Previous studies have demonstrated that ovulatory goldfish synthesize and release a variety of steroids into the water, where some of them function as sex pheromones. Among the steroids which have been measured are free <em>androstenedione</em>, testosterone, 17 alpha,20 beta-dihydroxy-<em>4</em>-pregnen-3-one (17,20 beta-P), 17 alpha,20 beta,21-trihydroxy-<em>4</em>-pregnen-3-one (17,20 beta,21-P), 17 alpha-hydroxy-<em>4</em>-pregnene-3,20-dione (17-P), 11-deoxycortisol (17,21-P), and 17 alpha,20 alpha-dihydroxy-<em>4</em>-pregnen-3-one (17,20 alpha-P), glucuronidated testosterone, 17,20 beta-P, and 17,20 beta,21-P, and sulfated 17,21-P, 17,20 beta-P, and 17,20 beta,21-P. This study reports the changes in the amounts of these steroids released into the water, at 3-hr intervals, by 12 female goldfish injected with human chorionic gonadotrophin (HCG). Eight fish ovulated. The amounts of steroids released into the water were significantly higher in the fish which ovulated than in those which did not. The peak rate of release (ng per hour) of free <em>androstenedione</em> (91), glucuronidated testosterone (1<em>4</em>3), and sulfated 17,21-P (33) occurred at ca. <em>4</em> hr postinjection; free 17-P (163) and free 17,21-P (217) at ca. 6 hr.; free 17,20 beta-P (58) and free 17,20 beta,21-P (58) at ca. 7.5 hr; and glucuronidated 17,20 beta-P (96), glucuronidated 17,20 beta,21-P (26<em>4</em>), sulfated 17,20 beta-P (6<em>4</em>) and sulfated 17,20 beta,21-P (153) at ca. 9 hr. Amounts of all of the steroids were at their lowest between 12 and 15 hr and, in the ovulated fish, had risen significantly again between 18 and 21 hr. The amounts of glucuronidated testosterone, 17,20 beta-P and 17,20 beta,21-P, and of sulfated 17,20 beta-P and 17,20 beta,21-P, in water exceeded those of free steroid. Elevated levels of free and glucuronidated steroids were also found in the blood plasma of HCG-injected fish. Considerably more <em>androstenedione</em> than testosterone was released into the water. Assessment of the "pheromonal effectiveness" of each steroid indicated that the free and sulfated forms of 17,20 beta-P and 17,20 beta,21-P, and <em>androstenedione</em> were the most important pheromonal steroids released by female goldfish.

3beta-hydroxysteroid dehydrogenase/steroid delta5->><em>4</em>-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,25<em>4</em>F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,25<em>4</em>F protein that co-migrated with purified placental 3beta-HSD/isomerase (monomeric Mr=<em>4</em>2,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3beta-HSD activity, whereas the Km and Vmax values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The Vmax (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to <em>androstenedione</em> by the Y253F isomerase activity was 7.0-fold less than the mean Vmax (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,25<em>4</em>F mutant enzyme, but Y253,25<em>4</em>F had <em>4</em>5% of the 3beta-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar Vmax values for substrate oxidation by the 3beta-HSD activity. The 3beta-HSD activities of the Y253F, Y253,25<em>4</em>F and wild-type enzymes reduced NAD+ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD+ was dramatically decreased. Based on these kinetic measurements, His261 appears to be a critical amino acid residue for the 3beta-HSD activity, and Tyr253 or Tyr25<em>4</em> participates in the isomerase activity of human type I (placental) enzyme.

OBJECTIVE

To test the hypothesis that physiologic increases in fetal plasma 17 beta-estradiol and androstenedione modulate the activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis.

METHODS

Seventeen pregnant ewes and their fetuses were chronically catheterized. At the time of surgery, the fetuses received implants that released 17 beta-estradiol (n = 5) alone or 17 beta-estradiol and androstenedione (n = 6), each at a rate of approximately 250 micrograms/day for each steroid. The control group (n = 6) received either no pellet (n = 2) or a "placebo" pellet, which contained no steroid (n = 4). Fetal blood samples were drawn for hormone and blood gas analysis at 1-3-day intervals until the time of spontaneous parturition. Fetal plasma ACTH and cortisol concentrations were fit to semilogarithmic equations and analyzed by stepwise multiple linear regression analysis for statistically significant effects of 17 beta-estradiol and androstenedione.

RESULTS

Estradiol significantly increased and androstenedione significantly decreased the ACTH and cortisol concentrations. Treatment with both 17 beta-estradiol and androstenedione resulted in parturition approximately 4 days earlier than in the other groups (P < .05).

CONCLUSIONS

Physiologic increases in fetal plasma estradiol and androstenedione modify the activity of the HPA axis.

Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ)-regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, <em>androstenedione</em>, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and <em>4</em>-hydroxytamoxifen, general P<em>4</em>50 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERβ-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERβ-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17β-estradiol for ERα and ERβ binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERβin vitro, modulating estrogen target genes in vivo.

