The effects of l-thyroxine and dexamethasone on steroid dynamics in male cynomologous monkeys

Male cynomologous monkeys (M. fascicularis) were infused with [3H]androgens, [14C]estrogens and [3H]cortisol before and after the administration of l-thyroxine, (l-T4) 150 micrograms/day for 6 wk, dexamethasone 8 mg every 8 h for 3 doses and dexamethasone 1.0 mg/day for 8 days. Blood samples were obtained before each of the infusions and analyzed for endogenous T, A, E1, E2 and F concentrations, % free T and % free E2, sex hormone-binding globulin (SHBG) and cortisol binding globulin (CBG) capacity. When l-T4 was being administered, T4 and triiodothyronine (T3) concentrations were also measured. Blood samples were obtained during the infusions and analyzed for radioactivity as testosterone (T), androstenedione (A), dihydrotestosterone (DHT), estradiol (E1), estrone (E2), and cortisol (F). All urine was collected for 96 h and an aliquot of the pooled urine was analyzed for radioactivity as estrone and estradiol glucuronide. The administration of l-T4 for 6 wk to 3 monkeys resulted in a marked rise in T4 and T3 levels, from 4.8 +/- 0.4 micrograms/dl and from 136 +/- 6 to 515 +/- 71 ng/dl, respectively. MCRT, MCRE2 and MCRE1 did not change, but MCRA values increased slightly and MCRF increased 2-3 fold. [rho]T.E2 did not change but [rho]A.E1BM showed a slight but significant increase. The inter-conversions between the androgens and between the estrogens were not altered. There was a 2-3-fold increase in SHBG and a decrease in %FT but no change in %FE2 or CBG. The concentrations of T, A and DHT rose but there was no trend in the levels of the estrogens. The administration of dexamethasone 8 mg every 8 h for 3 doses or 1 mg/day for 8 days caused no changes in the MCRs for T, A, E1 and E2 but did cause a significant decrease in MCRF. Measurement of splanchnic and peripheral tissue extractions before and after acute dexamethasone administration in 1 monkey showed that the decrease in MCRF was the result of a marked decrease, 11-2%, in splanchnic extraction of F. The extractions of T and E2 were relatively unaffected. The concentrations of T and F fell but E2 remained the same. % FT and % FE2 rose slightly and the concentrations of SHBG and CBG were unchanged. The androgen interconversions and estrogen interconversions were not affected but [rho]T,E2BM and [rho]A,E1BM showed slight decreases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Steroid dynamics in the rabbit

Male rabbits were infused at a constant rate with 3H-androstenedione/14C-estrone (n = 5) or 3H-testosterone/14C-estradiol-17 beta (n = 3) for 3 1/2 hr and blood samples were obtained over the last hour and analyzed for radioactivity as androstenedione (A), testosterone (T), estrone (E1), estradiol-17 beta (E2 beta) and estradiol-17 alpha (E2 alpha). The mean value for the metabolic clearance rate of androstenedione (MCRA) was 85 +/- 10 l/day/kg, which was significantly greater than the mean MCRE1 59 +/- 10 l/day/kg. MCRT, 42 +/- 8 l/day/kg, and MCRE2 beta, 45 +/- 9 l/day/kg were not different. The conversion ratio of androstenedione to testosterone (CRA,T) was greater than CRT,A but for the estrogens, CRE2 beta, E1 was greater than CRE1,E2 beta. CRE2 beta, E2 alpha was greater than CRE1,E2 alpha. The overall aromatization of androstenedione to estrone, the fraction of 3H-androstenedione infused into the blood and measured as 3H-estrone in blood [( rho]A,E1BB) was 0.0005 +/- 0.0001 and for [rho]T,E2 beta BB was 0.0012 +/- 0.0006. In the rabbit both sex hormone binding globulin (SHBG) and albumin binding may effect the MCRs, and peripheral aromatization of androgens occurs to a far lesser degree than in humans and primates.     

