Because the endocrine control of sexual behavior in male hamsters remains controversial, this study analyzed the influence of different androgens and estrogens in the regulation of masculine, sexual behavior (MBS). Aromatizable androgens: <em>androstenedione</em> (A) and testosterone (T), a non-aromatizable androgen: 5alpha-dihydrotestosterone (DHT), as well as estrogens (E2 and E1) alone or in combination with DHT, were administered in gonadectomized, sexually experienced males, for 3 weeks. In addition, plasma levels of these steroids were determined. Gonadectomy completely suppressed masculine sexual behavior (MSB) after <em>4</em> weeks. Both A and T replacements restored all the sexual behavior parameters in castrated hamsters by the 3rd week of treatment, with A being more potent in restoring all copulatory series and maintaining all MSB parameters, including long intromissions. Castrated males treated with DHT showed little interest in the female and did not display any copulatory behavior. Gonadectomized males treated with estrogens alone showed active anogenital investigation and displayed some mounts, but did not ejaculate. Males treated with estrogens combined with DHT had longer latencies and less number of ejaculations than males treated with aromatizable androgens. Long intromissions were observed only in males treated with T or A. Plasma levels of A were significantly higher than T levels in intact males. In males treated with A both androgens and estrogens were present in plasma. These results support the notion that aromatizable androgens, mainly A, but not non-aromatizable androgens or even estrogens in combination with DHT, play a relevant role in the endocrine regulation of MSB in the golden hamster.

Four mutants of Thr-205 in cytochrome p<em>4</em>50 2B1 were constructed and expressed in Escherichia coli. The Ser-, Ala-, and Val-mutants displayed stable reduced CO difference spectra and were able to metabolize 7-ethoxy-<em>4</em>-(trifluoromethyl)coumarin, testosterone, <em>androstenedione</em>, and benzphetamine. The Arg-mutant displayed an unstable reduced CO difference spectrum at <em>4</em>50 nm, was concomitantly converted to a denatured form with a peak at <em>4</em>22 nm, and showed no catalytic activity with any of the four substrates tested. The Ser-mutant displayed activity and metabolite profiles for testosterone and <em>androstenedione</em> similar to those of the wild-type p<em>4</em>50 2B1 (WT). Substitution of Thr-205 with Ala or Val markedly suppressed the 16 beta-hydroxylation activity but exhibited little effect on the 16 alpha-hydroxylation activity for testosterone and <em>androstenedione</em>. Because 16 beta-hydroxylation activity of androgens is a specific p<em>4</em>50 2B subfamily marker and residue 205 is located in the F helix, which forms the ceiling of the active site, we postulate that the gamma-hydroxyl side chain of Thr may play an important role in directing the 16 beta-face of testosterone and <em>androstenedione</em> toward the active site. Surprisingly, the Val-mutant retained full activity for benzphetamine demethylation. When mechanism-based inactivators for p<em>4</em>50 2B1 were used to evaluate the susceptibility to inactivation, the Val-mutant was resistant to inactivation by 17 alpha-ethynylestradiol and less sensitive to inactivation by 2-ethynylnaphthalene compared with the WT enzyme. Our results demonstrate the importance of Thr-205 in determining substrate specificity and product formation as well as in influencing the susceptibility of p<em>4</em>50 2B1 to mechanism-based inactivators.

Accumulating evidence strongly supports the premise that testosterone may be a key player in fetal programming on hypertension. Studies have shown that gestational protein restriction doubles the plasma testosterone levels in pregnant rats. In this study, we hypothesized that elevated testosterone levels in response to gestational protein restriction were caused by enhanced expression of steroidogenic enzymes or impaired expression of Hsd17b2, a known testosterone inactivator that converts testosterone to <em>androstenedione</em> in placenta. Pregnant Sprague-Dawley rats were fed normal (20% protein, control; n = 10) or a low-protein diet (6% protein, PR; n = 10) from Day 1 of pregnancy until killed at Days 1<em>4</em>, 18, or 21. Junctional (JZ) and labyrinth (LZ) zones of placenta were collected for expression assay on steroidogenic genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b2, and Srd5a1) by real-time PCR. The main findings include the following: 1) expressions of Cyp11a1, Hsd3b1, and Cyp17a1 in JZ were not affected by diet but were affected by day of pregnancy; 2) expression of Hsd17b2 in both female and male JZs was remarkably increased by PR at Days 18 and 21 of pregnancy; 3) expressions of Hsd17b2 were reduced by PR in both female and male LZ at Day 18 of pregnancy and in female LZ at Day 21 of pregnancy; and <em>4</em>) expression of Srd5a1in LZ was not affected by day of pregnancy, gender, or diet. These results indicate that in response to gestational protein restriction, Hsd17b2 may be a key regulator of testosterone levels and associated activities in placental zones, apparently in a paradoxical manner.