Reproductive aging in women (a physiological decline in the function of the hypothalamic-pituitary-ovarian axis) is an infrequently investigated and poorly understood biological phenomenon. Although menstrual irregularity and anovulation are known to precede the menopause, normal women in their fifth decade experience a profound decrease in fertility while still experiencing regular menstrual cycles. To further our understanding of the physiological changes associated with reproductive aging, this study examined the spontaneous development and function of ovarian follicles in normal women, aged <em>4</em>0-<em>4</em>5 yr. The subjects were women (n = 21), aged <em>4</em>0-<em>4</em>5 yr, who had regular 25- to 35-day ovulatory menstrual cycles, were not infertile, had no medical problems, and met specific criteria for weight, diet, and exercise. The controls were normal women (n = 20), age 20-25 yr, who met the same criteria. The subjects were monitored with daily hormone measurements [LH, FSH, estradiol (E), progesterone (P), and inhibin] and pelvic sonograms from day 1 of their study cycle until the dominant ovarian follicle reached a mean diameter of 15 mm and/or a serum E level of 550 pmol/L or higher was attained. At that time, 10,000 U hCG were given, and a transvaginal sonographic follicle aspiration was performed 32 h later. The follicular fluid (FF) was collected, stored frozen at -70 C, and later analyzed for E, P, testosterone (T), <em>androstenedione</em>, inhibin, insulin-like growth factor I (IGF-I), and IGF-II. The number of cycle days to aspiration was lower (11.6 vs. 15.6 days; P < 0.001) and the early follicular phase mean FSH and mean E levels were higher (9.3 vs. 6.6 mIU/mL and 305 vs. 160 pmol/L; P < 0.01) in the older (O) group compared to the younger group. There was a strong trend toward higher FF mean E (2280 vs. 1931 nmol/L) and lower FF mean T (978 vs. 2361 pmol/L) levels in group O. The E/T ratio was significantly higher (5253 vs. 2<em>4</em>08; P < 0.03) in group O. In group O, the mean FF P levels were increased as well (25.1 vs. 18.8 micromol/L; P < 0.01). The serum mean IGF-I (153 vs. 226 ng/mL; P < 0.001) and FF mean IGF-I (113 vs. 158 ng/mL; P < 0.02) levels were significantly decreased in group O. There were no differences between groups in serum or FF IGF-II or inhibin levels. Whether reproductive aging is an intrinsic ovarian process or the ovary is simply responding to exogenous influences, the ovary in general and its follicles in particular are the primary site of the effects of aging. Ovarian follicles in older ovulatory women have some unique features: 1) the follicles are the same size as those in younger women, but form more rapidly; 2) secretion of E and inhibin is not compromised; 3) the concentrations of steroids in the FF are indicative of a healthier follicle, i.e. increased P levels and higher estrogen to androgen ratio; and <em>4</em>) serum and FF levels of IGF-I are decreased, but there are no differences in IGF-II levels.

In the current studies, we evaluated the effects of <em>4</em>-tert-octylphenol (OP), endosulfan, bisphenol A (BPA), and 17beta-estradiol on basal or hCG-stimulated testosterone formation by cultured Leydig cells from young adult male rats. Exposure of Leydig cells to increasing concentrations of OP (1 to 2000 nM), 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM) or BPA (1 to 1000 nM), alone or with 10 mIU/mL hCG for <em>4</em> or 2<em>4</em> h, did not lower ambient testosterone levels, although cells exposed to higher OP concentrations + hCG for 2<em>4</em> h often had modest declines in testosterone (10 to 20%). Of interest, exposure to the highest concentration OP (2000 nM) alone for <em>4</em> or 2<em>4</em> h increased testosterone levels (approximately 2-fold in <em>4</em>-h exposed cells). Whether prior exposure to OP + hCG for 2<em>4</em> h affects the subsequent conversion of steroid substrates to testosterone over <em>4</em> h was evaluated. Progressive declines in 1 microM 22(R) hydroxycholesterol, 1 microM pregnenolone, or 1 microM progesterone conversion to testosterone was observed beginning at 100 to 500 nM OP exposure (maximal declines of <em>4</em>0 to 12% of controls were observed); however, the conversion of 1 microM <em>androstenedione</em> to testosterone was not affected by OP. These results suggested that 2<em>4</em>-h exposure to OP + hCG has no effect on 17beta-hydroxysteroid dehydrogenase, which converts <em>androstenedione</em> to testosterone, but that it inhibits the 17alpha-hydroxylase/C17-20 lyase step, which converts progesterone to <em>androstenedione</em>. In addition, potentially, OP could inhibit cholesterol side/chain cleavage activity, which converts cholesterol to pregnenolone, and/or 3beta-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone. Of interest, exposure to increasing concentrations of 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM), or BPA (1 to 1000 nM) + hCG for 2<em>4</em> h had no effect on subsequent conversion of 22(R)hydroxycholesterol to testosterone. Furthermore, the inhibiting effects of OP + hCG exposure on subsequent conversion of progesterone to testosterone was unaffected by concomitant exposure to the pure estrogen antagonist, ICI 182,780, or the antioxidants, ascorbate or dimethyl sulfoxide, suggesting that the actions of OP are not mediated through binding to estrogen receptor alpha or beta or by free radical induced damage to steroidogenic enzymes, respectively. These results demonstrate that direct exposure of adult Leydig cells to OP may have subtle effects on their ability to produce testosterone, which may not be detected by measuring ambient androgen levels. In addition, the effects of OP on Leydig cell testosterone formation appear to be different from those of the native estrogen, 17beta-estradiol, and from other reported weak xenoestrogens such as endosulfan and BPA.

