The isolation and cloning of a full-length cDNA insert complementary to mRNA encoding human aromatase system cytochrome P-<em>4</em>50 is reported. The insert contains an open reading frame encoding a protein of 503 amino acids. This gene is clearly a member of the cytochrome P-<em>4</em>50 gene superfamily, because the sequence contains regions of marked homology to those of other members, notably a putative membrane-spanning region, I helix, Ozols, and heme-binding regions. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental aromatase system cytochrome P-<em>4</em>50, as detected by immunoblot analysis, and catalyzed the aromatization of <em>androstenedione</em>, testosterone, and 16 alpha-hydroxy<em>androstenedione</em>. This activity was inhibited by the known aromatase inhibitors, <em>4</em>-hydroxy<em>androstenedione</em> and econazole. Thus the several steps involved in the aromatization reaction appear to be catalyzed by a single polypeptide chain, which can metabolize the three major physiological substrates.

3 beta-hydroxysteroid dehydrogenase/delta 5 - delta <em>4</em> isomerase (3 beta-HSD) activity was measured in primary dissociated cell cultures prepared from telencephalons of developing zebra finches. 3 beta-HSD activity was confirmed after cultures were incubated with [7-3H]pregnenolone (Preg) or (1,2,6,7-3H-) dehydroepiandrosterone (DHEA) and 3H-progesterone (Prog) and 3H-<em>androstenedione</em> (AE) were detected in the medium. Product identity was confirmed by recrystallizations and by HPLC analysis. When DHEA was used as substrate, 3H-estradiol and 3H-estrone were also detected in the culture medium, presumably derived from the aromatization of 3H-AE or 3H-T produced from 3H-DHEA. To test this idea, cultures were incubated with 3H-DHEA together with radioinert AE or with fadrozole HCl, a potent and specific aromatase inhibitor. In the presence of radioinert AE, 3H-AE increased but metabolites of 3H-AE decreased in the media; in the presence of fadrozole, 3H-estrogens decreased but 3H-AE and its androgenic metabolite 3H-5 beta-androstanedione increased. These data demonstrate 3 beta-HSD activity in the songbird brain. The presence of Prog and estradiol in these cultures suggest that Preg and DHEA can potentially serve as substrates for the ultimate formation of active sex steroids in the songbird telencephalon.

The mRNA expression patterns of the androgen receptor and the androgen metabolizing enzymes 3beta-hydroxysteroid dehydrogenase/Delta(5-<em>4</em>)-isomerase, 17beta-hydroxysteroid dehydrogenase, 5alpha-reductase, and 3alpha-hydroxysteroid dehydrogenase were investigated in three different cell populations originating from human skin, SZ95 sebocytes, HaCaT keratinocytes, and MeWo melanoma cells, by means of reverse transcription polymerase chain reaction. Restriction analysis of cDNA fragments was performed to identify isozymes of 3beta-hydroxysteroid dehydrogenase/Delta(5-<em>4</em>)-isomerase and 3alpha-hydroxysteroid dehydrogenase. In addition, 3H-dihydroepiandrosterone and 3H-testosterone were used as substrates to determine the metabolic activity of these enzymes in SZ95 sebocytes, primary sebocyte cultures, and HaCaT keratinocytes. Furthermore, the effects of the selective 5alpha-reductase type 1 and 2 inhibitors, <em>4</em>,7beta-dimethyl-<em>4</em>-aza-5alpha-cholestan-3-one and dihydrofinasteride, respectively, and of the 3beta-hydroxysteroid dehydrogenase/Delta(5-<em>4</em>)-isomerase inhibitor cyproterone acetate on androgen metabolism were investigated. Androgen receptor mRNA was detected in SZ95 sebocytes and HaCaT keratinocytes but not in MeWo melanoma cells, whereas 3beta-hydroxysteroid dehydrogenase/Delta(5-<em>4</em>)-isomerase isotype 1 mRNA and metabolic activity were only found in SZ95 sebocytes. The enzyme activity could be inhibited by cyproterone acetate. Type 2 17beta-hydroxysteroid dehydrogenase, type 1 5alpha-reductase, and 3alpha-hydroxysteroid dehydrogenase mRNA were expressed in all three cell populations tested, whereas type 3 17beta-hydroxysteroid dehydrogenase mRNA could only be detected in SZ95 sebocytes. The major metabolic steps of testosterone in SZ95 sebocytes, primary sebocyte cultures, and HaCaT keratinocytes were its conversion to <em>androstenedione</em> by 17beta-hydroxysteroid dehydrogenase and further to 5alpha-androstanedione by 5alpha-reductase. The type 1 5alpha-reductase selective inhibitor <em>4</em>,7beta-dimethyl-<em>4</em>-aza-5alpha-cholestan-3-one, but not the type 2 selective inhibitor dihydrofinasteride, inhibited 5alpha-reductase at low concentrations in SZ95 sebocytes and HaCaT keratinocytes. 5alpha-androstanedione was degraded to androsterone by 3alpha-hydroxysteroid dehydrogenase, which exhibited a stronger activity in HaCaT keratinocytes than in SZ95 sebocytes and in primary sebocyte cultures. Lower levels of 5alpha-dihydrotestosterone and 5alpha-androstanediol were also detected in all cells tested. Our investigations show that specific enzyme expression and activity in cultured sebocytes and keratinocytes seem to allocate different duties to these cells in vitro. Sebocytes are able to synthesize testosterone from adrenal precursors and to inactivate it in order to maintain androgen homeostasis, whereas keratinocytes are responsible for androgen degradation.