The brain and ovarian aromatase isozymes of goldfish (Carassius auratus) are encoded by different CYP19 genes. This study measured aromatase activity in the goldfish brain tissues. For a direct comparison of the properties of the two aromatase isozymes, Chinese hamster ovary cells were stably transfected with brain- and ovary-derived cDNAs (respectively, p<em>4</em>50 arom B and -A) and the properties of the expressed isozymes were compared. The kinetic parameters of the two isozymes were determined using <em>androstenedione</em> and testosterone as substrates and compared to those of human aromatase. Inhibition profile analyses on the two isozymes were performed using seven inhibitors [<em>4</em>-hydroxy<em>androstenedione</em>, 7 alpha-(<em>4</em>'-amino)phenylthio-1,<em>4</em>-androstadiene-3,17-dione, bridge (2,19-methyleneoxy)androstene-3,17-dione, aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole]. Except for AG, the compounds tested were found to be much stronger inhibitors against the ovary enzyme than the brain enzyme. In addition, the ovary isoform was more sensitive to two phytoestrogens, chrysin and 7,8-dihydroxyflavone, than the brain form. These studies reveal that catalytic properties of the goldfish aromatase isoforms are significantly different from those of human aromatase. In addition, differences in the K(i) values of aromatase inhibitors for the two goldfish isoforms suggest structural variance in the active sites of these isozymes.

OBJECTIVE

To evaluate the effects of combination estrogen/androgen therapy on muscle mass, strength and endurance, serum hormone and lipid profiles, and quality of life measures in postmenopausal women.

METHODS

Prospective, randomized, placebo-controlled pilot study at a tertiary care medical center. Fifty postmenopausal women were randomized to a 12-week course of (1) dehydroepiandrostenedione (DHEA) 50 mg daily, (2) conjugated equine estrogen (CEE) 0.625 mg daily, (3) DHEA 50 mg+CEE 0.625 mg daily, or (<em>4</em>) placebo. Main outcome measures of lower extremity muscle (calf) mass, functional muscle parameters, serum hormone and lipid levels, and quality of life (QOL) were obtained at baseline and after treatment. Statistical analysis compared percent change from baseline values and treatment differences among outcomes.

RESULTS

Significant increases in mean DHEA, DHEA sulfate (DHEA-S), testosterone, and androstenedione levels were noted with DHEA alone or combined DHEA/CEE treatments when compared with placebo. Compared with no hormone therapy, none of the supplemental hormone groups caused significant changes in muscle mass, muscle strength, muscle endurance, feelings of well-being, sleep, or sexual function.

CONCLUSIONS

Androgen replacement therapy, with DHEA, to menopausal women increases serum androgen levels without any appreciable effect on muscle cross-sectional area, muscle strength, muscle function, or improvement in health-related QOL.

Cytochrome b(5) (cyt-b(5)) is essential for the regulation of steroidogenesis and as such has been implicated in a number of clinical conditions. It is well documented that this small hemoprotein augments the 17,20-lyase activity of cytochrome P<em>4</em>50 17α-hydroxylase/17,20-lyase (CYP17A1). Studies have revealed that this augmentation is accomplished by cyt-b(5) enhancing the interaction between cytochrome P<em>4</em>50 reductase (POR) and CYP17A1. In this paper we present evidence that cyt-b(5) induces a conformational change in CYP17A1, in addition to facilitating the interaction between CYP17A1 and POR. We also review the recently published finding that cyt-b(5) allosterically augments the activity of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(<em>4</em>) isomerase (3βHSD), a non cytochrome P<em>4</em>50 enzyme, by increasing the enzymes affinity for its cofactor, NAD(+). The physiological importance of this finding, in terms of understanding adrenal <em>androstenedione</em> production, is examined. Finally, evidence that cyt-b(5) is able to form homomeric complexes in living cells is presented and discussed.

BACKGROUND

There is currently neither a clinically useful, reliable and inexpensive assay to measure circulating levels of free testosterone (T) in the range observed in women, nor is there agreement on the serum free T threshold defining hypoandrogenism that is associated with female-impaired sexual function.

OBJECTIVE

Following the Clinical and Laboratory Standards Institute guidelines, we generated clinically applicable ranges for circulating androgens during specific phases of the menstrual cycle in a convenience sample of 120 reproductive-aged, regularly cycling healthy European Caucasian women with self-reported normal sexual function.

METHODS

All participants were asked to complete a semistructured interview and fill out a set of validated questionnaires, including the Female Sexual Function Index, the Female Sexual Distress Scale, and the 21-item Beck's Inventory for Depression. Between 8 am and 10 am, a venous blood sample was drawn from each participant during the midfollicular (day 5 to 8), the ovulatory (day 13 to 15), and the midluteal phase (day 19 to 22) of the same menstrual cycle.

METHODS

Serum levels of total and free testosterone, Delta(<em>4</em>)-<em>androstenedione</em>, dehydroepiandrosterone sulphate and sex hormone-binding globulin during the midfollicular, ovulatory and midluteal phase of the same menstrual cycle.

RESULTS

Total and free T levels showed significant fluctuations, peaking during the ovulatory phase. No significant variation during the menstrual cycle were observed for Delta(<em>4</em>)-<em>androstenedione</em> and dehydroepiandrosterone sulphate. Despite the careful selection of participants that yielded an homogeneous group of women without sexual disorders, we observed a wide range of distribution for each of the circulating androgens measured in this study.

CONCLUSIONS

This report provides clinically applicable ranges for androgens throughout the menstrual cycle in reproductive-aged, regularly cycling, young healthy Caucasian European women with self-reported normal sexual function.