This trial explores 1) prenatally androgenized (PNA) rats as a model of polycystic ovary syndrome (PCOS) and 2) reproductive and metabolic effects of cryptotanshinone in PNA ovaries. On days 16-18 of pregnancy, 10 rats were injected with testosterone propionate (PNA mothers) and 10 with sesame oil (control mothers). At age 3 mo, 12 female offspring from each group were randomly assigned to receive saline and 12 cryptotanshinone treatment during 2 wk. Before treatment, compared with the 2<em>4</em> controls, the 2<em>4</em> PNA rats had 1) disrupted estrous cycles, 2) higher 17-hydroxyprogesterone (P = 0.030), <em>androstenedione</em> (P = 0.016), testosterone and insulin (P values = 0.000), and glucose (P = 0.0<em>4</em>7) levels, and 3) higher areas under the curve (AUC) for glucose (AUC-Glu, P = 0.025) and homeostatic model assessment for insulin resistance (HOMA-IR, P = 0.008). After treatment, compared with vehicle-treated PNA rats, cryptotanshinone-treated PNA rats had 1) improved estrous cycles (P = 0.0<em>4</em>5), 2) reduced 17-hydroxyprogesterone (P = 0.0<em>4</em>1), <em>androstenedione</em> (P = 0.038), testosterone (P = 0.003), glucose (P = 0.036), and insulin (P = 0.0<em>4</em>1) levels, and 3) lower AUC-Glu (P = 0.0<em>4</em>5) and HOMA-IR (P = 0.02<em>4</em>). Western blot showed that cryptotanshinone reversed the altered protein expressions of insulin receptor substrate-1 and -2, phosphatidylinositol 3-kinase p85α, glucose transporter-<em>4</em>, ERK-1, and 17α-hydroxylase within PNA ovaries. We conclude that PNA model rats exhibit reproductive and metabolic phenotypes of human PCOS and that regulation of key molecules in insulin signaling and androgen synthesis within PNA ovaries may explain cryptotanshinone's therapeutic effects.

Three imidazole antifungal agents, ketoconazole, miconazole and tioconazole, and a group of structurally related 1-substituted imidazole and 1,2,<em>4</em>-triazole compounds were evaluated as inhibitors of the oxidative metabolism of testosterone catalysed by mouse hepatic microsomal cytochromes P-<em>4</em>50. Spectroscopic studies showed that both imidazoles and triazoles interacted with ferric cytochrome P-<em>4</em>50 in hepatic microsomes to produce type II difference spectra which could be distinguished by their different absorbance maxima; <em>4</em>29-<em>4</em>30 nm and <em>4</em>25-<em>4</em>26 nm respectively. Compound <em>4</em>, which possesses both types of functional group, produced a spectrum which resembled that of imidazole compounds, indicating that the imidazole moiety had a higher affinity than the triazole for the haem of cytochromes P-<em>4</em>50 present in microsomes. The test compounds differentially inhibited regio- and stereo-specific testosterone metabolism and the pattern of inhibition varied with the 1-substituent on the azole ring. Ketoconazole was a potent inhibitor of testosterone 6 beta-hydroxylation (IC50 0.08 microM) but was considerably less active against other hydroxylations and 17 beta-oxidation to <em>androstenedione</em> (IC50 range 13 to greater than 100 microM). In contrast, tioconazole (IC50 range 0.18 to 3.3 microM) and miconazole (IC50 range 0.15 to 10 microM) were relatively non-selective. Compounds 1 and 2, which differed from each other only in the type of azole ring, were most active against 16 beta-hydroxylation. The triazole analogue (compound 2) was a significantly more potent inhibitor of 16 beta-hydroxylation than the imidazole (compound 1), equipotent against <em>androstenedione</em> formation and less active against the other hydroxylations. Two relatively polar bis-azole analogues (compounds 3 and <em>4</em>) were most active against <em>androstenedione</em> formation; however, in general they were less inhibitory than the lipophilic azoles. We conclude that azole antifungal agents of differing structure show different patterns of selective interaction with cytochromes P-<em>4</em>50, a phenomenon primarily dependent on the 1-substituent on the azole ring, but also modulated to a lesser extent by the type of azole ring (imidazole or triazole).

Women with polycystic ovary syndrome (PCOS) have reduced GnRH sensitivity to suppression by ovarian steroids, which can be ameliorated by androgen blockade. We studied nine PCOS women and nine controls to determine whether metformin could change feedback inhibition by estradiol (E(2)) and progesterone (P). LH was measured every 10 min, and FSH, E(2), P, and testosterone (T) were measured every 2 h. Frequently sampled iv glucose tolerance test was performed at the end of each admission. After the first admission, metformin (500 mg, three times a day) was started. The second admission occurred on d 8-11 of the next menstrual cycle in controls and on d 28 in PCOS patients. Patients subsequently took E(2) and P for 1 wk until the third admission. At baseline, PCOS women had higher T, free T, <em>androstenedione</em>, and estrone. After <em>4</em> wk of metformin, controls had a slight reduction in total T, but free T was unchanged. However, PCOS patients had reduced insulin, T, and E(2), and increased LH mean/amplitude and FSH. After ovarian steroids, controls had a greater reduction in LH pulse frequency than PCOS (61 vs. 25%). These results suggest that the beneficial effects of metformin on ovulatory function in obese PCOS women are probably not mediated by enhanced hypothalamic sensitivity.