Alterations in sex steroids and gonadotropins in post-menopausal women subsequent to long-term mifepristone administration

Long-term administration of progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) has been proposed as a novel hormonal therapy for various hormone dependent maladies. We studied the long-term endocrine effects of mifepristone on the kinetics of estradiol (E(2)) and its precursors, and on gonadotropin levels in five postmenopausal women treated for unresectable meningioma with mifepristone [200 mg/day] for at least 15 months. Serum samples were analyzed for LH, FSH and SHBG with fluoroimmunoassay; androstenedione (A), testosterone (T), estrone (E(1)) and E(2) were measured with radioimmunoassay (RIA). Serum levels of mifepristone were measured using both RIA and high performance-liquid chromatography (HPLC). Serum levels (mean +/- SD) of LH and FSH were suppressed from pretreatment values of 32 +/- 16 and 65 +/- 30 IU/l to 13 +/- 7 and 33 +/- 16 IU/l at 6 months (P < 0.05), respectively. Serum (mean +/- SD) A, T, E(1), and E(2) were increased from initial values of 6.9 +/- 0.9 nmol/l, 1.2 +/- 0.3 nmol/l, 77 +/- 25 pmol/l, and 29 +/- 14 pmol/l to 6 month values of 13.1 +/- 5.6 nmol/l, 1.8 +/- 0.6 nmol/l, 178 +/- 60 pmol/l, and 45 +/- 22 pmol/l (n.s.). The correlation coefficients between the levels of A, T, E(1), and E(2) were statistically significant, whereas the ratios of T/A, E(1)/A, E(2)/E(1), and E(2)/T remained unchanged. The levels of SHBG remained stable, and ranged from 48 +/- 10 to 65 +/- 9 nmol/l (mean +/- SD). Thus, prolonged mifepristone treatment marginally increased the serum levels of A, T, E(1) and E(2). These effects of mifepristone are likely due to its antiglucocorticoid effect and thus increased secretion of adrenal A. Serum levels of LH and FSH declined. The serum levels of gonadotropins and those of T, E(1) and E(2) were inversely, yet significantly, correlated. Therefore the decrease in LH and FSH might reflect the slightly increased levels of T, E(1) and E(2). However, the lack of change in SHBG and the low E(2) levels suggest that enhanced systemic estrogen effects are unlikely during long-term mifepristone treatment.     

Utilization of maternal and fetal androstenedione for placental estrogen production at mid and late baboon pregnancy.

The present study determined the placental and whole-body metabolism of androstenedione originating in the maternal and fetal compartments of the pregnant baboon at mid (day 100; n = 4) and late (day 165; n = 3) gestation (term = day 184) in untreated animals and at midgestation in animals (n = 3) treated with pellets (50 mg) of androstenedione inserted at 8-day intervals in the mother between days 70 and 100 of gestation. Baboons were anesthetized with ketamine-halothane-nitrous oxide, blood samples obtained from maternal, uterine, fetal and umbilical vessels during constant infusion of [3H] or [14C]androstenedione via the fetal or maternal circulation, respectively, and radiolabeled precursor/products in plasma purified by HPLC. The metabolic clearance rate (MCR; 1/day/kg body wt) of androstenedione in the mother was similar at mid (81 +/- 6) and late (69 +/- 12) gestation and was unaltered by treatment with androstenedione (92 +/- 17). Fetal MCR of androstenedione was 3-fold greater (P less than 0.05) than in the mother and was similar in the three treatment groups. In the maternal compartment, the conversion ratio of androstenedione to estradiol (range 26-37%) exceeded (P less than 0.05) that to testosterone (range 15-19%) which exceeded (P less than 0.05) that to estrone (range 7-14%), a pattern unaffected by stage of gestation or treatment with androstenedione in vivo. Similar results were observed in the fetal compartment although values for each conversion were always 3-4-fold lower (P less than 0.05) than in the maternal compartment. Regardless of stage of gestation or treatment with androstenedione, [14C]estradiol in the uterine vein (95 +/- 15 cpm/ml) exceeded (P less than 0.05) that in the umbilical vein (3 +/- 1) indicative of preferential secretion of estradiol to the maternal compartment. In contrast, the concentration of [14C]estrone in uterine (15 +/- 4) and umbilical (18 +/- 4) vessels were similar indicating that estrone was secreted equally into the mother and fetus. Similar observations were noted for respective values for [3H]estrogens derive from fetal [3H]androstenedione. Placental extraction of fetal androstenedione (range 86-93%) exceeded (P less than 0.05) that for androstenedione originating in the mother (range 44-54%) and neither were affected by stage of gestation or treatment with androstenedione in vivo. Less than 1% of fetal [3H]androstenedione reached the maternal circulation unaltered, presumably due to placental catabolism. Similarly, the concentration of maternally-derived [14C]androstenedione present in fetal plasma (less than 5%) was minimal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reexamination of testosterone, dihydrotestosterone, estradiol and estrone levels across the menstrual cycle and in postmenopausal women measured by liquid chromatography-tandem mass spectrometry