We have developed an estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line growing in 96-well microtiter plates. Alkaline phosphatase enzyme activity (AlkP) in these cells is markedly stimulated by estrogens, and this enzyme can be easily quantified in situ using a chromogenic substrate. These cells are very sensitive to estrogens; estradiol induces AlkP at levels as low as 10(-12) M. Antiestrogens completely block the action of estradiol. Various estrogens stimulate AlkP with potencies comparable to those achieved in vivo. The induction of AlkP is specific for estrogens; no other type of steroid, including androgens, progestins, mineralocorticoids, or glucocorticoids produce this effect. The stimulation of AlkP in Ishikawa cells is specific for estrogens, is highly reproducible and sensitive, and permits large numbers of samples to be assayed with ease. We have used this assay to investigate the estrogenic action of the adrenal delta 5-3 beta-hydroxysteroids. While pregnenolone is inactive, dehydroepiandrosterone and its sulfate ester induce AlkP slightly. However, the C19 steroid, 5-androstene-3 beta, 17 beta-diol is considerably more estrogenic in this assay, although it stimulates Ishikawa AlkP with a potency of 1/30,000 that of estradiol. The stimulation by 5-androstene-3 beta,17 beta-diol is inhibited by antiestrogens, but it is not blocked by the delta 5-3 beta-hydroxysteroid isomerase/dehydrogenase inhibitor, cyanoketone, or by the aromatase inhibitor, <em>4</em>-hydroxy-<em>androstenedione</em>. Thus, neither conversion to a delta <em>4</em>-3-ketone nor aromatization is required for the action of this unusual estrogen.

By converting <em>androstenedione</em> to estrone, or testosterone to estradiol, aromatase is a key enzyme in estrogen biosynthesis. Encoded by a single gene CYP19, aromatase is expressed in various tissues, including ovary, placenta, bone, brain, skin, and adipose tissue, via partially tissue-specific promoters, and is essential for normal estrogen-dependent physiological functions. In disease-free breast tissue, aromatase mRNA is primarily transcribed from the weak promoter I.<em>4</em> and maintained at low levels in breast adipose stromal fibroblasts. In breast cancer a distinct set of aromatase promoters, i.e. I.3, II, and I.7, is activated, leading to a marked increase in aromatase expression in breast tumors and breast adipose tissue adjacent to a breast tumor, and a consequent local overproduction of estrogen that promotes growth and progression of breast cancer. In addition, the total amount of promoter I.<em>4</em>-specific aromatase transcript in breast adipose fibroblasts may also be increased due to both cytokine-induced desmoplastic reaction and cytokine-stimulated promoter I.<em>4</em> activity in breast cancer. Targeting aromatase has proven beneficial in treating breast cancer, since aromatase inhibitors are the most effective endocrine treatment of breast cancer to date. However, aromatase inhibitors cause major side effects such as bone loss and abnormal lipid metabolism, due to indiscriminate reduction of aromatase activity in all expression sites of the body. Therefore, inhibition of aromatase expression via breast cancer-associated aromatase promoters is a useful strategy to selectively block local aromatase production, and hence estrogen synthesis, in breast cancer. This review will summarize the significant findings on regulation of the breast cancer-associated aromatase promoters, and highlight the discovery of chemical compounds and nuclear receptor ligands that specifically inhibit activation of these aromatase promoters. Clinical side effects of these agents require development of new drugs with better specificity and efficacy, and epigenetic therapies with breast cancer tissue-selective aromatase siRNA-conjugated nanoparticles.

