal onset, malignant granulosa cell (GC) tumors of the ovary is inherited in SWR/Bm and certain SWR-related SWXJ recombinant inbred strains of mice. In some SWXJ strains, GC tumors occur spontaneously (spontaneous strains), and in others GC tumors can only be induced by treatment with dehydroepiandrosterone (DHEA-dependent strains). A gene controlling susceptibility to both spontaneous and DHEA-induced GC tumorigenesis, Gct, has been assigned to Chromosome <em>4</em>. Additional research on the role of steroids in GC tumorigenesis has revealed a second gene controlling response to C19 androgenic steroids. Spontaneous strains showed increased tumor frequency after treatment with testosterone (T), whereas DHEA-dependent strains showed no GC tumors following T treatment. Within treatment groups, serum steroid data from DHEA, T, and control treated mice showed no consistent differences between spontaneous and DHEA-dependent strains with respect to progesterone, DHEA, <em>androstenedione</em>, dihydrotestosterone, T, estrone, or estradiol. Thus, observed differences in GC tumor responsiveness to exogenous steroids were not due to different patterns of steroid metabolism among spontaneous and DHEA-dependent strains. Further studies on the range of effective C19 steroids were conducted using one spontaneous and one DHEA-dependent strain. The spontaneous strain showed increased GC tumor frequency in response to dihydrotestosterone and androsterone treatment, whereas the DHEA-dependent strain showed no response. This result suggests that spontaneous strains may be sensitive to a broad range of C19 steroids. To determine whether genetic differences in endogenous steroid levels have a role in spontaneous GC tumorigenesis, serum steroid levels were measured in SWR/Bm and SJL/Bm progenitor strains during the developmental period of risk between 22 and 38 days of age. With the exception of transiently increased DHEA at 22 days, there were no consistent differences in steroid levels analyzed. Thus, serum steroid profiles were not reliably prognostic for GC tumorigenesis. In conclusion, GC tumor induction in response to T treatment has co-segregated with susceptibility to spontaneous GC tumors in the SWXJ recombinant inbred strains. Thus, the second gene in our ovarian granulosa cell tumor model regulates responsiveness to T. We propose to name this gene spontaneous ovarian tumorigenesis (Sot), with alleles for susceptibility (s) carried by spontaneous strains and resistance (r) carried by DHEA-dependent strains.

In July 1998, Cortef oral suspension (Pharmacia & Upjohn) was reformulated changing the suspending agent tragacanth to xanthan gum. We subsequently observed suboptimal control of hormone levels in a group of children with classic congenital adrenal hyperplasia, despite increasing doses of Cortef suspension and stringent instructions to parents regarding shaking of the bottles of medication. Nineteen children receiving Cortef and fludrocortisone therapy were changed to hydrocortisone tablets and fludrocortisone, with a 10 percent reduction in hydrocortisone dose. A significant decrease in 17-hydroxyprogesterone (235 +/- 120 vs. 27 +/- 7 nmol/L; p</=0.001) and <em>androstenedione</em> (18.9 +/- 18.0 vs. 3.5 +/- 3.5 nmol/L; p=0.002) was observed <em>4</em>-6 weeks later. Twenty-one percent (<em>4</em>/19) had 17-hydroxyprogesterone and <em>androstenedione</em> levels at or below the detection limit of the assay. Despite a significant reduction in glucocorticoid dose (19.6 +/- <em>4</em>.7 vs. 17.6 +/- 3.9 mg/M(2)/day; p<0.001), eight children experienced significant weight gain and appetite increase, three experienced trouble sleeping, four experienced moodiness, and three developed hypertension requiring a decrease in fludrocortisone therapy. Hydrocortisone dose was further decreased to 15.2 +/- 2.6 mg/M(2)/day with resolution of symptoms. We conclude that Cortef suspension and hydrocortisone tablets are not bioequivalent and the reformulated form of hydrocortisone oral suspension was inadequate in the control of children with congenital adrenal hyperplasia. Cortef suspension has been recalled as a result of these data.

To examine the relationship between whole prostatic dihydrotestosterone (DHT) concentrations and epithelial and stromal protein synthesis, patients awaiting surgery for benign prostatic hypertrophy were given medication to reduce circulating and tissue androgen levels for comparison to levels in untreated patients. The medications, either megestrol acetate, a progestational antiandrogen, or megestrol acetate plus dexamethasone, were given for 1 week before surgery. Aliquots of prostate tissue obtained at surgery were assayed for DHT. The remainder of the tissue was separated into stromal and epithelial cells using enzymatic separation; the incorporation of both [3H] proline into stromal protein and [3H]leucine into epithelial cell protein was then measured. Over a wide range of tissue DHT concentrations, DHT correlated significantly with [3H]proline incorporation into stromal protein (r = 0.58). Likewise, epithelial cell incorporation of [3H]leucine into protein correlated significantly with tissue DHT concentrations, with a r value of 0.79. In the case of stromal cells, no additional effects of dexamethasone given with megestrol acetate were found on stromal protein synthesis. Epithelial cell [3H]leucine incorporation into protein was paradoxically enhanced by dexamethasone plus megestrol acetate compared to the latter alone, despite the fact that tissue DHT and circulating adrenal androgens, dehydroepiandrosterone sulfate and delta <em>4</em>-<em>androstenedione</em>, were not significantly different from values obtained after treatment with megestrol acetate alone. These studies support the conclusion that there is a dominant role of DHT in regulating protein synthesis in both prostatic epithelial and stromal cells. Dexamethasone appears to have a growth-stimulating effect on prostatic epithelial cells.