Measuring serum androgen levels in women has been challenging due to limitations in method accuracy, precision sensitivity and specificity at low hormone levels. The clinical significance of changes in sex steroids across the menstrual cycle and lifespan has remained controversial, in part due to these limitations. We used validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays to determine testosterone (T) and dihydrotestosterone (DHT) along with estradiol (E2) and estrone (E1) levels across the menstrual cycle of 31 healthy premenopausal females and in 19 postmenopausal females. Samples were obtained in ovulatory women in the early follicular phase (EFP), midcycle and mid luteal phase (MLP). Overall, the levels of T, DHT, E2 and E1 in premenopausal women measured by LC-MS/MS were lower overall than previously reported with immunoassays. In premenopausal women, serum T, free T, E2, E1 and SHBG levels peaked at midcycle and remained higher in the MLP, whereas DHT did not change. In postmenopausal women, T, free T, SHBG and DHT were significantly lower than in premenopausal women, concomitant with declines in E2 and E1. These data support the hypothesis that the changes in T and DHT that occur across the cycle may reflect changes in SHBG and estrogen, whereas in menopause, androgen levels decrease. LC-MS/MS may provide more accurate and precise measurement of sex steroid hormones than prior immunoassay methods and can be useful to assess the clinical significance of changes in T, DHT, E2 and E1 levels in females.     

Estrogen dynamics in the female rhesus monkey

The metabolic clearance rates (MCR) and interconversions [( rho]BB) values for estrone (E1) and estradiol (E2) in female rhesus (Macaca mulatta) monkeys on Days 9, 14, and 23 of the menstrual cycle were measured using constant infusions of [3H] estradiol and [14C] estrone. The menstrual cycles in these monkeys were reproduced by using Silastic capsules of E2 and progesterone after bilateral ovariectomy. The serum levels of E2 and progesterone were measured by radioimmunoassay and were similar to those for the intact menstrual cycle. The MCR of E2 on Day 14 (52.8 +/- 6.8 l/day/kg) was significantly greater (p less than 0.05) than that measured on Day 9 (31.1 +/- 3.6 l/day/kg) or Day 23 (35.4 +/- 2.1 l/day/kg). The MCR of E1 was also different (p less than 0.05) on Day 14 (77.6 +/- 14.9 l/day/kg) compared to the values on Days 9 and 23 (50.2 +/- 4.9 and 48.2 +/- 3.9 l/day/kg, respectively. There was no change in percentage of free E2, percentage of albumin-bound E2, or sex hormone-binding globulin levels on those 3 days of the cycle. The interconversions between E2 and E1 were not influenced by the day of the cycle. We conclude that the high levels of E2 occurring at the time of the E2 peak result in increases in the MCRs of both E2 and E1 that are not associated with changes in the pattern of protein-binding or in the activity of the 17 beta-hydroxy steroid dehydrogenase.     

Influence of progestins on serum hormone levels in postmenopausal women with advanced breast cancer--I. General findings

The influence of oral high dose progestin (medroxyprogesterone acetate, MPA and megestrol acetate, MA) treatment on serum hormone levels was studied in ten postmenopausal women with advanced breast cancer. The gonadotropins and ACTH were significantly reduced by greater than 50 and 23%, respectively. Serum cortisol, DHEAS, androstenedione and testosterone were all significantly reduced (mean reduction between 64 and 76%), while serum estrone, estradiol and estrone sulfate were significantly reduced by 20-30%. Sex hormone binding globulin (SHBG) and corticosteroid binding globulin (CGB) were reduced by 68 and 25%, respectively. Although the dose of MA used (160 mg/day) was only 1/6 of the MPA dose (1000 mg/day), the mean serum level of MA was 2-fold higher than the mean serum level of MPA. MPA treatment gave a more pronounced suppression of SHBG than MA treatment, while estrone sulfate levels were more suppressed by MA. These findings suggest a differential effect of MPA and MA on certain plasma hormones, possibly of importance for understanding the mechanism of action of the two drugs. The reduction of estrone sulfate may be beneficial for the action of MA against breast cancer.     