Uterine leiomyomata are steroid hormone dependent tumors which possess receptors for estrogen (ER) and progesterone (PR). We reasoned that an antiprogesterone (RU <em>4</em>86) may induce regression of leiomyomata by withdrawal of progesterone action and/or by its interference of estrogen action. Accordingly, we examined the effects of daily administration of RU <em>4</em>86 (50 mg) for a period of 3 months in 10 patients with uterine leiomyomata and regular menstrual cycles. Baseline ultrasound examinations were obtained and repeated monthly during treatment as a measure of leiomyomata volume. Hormonal parameters were monitored by blood samples obtained prior to treatment and daily for 7 days, weekly for <em>4</em> weeks and monthly for the duration of therapy. Myomectomy or hysterectomy was performed in 6 of 10 patients at the end of treatment. Leiomyomata and myometrial tissue was obtained for immunocytochemical analysis of ER and PR protein. Amenorrhea was induced in all patients during treatment. Leiomyomata volume (mean +/- SE) decreased 21.9 +/- <em>4</em>.8% after <em>4</em> weeks, 39.5 +/- 6.6% (P < 0.001) after 8 weeks, and <em>4</em>9.0 +/- 9.2% (P < 0.001) after 12 weeks of treatment compared to pretreatment measurements. Serum LH levels (P < 0.005), but not FSH levels, more than doubled during the first 3 weeks of treatment with a concomitant increase in serum <em>androstenedione</em> (P < 0.006) and testosterone (P < 0.0001) levels. These elevated hormonal levels returned to baseline at <em>4</em> weeks without further changes during the remainder of treatment. A significant rise in serum dehydroepiandrosterone sulfate (P < 0.0001) and cortisol (P < 0.01) was seen at 12 weeks, suggesting an antiglucocorticoid effect of RU <em>4</em>86 has occurred. Serum estradiol, estrone, progesterone, sex hormone binding protein, thyroid-stimulating hormone, and PRL were unchanged from early follicular phase values. PR but not ER immunoreactivity was significantly reduced in both leiomyomata and myometrium after RU <em>4</em>86 treatment compared with tissues from untreated patients, suggesting that regression of tumors may be attained through a direct antiprogesterone effect. However, an alteration in ER functionality cannot be excluded. We conclude that RU <em>4</em>86 is well tolerated, safe, and effective; thus, it may prove to be a novel mode of management for uterine leiomyomata.

Serum gonadotropin, estrogen, and androgen levels were measured in samples obtained from 19 patients with polycystic ovarian disease (PCO) and from 10 normal women on day 2 to <em>4</em> of their menstrual cycles. In patients with PCO, the mean (plus or minus S.E.) concentration was significantly higher (P smaller than 0.001) than the concentrations found in the normal subjects for LH (35 plus or minus <em>4</em>.6 vs. 12.7 plus or minus 2.6 m.I.U. per milliliter), but not for FSH (10.3 plus or minus 0.7 vs. 8.7 plus or minus 0.9 m.I.U. per milliliter). Estrone (E1) levels (92 plus or minus <em>4</em> vs. 52 plus or minus 5 pg. per milliliter) were also significantly higher (P smaller than 0.001), while estradiol (E2) concentrations (58 plus or minus <em>4</em> vs. 63 plus or minus 8 pg. per milliliter) were comparable. Testosterone (T) (<em>4</em>68 plus or minus <em>4</em>1 vs. 325 plus or minus 3<em>4</em> pg. per milliliter, P smaller than 0.05), <em>androstenedione</em> (delta) (2,083 plus or minus 138 vs. 1,123 plus or minus 153 pg. per milliliter, P smaller than 0.001), and dehydroepiandrosterone sulfate (3.<em>4</em> plus or minus 0.<em>4</em> vs. 2.0 plus or minus 0.37 mug per milliliter, P smaller 0.02) were also significantly increased over the values in normal controls. The mean dehydroepiandrosterone (DHEA) was elevated in the patients with PCO (11.3 plus or minus 1.7 vs. 7.5 plus or minus 1.2 mug per milliliter), but was not significantly different. A positive correlation was found between LH levels and both E2 and E1 concentrations in the patients with PCO. These data show a distinct profile of gonadotropin, estrogen, and androgen levels in patients with PCO.

Precocious pubarche in girls is often preceded by low weight at birth and followed by hirsutism, ovarian hyperandrogenism, and oligomenorrhea in adolescence, the latter usually being accompanied by dyslipidemia and hyperinsulinism, which are, in turn, two major risk factors for cardiovascular disease in later life. We hypothesized that insulin resistance may be a key pathogenetic factor in this sequence. We tested the hypothesis by assessing the effects of an insulin-sensitizing agent, metformin, given at a daily dose of 1275 mg for 6 months to 10 nonobese adolescent girls (mean age, 16.8 yr; body mass index, 21.9 kg/m2; birth weight, 2.7 kg) with hirsutism, ovarian hyperandrogenism (diagnosis by GnRH agonist test), oligomenorrhea, dyslipidemia, and hyperinsulinemia after precocious pubarche. Before the metformin trial, longitudinal studies in these girls had shown that hyperinsulinism was present at prepubertal diagnosis of precocious pubarche, and that it increased markedly in late puberty or early postmenarche. Metformin treatment was well tolerated and was accompanied by a marked drop in hirsutism score, insulin response to oral glucose tolerance test, free androgen index, and baseline testosterone, <em>androstenedione</em>, dehydroepiandrosterone, and dehydroepiandrosterone sulfate levels (all P < 0.01). During metformin treatment, the LH and 17-hydroxyprogesterone hyperresponses to GnRH agonist were attenuated (P < 0.01); serum triglyceride, total cholesterol, and low density lipoprotein cholesterol levels decreased; and high density lipoprotein cholesterol rose. All girls reported regular menses within <em>4</em> months. Withdrawal of metformin treatment was followed, within 3 months, by a consistent reversal toward pretreatment conditions. In conclusion, metformin treatment reduced hyperinsulinemia, hirsutism, and hyperandrogenism; attenuated the LH and 17-hydroxyprogesterone hyperresponses to GnRH agonist; improved the atherogenic lipid profile; and restored eumenorrhea in nonobese adolescent girls with a history of precocious pubarche. These observations corroborate the idea that insulin resistance may indeed be a prime factor underpinning the sequence from reduced fetal growth, through precocious pubarche, to adolescent endocrinopathies that are reminiscent of so-called polycystic ovary syndrome.