We wished to establish an in vitro culture system to examine gene expression in the context of differentiated function with rhesus monkey syncytiotrophoblasts. Chorionic villous tissue from placentas obtained at cesarean section was dispersed with trypsin and DNase and fractionated on a 5-70% Percoll gradient. When placed in culture, cells from a mononuclear fraction demonstrated to be very highly enriched (95-97% pure) for cytotrophoblasts aggregated and began to form syncytia within 2<em>4</em> h in culture, reminiscent of placental syncytiotrophoblast formation. The migration and fusion of individual cytotrophoblasts to form multinuclear syncytia were documented with time-lapse video microscopy. Incorporation studies with tritiated thymidine supported the conclusion from videomicroscopy that syncytia form by the fusion of individual cells and the addition of mononuclear cells to existing syncytia, rather than by endomitosis. The syncytiotrophoblast marker pregnancy-specific beta 1-glycoprotein (SP1) was immunocytochemically identified in both intact placenta and cultured syncytiotrophoblast cells. With cells isolated from placentas obtained on day 28, 50, 70, or 1<em>4</em>0 of pregnancy, treatment with 8-bromo-cAMP increased both rhesus monkey CG alpha-subunit (mCG alpha) and chorionic somatomammotropin (mCS) mRNA levels by an average of <em>4</em>-fold. Increases of up to 2.5-fold were seen with mCG alpha mRNA in as little as 2 h after treatment, with a statistically significant average response seen within 6 h. The response with mCS required at least 2<em>4</em> h before a significant effect was seen. Actin mRNA levels were generally unchanged or suppressed by this treatment, indicating that the effect of 8-bromo-cAMP is relatively specific for the hormone mRNAs. Treatment of syncytiotrophoblasts with dexamethasone, but not progesterone or <em>androstenedione</em>, resulted in an approximately <em>4</em>-fold increase in mCG alpha mRNA levels within 6 h of treatment. Steroid treatment did not affect mCS mRNA levels. Treatment with <em>4</em>.5-<em>4</em>00 nM GnRH or 0.1 to 100 ng/ml basic fibroblast growth factor likewise had no effect on the level of either mRNA, suggesting that any actions of these factors on hormone secretion are not effected via changes in steady state mRNA. These results communicate that the expression of the mRNAs for rhesus monkey CG alpha and CS in syncytiotrophoblast are regulated by steroid hormone- and cAMP-mediated pathways.

The metabolism of [3H]<em>androstenedione</em> by human alveolar macrophages was investigated. Alveolar macrophages were obtained by bronchopulmonary lavage by use of a heparinized saline solution devoid of Ca++ and Mg++. After purification, the macrophages were incubated at 37 C in RPMI-16<em>4</em>0 medium that contained glucose and [1,2,6,7-3H]<em>androstenedione</em> under various experimental conditions. Control incubations were conducted without macrophages. After incubation, 1<em>4</em>C-labeled steroids that corresponded to the metabolites were added as internal recovery standards. The metabolites were characterized by chromatography and crystallization to constant 3H to 1<em>4</em>C ratios. Human alveolar macrophages convert [3H]<em>androstenedione</em> to 5 alpha-androstane-3,17-dione, testosterone, 5 alpha-dihydrotestosterone, androsterone, and isoandrosterone. Unidentified polar metabolites also were formed. Therefore, the following enzymes are present in these cells: 5 alpha-reductase, 17 beta-hydroxysteroid dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, and unknown hydroxylase(s). The rates of formation of the principal metabolites, 5 alpha-androstanedione and testosterone, remained linear up to <em>4</em> h of incubation and with macrophage number up to 1.5 X 10(7) cells/ml. These findings suggest that alveolar macrophages may be involved in the peripheral metabolism of <em>androstenedione</em> to potent androgens in man. It is possible that androgens, formed from blood-borne <em>androstenedione</em> within alveolar macrophages, may modulate phagocytic and other activities in these cells.

The annual reproductive cycle of oyster Crassostrea gigas depends on environmental factors, but its endocrine regulations are still unknown. Sexual steroids play important roles at this level in vertebrates, and some estradiol effects have been described in invertebrates such as bivalve mollusks. To question these roles in invertebrates, we studied androgen metabolism in C. gigas. Incubations of tissue homogenates with 1<em>4</em>C-steroids such as <em>androstenedione</em> (A), testosterone (T), estrone (E1) and estradiol (E2), followed by TLC and HPLC, provide evidence for 17beta-hydroxysteroid dehydrogenases (17beta-HSDs, conversions of A into T, T into A, E1 into E2 and E2 into E1) and aromatase-like (A into E1) activities. The latter activity was further characterized by tritiated water release assay; it was time- and temperature-dependent. Furthermore, this oyster aromatase-like activity was inhibited by <em>4</em>-hydroxy<em>androstenedione</em> (IC(50) 0.<em>4</em>56 microM) and by other pharmacological compounds including specific cytochrome P<em>4</em>50 inhibitors (MR20<em>4</em>9<em>4</em>, miconazole) and a marine pollutant (tributyltin).

The aim was to investigate whether reference cultures and fresh isolates of Treponema denticola are able to 5alpha-reduce and further metabolise testosterone, <em>4</em>-<em>androstenedione</em>, progesterone, corticosterone, cortisol or cholesterol. Two reference and five freshly isolated cultures of T. denticola were incubated with either radiolabeled or unlabeled steroid substrates; in the first case products were identified by thin layer chromatography and in the latter by gas chromatography-mass spectroscopy. All the substrates were 5alpha-reduced. Both reference cultures and fresh isolates of T. denticola presented 3beta- and 17beta-hydroxy steroid dehydrogenase activity. It was concluded that T. denticola was capable of steroid metabolism and hypotheses are discussed regarding the in vivo function of this metabolism including, T. denticola utilising host supplied steroids as growth factors and T. denticola steroid metabolism acting as a virulence factor.