Rates of dissociation of sex steroid hormones from human sex hormone-binding globulin: a reassessment

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure the rates of dissociation of dihydrotestosterone (DHT), testosterone (T), and estradiol (E2) from their binding proteins in human serum at 37 degrees C. Because measurements were begun after each hormone had fully (greater than 99%) dissociated from albumin, the observed rates of dissociation correspond to the rates of dissociation of the sex hormone-binding globulin (SHBG)-hormone complexes. The dissociation rate constants of the hormone-SHBG complexes were determined to be 0.016 +/- 0.001, 0.056 +/- 0.002, and 0.083 +/- 0.003 s-1 for DHT, T, and E2, respectively, corresponding to half-times of dissociation (t1/2) of 43, 12 and 8.4 s, respectively. The physiological significance of these findings can best be appreciated by comparing these t1/2 s with the capillary and sinusoidal transit times of various tissues (less than 1 s to approximately 10 s).     

Self-administration of estrogen and dihydrotestosterone in male hamsters

Anabolic-androgenic steroids (AAS) are drugs of abuse. Previous studies have shown that male and female hamsters self-administer testosterone (T) and other AAS, suggesting that androgens are reinforcing in a context where athletic performance is irrelevant. AAS are synthetic derivatives of T, which may be aromatizable to estrogen and/or reducible to dihydrotestosterone (DHT). However, we do not know which metabolites of T are reinforcing. To determine if DHT, estradiol (E(2)), or DHT + E(2) are reinforcing, we tested intracerebroventricular (icv) self-administration in male hamsters. The hypothesis was that androgen reinforcement is sensitive to both androgenic and estrogenic T metabolites. If so, hamsters would self-administer DHT, E(2), and DHT + E(2). Twenty four castrated male hamsters (n = 8/group) received icv cannulas and sc T implants for physiologic androgen replacement. One week later, hamsters self-administered DHT (0.1, 1.0, 2.0 microg/microl), E(2) (0.001, 0.01, 0.02 microg/microl), or DHT + E(2), each for 8 days in increasing concentration (4 h/day). Operant chambers were equipped with an active and inactive nose-poke. At the medium concentration, hamsters self-administered DHT (active nose-poke: 47.9 +/- 13.9 responses/4 h vs. inactive: 18.7 +/- 4.8), E(2) (active: 44.8 +/- 14.9 vs. inactive: 16.6 +/- 2.6), and DHT + E(2) (active: 19.1 +/- 2.4 vs. inactive: 10.4 +/- 2.4, P < 0.05). At the highest concentration, males self-administered DHT (active: 28.3 +/- 7.7 vs. inactive: 15.0 +/- 3.5, P < 0.05) and DHT + E(2) (active: 22.6 +/- 3.8 vs. inactive: 11.6 +/- 2.5, P < 0.05), but not E(2). Hamsters did not self-administer the lowest concentrations of DHT, E(2), or DHT + E(2). These results support our hypothesis that both androgenic and estrogenic T metabolites are reinforcing. Together, they do not exert synergistic effects.     

Estimation by gas chromatography-mass spectrometry with selected ion monitoring of urinary excretion rates of 3 alpha-androstanediol during/after i.v. administration of 13C-labelled testosterone in man

To study in vivo the conversion of testosterone (T) into its metabolites, dihydro-testosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta diol (3 alpha-Diol) the urinary excretion rates of these steroids were determined by mass spectrometry in 6 healthy men during/after the i.v. infusion (t = 4 h) of 20 mg [13C]testosterone. In addition, plasma concentrations of T, DHT and 3 alpha-Diol were determined by radioimmunoassay. During steady state conditions at the end of the 4-h infusion of [13C]T the increase in the plasma concentrations of T from, basal, 405 +/- 140 ng/dl to 4205 +/- 804 ng/dl was paralleled by an increase in the plasma concentrations of DHT to 106.4 +/- 62.5 ng/dl) (basal: 30.8 +/- 21.8 ng/dl), and of 3 alpha-Diol to 32.2 +/- 12.5 ng/dl (basal: 12.5 +/- 13.9 ng/dl). Plasma concentrations of T, DHT and 3 alpha-Diol then returned to basal concentrations within 24 hours. Using mass-spectrometry we found a cumulative renal excretion of 13C-labelled T of 15.6 +/- 9.6 micrograms/24 h, equivalent to 0.08 +/- 0.05% of the infused amount (20 mg) of [13C]T. Whereas urinary excretion of [13C]DHT was below the level of detection by mass-spectrometry the cumulative excretion of [13C]3 alpha-Diol was 67.7 +/- 19.9 micrograms/24 hours which is equivalent to 0.3 +/- 0.1% of the infused dose of 13C-labelled testosterone. These data suggest that the determination of urinary 3 alpha-Diol by mass-spectrometry during/after the infusion of stable-labelled testosterone represents an alternative to the use of radioactive label for turnover studies.     