Tumor necrosis factor-alpha (TNF) blocks LH-stimulated <em>androstenedione</em> production by immature rat theca-interstitial cells (TIC) in vitro. The mechanism for TNF inhibition of LH-induced <em>androstenedione</em> is unknown and was investigated. LH stimulation of <em>androstenedione</em> synthesis in TIC is mediated via a cAMP-dependent signaling pathway. LH-stimulated cAMP in TIC-conditioned medium was reduced in a biphasic manner by TNF at 1 and <em>4</em>8 h, but not at <em>4</em> and 2<em>4</em> h. To determine whether inhibition of cAMP resulted from TNF interference of LH binding, TIC were given TNF for 2<em>4</em> and <em>4</em>8 h, and LH binding was determined. TNF inhibited LH binding at 2<em>4</em> and <em>4</em>8 h. Scatchard analysis revealed a TNF-induced decrease in LH receptor number without altered affinity. TIC were given TNF and cAMP analogs [N6-benzoyl-cAMP, 8-thiomethyl-cAMP, 8-(6-aminohexyl)amino-cAMP, and N6-2'-O-(Bu)2cAMP], which selectively activate cAMP-dependent protein kinase (PKA) type I and/or PKA type II, respectively. At <em>4</em>8 and 96 h, TNF blocked <em>androstenedione</em> production stimulated by all combinations of cAMP analogs; however, <em>androstenedione</em> synthesis recovered by <em>4</em>8 h after removal of TNF. Peak PKA activity in TIC was observed at 30 min in the presence of LH or cAMP analogs. LH- or cAMP analog-directed PKA activity was inhibited after concomitant exposure to TNF; however, a 2<em>4</em>-h pretreatment with TNF did not affect cAMP analog-stimulated PKA activity. The results indicate that in the modulation of steroidogenesis, TNF acts at multiple sites in the PKA pathway. First, TNF suppresses LH-stimulated cAMP production by TIC. Secondly, inhibition of cAMP may result from TNF attenuation of LH binding, and thirdly, TNF inhibits PKA activity of TIC and, thus, attenuates <em>androstenedione</em> production.

Twelve site-directed mutants of rat cytochrome P<em>4</em>50 2B1 distributed over seven positions and four putative substrate recognition sites (SRS) were constructed and expressed in COS cells. Function was examined using <em>androstenedione</em> and testosterone as substrates. Substitutions at positions 303, 360, and <em>4</em>73 did not markedly affect the regio- or stereoselectivity of androgen metabolism, whereas mutants in positions 206 (SRS-2), 302 (SRS-<em>4</em>), and 363 and 367 (SRS-5) exhibited markedly different steroid metabolite profiles compared with parental P<em>4</em>50 2B1. In particular, the Phe-206->>Leu substitution conferred androgen 6 alpha- and testosterone 7 alpha-hydroxylase activities, and the Thr-302->>Ser substitution suppressed androgen 16 beta-hydroxylation in favor of <em>androstenedione</em> 16 alpha- and testosterone 15 alpha-hydroxylation. Replacement of Val-363 or Val-367 with Ala conferred androgen 15 alpha-hydroxylase and 6 beta-hydroxylase activities, respectively, and suppressed susceptibility to mechanism-based inactivation by the P<em>4</em>50 2B1-selective chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. The Val-367->>Ala mutant was also resistant to chloramphenicol itself. The Leu mutant at position 363 exhibited increased specificity for <em>androstenedione</em> and testosterone 16 beta-hydroxylation, whereas the Leu mutant at position 367 exhibited decreased stereospecificity. Most interestingly, the size of key residues identified plays a critical role in governing steroid hydroxylation from the alpha-face or beta-face and hydroxylation on the D-ring or the B-ring.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

Insulin resistance, infertility, and hirsutism, common characteristics of polycystic ovary syndrome (PCOS), improve with even modest weight loss. Optimal dietary treatment for PCOS is not known.

OBJECTIVE

We compared the effects of acute protein administration with those of glucose challenges on hormones related to obesity and insulin resistance (ie, cortisol and insulin), hirsutism [ie, dehydroepiandosterone (DHEA) and androstenedione], and hunger (ie, ghrelin).

METHODS

Patients with PCOS (n = 28; aged 26 +/- 2 y) were tested with a 5-h oral-glucose-tolerance test (OGTT) and a euvolemic, euenergetic protein challenge.

RESULTS

Glucose ingestion caused larger fluctuations in blood glucose and more hyperinsulinemia than did protein (P < 0.01, overall treatment-by-time interaction). During the protein challenge, cortisol and DHEA declined over 5 h. During OGTT, cortisol and DHEA increased after the third hour and began to show significant divergence from protein from the fourth hour (P <or= 0.01). During OGTT, 18 patients who had a blood glucose nadir of <69 mg/dL had elevated cortisol (baseline: 10.4 +/- 0.4; nadir: 5.9 +/- 0.1; peak: 12.7 +/- 0.9 microg/dL) and DHEA (baseline: 15.6 +/- 1.3; nadir: 11.2 +/- 1.0; peak: 24.6 +/- 1.6 ng/mL) (P < 0.01), whereas the remaining 10 patients with a glucose nadir of 76 +/- 2 mg/dL had no increase in adrenal steroids. Both glucose and protein suppressed ghrelin (from 935 +/- 57 to 777 +/- 51 pg/mL and from 948 +/- 60 to 816 +/- 61 pg/mL, respectively). After glucose ingestion, ghrelin returned to baseline by 4 h and increased to 1094 +/- 135 pg/mL at 5 h. After the protein challenge, ghrelin remained below the baseline (872 +/- 60 pg/mL) even at 5 h. The overall treatment effect was highly significant (P < 0.0001).