Intracranial hypertension in the absence of an intracranial mass lesion or hydrocephalus can be caused by an alteration of one or more of the four determinants of cerebrospinal fluid (CSF) pressure: (1) intrasagittal sinus pressure, (2) resistance of arachnoid villi to the egress of CSF, (3) rate of production of CSF, and (<em>4</em>) compliance of the CSF space. The pseudotumor cerebri syndrome of obese young women may be caused partially by increased CSF production. Estrone (produced by the conversion of <em>androstenedione</em> by adipocytes) probably induces the menstrual irregularities of these women and may affect CSF secretion. Pseudotumor cerebri syndromes associated with other conditions may be separated into pathogenic categories.

To discern the pharmacological effects of dehydroepiandrosterone (DHEA) in older women with low endogenous DHEA and DHEA sulfate (DS), 1600 mg/day (in four divided doses) were administered orally to six postmenopausal women for 28 days in a double blind placebo-controlled cross-over study. Serum concentrations of androgens after the first <em>4</em>00-mg dose of DHEA increased rapidly and reached a maximum at 180-2<em>4</em>0 min, resulting in increases over baseline of 6-fold for DHEA (5.8 +/- 2.1 to 28.8 +/- 5.5 nmol/L), 12-fold for DS (3.0 +/- 1.6 to 28.2 +/- <em>4</em>.6 mumol/L) and <em>androstenedione</em> (1.<em>4</em> +/- 0.3 to 19.5 +/- 9.8 nmol/L), 2.5-fold for testosterone (0.7 +/- 0.1 to 2.2 +/- 0.6 nmol/L), and 15-fold for dihydrotestosterone (0.2 +/- 0.06 to 2.73 +/- 1.0 nmol/L), but estrone, estradiol, and sex hormone-binding globulin (SHBG) were unchanged. Assessment at weekly intervals revealed a further increase in all androgens which was maximal at 2 weeks and remained markedly elevated, although it declined somewhat by <em>4</em> weeks. The increments observed after 2 weeks of DHEA administration reached 15-fold for DHEA (71.9 +/- 1<em>4</em>.2 nmol/L), 9-fold for testosterone (6.5 +/- 1.7 nmol/L), and 20-fold for DS (65.1 +/- 1<em>4</em>.9 nmol/L), <em>androstenedione</em> (30.5 +/- 11.5 nmol/L), and dihyrotestosterone (3.8 +/- 1.5 nmol/L). Both estrone and estradiol showed a progressive increase to 2-fold the basal value at <em>4</em> weeks. Integrated SHBG and thyroid binding globulin levels decreased (P less than 0.05) during DHEA treatment. However, LH, FSH, body weight, and percent body fat, as measured by underwater weighing, were unaltered during the <em>4</em>-week experiment. A marked decline of 11.3% (P less than 0.05) in serum cholesterol and 20.0% (P less than 0.05) in high density lipoprotein noted within the first week of DHEA administration persisted for the 28-day period and was accompanied by a nonsignificant downward trend in low density lipoprotein, very low density lipoprotein, and triglycerides. Peak insulin levels during the 3-h oral glucose tolerance test were significantly higher (P less than 0.05) after the 28 days of DHEA (1126 +/- 165 vs. 7<em>4</em>6 +/- 165 pmol/L) and were accompanied by a 50% increase in the integrated insulin response (P less than 0.01) without a significant change in fasting glucose insulin or glucose-6-phosphate dehydrogenase values.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

BACKGROUND

To investigate the effectiveness and safety of pioglitazone (<em>4</em>5 mg/day) on clinical and endocrine-metabolic features of polycystic ovary syndrome (PCOS), we studied 18 obese PCOS patients, classified as normoinsulinaemic (N-PCOS, n = 6) and hyperinsulinaemic (H-PCOS, n = 12) according to their insulin secretion.

METHODS

Evaluation of clinical signs, hormonal and lipid profile assays, oral glucose tolerance tests and euglycaemic hyperinsulinaemic clamps were performed at baseline and after 2 (visit 2), <em>4</em> (visit 3) and 6 (visit <em>4</em>) months of treatment.

RESULTS

Body weight, body fat distribution and blood pressure remained stable throughout the treatment; hirsutism and acne significantly improved (P < 0.001 for visits 3 and <em>4</em> versus baseline) in both groups. A restoration of menstrual cyclicity was observed at visit <em>4</em> in 83% (P < 0.001) of H-PCOS and in 33% of N-PCOS. A decrease in LH, LH/FSH ratio, androstenedione and 17-hydroxy-progesterone was observed in both groups, attaining statistical significance in H-PCOS. A significant amelioration of insulin secretion, sensitivity and clearance was obtained in H-PCOS. A trend towards improvement was observed in lipid assessment of both groups. Therapy was well-tolerated.