Effect of estrogen on the metabolism of cortisol and cortisone in the baboon fetus at midgestation

To determine whether the metabolism of cortisol (F) and cortisone (E) in the baboon fetus is regulated by estrogen, fetal interconversion of F/E was measured at midgestation after an experimental increase in placental estradiol (E2) production. Six baboons (Papio anubis) received increasing numbers of androstenedione implants (50 mg) inserted s.c. at 8-day intervals between Days 70 and 100 of gestation (term = Day 184) to elevate the production of estrogen; controls (N = 8) received no treatment. On Day 100 of gestation, each animal was anesthetized with ketamine:halothane/nitrous oxide, the fetus was exteriorized and [3H] F/[14C] E was infused via a fetal femoral vein for 70 min. Blood samples were then obtained from the contralateral fetal femoral vein, the umbilical vein/artery, and a maternal saphenous vein. After purification of F and E, the metabolic clearance rate (MCR), peripheral interconversion, and placental extraction of F and E were calculated. Maternal serum E2 concentrations (ng/ml; mean +/- SE) between Days 80 and 100 of gestation were greater (p less than 0.01) in androstenedione-treated baboons (2.2 +/- 0.2) than in untreated controls (1.2 +/- 0.1). Although the MCR of F was similar in control (5.2 +/- 0.3 1/day) and treated (7.7 +/- 1.0 1/day) animals, the MCR of E (13.5 +/- 2.0 1/day) was increased (25.8 +/- 2.5 1/day; p less than 0.05) by androstenedione treatment. Placental extraction of F (59 +/- 9%) was lower (p less than 0.01) than that of E (82 +/- 5%) in untreated baboons and was not affected by androstenedione treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum protein binding characteristics of cyproterone acetate, gestodene, levonorgestrel and norethisterone in rat, rabbit, dog, monkey and man

Protein binding characteristics including percentage of total binding, total binding capacity (pmol/mg protein), degree of specific binding, competition with dihydrotestosterone (DHT) and estradiol (E2) binding sites and dissociation constants (Kd) of low and high affinity binding sites were investigated for the progestins cyproterone acetate (CPA), gestodene (G), norethisterone (NET) and levonorgestrel (LN) in serum or plasma pools from man and four laboratory animal species (rat, rabbit, dog and monkey). Serum pools from animals were constructed from samples obtained either prior to or 1 day after pretreatment with ethinyl estradiol (EE2) (5 micrograms/kg/day for 7 days). Human plasma pools differed by SHBG levels (normal/induced). All serum pools were characterized by protein content and distribution. Equilibrium dialysis or dextran-coated charcoal (DCC) methods were used to separate bound and free steroids labelled with tritium. All progestins were highly (greater than 80%) bound to proteins in all undiluted samples. Total binding capacity was highest in rat and lowest in monkey. Human plasma showed a capacity of 1.5-2.1 microgram steroid/ml. In man, monkey and rabbit LN and G were specifically bound to the same degree as DHT, whereas NET binding was 50% lower. Specific binding of CPA to dog serum was 2-3 times higher than for other steroids. Two (high and low affinity) binding sites were found for LN, G and NET in man, monkey and rabbit and in dog for LN. Kd values for high affinity binding ranged from 3.5 (G in man) to 23 (NET in man) x 10(-9)M. Kd values of low affinity binding varied from 0.5 (CPA in dog) to 4 (NET in man) x 10(-6)M. E2 and DHT competition experiments confirmed the concept of SHBG as a carrier protein of 19-nor-progestins and DHT and its occurrence in man, monkey and rabbit. A sex hormone binding protein (SBP) in the dog seems to be responsible for the relatively high specific binding of CPA. SHBG is inducible by means of EE2 in man and monkey, but not in rabbit. EE2 may induce SBP in the dog. Comparison of in vitro Kds (high affinity binding) and in vivo metabolic clearance rates showed the same rankings for LN, G and NET in man, monkey and rabbit.     