CONCLUSIONS

Glucose ingestion caused significantly more hyperinsulinemia than did protein, and it stimulated cortisol and DHEA. Protein intake suppressed ghrelin significantly longer than did glucose, which suggested a prolonged satietogenic effect. These findings provide mechanistic support for increasing protein intake and restricting the simple sugar intake in a PCOS diet.

Sex steroid hormones such as estrogens and androgens are involved in the development and differentiation of the breast tissue. The activity and concentration of sex steroids is determined by the availability from the circulation, and on local conversion. This conversion is primarily mediated by aromatase, steroid sulfatase, and 17β-hydroxysteroid dehydrogenases. In postmenopausal women, this is the primary source of estrogens in the breast. Up to 70-80% of all breast cancers express the estrogen receptor-α, responsible for promoting the growth of the tissue. Further, 60-80% express the androgen receptor, which has been shown to have tissue protective effects in estrogen receptor positive breast cancer, and a more ambiguous response in estrogen receptor negative breast cancers. In this review, we summarize the function and clinical relevance in cancer for 17β-hydroxysteroid dehydrogenases 1, which facilitates the reduction of estrone to estradiol, dehydroepiandrosterone to androstendiol and dihydrotestosterone to 3α- and 3β-diol as well as 17β-hydroxysteroid dehydrogenases 2 which mediates the oxidation of estradiol to estrone, testosterone to <em>androstenedione</em> and androstendiol to dehydroepiandrosterone. The expression of 17β-hydroxysteroid dehydrogenases 1 and 2 alone and in combination has been shown to predict patient outcome, and inhibition of 17β-hydroxysteroid dehydrogenases 1 has been proposed to be a prime candidate for inhibition in patients who develop aromatase inhibitor resistance or in combination with aromatase inhibitors as a first line treatment. Here we review the status of inhibitors against 17β-hydroxysteroid dehydrogenases 1. In addition, we review the involvement of 17β-hydroxysteroid dehydrogenases <em>4</em>, 5, 7, and 1<em>4</em> in breast cancer.

Ovarian tissue cryopreservation is the only proven option for fertility preservation in female cancer patients who are prepubertal or require immediate treatment. However it remains unclear which cryopreservation protocol is best in cases where the tissue may contain cancerous cells, as these should be matured in vitro rather than autografted. This study evaluated different cryoprotectant exposure times and whether the addition of synthetic polymers (Supercool X-1000, Z-1000 and polyvinylpyrrolidone [PVP K-12]) to the vitrification solution is beneficial to tissue morphology, cellular proliferation and subsequent in vitro function of secondary follicles. Pieces of macaque (n=<em>4</em>) ovarian cortex were exposed to vitrification solution containing glycerol (25%, v/v) and ethylene glycol (25%, v/v) for 3 or 8 min, without (V3, V8) or with (VP3, VP8) polymers (0.2% [v/v] X-1000, 0.<em>4</em>% Z-1000 and 0.2% PVP). Fresh and vitrified tissues were fixed for histology and phosphohistone H3 (PPH3) analysis, or used for secondary follicle isolation followed by encapsulated 3D culture. Five-week follicle survival and growth, as well as steroid hormones (estradiol [E(2)], progesterone, <em>androstenedione</em>) were measured weekly. Morphology of the stroma and preantral follicles as well as PPH3 expression, was preserved in all vitrified tissues. Vitrification with polymers and shorter incubation time (VP3) increased in vitro follicle survival and E(2) production compared to other vitrified groups. Thus, a short exposure of macaque ovarian tissue to a vitrification solution containing synthetic polymers preserves morphology and improves in vitro function of secondary follicles.

BACKGROUND

The objective of the study was to assess the therapeutic effects of rosiglitazone in overweight women with polycystic ovary syndrome (PCOS).

METHODS

A double-blind, placebo-controlled study was conducted on 30 (BMI>> 25 kg/m2, mean age 29.1 +/- 1.2 years) overweight women with PCOS treated with rosiglitazone or placebo for <em>4</em> months. Waist-to-hip ratios (WHRs), serum concentrations of sex hormones and binding proteins, blood glucose, serum insulin and serum C-peptide during a 75-g oral glucose tolerance test (OGTT), first-phase insulin secretion as determined by an intravenous glucose tolerance test (IVGTT), M values (expressing insulin sensitivity using a euglycaemic clamp) and calorimetric data were assessed at 0 and <em>4</em> months of treatment.

RESULTS

Rosiglitazone improved menstrual cyclicity, increased serum sex hormone-binding globulin (SHBG) levels and decreased serum levels of androstenedione, 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEA-S). Glucose tolerance [expressed as AUC(glucose) during the OGTT] improved (P = 0.002) and peripheral insulin response (expressed as AUC(insulin)) decreased (P = 0.00<em>4</em>) in the rosiglitazone group (ROSI group). M value improved in the ROSI group from 33.<em>4</em> +/- 3.27 to <em>4</em>0.0 +/- 5.51 micromol/kg min (P = 0.0<em>4</em>).

CONCLUSIONS

Rosiglitazone, by improving menstrual cyclicity, hyperandrogenism, insulin resistance and hyperinsulinaemia, represents an alternative treatment for overweight anovulatory women with PCOS and no pregnancy desire.

OBJECTIVE

To compare the efficacy of rosiglitazone and clomiphene citrate (CC) with metformin and CC in women with CC-resistant polycystic ovary syndrome (PCOS).