CONCLUSIONS

Present data suggest that there is a selective effect, partially independent of insulin secretion, of pioglitazone on the clinical and hormonal disturbances of PCOS.

High pressure liquid chromatographic systems capable of resolving at least 28 known and potential metabolites of 17 beta-hydroxy-<em>4</em>-androsten-3-one (testosterone) and <em>4</em>-androstene-3,17-dione (<em>androstenedione</em>) were used to quantitatively assess the metabolism of the two steroids in monooxygenase systems reconstituted with five purified rat liver cytochrome P-<em>4</em>50 isozymes. Cytochromes P-<em>4</em>50a, -b, -c, -d, and -e catalyzed the oxidation of testosterone at overall rates of 21, 27, 2, 0.7, and 3 nmol/min/nmol of cytochrome P-<em>4</em>50, respectively; while the corresponding rates for total <em>androstenedione</em> metabolism were 12, 62, 1.5, 0.3, and 5. Cytochrome P-<em>4</em>50a catalyzed the oxidation of testosterone and <em>androstenedione</em> almost exclusively to their respective 7 alpha-hydroxy metabolites. Cytochrome P-<em>4</em>50b catalyzed the oxidation of testosterone to <em>androstenedione</em> and 16 alpha- and 16 beta-hydroxytestosterone in approximately equal molar ratios. However, this same hemoprotein exhibited a marked stereoselectivity in the metabolism of <em>androstenedione</em> since the molar ratio of 16 alpha- and 16 beta-hydroxy<em>androstenedione</em> was greater than 1:10. Cytochrome P-<em>4</em>50e catalyzed the oxidation of both steroids to the same products as cytochrome P-<em>4</em>50b, but at approximately 10% of the rate. Cytochromes P-<em>4</em>50c and P-<em>4</em>50d catalyzed the oxidation of testosterone and <em>androstenedione</em> regio- and stereospecifically to their respective 6 beta-hydroxy metabolites. These results indicate that certain cytochrome P-<em>4</em>50 isozymes show marked positional specificity in the metabolism of both testosterone and <em>androstenedione</em>, and that the rate as well as stereoselectivity of the oxidative reactions can be markedly dependent on subtle differences in the structure of the steroid substrate.

The interconversion of estrone (E1) and 17 beta-estradiol (E2), <em>androstenedione</em> (<em>4</em>-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3 beta,17 beta-diol is catalyzed by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). The enzyme 17 beta-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17 beta-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17 beta-HSD mRNA in some human tissues. Enzymatic activity was measured using 1<em>4</em>C-labeled E1, E2, <em>4</em>-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highest level of 17 beta-HSD activity, low but significant levels of E2 as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E2----E1, T----<em>4</em>-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17 beta-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.

It is currently believed that the postmenopausal ovary remains a gonadotropin-driven, androgen-producing gland. However, the adrenal contribution to circulating androgen levels may explain some conflicting results previously reported. In addition, the steroidogenic potential and gonadotropin responsiveness of the postmenopausal ovary have not been recently reassessed. Plasma T, bioavailable T, free T, <em>androstenedione</em> (Adione), and dehydroepiandrosterone sulfate levels were measured in postmenopausal or ovariectomized women with complete adrenal insufficiency, compared with women with intact adrenals. A stimulation human chorionic gonadotropin test (on d 0, 3, and 6) was performed in postmenopausal women with adrenal insufficiency. Dexamethasone was administered for <em>4</em> d in postmenopausal women with intact adrenals. Intraovarian T and <em>androstenedione</em> were also measured in homogenates of ovarian tissue from postmenopausal women. Immunocytochemistry was performed on postmenopausal ovaries and premenopausal controls to detect the presence of steroidogenic enzymes (P-<em>4</em>50 aromatase, P-<em>4</em>50 SCC, 3beta HSD, and P-<em>4</em>50 C17) and gonadotropin receptors. Plasma androgen levels were below or close to the limit of the assay in all women with adrenal insufficiency. They were similar in postmenopausal and oophorectomized women with normal adrenals. No hormonal changes were observed after human chorionic gonadotropin injections in women with adrenal insufficiency. In contrast, a dramatic decrease of all steroids was observed after dexamethasone administration in postmenopausal women with intact adrenals. Intraovarian T and <em>androstenedione</em> levels were negligible in postmenopausal ovarian tissue. P-<em>4</em>50 aromatase was absent from the 17 ovaries studied, and the enzymes for androgen biosynthesis were either absent (n = 13) or present in very low amounts (n = <em>4</em>). In all the postmenopausal ovaries, FSH and LH receptors were completely absent. In the absence of adrenal steroids, postmenopausal women have no circulating androgens. This result is consistent with the immunocytochemical studies showing the almost constantly absent steroidogenic enzymes and LH receptors in the postmenopausal ovary. Thus, the climacteric ovary is not a critical source of androgens. The arrest of androgen secretion after menopause may impact significantly on women's health.