Plasma steroids in benign prostatic hypertrophy and carcinoma of the prostate

Cortisol, 17-hydroxyprogesterone (17-OH-P), progesterone, testosterone, 5 alpha-dihydrotestosterone (DHT), estrone (E1) and estradiol (E2) were determined by RIA after chromatographic separation of steroids on Sephadex LH-20 columns, in 54 hospitalized patients with benign prostatic hyperplasia (BPH) and in 32 hospitalized patients with prostatic carcinoma (PCA) (T34, N01, M01). The patients' values were compared with those of 63 age-matched controls. Increased cortisol and DHT levels, subnormal estrogen and 17-OH-P values and normal progesterone level were found in both benign and malignant groups. Higher mean values for testosterone, and T/DHT ratio and lower mean values for E2/T ratio were found in PCA, as compared with those in BPH. An age invariance of cortisol, testosterone, T/DHT ratio and estradiol was found in both BPH and PCA, instead of the age dependence found in normal subjects. The normal relations between testosterone and its precursor (17-OH-P) and its peripheral metabolite (E2), respectively, and the normal relation between estrone and 17-OH-P were not evident in either BPH or PCA group. The normal direct relation between testosterone and DHT has been found in both patient groups.     

Characteristics of estrogen-2/4-hydroxylase of porcine ovarian follicles: influence of steroidal and non-steroidal agents on the activity of the enzyme in vitro

The conversion of [3H]estradiol to 2-hydroxyestradiol (2-OH-E2) by homogenates of porcine ovarian follicles was assayed in vitro in the presence and absence of 10 and 100 microM concentrations of the following potential substrates or inhibitors of estrogen-2/4-hydroxylase (E-2/4-H): (1) estrogens; estrone (E1), estriol (E3) and 17 alpha-estradiol (17 alpha-E2), (2) catecholestrogens; 2-hydroxyestradiol (2-OH-E2), 4-hydroxyestradiol (4-OH-E2) and 2-hydroxyestrone (2-OH-E1); (3) 2-methoxyestradiol (2-MeO-E2); (4) halogenated estrogens; 2-bromoestradiol, (2-Bromo-E2) 4-bromoestradiol and 2,4-dibromoestradiol; (5) androgens; testosterone (T), dihydrotestosterone (DHT) and androstenedione; (6) progesterone; (7) epinephrine; (8) inhibitors of steroid aromatase; aminoglutethimide and 4-hydroxyandrostenedione and (9) SKF 525A, an inhibitor of cytochrome P-450. Progesterone and 2-Bromo-E2 were the two most effective inhibitors (2-OH-E2 formation = 4 and 5% of control at 100 microM and 29.6 and 17.4% at 10 microM of progesterone and 2-Bromo-E2, respectively). 2-MeO-E2 at 100 microM was nearly as effective as progesterone in inhibiting E-2/4-H activity but only caused about 50% inhibition at 10 microM. The three catecholestrogens reduced 2-OH-E2 formation to about the same degree (21-23% of control at 100 microM). The 2,4-dibromo-E2 was equipotent with the catecholestrogens while 4-bromo-E2 was about half as effective. The phenolic estrogens, potential substrates for the enzyme, reduced 2-OH-E2 formation to different degrees, with E3 being the most effective. Among the androgens, DHT was almost as effective an inhibitor as the catecholestrogens, T was about half as effective while androstenedione had no effect. Epinephrine and the two inhibitors of aromatase did not inhibit E-2/4-H activity. SKF 525A inhibited E-2/4-H activity but with a potency only about 1/10th that reported for liver.     

Effect of an infusion of Gn-RH upon levels of sex hormones in prepubertal and pubertal girls: evidence for relative ovarian insensitivity.