METHODS

Randomized controlled trial (RCT).

METHODS

A university teaching hospital in Jeddah, Saudi Arabia.

METHODS

Twenty-five women with CC-resistant PCOS.

METHODS

Twelve women were assigned to the rosiglitazone and CC group, and 13 women were assigned to the metformin and CC group for three treatment cycles. The first cycle was started on the first day of the period with either rosiglitazone (<em>4</em> mg twice daily) or metformin (500 mg three times daily) and continued for three cycles. Clomiphene citrate (100 mg) from the third day for 5 days was added to each cycle.

METHODS

Ovulation rate, number of follicles and estradiol (E2) on day 12 of the cycle, pregnancy rate, and changes in fasting glucose, serum insulin, HbA(1C), total testosterone (T), free T, luteinizing hormone (LH), follicle-stimulating hormone (FSH), dehydroepiandrosterone sulfate (DHEAS), delta<em>4</em>-androstenedione (delta<em>4</em>-A), sex hormone-binding globulin (SHBG), insulin-like growth factor (IGF)-1, insulin-like growth factor binding protein (IGFBP)-1, and IGFBP-3.

RESULTS

No significant differences were found in the baseline characteristics of both groups. Ovulation rate was significantly higher in the rosiglitazone and CC group (18 out of 28 cycles [[6<em>4</em>.3%]]) than the metformin and CC group (12 out of 33 cycles [[36.<em>4</em>%]]) (P=.035). Similarly, statistically significant differences were found in the number of follicles>> or =1<em>4</em> mm in the rosiglitazone and CC group (2.2 +/- 1) compared with the metformin and CC group (1.1 +/- 0.9) (P=.02) and E2 on day 12 of the cycle in the rosiglitazone and CC group (1,991 +/- 1,389 pmol/L) compared with the metformin and CC group (5<em>4</em>8 +/- 327) (P<.001). The pregnancy rate was also higher in the rosiglitazone and CC group (6 out of 12 [[50%]] women) than the metformin and CC group (5 out of 13 [[38.5%]] women), but did not reach statistical significance (P=.58). Both groups showed no significant changes in fasting plasma glucose or HbA(1C) or IGFBP-3 values. However, in both groups, fasting serum insulin, total T, free T, LH, DHEA-S, delta<em>4</em>A, and IGF-1 levels decreased significantly, and SHBG and IGFBP-1 exhibited significant increases.

CONCLUSIONS

These findings suggest that short-term use of rosiglitazone and CC is more efficacious than metformin and CC in ovulation induction in women with CC-resistant PCOS.

We have studied the effects of human interleukin-1 beta on steroidogenesis in cultured immature rat Leydig cells. In the presence of low concentrations of LH or in its absence interleukin-1 beta markedly stimulates the production of C19-steroids (testosterone and <em>androstenedione</em>) and C21-steroids (progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxypregn-<em>4</em>-en-3-one). In the presence of maximally effective concentrations of LH, on the contrary, interleukin-1 beta inhibits C19-steroid production by provoking a block at the level of the 17,20-desmolase. These actions were observed with similar low doses of interleukin-1 beta (ED50 = 1 U/ml), but the stimulatory effects are evident within the first 2 h of incubation whereas the inhibitory actions appeared after a latent period of 6 h. None of the effects of interleukin-1 beta is accompanied by measurable changes in cAMP output, and the effects are much less pronounced in freshly isolated Leydig cells than in cultured cells. At maximally effective doses the effects of interleukin-1 beta are additive with those of a number of other Leydig cell agonists: LHRH, epidermal growth factor, arginine vasopressin and Sertoli cell-derived factor(s), suggesting that these agonists act by mechanisms different from that of interleukin-1 beta. The possibility is considered that Leydig cells may act as target cells for interleukin-1 beta derived from testicular macrophages or for interleukin-1-like factors derived from testicular tubules.

OBJECTIVE

The aim of the current study is to investigate the influence of Tribulus terrestris extract on androgen metabolism in young males.

METHODS

Twenty-one healthy young 20-36 years old men with body weight ranging from 60 to 125 kg were randomly separated into three groups-two experimental (each n=7) and a control (placebo) one (n=7). The experimental groups were named TT1 and TT2 and the subjects were assigned to consume 20 and 10 mg/kg body weight per day of Tribulus terrestris extract, respectively, separated into three daily intakes for <em>4</em> weeks. Testosterone, <em>androstenedione</em> and luteinizing hormone levels in the serum were measured 2<em>4</em> h before supplementation (clear probe), and at 2<em>4</em>, 72, 2<em>4</em>0, <em>4</em>08 and 576 h from the beginning of the supplementation.

RESULTS

There was no significant difference between Tribulus terrestris supplemented groups and controls in the serum testosterone (TT1 (mean+/-S.D.: 15.75+/-1.75 nmol/l); TT2 (mean+/-S.D.: 16.32+/-1.57 nmol/l); controls (mean+/-S.D.: 17.7<em>4</em>+/-1.09 nmol/l) (p>0.05)), <em>androstenedione</em> (TT1 (mean+/-S.D.: 1.927+/-0.126 ng/ml); TT2 (mean+/-S.D.: 2.026+/-0.256 ng/ml); controls (mean+/-S.D.: 1.952+/-0.236 ng/ml) (p>0.05)) or luteinizing hormone (TT1 (mean+/-S.D.: <em>4</em>.662+/-0.27<em>4</em>U/l); TT2 (mean+/-S.D.: <em>4</em>.103+/-0.869U/l); controls (mean+/-S.D.: <em>4</em>.170+/-0.<em>4</em>06U/l) (p>0.05)) levels. All results were within the normal range. The findings in the current study anticipate that Tribulus terrestris steroid saponins possess neither direct nor indirect androgen-increasing properties. The study will be extended in the clarifying the probable mode of action of Tribulus terrestris steroid saponins.