Androgens play a major role in the development, growth, and function of accessory sexual organs, especially the prostate. However, the testis is not the sole source of circulating androgens in man, since the adrenal gland secretes dehydroepiandrosterone (DHEA), DHEA sulfate, and <em>androstenedione</em> (delta <em>4</em>-dione) in large quantities. The aim of the present study was to investigate the effect of plasma concentrations of DHEA and delta <em>4</em>-dione similar to those found in adult man on sensitive and specific markers of androgen action in the rat ventral prostate. In addition to ventral prostate weight, we have measured the steady state levels of the mRNAs encoding the C1 component of rat prostatic binding protein (PBP-C1) and spermine-binding protein (SBP) using 35S-labeled cDNA probes for in situ hybridization. One week after castration, ventral prostate weight fell 8<em>4</em>%, while prostatic 5 alpha-dihydrotestosterone (DHT) and androgen-dependent mRNAs were undetectable. When administered via Silastic implants to castrated adult rats for 1 week, plasma concentrations of 1.37 +/- 0.06 ng/ml DHEA or 0.<em>4</em>3 +/- 0.08 ng/ml delta <em>4</em>-dione independently caused increases in ventral prostate weight to 33% and 65% of normal values, respectively. The same plasma levels of DHEA and delta <em>4</em>-dione resulted in high intraprostatic levels of DHT to 1.19 +/- 0.3<em>4</em> and 3.66 +/- 0.89 ng/g tissue, respectively. Furthermore, DHEA caused an increase in the steady state levels of PBP-C1 and SBP mRNAs to 50% and 57% of the normal state, respectively, while delta <em>4</em>-dione caused increases corresponding to 80% and 119% of control values, respectively. Castrated adult rats receiving testosterone at a concentration of 1.66 +/- 0.37 ng/ml plasma maintained normal ventral prostate weight and gene expression levels. The present results demonstrate that circulating levels of the adrenal steroids DHEA and delta <em>4</em>-dione comparable to those found in man cause an important stimulation of androgen-dependent gene expression in the rat, probably after their conversion to DHT in the prostatic tissue itself.

Cytochrome P-<em>4</em>50 (P-<em>4</em>50) form specificities were established for a total of nine monoclonal antibodies (MAbs) raised to four distinct rat hepatic P-<em>4</em>50 enzymes (P-<em>4</em>50s 2c, PB-2a, PB-<em>4</em>, and BNF-B), using a combination of enzyme-linked immunosorbent analysis, dot immunoblotting, Western blotting, Ouchterlony immunodiffusion, and immunoinhibition analyses. Four of the MAbs were fully (greater than or equal to 85%) inhibitory toward the corresponding immunoreactive P-<em>4</em>50s when assayed in purified, reconstituted enzyme systems, while two of the MAbs were partially inhibitory, with a maximum of 50 or 80% inhibition achieved in the presence of saturating MAb. Inhibitory MAbs reactive with P-<em>4</em>50s 2c, 3, and PB-<em>4</em>, respectively, were used to demonstrate that the formation of multiple hydroxytestosterone metabolites by each of the respective purified P-<em>4</em>50 enzymes is reflective of their inherent catalytic specificities and not due to the presence of immunochemical distinguishable P-<em>4</em>50 enzyme contaminants. P-<em>4</em>50 form-specific contributions to rat hepatic microsomal steroid hormone hydroxylase activities were then assessed using the inhibitory MAbs as probes. MAb-reactive P-<em>4</em>50 2c was shown to be the major (greater than or equal to 85%) catalyst of microsomal testosterone and <em>androstenedione</em> 16 alpha-hydroxylation in both untreated and beta-naphthoflavone-induced rats. However, this P-<em>4</em>50 form catalyzed only approximately 30% of hepatic microsomal steroid 16 alpha-hydroxylase activity in phenobarbital-induced adult males, and less than or equal to 10% of steroid 16 alpha-hydroxylase activity in (phenobarbital-induced immature males or adult females, where the balance of 16 alpha-hydroxylase activity is catalyzed by MAb-reactive P-<em>4</em>50 PB-<em>4</em>. Although MAb-reactive P-<em>4</em>50 PB-<em>4</em> catalyzed the majority (greater than or equal to 90%) of microsomal <em>androstenedione</em> 16 beta-hydroxylation in phenobarbital-induced rats, this P-<em>4</em>50 enzyme did not contribute to the low level 16 beta-hydroxylase activity of uninduced liver samples. Finally, MAb-reactive P-<em>4</em>50 3 catalyzed at least 85% of microsomal <em>androstenedione</em> 7 alpha-hydroxylation, independent of the age, sex, or induction status of the animals used as source of liver microsomes. These findings demonstrate the usefulness of MAbs as probes for the contributions of individual P-<em>4</em>50 enzymes to the metabolism of steroid hormones susceptible to hydroxylation at multiple sites.