The question of whether to take hormone therapy (HT) will impact every woman as she enters reproductive senescence. In women, studies suggest that ovarian hormone loss associated with menopause has deleterious cognitive effects. Results from clinical studies evaluating whether estrogen-containing HT mitigates these effects, and benefits cognition, are discrepant. Type of menopause, surgical vs. transitional, impacts cognitive outcome in women. However, whether type of menopause impacts cognitive effects of HT has not been methodically tested in women or an animal model. We used the <em>4</em>-vinylcyclohexene diepoxide rodent model of ovarian follicle depletion, which mimics transitional menopause, and the traditional rat model of menopause, ovariectomy, to cognitively test the most commonly prescribed estrogen therapy in the United States, conjugated equine estrogens (Premarin). Here we show conjugated equine estrogens benefited cognition in surgically menopausal rats, but, in contrast, impaired cognition in transitionally menopausal rats. <em>Androstenedione</em>, released from the residual transitional menopausal ovary, was positively associated with impaired performance, replicating our previous findings in <em>4</em>-vinylcyclohexene diepoxide animals. The current findings are especially salient given that no clinical study testing cognition has methodically separated these two populations of menopausal women for analysis. That we now show surgical vs. transitional modes of menopause result in disparate cognitive effects of HT has implications for future research and treatments optimizing HT for menopausal women.

BACKGROUND

Current immunoassays for analysis of plasma androgens in children have several limitations due to antibody-specific variations of data and normal ranges. Mass spectrometry-based methods are available for individual steroids but need complex sample preparation and report only fragmentary reference data for the pediatric population.

OBJECTIVE

Our objective was to develop a state of the art sensitive and specific tandem mass spectrometry method for high-throughput simultaneous determination of plasma concentrations of androstenedione (A), testosterone (T), and dihydrotestosterone (DHT) and to report age-, sex-, and pubertal stage-specific reference levels for these steroids in children aged 0-18 yr.

METHODS

Plasma (100 microl) was mixed with internal standard and extracted by solid-phase extraction. Androgens were measured by ultrapressure liquid chromatography tandem mass spectrometry. Samples of 138 boys and 131 girls with neither signs of endocrine nor systemic disease were considered for the generation of reference data. The following age groups were used: less than 1 wk, 2 wk to 2 months, 3-5 months, 6-11 months, 1-3 yr, 4-6 yr, 7-9 yr, 10-12 yr, 13-15 yr, and over 16 yr.

RESULTS

Lower quantification limit was 2.9 ng/dl (0.1 nmol/liter) for A, T, and DHT. No relevant interference with other steroids was detected. Reference data for A, T, and DHT are reported as functions of age, sex, pubertal maturation, and testicular volume.

CONCLUSIONS

Simplicity, velocity, sensitivity, specificity, and the availability of pediatric reference data allow application of our new method in clinical routine as well as in research settings.

We have examined the effect of androst-5-ene-3 beta,17 beta-diol (delta 5-diol) and its precursors, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 3 beta-sulfate (DHEAS), on the growth of the estrogen-sensitive human breast cancer cell line, ZR-75-1. While the cell number was increased up to <em>4</em>-fold by maximal concentrations of estradiol, delta 5-diol maximally stimulated cell proliferation by approximately 3-fold. Since the half-maximal stimulation achieved by delta 5-diol is observed at 2.5 nM and the normal range of plasma concentrations of this steroid in women is 1 to 3 nM, it is most likely that the stimulatory effect of delta 5-diol has physiological significance. DHEA and DHEAS were much less effective than delta 5-diol in stimulating the proliferation of ZR-75-1 cells, the maximal effect on cell number being 75% at the maximal dose used, namely 10 microM. The mitogenic effects of estradiol and delta 5-diol were competitively inhibited by the antiestrogen LY156758 (keoxifene), while the effects of DHEA and DHEAS were completely abolished by the antiestrogen. The effects of DHEA and delta 5-diol on cell proliferation are not likely to be mediated via their conversion to estrone or estradiol, since <em>androstenedione</em> had no effect, while testosterone and dihydrotestosterone decreased cell number by about 20%. The number of specific progesterone binding sites was increased 3.7-, 3.2-, and 2.0-fold by delta 5-diol, DHEA, and DHEAS, respectively. The relative potency of the C19-delta 5-steroids to increase the number of progesterone-specific binding sites was comparable to their ability to stimulate cell proliferation. Direct competition experiments performed with intact cells in monolayer culture showed that, under conditions of minimal metabolism, only delta 5-diol could significantly compete with estradiol for cellular estrogen-specific binding sites with an apparent dissociation constant of 11 nM, thus suggesting that physiological concentrations of C19-delta 5-steroids of adrenal origin could exert an estrogenic stimulation of breast tumor growth without involvement of the aromatase pathway. The present data suggest not only that estrone derived from <em>androstenedione</em> could play a role in estrogen-sensitive breast cancer in women but that delta 5-diol could well be the most important estrogen in breast cancer in women.

OBJECTIVE

To evaluate the effects of metformin administration on spontaneous LH episodic release in a group of nonobese polycystic ovary (PCOS) patients.

METHODS

Controlled clinical study.

METHODS

PCOS patients in a clinical research environment.

METHODS

Twenty nonobese PCOS patients were enrolled after informed consent.