Serum sex hormone-binding globulin (SHBG), testosterone, non-SHBG-bound testosterone, <em>androstenedione</em>, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and cortisol were measured in 58 homosexual men seropositive for human immunodeficiency virus (HIV), all clinically asymptomatic (Centers for Disease Control 1993 classification stage A). The HIV patients were divided into four groups according to the CD<em>4</em> lymphocyte count--group 1 (more than 500/microliters, N = 1<em>4</em>), group 2 (between 350 and 500/microliters, N = 16), group 3 (between 200 and 3<em>4</em>9/microliters, N = 22) and group <em>4</em> (less than 200/microliters, N = 6)--and compared with 11 antibody-negative men as controls. The SHBG levels were significantly increased in groups 1, 2, 3 (p < 0.01) and <em>4</em> (p < 0.05) compared with controls, with no differences between groups of patients. Compared with controls, testosterone concentrations were significantly lower in group <em>4</em> (p < 0.05) and non-SHBG-bound testosterone levels were significantly lower in groups 1 (p < 0.05), 2 (p < 0.01), 3 (p < 0.001) and group <em>4</em> (p < 0.001); DHT and <em>androstenedione</em> levels were significantly lower in group <em>4</em> (p < 0.05) and DHEA levels were significantly lower in group 2, group 3 (p < 0.01) and group <em>4</em> (p < 0.05) than in controls. Cortisol levels were significantly increased in groups 1 and <em>4</em> (p < 0.05) and FSH and LH concentrations were not significantly higher in HIV-infected men than in controls. Also, the DHEA, <em>androstenedione</em>, non-SHBG-bound testosterone and DHT levels were correlated with CD<em>4</em> cell counts, showing that hypogonadism occurs as the CD<em>4</em> lymphocytes decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis. Follicles consist of oocytes and two primary steroidogenic cell types, the granulosa cells, and the theca cells. Immortalized human granulosa cells are essential for researching the mechanism of steroidogenesis and folliculogenesis. We obtained granulosa cells from a 35-yr-old female and immortalized them by lentivirus-mediated transfer of several genes so as to establish a human nonluteinized granulosa cell line (HGrC1). We subsequently characterized HGrC1 and investigated its steroidogenic performance. HGrC1 expressed enzymes related to steroidogenesis, such as steroidogenic acute regulatory protein, CYP11A, aromatase, and gonadotropin receptors. Stimulation with FSH increased the mRNA levels of aromatase, which consequently induced the aromatization of <em>androstenedione</em> to estradiol. Activin A increased the mRNA levels of the FSH receptor, which were synergistically up-regulated with FSH stimulation. HGrC1 also expressed a series of ligands and receptors belonging to the TGF-β superfamily. A Western blot analysis showed that bone morphogenetic protein (BMP)-<em>4</em>, BMP-6, and BMP-7 phosphorylated small mother against decapentaplegic (Smad)1/5/8, whereas growth differentiation factor-9 phosphorylated Smad2/3. BMP-15 and anti-Müllerian hormone phosphorylated Smad1/5/8 while also weakly phosphorylating Smad2/3. These results indicate that HGrC1 may possess the characteristics of granulosa cells belonging to follicles in the early stage. HGrC1 might also be capable of displaying the growth transition from a gonadotropin-independent status to gonadotropin-dependent one.

Dairy cattle are susceptible to heat stress-induced reductions in fertility; however, direct effects of hyperthermia on specific reproductive functions are difficult to determine in vivo. The objective of this experiment was to examine the effect of elevated temperature in vitro on follicular steroidogenesis, to gain insight into specific follicular responses associated with heat stress. Dominant follicles were obtained from Holstein heifers on day 6 post-estrus (luteal phase; n = <em>4</em>) or day 8, 36 h after an injection with 25 mg PGF(2alpha) to induce regression of the corpus luteum (follicular phase; n = <em>4</em>). Pieces of follicle wall were isolated from dominant follicles and cultured for 96 h with 0, 2 or 100 ng/ml LH or FSH at 37, 39 or <em>4</em>1 degrees C. Concentrations of <em>androstenedione</em>, estradiol and progesterone were determined in culture media collected every 2<em>4</em>h. During the last <em>4</em>8 h of culture, basal secretion of <em>androstenedione</em> and estradiol by pieces of follicle wall was lower at <em>4</em>1 degrees C than at 37 or 39 degrees C (P < 0.05). In contrast, cumulative secretion of progesterone by pieces of follicle wall in medium alone was higher at <em>4</em>1 degrees C than at 37 or 39 degrees C (P < 0.05). Pieces of follicle wall responded to treatment with both low (2 ng/ml) and high (100 ng/ml) doses of gonadotropins at all temperatures. However, gonadotropin-induced secretion of <em>androstenedione</em> and estradiol was generally lower, whereas gonadotropin-induced secretion of progesterone was higher at <em>4</em>1 degrees C and sometimes at 39 than at 37 degrees C. The changes in basal steroidogenesis and in responses to gonadotropins suggest that follicular cells begin to luteinize at elevated temperatures in vitro. Premature luteinization of follicular cells in vivo has been associated with reduced fertility in cattle with persistent follicles, suggesting that the premature differentiation of follicular cells observed in the current study may be responsible, in part, for the reduced fertility of dairy cattle under heat-stressed conditions.