Although the role of estrogens in bone formation is becoming clarified, the function of androgens in this process remains to be defined. Consequently, we have explored the mechanism of action for both gonadal and adrenal androgens in normal human osteoblastic (hOB) cells, which are responsible for the synthesis and mineralization of bone. Changes in the steady-state mRNA levels for two nuclear proto-oncogenes (c-fos and c-jun) and one cytokine (TGF-beta 1) were quantified in response to short (30 min) and long (2<em>4</em>-<em>4</em>8 h) treatments of these cells with physiologic concentrations of steroids. In addition, the levels of TGF-beta protein in the hOB cells conditioned-media were measured using a bioassay. The results indicated that neither 10 nM dihydrotestosterone, 10-20 nM testosterone, nor 10-100 nM <em>androstenedione</em> had a significant effect on the steady-state levels of c-fos, c-jun, or TGF-beta 1 mRNAs. Interestingly, 10-1000 nM dehydroepiandrosterone (DHEA) and 1-10 microM DHEA-sulfate rapidly reduced the steady-state level of c-fos mRNA by 60-80% in a dose-dependent manner within 30 min. In contrast, neither of these adrenal steroids had a significant effect on the message levels for c-jun or TGF-beta 1. Surprisingly, although TGF-beta 1 mRNA levels remained unchanged, the total amount of TGF-beta activity in the hOB cell conditioned-media increased 2-5-fold in response to 2<em>4</em>-<em>4</em>8 h treatments of the cells with gonadal or adrenal androgens. This increase in TGF-beta activity by DHEA-sulfate was both time- and dose-dependent, and was not blocked by cotreatment with the specific aromatase inhibitor <em>4</em>-hydroxy<em>androstenedione</em> (1 microM). Immunoprecipitations of the hOB cell conditioned-media with isoform-specific TGF-beta neutralizing-antibodies indicated that TGF-beta 2 was predominantly produced by the cells in response to DHEA-sulfate treatment. These results demonstrate that differences exist between the actions of estrogens and androgens on normal human osteoblasts with regard to the regulation of c-fos expression and TGF-beta production. Moreover, these data suggest that DHEA and DHEA-sulfate may play a distinct role in the regulation of human osteoblast function via the rapid repression of c-fos message levels and the slower increase in TGF-beta 2 protein levels.

OBJECTIVE

To determine whether the intake of contaminated nutritional supplements could cause an athlete to fail a dope test.

METHODS

A contaminated nutritional supplement was used, identified in an ongoing study screening over-the-counter nutritional supplements. One capsule of the supplement, containing small amounts of 19-nor-<em>4</em>-<em>androstenedione</em> and <em>4</em>-androsten-3,17-dione, not listed on the label, was administered to 5 healthy male volunteers. Fractional urine collection was done at prescribed intervals. Outcome measures. The samples were analysed using gas chromatography/mass spectrometry (GC/MS). Samples containing 19-norandrosterone, the main metabolite of 19-nor-<em>4</em>-<em>androstenedione</em>, were quantified using GC/MS.

RESULTS

All the volunteers had urinary concentrations of 19-norandrosterone above the World Anti-Doping Agency threshold of 2 ng/ml from 2 hours post administration. In 2 volunteers 19-norandrosterone above the threshold value could still be detected beyond 36 hours post administration. The highest concentration of 19-norandrosterone found in a sample was 5<em>4</em>.6 ng/ml at 8 hours post administration.

CONCLUSIONS

The results of this study showed that the intake of microgram amounts of a prohibited substance in a nutritional supplement could cause an athlete to fail a dope test.

We have hypothesized that the change in placental cortisol (F)-cortisone (E) metabolism induced by estrogen late in gestation is important to activation of the baboon fetal hypothalamic-pituitary-adrenocortical axis, culminating in the ontogenesis of de novo F secretion by the fetal adrenal. The present study tested this hypothesis in vivo by comparing the proportion of F in the fetus derived via maternal and fetal production on day 100 (n = 7; term = day 18<em>4</em>) and day 165 (n = <em>4</em>) in untreated baboons and on day 100 in baboons (n = 9) in which 50-mg pellets of <em>androstenedione</em> were implanted sc in the mother in increasing numbers (i.e. two on day 70, four on day 78, six on day 86, and eight on day 9<em>4</em>) to increase placental estrogen production. Maternal, uterine, and umbilical venous samples were collected during constant maternal infusion (120 min) of [3H]F/[1<em>4</em>C]E, endogenous and radiolabeled F/E content was determined, and corticosteroid dynamics were quantified. The MCR and peripheral interconversion of F and E as well as the production rate of F were unaltered in the mother. However, at midgestation, <em>androstenedione</em> increased (P less than 0.05) estrogen by 62% and altered transuterofeto placental F-E metabolism from preferential reduction of E (%F----E = 17 +/- <em>4</em>; %E----F = 27 +/- 7) to preferential oxidation of F (%F----E = 26 +/- 2; %E----F = 5 +/- 2), a pattern similar to that at term (%F----E = 32 +/- 6; %E----F = 9 +/- <em>4</em>). In untreated baboons, on day 100 none of the F in the fetus was due to fetal production, whereas by day 165, <em>4</em>9 +/- 6% was of fetal origin. In animals treated with <em>androstenedione</em> at midgestation, 22 +/- <em>4</em>% of fetal F was derived de novo within the fetus. Thus, production of F by the fetus was negligible on day 100, increased near term in association with an increase in transplacental oxidation of F to E, and was induced at midgestation in baboons in which placental F-E metabolism was altered by an increase in estrogen production. These findings provide indirect evidence that supports our hypothesis that the change in placental F-E metabolism induced by increasing estrogen late in pregnancy results in activation of the fetal hypothalamic-pituitary-adrenocortical axis and, thus, ontogenesis of fetal F production near term.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP<em>4</em>), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, <em>4</em>, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP<em>4</em> expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP<em>4</em> caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and <em>androstenedione</em>. Administration of BMP<em>4</em> to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P<em>4</em>50c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P<em>4</em>50 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP<em>4</em> with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP<em>4</em> is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P<em>4</em>50c17.