Changes in levels of sex steroids and gonadotropins were measured in 16 normal prepubertal and 15 pubertal girls prior to and after a 3 hour infusion of 100 microgrm synthetic gonadotropin releasing hormone (Gn-RH). Plasa estradiol (E2) concentrations rose significantly (p less than 0.02) from 29.7 +/- 4.6 (SE) pg/ml in the basal period to to 46.8 +/- 7.1 at the end of the infusion in the pubertal girls but were unchanged in the prepubertal girls. Estrone (E1), progesterone (P), 17-HYDROXYPROGESTERONE (17OHP), TESTOSTERONE (T), DIHYDROTESTOSTERONE (DHT), and androstenedione (A), dehydroepiandrosterone (DHA) and dehydroepiandrosterone sulfate (DHAS) levels were not altered in either maturity group. Basal plasma E2, E1, T, DHT, DHA and DHAS concentrations significantly correlated with the releasable pool of LH evoked by Gn-RH from the pituitary gonadotropes. We conclude: 1) The ovary is not highly and rapidly responsive to transient elevations of endogenous gonadotropin, and 2) Adrenal androgens may to some extent modulate the maturation of the hypothalamic-pituitary-gonadal system, at least as reflected by the pituitary response to exogenous Gn-RH.     

Steroid metabolism in the gonads of a patient with testicular feminization. II. Metabolism of c19- and c18-steroids in vitro.

The metabolism of C19- and C18-steroids, in particular, the aromatization of androstenedione and testosterone, the interconversion of androgens to estrogens and the 5alpha-reductase activity of a right abdominal (r) and a left inguinal (l) testis of a patient with testicular feminization, are reported. Aromatization and 5alpha-reductase activity were also evaluated in tissue from the left ductus diferens (ld). The following results were obtained: 1. aromatization of androstenedione to estrone 2.52% (r), 0.02% (l), 0.94% (ld); 2. aromatization of testosterone to estradiol 0.58% (r), 2.88% (l); 3. conversion of androstenedione to testosterone 95.65% (r), 98.07% (l); 4. conversion of testosterone to androstenedione 33.14% (r), 53.65% (l); 5. conversion of estrone to estradiol85.29% (r), 100% (l), 6. conversion of estradiol to estrone 33.12% (r), 32.33% (l); 7.5alpha-reduction of testosterone to 5alpha-dihydrotestosterone 12.01% (r), 13.64% (l) and 4.10% (ld). A lack of 5alpha-reductase activity was not found in the tissues examined as stated in the literature. Estrogen production in these testes was demonstrated by the aromatization of androstenedione and testosterone to estrone and estradiol and is reflected in the difference of the estradiol concentration measured in spermatic and peripheral blood of the same patient (168 versus 33 pg/ml).     

Formation of estrogen glucuronides by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization

The formation of steroid glucuronides by human granulosa cells isolated from human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG)-stimulated cycles for in vitro fertilization was studied. From granulosa cells in suspension, 5 x 10(-7) M androstenedione was converted into estradiol (2.50 +/- 0.21 ng/ml), estrone (1.84 +/- 0.16 ng/ml), estradiol glucuronide (0.38 +/- 0.07 ng/ml), as well as estrone glucuronide (0.24 +/- 0.04 ng/ml). When 5 x 10(-7) M estradiol was incubated, estrone (15.5 +/- 0.9 ng/ml) and estradiol glucuronide (0.12 +/- 0.05 ng/ml) were detected in medium. Using the same preparation of granulosa cells, we have observed that androsterone could uniquely be transformed into androstane-3 alpha, 17 beta-diol (1.42 +/- 0.56 ng/ml), and only low amounts of steroid glucuronides could be detected. Since the formation of steroid glucuronides was extremely small when granulosa cells in suspension were used, we subsequently studied granulosa cells in culture. When 5 x 10(-7) M estradiol was added, estrone (7.8 +/- 1.3 ng/ml) and estradiol glucuronide (0.68 +/- 0.08 ng/ml) were formed. The addition of follicle-stimulating hormone did not cause a further increase in estrone or estradiol glucuronide levels. As observed with granulosa cells in suspension, incubation with androsterone led to the formation of androstane-3 alpha, 17 beta-diol (24.2 +/- 0.07 ng/ml). Our data demonstrated the presence of glucuronyltransferase in human granulosa cells obtained from preovulatory follicles of hMG/hCG-treated women. In addition, since the conversion of androsterone into C-19 steroid glucoronide was relatively small, the present finding also indicates that the glucoronyltransferase enzymatic activity in granulosa-luteal cells preferentially conjugated estrogens.     