The prevalence of male reproductive disorders and testicular cancer is steadily increasing. Because the exposure to chemicals disrupting natural hormone action has been associated with these diseases, it is important to identify endocrine disrupting chemicals (EDCs) and their targets of action. Here, a 3D-structural database that can be applied for virtual screening approaches to facilitate the identification of EDCs was constructed. The database was screened using pharmacophores of 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), which catalyzes the last step of testosterone synthesis in testicular Leydig cells and plays an essential role during male sexual development. Among other chemicals, benzophenone (BP) UV-filters were predicted as potential 17beta-HSD3 inhibitors. Biological analyses revealed (2,<em>4</em>-dihydroxyphenyl)-phenylmethanone (also known as benzophenone-1, BP-1) as an inhibitor of human 17beta-HSD3 (IC(50) 1.05microM). BP-1 also efficiently blocked conversion of <em>androstenedione</em> to testosterone by mouse and rat 17beta-HSD3 in whole-organ enzyme assays. Moreover, BP-1 antagonized the testosterone-dependent activation of androgen receptors (IC(50) 5.7microM), suggesting synergistic anti-androgenic effects of BP-1 by preventing testosterone formation and blocking receptor activation. In addition, analyses of several commonly used UV-filters on estrogen- and androgen-metabolizing 17beta-HSD enzymes revealed 3-benzylidene camphor (3-BC) and <em>4</em>-methylbenzylidene camphor (<em>4</em>-MBC) as low micromolar 17beta-HSD2 inhibitors. In conclusion, screening of virtual chemical structure libraries can facilitate the identification of compounds interfering with hormone action. The potential disruption of 17beta-HSD enzyme function by the UV-filters BP-1, 3-BC and <em>4</em>-MBC requires further investigation and should be considered for safety assessment of these chemicals.

A comparative approach was taken in this study to evaluate androgen (<em>androstenedione</em> and testosterone) metabolism in three invertebrate species: the gastropod Marisa cornuarietis, the amphipod Hyalella azteca, and the echinoderm Paracentrotus lividus. The existence of 17beta/3beta-hydroxysteroid dehydrogenase (HSD) and 5alpha-reductase catalyzed reactions was demonstrated in all three species. <em>Androstenedione</em> was primarily converted to 5alpha-androstanedione in M. cornuarietis, while it was primarily metabolized to testosterone in P. lividus and H. azteca. In addition, and consistent with vertebrate findings, tissue specific pathways and sexual dimorphism in androgen metabolism were observed. Namely, testosterone was metabolized to dihydrotestosterone in P. lividus gonads (via 5alpha-reductase), and metabolized to <em>4</em>-androstene-3beta,17beta-diol in the digestive tube (via 3beta-hydroxysteroid dehydrogenase). Furthermore, the synthesis of 17beta-reduced metabolites of <em>androstenedione</em> (testosterone and dihydrotestosterone) was 3- to <em>4</em>-fold higher in males of M. cornuarietis than in females. Organotin compounds, which have been shown to interfere with some aspects of androgen metabolism, had no major effect on testosterone metabolism in any of the three species. Fenarimol enhanced 5alpha-reductase-mediated catalysis in gonads of P. lividus. Overall, results demonstrate the ubiquity of some androgen biotransformation processes in invertebrates and reveals interphyla differences in androgen metabolic pathways, and different sensitivity of these pathways to some xenobiotics.

Groups of Atlantic cod and rainbow trout were treated (ip) with beta-naphthoflavone (BNF), phenobarbital, or peanut oil (controls), and properties of the hepatic xenobiotic and steroid metabolizing enzyme systems were evaluated. In both species, BNF treatment resulted in significant induction of microsomal 7-ethoxycoumarin O-deethylase, 7-ethoxyresorufin O-deethylase, biphenyl <em>4</em>-hydroxylase, and phenanthrene oxidation, especially at the 1,2-position. Immunochemical studies with rabbit IgG prepared against the major BNF-inducible cytochrome P-<em>4</em>50 in cod, P-<em>4</em>50c, revealed increased amounts of immunoreactive protein in liver slices from both species after BNF treatment. The molecular weight of the induced protein was approximately 58,000 Da, as shown by Western blotting. When titrating biphenyl <em>4</em>-hydroxylation, however, the antibodies distinguished between the two species, inhibiting the activity of BNF-induced cod 90% and that of rainbow trout <em>4</em>0% at 10 mg IgG/nmol P-<em>4</em>50. Furthermore, cytochrome b5 content and UDP-glucuronyltransferase activity were significantly induced only in rainbow trout, whereas the specific content of cytochrome P-<em>4</em>50 was significantly increased only in cod. Differences between the two species were observed in the levels of constitutive activities, the amount of induction, and in the regioselectivity of phenanthrene oxidation and <em>androstenedione</em> metabolism. Treatment with phenobarbital showed no effect on any of the parameters investigated in either species. The results show that although there are many common features of the hepatic xenobiotic and steroid biotransformation systems of the two teleosts, certain species characteristics exist in constitutive properties and induction responses.