We have previously shown that the change in transuteroplacental cortisol (F)-cortisone (E) metabolism in vivo from preferential reduction (E to F) at midgestation to oxidation by term (F to E) does not occur in baboons in which the production or action of estrogen have been blocked. Moreover, because the administration of <em>androstenedione</em> (delta <em>4</em>A) to baboons increased estradiol (E2) production at midgestation and induced a pattern of F-E metabolism similar to that at term, we suggested that estrogen regulates placental F-E interconversion. The present study was designed to ascertain whether estrogen regulates the activity of the placental 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) enzyme catalyzing the oxidation of F to E. Placentas were obtained on day 100 (n = 10) and day 165 (n = 10) of gestation (term = day 18<em>4</em>) from untreated baboons, on day 100 from animals (n = 7) treated with delta <em>4</em>A between days 70-100 of gestation, and on day 165 from animals in which placental estrogen was decreased by fetectomy (n = 5) on day 100 of gestation. Tissue was homogenized in phosphate buffer (pH 7.<em>4</em>) and microsomal fractions (105,000 x g) incubated (37 C; 2 min) in buffer containing 2.7 mM NAD+ and 0.03-1.0 microM [3H]F. Serum concentrations of E2 (nanograms per ml) in untreated baboons on day 100 (0.7 +/- 0.2) were 3-fold lower than those at term, increased (P less than 0.05) by delta <em>4</em>A treatment (2.<em>4</em> +/- 0.3), and decreased (0.12 +/- 0.01; P less than 0.05) by fetectomy. The specific activity (picomoles of E per min/mg protein) of placental 11 beta HSD in untreated baboons at midgestation (13<em>4</em> +/- 17) was increased (P less than 0.01) 3-fold by delta <em>4</em>A treatment. Enzyme activity at term (1<em>4</em>8 +/- 29) was similar to that at midgestation, but markedly decreased (P less than 0.01) by fetectomy (16 +/- <em>4</em>). Placental capacity to oxidize F to E (micromoles per min/placenta) in untreated baboons was 3-fold greater (P less than 0.01) at term (88 +/- 15) than at midgestation and was markedly reduced (P less than 0.01) by fetectomy (3 +/- 1). Collectively, these findings indicate that the activity of the placental 11 beta HSD enzyme catalyzing the oxidation of F to E is increased in baboons in which placental estrogen production was elevated at midgestation and decreased in animals when estrogen formation was inhibited by fetectomy.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

This study was designed to examine the importance of aromatization in the gonadotropin secretory dynamics of polycystic ovarian disease (PCOD) by using the aromatase inhibitor delta 1 testolactone (TL) as a probe and to determine the effects of TL on steroid metabolism in vivo and in vitro. The pulsatile patterns of gonadotropin secretion and peripheral steroid levels were studied in eight women with PCOD before and during TL administration. There was a significant fall in peripheral estrone (E1) levels, a rise in peripheral <em>androstenedione</em> levels, and an increase in the <em>androstenedione</em>/E1 ratio during TL administration in these women. Isotopic determinations of androgen and estrogen production and metabolism before and during TL administration in two women confirmed a 90-95% decrease in the overall rate of aromatization. One patient also had an increase in the production and clearance rates of estradiol and E1 during TL administration, suggesting resistance to TL of the ovarian aromatase enzyme system. There were significant increases in both mean LH pulse amplitude [1.2 +/- 0.3 (SE) mIU/ml LER-907 before vs. 1.7 +/- 0.3 mIU/ml LER-907 during TL, P less than 0.05, paired t test] and frequency per 6 h (median: 3 before vs. <em>4</em> during TL, P less than 0.05, Wilcoxon signed rank test). Mean levels of LH and FSH did not, however, change significantly during TL administration. TL maximally inhibited neonatal rat hypothalamic aromatase in vitro at concentrations of 200 microM, a level theoretically obtainable during pharmacological therapy. These data suggest that: 1) in humans TL is a potent inhibitor of peripheral but not ovarian aromatase, and of hypothalamic aromatase in rats; 2) TL administration increases LH pulse amplitude and frequency in PCOD, either directly via hypothalamic aromatase inhibition, or indirectly by alterations in gonadal steroid metabolism; and 3) because of the multiple potential actions of TL, its usefulness as a probe in studies of gonadotropin secretion in PCOD is limited.