Aromatase activity in human skin fibroblasts: characterization by an enzymatic method

Human skin fibroblasts were incubated for 24 h with 10(-6) M androstenedione and the estrone + estradiol released in the culture medium were measured by an enzymatic assay. Aromatase activity was expressed as pmol (estrone + estradiol) formed in the medium per mg cell protein per day. Using this method we were able to investigate the kinetic properties of aromatase in different cell strains and its stimulation by dexamethasone. Values of 92 nM and 9.1 pmol/mg protein/day were obtained respectively for Km and Vmax in cultured fibroblasts derived from genital skin of normal prepubertal subjects. In patients with complete androgen insensitivity syndrome CAIS, the Km was 156 nM and the Vmax 42 pmol/mg protein/day. Aromatase activity varied from 7.9 +/- 1.2 pmol/mg protein/day (mean +/- SD; n = 19) in normal prepubertal boys to 24.5 +/- 4.7 pmol/mg protein/day (mean +/- SD; n = 11) in those from normal postpubertal boys. The values were even higher in fibroblasts cultured from genital skin of prepubertal patients with CAIS. Cell concentrations did not modify the pattern of estrogen formation and aromatase activity did not vary with serial subcultures. The stimulatory effect of dexamethasone on aromatase activity in cultured fibroblasts was measured after preincubation of the cells for 48 h with dexamethasone, by determining estrogen formation after 24 h incubation of the cells with androstenedione 10(-6) M using this enzymatic method. This data suggest that aromatase activity measured in cultured fibroblasts could be a useful tool for studying extraglandular estrogen formation in physiological and pathological conditions.     

The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones

Sex hormone-binding globulins (SHBG) is a plasma glycoprotein that binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to investigate the role of steroids in the interaction of SHBG with its receptor. Because the probe for the interaction of SHBG with its receptor is 125I-SHBG, we first showed that 125I-SHBG binds [3H]dihydrotestosterone (DHT) at 4 degrees C and 37 degrees C with KD values similar to those published previously for pure radioinert SHBG. 125I-SHBG could be prevented from binding to its receptor by a variety of steroids whose relative inhibitory activity (dihydrotestosterone much greater than 2-methoxyestradiol greater than testosterone greater than estradiol much greater than methyltrienolone greater than cortisol) was almost identical to their relative ability to bind to SHBG. Because significant binding of [3H]DHT to the SHBG receptor could not be demonstrated, steroid inhibition of SHBG binding must be noncompetitive. If steroids bound to SHBG prevent binding to the SHBG receptor, then liganded SHBG should have a higher apparent KD for its receptor than unliganded SHBG. This is the case. The KD was 0.86 +/- 0.25 nM for the high affinity receptor site using liganded SHBG and 0.19 +/- 0.024 nM for unliganded SHBG. Thus, only liganded SHBG assumes a conformation that prohibits interaction with the SHBG receptor. However, when unliganded SHBG was prebound to its receptor, it retained its ability to bind [3H] DHT. The model that emerges from these observations is as follows. Unliganded SHBG can bind either steroids or receptor in a reversible reaction; SHBG bound to a steroid cannot bind to the receptor, but unliganded SHBG that first binds to the receptor can subsequently bind steroids.     

Metabolism of androstenedione by human ovarian tissues in vitro with particular reference to reductase and aromatase activity

The ability of granulosa and theca cells of the human ovarian follicle at different stages of development, as well as stromal and luteal tissues from human ovaries to metabolize androstenedione (delta 4) to testosterone (T), dihydrotestosterone (DHT), estrone (E1) and estradiol (E2) with or without exposure to additional amounts of folicle-stimulating hormone was investigated by in vitro experiments. The results show that all the aforementioned ovarian tissues metabolized delta 4 to DHT. Indeed, with the exception of estrogen-secreting granulosa cells from large antral follicle (greater than 10 mm diameter) and possibly also luteal tissue from mid-luteal phase ovaries, the various ovarian tissues preferentially metabolized delta 4 to DHT instead of E (E1 + E2). Although thecal tissue is a major source of delta 4 in human ovaries it is concluded that the granulosa cells do not interact with the theca for the synthesis of E as the follicle enlarges from 1 to 10 mm in diameter. Indeed, excessive thecal delta 4 during this growth phase probably inhibits normal follicular development. However, as the follicle enlarges beyond 10 mm in diameter, and as the granulosa cells begin to preferentially metabolize delta 4 to E, the two cell-types of the follicle may increasingly interact to enhance the follicular output of E.