METHODS

All patients underwent hormonal evaluations and a pulsatility study (sampling every 10 minutes for <em>4</em> hours) before and at the sixth month of therapy (metformin, 500 mg, p.o. b.i.d.). Ultrasound examinations and Ferriman-Gallwey scoring were also performed.

METHODS

Measurements of plasma LH, FSH, estradiol (E(2)), androstenedione (A), 17-hydroxy-progesterone (17-OHP), and testosterone (T), glucose, insulin, and C-peptide concentrations.

RESULTS

After 6 months of metformin administration, the plasma LH, 17-OHP, A, and T levels and LH/FSH ratio were significantly reduced. Insulin sensitivity, expressed as the glucose-to-insulin ratio, was significantly improved under glucose load after 6 months of treatment. Spontaneous LH episodic release showed a significant reduction in pulse amplitude with no changes in pulse frequency. Menstrual cyclicity was restored in all amenorrheic and oligomenorrheic women. The ovarian volume and Ferriman-Gallwey scores also were significantly reduced.

CONCLUSIONS

Metformin administration improves reproductive axis functioning in hyperandrogenic nonobese PCOS patients. By acting on the ovary and restoring normal ovarian activity, metformin positively modulates the reproductive axis (namely GnRH-LH episodic release).

17Beta-hydroxysteroid dehydrogenase-3 (17betaHSD3) deficiency is an autosomal recessive form of male pseudohermaphroditism caused by mutations in the HSD17B3 gene. In a nationwide study on male pseudohermaphroditism among all pediatric endocrinologists and clinical geneticists in The Netherlands, 18 17betaHSD3-deficient index cases were identified, 12 of whom initially had received the tentative diagnosis androgen insensitivity syndrome (AIS). The phenotypes and genotypes of these patients were studied. Endocrine diagnostic methods were evaluated in comparison to mutation analysis of the HSD17B3 gene. RT-PCR studies were performed on testicular ribonucleic acid of patients homozygous for two different splice site mutations. The minimal incidence of 17betaHSD3 deficiency in The Netherlands and the corresponding carrier frequency were calculated. Haplotype analysis of the chromosomal region of the HSD17B3 gene in Europeans, North Americans, Latin Americans, Australians, and Arabs was used to establish whether recurrent identical mutations were ancient or had repeatedly occurred de novo. In genotypically identical cases, phenotypic variation for external sexual development was observed. Gonadotropin-stimulated serum testosterone/<em>androstenedione</em> ratios in 17betaHSD3-deficient patients were discriminative in all cases and did not overlap with ratios in normal controls or with ratios in AIS patients. In all investigated patients both HSD17B3 alleles were mutated. The intronic mutations 325 + <em>4</em>;A->>T and 655-1;G->>A disrupted normal splicing, but a small amount of wild-type messenger ribonucleic acid was still made in patients homozygous for 655-1;G->>A. The minimal incidence of 17betaHSD3 deficiency in The Netherlands was shown to be 1: 1<em>4</em>7,000, with a heterozygote frequency of 1:135. At least <em>4</em> mutations, 325 + <em>4</em>;A->>T, N7<em>4</em>T, 655-1;G->>A, and R80Q, found worldwide, appeared to be ancient and originating from genetic founders. Their dispersion could be reconstructed through historical analysis. The HSD17B3 gene mutations 326-1;G->>C and P282L were de novo mutations. 17betaHSD3 deficiency can be reliably diagnosed by endocrine evaluation and mutation analysis. Phenotypic variation can occur between families with the same homozygous mutations. The incidence of 17betaHSD3 deficiency is 0.65 times the incidence of AIS, which is thought to be the most frequent known cause of male pseudohermaphroditism without dysgenic gonads. A global inventory of affected cases demonstrated the ancient origin of at least four mutations. The mutational history of this genetic locus offers views into human diversity and disease, provided by national and international collaboration.