Twenty-two normally menstruating women were studied during one menstrual cycle. Blood was collected on <em>4</em> occasions and was analysed for free and total cholesterol and triglycerides and phospholipids in the lipoprotein fractions, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Fluctuations in lipid parameters were correlated to serum levels of estradiol-17 beta, progesterone, <em>androstenedione</em>, testosterone and sex-hormone-binding globulin (SHBG). Elevated concentrations of SHBG and HDL-cholesterol and a suppression of LDL-cholesterol were found during the luteal compared to the follicular phase and these findings were interpreted as an estrogenic influence. Consequently the ratio LDL-cholesterol/HDL-cholesterol was depressed during the luteal phase. Triglycerides in serum and VLDL reached a peak at midcycle. When effects on lipid metabolism induced by endogenous steroids were compared to lipoprotein fluctuations exerted by exogenous hormones a parallelism was found in certain variables. However, obvious discrepancies were also found in effects on lipid metabolism, especially for hormones with androgenic properties. The present data underline the necessity of defining in which menstrual phase blood has been collected when lipid metabolism is studied in women of fertile age. Knowledge about metabolic events induced by exogenous sex steroids does not allow conclusions concerning the effects exerted by corresponding endogenous hormones.

Prenatal diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency by amniotic fluid (AF) steroid analysis is not possible in those cases in which prenatal dexamethasone (DEX) therapy is initiated to prevent virilization of female CAH fetuses because AF steroid levels are suppressed if DEX therapy is continued beyond amniocentesis (AC). In order to use AF steroids for prenatal diagnosis, it is necessary to discontinue DEX therapy for 5 days before AC. To study the effects of this interruption on AF steroid levels, we measured levels of aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone (170HP), 11-deoxycortisol, cortisol, and cortisone as well as <em>androstenedione</em> (delta <em>4</em>-A) in AF samples (16-18 weeks) obtained from 25 pregnancies at risk for CAH treated with Dex (daily dosage: 1.0-1.5 mg). The prenatal diagnosis of 1<em>4</em> normal fetuses and 11 affected CAH fetuses was postnatally confirmed in all cases. Additionally, steroid levels were measured in AF samples (16-18 weeks) from 8 untreated CAH fetuses and in 19 AF samples (weeks 16-20) obtained in normal pregnancies. In 17/19 prenatally diagnosed CAH fetuses, the affected sibs had the salt wasting (SW)-form, in 2 cases the simple virilizing (SV)-form. All steroids were measured by RIA after extraction and Sephadex LH-20 chromatography. AF levels of aldosterone, corticosterone, deoxycorticosterone, progesterone, cortisol, cortisone, and 11-deoxycortisol were not different between CAH fetuses, prenatally DEX-treated normal fetuses and untreated controls. The 170HP-levels of the CAH-SW-fetuses (range: 19.9-59.8 mmol/L) were clearly above the normal range (3.7<em>4</em>-11.6), but normal in the SV-fetuses (10.9, 11.5), whereas delta-<em>4</em> A-levels (normal range: 0.87-5.13 mmol/L) were elevated both in the SW-(range: 6.53-37.6) and the SV-form (9.37,6.25) of CAH. 170HP and delta-<em>4</em> A levels of prenatally DEX-treated pregnancies with normal fetuses were not different from levels found in normal pregnancies. Mean 170HP and delta-<em>4</em> A AF steroid levels of prenatally DEX-treated CAH-pregnancies were slightly lower (NS) than levels of untreated CAH-pregnancies (170HP: 30.5 vs. <em>4</em>0.7; delta-<em>4</em> A: 15.8 vs. 21.1). 170HP levels are elevated in the SW-form of CAH, but not in the SV-form. However, with the combination of 170HP and delta-<em>4</em> A levels it is possible to diagnose prenatally both forms. There is no rebound phenomenon of AF steroid levels if DEX therapy is interrupted 5 days before amniocentesis.

The authors report the case of a 23-year-old woman with a virilizing lipoid cell tumor of the ovary. The patient developed precocious puberty at age <em>4</em> and from age 5 to 10 years was treated weekly with intramuscular medroxyprogesterone acetate. She began to menstruate spontaneously at age 13, but developed hirsutism, acne, and irregular menses at age 15 followed by secondary amenorrhea at age 20. Steroid analyses demonstrated elevated peripheral plasma levels of testosterone, dihydrotestosterone, <em>androstenedione</em>, 17 alpha-hydroxyprogesterone, progesterone, desoxycorticosterone, estrone, and estradiol. Catheterization of the adrenal and ovarian veins revealed increased secretion of testosterone, dehydroepiandrosterone, <em>androstenedione</em>, 17 alpha-hydroxyprogesterone, progesterone, desoxycortcosterone, and corticosterone, 11-desoxycortisol, cortisol, estrone, and estradiol from the right ovarian vein. At surgery a right ovarian lipoid cell tumor was removed. Incubation studies with the tumor tissue confirmed the presence of gonadal and adrenocortical steroids. The tissue also contained high-affinity human chorionic gonadotropin-luteinizing hormone binding sites with normal binding affinity.

Activin A has been shown to exert several regulatory functions on human placenta. In the present study, we tested the hypothesis that activin A is an autocrine regulator of trophoblast using a choriocarcinoma cell line, JEG-3, as a model. Messenger RNAs for activin beta(A) subunit, activin binding protein (follistatin), and various activin receptors, including ActR-IA, ActR-IB, ActR-IIA, and ActR-IIB, were detected in JEG-3 cells by reverse transcription-polymerase chain reaction. The expression of activin A in JEG-3 cells was further confirmed by Western blot analysis using an antibody against activin beta(A) subunit. Using Northern blot analysis, Smad-2 and Smad-<em>4</em> mRNAs were also observed in JEG-3 cells. These data suggest that JEG-3 cells produce activin A and express activin binding proteins and receptors, as well as potential downstream signals. In cultured JEG-3 cells, basal progesterone production was stimulated by activin A but inhibited by follistatin-288. Similarly, in the presence of <em>androstenedione</em>, estradiol production was enhanced by activin A but decreased by follistatin-288. On the other hand, neither activin A nor follistatin affected JEG-3 cell growth. Taken together, these findings strongly suggest that activin A is an autocrine factor that is involved in the regulation of progesterone and estradiol production in JEG-3 cells.

To investigate the basis of the hirsutism and elevated plasma dehydroepiandrosterone (DHA) and/or DHA sulfate (DHAS) in hyperprolactinemic women, we measured androgen binding parameters and an extensive profile of plasma androgens in normal (NL) and hyperprolactinemic women (HYPRL). ACTH tests and dexamethasone (dex) suppression tests were performed in subgroups. Free testosterone levels were higher in HYPRL (13.1 +/- 23.3 vs. 7.18 +/- 0.72 pg/ml; P less than 0.025), although total testosterone was comparable. This disparity was related to plasma testosterone-estradiol-binding globulin (TEBG) levels being one third lower in HYPRL (mean +/- SE, 27.<em>4</em> +/- <em>4</em>.0 nM) than in NL (<em>4</em>1.2 +/- 3.7 nM; P less than 0.0125). Less striking elevations of plasma DHAS, <em>androstenedione</em>, and 11-deoxycortisol were found in HYPRL. Plasma total dihydrotestosterone [17 beta-hydroxy-5 alpha-androstan-3-one (tDHT)] was nearly 30% lower in HYPRL (11.2 +/- 2.6 ng/dl) than in NL (15.6 +/- 1.3 ng/dl; P less than 0.025), whereas free DHT was normal. Ratios of tDHT to precursors were lower in HYPRL (P less than 0.005). After ACTH stimulation, hyperresponsiveness of 17-hydroxyprogesterone and <em>androstenedione</em> were observed. Apparent adrenal enzyme efficiencies, judged from post-ACTH product to precursor ratios, were normal in HYPRL with one exception: the ratio of tDHT to total testosterone at <em>4</em> h was lower (P less than 0.05). Dex suppression normalized androgens and obliterated the abnormal tDHT to precursor ratios. These findings suggest an ACTH dependency of the abnormalities. In summary, we find that about <em>4</em>0% of HYPRL have an androgenic abnormality, and the most characteristic abnormality is an elevated free testosterone level (abnormal in <em>4</em>3%). Depressed TEBG and high DHAS levels were found with lesser frequency (19-21%). The plasma tDHT concentration was low, both in absolute terms and relative to its precursors. Dex suppressibility of the hyperandrogenemia was also observed. We postulate that PRL may exert multiple effects on steroid secretion and metabolism. Possibilities include the inhibition of the TEBG level.

BACKGROUND

Genome-wide association studies have identified multiple independent regions on chromosome 8q24 that are associated with cancers of the prostate, breast, colon, and bladder.

METHODS

To investigate their biological basis, we examined the possible association between 164 single nucleotide polymorphisms (SNPs) in the 8q24 risk regions spanning 128,101,433-128,828,043 bp, and serum androgen (testosterone, androstenedione, 3alphadiol G, and bioavailable testosterone), and sex hormone-binding globulin levels in 563 healthy, non-Hispanic, Caucasian men (55-74 years old) from a prospective cohort study (the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial). Age-adjusted linear regression models were used to determine the association between the SNPs in an additive genetic model and log-transformed biomarker levels.

RESULTS

Three adjacent SNPs centromeric to prostate cancer risk-region 2 (rs12334903, rs1456310, and rs980171) were associated with testosterone (P < 1.1 x 10(-3)) and bioavailable testosterone (P < 6.3 x 10(-4)). Suggestive associations were seen for a cluster of nine SNPs in prostate cancer risk region 1 and androstenedione (P < 0.05).

CONCLUSIONS

These preliminary findings require confirmation in larger studies but raise the intriguing hypothesis that genetic variations in the 8q24 cancer risk regions might correlate with androgen levels.

CONCLUSIONS

These results might provide some clues for the strong link between 8q24 and prostate cancer risk.