An important source of androgens in the human prostate are those synthesized locally from the inactive adrenal precursor dehydroepiandrosterone (DHEA) and its sulfated derivative DHEA-S. Three beta-HSD (hydroxysteroid dehydrogenase) converts DHEA into <em>androstenedione</em> (<em>4</em>-dione), whereas type 5 17beta-HSD catalyzes the reduction of <em>4</em>-dione into testosterone in the human prostate and other peripheral intracrine tissues. In the present study, we have used two complementary approaches, namely in situ hybridization and immunocytochemistry, to identify the cells that contain the type 5 17beta-HSD messenger RNA and enzyme in human benign prostatic hyperplasia (BPH). Localization of 3beta-HSD and of the androgen receptor (AR) was also investigated by immunostaining in the same tissue. To find out whether there are any differences between BPH and normal prostate tissue, the localization of type 5 17beta-HSD was reexamined by immunocytochemistry in the normal human prostate samples and also in normal prostate epithelial cell line (PrEC). The in situ hybridization results obtained with a tritiated uridine triphosphate (3H-UTP)-labeled type 5 17beta-HSD riboprobe are in agreement with the immunostaining data obtained with a specific antibody to the enzyme. The immunostaining results obtained from normal prostate tissue and BPH were found to be similar. Thus, in the glandular epithelium, basal cells highly express the messenger RNA and the enzyme, whereas luminal cells show a much lower and variable level of expression. In the stroma and walls of blood vessels, fibroblasts and the endothelial cells lining the blood vessels show positive staining. Similar results are observed when the cellular distribution of 3beta-HSD is investigated. AR immunoreactivity, however, shows a different distribution because, in the epithelium, most of the nuclei of basal cells are negative, whereas the majority of nuclei of the luminal cells show positive staining. A strong reaction for AR is also found in most stromal cell nuclei and in the nuclei of most endothelial cells, as well as in some other cells of the walls of blood vessels. In conclusion, human type 5 17beta-HSD, as well as 3beta-HSD, are highly expressed, not only in the basal epithelial cells and stromal fibroblasts but also in the endothelial cells and fibroblasts of the blood vessels. AR, on the other hand, is highly expressed in the luminal cells. The present data suggest that DHEA is transformed in the basal cells of the glandular epithelium into <em>4</em>-dione by 3beta-HSD and then into testosterone by type 5 17beta-HSD, whereas dihydrotestosterone is synthesized in the luminal cells after diffusion of testosterone from the underlying layer of basal cells. The potential role of androgen formation and action in blood vessels is unknown and opens new avenues of investigation for a better understanding of the multiple roles of androgens.

Finasteride, a 5 alpha-reductase inhibitor, was administered to normal male volunteers in a blinded placebo-controlled study at daily oral doses of 25, 50, and 100 mg for 11 days (part 1) and daily oral doses of 0.0<em>4</em>, 0.12, 0.2, and 1.0 mg for 1<em>4</em> days (part 2). Results from part 1 showed a significant reduction in dihydrotestosterone (DHT) at all doses and a significant increase in both testosterone (T) and delta <em>4</em>-<em>androstenedione</em> at the 50- and 100-mg doses. No change was seen in LH, FSH, cortisol, or estradiol levels. Serum lipids, including total cholesterol, low density lipoprotein, high density lipoprotein, and triglycerides were not affected by treatment. Results from part 2 again showed significant reduction in DHT at all doses. DHT levels returned to pretreatment values within 1<em>4</em> days of discontinuing treatment. Significant increases in T were observed only in the 1.0 mg group and only during the first 8 days of treatment. The T/DHT ratio increased with all doses and returned to baseline when drug was discontinued. The DHT metabolites and androstanediol glucuronide and androsterone glucuronide were significantly reduced at all doses. There were no significant adverse experiences reported during part 1 or 2. In conclusion, finasteride is well tolerated by normal volunteers and results in significant suppression of serum DHT at all doses tested.

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M <em>androstenedione</em>, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of <em>4</em> X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.

Seborrhoea and acne are exclusively human diseases and sebaceous gland differentiation is species specific. Therefore, fundamental research on human sebaceous cell function and control requires human in vitro models. The human sebocyte culture model, introduced in 1989, has been used in several studies to elucidate sebaceous gland activity and its regulation at the cellular level. Cultured human sebocytes have been shown to preserve important sebocytic characteristics, although they undergo an incomplete terminal differentiation in vitro. In vitro synthesis of free fatty acids without bacterial involvement and marked interleukin 1 alpha expression at the mRNA and protein levels with no further induction by lipopolysaccharides lead to the assumption that human sebocytes may initiate acne lesions by an intrinsic mechanism. Androgens affected sebocyte activity in vitro in a manner dependent on the localization of the sebaceous glands. In vitro stimulation of sebocyte proliferation by androgens could be completely abolished by spironolactone. Cultured sebocytes strongly expressed type 1 5 alpha-reductase and metabolized testosterone to <em>androstenedione</em>, 5 alpha-androstanedione, 5 alpha-dihydrotestosterone, androsterone and 5 alpha-androstanediol, whereas the levels of 5 alpha-reductase activity were probably not feedback regulated. <em>4</em>,7 beta-Dimethyl-<em>4</em>-aza-5 alpha-cholestan-3-one, a type 1 5 alpha-reductase inhibitor, induced an early, marked down-regulation of 5 alpha-reductase activity in human sebocytes in vitro, while hydrofinasteride, a type 2 inhibitor, required 10(3)-fold higher concentrations to induce similar effects. Stimulation of sebocyte proliferation by insulin, thyroid-stimulating hormone and hydrocortisone indicates that the hormonal control of the sebaceous gland could be a complex mechanism. Retinoids inhibited sebocyte proliferation in a dose-dependent manner and down-regulated lipid synthesis and sebocyte differentiation in vitro. Isotretinoin was the most potent compound. On the other hand, vitamin A was found essential for sebocyte activity and differentiation in vitro and could be partially substituted by synthetic retinoids. The inhibitory effect of isotretinoin on sebocyte proliferation was barely affected by the presence of vitamin A. The low persistent isotretinoin levels or, more likely, the considerably elevated tretinoin concentrations detected in human sebocytes after treatment with isotretinoin in vitro may be responsible for the inhibitory effect of this compound on sebocyte activity.