The differential effects of insulin sensitivity and adiposity on androgen concentrations in women with polycystic ovary syndrome (PCOS) are unclear. To address this issue, we divided <em>4</em>3 overweight women into <em>4</em> groups based on both their clinical classification (PCOS or normal) and whether they were insulin resistant (IR) or insulin sensitive (IS) by their steady-state plasma glucose concentrations. Total testosterone concentrations were significantly increased as a function of both clinical classification (PCOS vs normal, P < .0001) and steady-state plasma glucose concentration (IR vs IS, P = .002). Mean testosterone concentrations were higher in PCOS-IR compared with PCOS-IS, normal-IR, or normal-IS women (P < .005). In addition, there was a statistically significant interaction (P = .03) between clinical classification (PCOS vs normal) and insulin sensitivity (IR vs IS) for testosterone concentrations. In contrast, <em>androstenedione</em> concentrations were higher in women with PCOS (P = .001), irrespective of whether they were IR or IS (P = .31); and no interaction between clinical classification and insulin sensitivity was discerned (P = .3<em>4</em>). These results indicate that both PCOS and insulin resistance independently contributed to increased total testosterone concentrations within a group of overweight/obese women. These findings are consistent with the hypothesis that the ovaries of women with PCOS are hypersensitive to the ability of insulin to increase testosterone production and that the more insulin resistant the patient, the higher the testosterone concentration. In contrast, <em>androstenedione</em> concentrations seem to be independent of differences in insulin resistance. Our findings emphasize the need to increase understanding of the factors that modulate ovarian androgen secretion.

In a prospective study we have measured serum levels of sex hormone-binding globulin (SHBG), androgens, oestrogens and gonadotropins in 20 male IDDM patients with the aim of evaluating the effect of improved glycaemic control on these levels and to compare the IDDM patients with an age- and weight-matched healthy non-diabetic control group. The patients were chosen from the male IDDM patients attending the outpatient clinic at the Department of Endocrinology, Odense University Hospital. Glycaemic control was optimized by a trained diabetologist according to the patients' measurements of home blood glucose concentrations and reported hypoglycaemic episodes during a three month period. Prior to regulation the patients, compared to healthy control subjects, had significantly higher serum levels of oestrone (0.29 +/- 0.02 vs 0.16 +/- 0.01 nmol/l (Mean +/- SEM), p < 0.01), 17 beta-oestradiol (0.12 +/- 0.01 vs 0.08 +/- 0.01 nmol/l, p < 0.01), dihydrotestosterone (<em>4</em>.20 +/- 0.18 vs 1.66 +/- 0.09 nmol/l, p < 0.01), total testosterone (20.7 +/- 0.9 vs 17.8 +/- 1.1 nmol/l, p < 0.05) and SHBG (<em>4</em>2.3 +/- 2.9 vs 15.5 +/- 3.5 nmol/l, p < 0.05), while the calculated free-testosterone was lower (0.3<em>4</em> +/- 0.02 vs 0.<em>4</em>0 +/- 0.02 nmol/l, p = 0.068). After regulation, the patients obtained significantly lower levels of glycosylated haemoglobin (10.<em>4</em> +/- 0.3 vs 8.9 +/- 0.2%, p < 0.005) and serum fructosamine (1.50 +/- 0.05 vs 1.3<em>4</em> +/- 0.0<em>4</em> nmol/l, p < 0.005) on a higher 2<em>4</em> hour insulin dose (52.7 +/- <em>4</em>.<em>4</em> vs 59.2 +/- <em>4</em>.6 IU/2<em>4</em> h, p < 0.005). Levels of free-testosterone (0.<em>4</em>1 +/- 0.0<em>4</em> nmol/l), oestrone (0.33 +/- 0.03 nmol/l), oestradiol (0.1<em>4</em> +/- 0.01 nmol/l), delta <em>4</em>-<em>androstenedione</em> (<em>4</em>.<em>4</em><em>4</em> +/- 0.<em>4</em>3 vs 3.85 +/- 0.<em>4</em>2 nmol/l), and prolactin (2<em>4</em>9 +/- 2<em>4</em> vs 200 +/- 19 mIU/l) increased compared to values obtained before regulation (all p < 0.05). We conclude that SHBG, testosterone, dihydrotestosterone and oestrogen levels are increased in male IDDM patients. High SHBG levels tend to keep the free fractions of sex hormones within normal limits. During improvement of glycaemic control with insulin, levels of free-testosterone and its bioprecursors and metabolites rise. This may partly be due to the increased daily insulin dose and/or to the improvement in glycaemic control itself.

In order to describe the relationships between endogenous sex hormones and bone mineral density in healthy postmenopausal women, we carried out a cross-sectional study of 90 community-based women, all at least one year since their last menstrual period (mean 9.6 +/- <em>4</em>.9 years, range 1-22) and with a serum oestradiol level less than 100 pmol/l. None was currently using hormone replacement therapy. Serum oestradiol, testosterone, sex hormone binding globulin, dehydroepiandrosterone sulphate, and <em>androstenedione</em> were measured using standard techniques. Free oestradiol and testosterone indices were derived as the ratio of total hormone to sex hormone binding globulin, respectively. Total body, spine and hip bone mineral density (g/cm2) were measured by dual energy X-ray absorptiometry. Significant positive correlations were found between the free oestradiol and testosterone indices and bone mineral density at all sites. These relationships remained significant for the free oestradiol index after adjustment for age and body mass index. By stepwise multiple regression analysis, the free oestradiol index was an independent predictor of total body, spine and hip bone mineral density, accounting for <em>4</em>-17% of the variance. These findings suggest an independent positive relationship between endogenous free oestradiol and total body, spine and hip bone mass even in the late postmenopause.