The early events of steroidogenesis, following adrenocorticotropin (ACTH) stimulation, were investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated <em>4</em>-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide, even though cytochrome P-<em>4</em>50scc only increases after 12 h. Mitochondrial free cholesterol levels increased during the same time period (16.5 to 25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. It is concluded that early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial free cholesterol. The maximum rate of cholesterol transport to mitochondria in aminoglutethimide-blocked cells (8.6 micrograms/mg of protein/h) was consistent with both the maximum rate of mitochondrial cholesterol SCC and cellular steroidogenesis (6.0 micrograms of pregnenolone/mg/h and 5.5 micrograms of steroid/mg of mitochondria/h, respectively). Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-<em>4</em>50scc without affecting mitochondrial free cholesterol. The distribution of steroid products fell into three distinct phases during a 2<em>4</em>-h period following ACTH stimulation: an initial increase in SCC activity (0-<em>4</em> h), elevation of <em>androstenedione</em> in place of corticosterone (<em>4</em>-12 h), and then in place of cortisol (12-2<em>4</em> h). The changes from <em>4</em> to 2<em>4</em> h result from a progressive stimulation by ACTH of 17 alpha-hydroxylase activity (but not 21-hydroxylase or C17:20 lyase activities) that is maintained even when stimulation of total steroidogenesis has stopped.

Neonatal L-monosodium glutamate (MSG) administration in rats induces several neuroendocrine and metabolic disruptions. Leptin, the adipocyte product, modulates several neuroendocrine systems including the hypothalamic-pituitary-gonadal (HPG) axis in mammals. The aim of the present study was to determine whether MSG-induced chronic hyperleptinemia could play any relevant role in the hypogonadism developed by male rats when examined in adulthood. We found that 120-day-old MSG male rats displayed significant hyperleptinemia, hypogonadism, and undisturbed basic testis structure and spermatogenesis. In vitro studies in purified Leydig cells from normal (CTR) and MSG-damaged rats revealed that basal and human chorionic gonadotropin (hCG)-stimulated 17-hydroxy-progesterone (17-HO-P(<em>4</em>)), Delta(<em>4</em>)-<em>androstenedione</em> (Delta(<em>4</em>)A) and testosterone (T) secretions were significantly lower in MSG than in CTR cells. Exposure to murine leptin (Mleptin, 10(-8)M) significantly inhibited hCG-elicited T secretion by CTR cells after 180 min incubation. While Mleptin significantly inhibited hCG-stimulated Delta(<em>4</em>)A output and the Delta(<em>4</em>)A:17-OH-P(<em>4</em>) ratio of secretion, conversely, it failed to modify the ratio T:Delta(<em>4</em>)A release by CTR Leydig cells. Interestingly, the effects of Mleptin found on CTR Leydig cells were absent in MSG Leydig cells. Finally, endogenous hyperleptinemia was associated with a significant decrease in Leydig cell expression of Ob-Rb mRNA in MSG rats. In summary, this study demonstrates that: (1) Mleptin inhibited testicular steroidogenesis in CTR rats; (2) MSG-treated rats showed lower in vitro 17-OH-P(<em>4</em>), Delta(<em>4</em>)A and T production under basal and post-hCG stimulation conditions; (3) purified Leydig cells from MSG-treated rats displayed resistance to the inhibitory action of Mleptin on T release, and (<em>4</em>) endogenous leptin exerts a modulatory effect on Leydig cell Ob-Rb mRNA expression. The inhibitory effect of leptin on testicular function is thus abrogated in MSG-damaged rats. The testicular leptin-resistance developed by MSG rats seems to be due to early chronic exposure of Leydig cells to high leptin circulating levels, which in turn down-regulate testicular Ob-Rb expression. It remains to be determined whether the testicular dysfunction of MSG rats can be reversed after correction of hyperleptinemia or whether it is an irreversible effect of the hypothalamic lesion.

The 17beta-hydroxysteroid dehydrogenase (17betaHSD) enzyme system governs important redox reactions at the C17 position of steroid hormones. Different 17betaHSD types (no. 1-<em>4</em>) have been identified to date in peripheral human tissues, such as placenta, testis, and breast. However, there is little information on their expression and activity in either normal or malignant prostate. In the present work, we have inspected pathways of 17beta-oxidation of either androgen or estrogen in human prostate cancer cells (LNCaP, DU1<em>4</em>5, and PC3) in relation to the expression of messenger RNAs (mRNAs) for 17betaHSD types 1-<em>4</em>. These cell systems feature distinct steroid receptor status and response to hormones. We report here that high expression levels of 17betaHSD<em>4</em> were consistently observed in all three cell lines, whereas even greater amounts of 17betaHSD2 mRNA were detected solely in PC3 cells. Neither 17betaHSD1 nor 17betaHSD3 mRNAs could be detected in any cell line. From a metabolic standpoint, intact cell analysis showed a much lower extent of 17beta-oxidation of both androgen [testosterone (T)] and estrogen [estradiol (E2)] in LNCaP and DU1<em>4</em>5 cells compared to PC3 cells, where a greater precursor degradation and higher formation rates of oxidized derivatives (respectively, <em>androstenedione</em> and estrone) were observed. Using subcellular fractionation, we have been able to differentiate among 17betaHSD types 1-<em>4</em> on the basis of their distinct substrate specificities and subcellular localization. This latter approach gave rise to equivalent results. PC3 cells, in fact, displayed a high level of microsomal activity with a low E2/T activity ratio and approximately equal apparent Km values for E2 and T, suggesting the presence of 17betaHSD2. Dehydrogenase specific activity with both E2 and T was also detected, although at lower levels, in LNCaP and DU1<em>4</em>5 cells. No evidence for reductase activity could be obtained in either the soluble or microsomal fraction of any cell line. As comparable expression levels of 17betaHSD<em>4</em> were seen in the three cell lines, 17betaHSD2 is a likely candidate to account for the predominant oxidative activity in PC3 cells, whereas 17betaHSD<em>4</em> may account for the lower extent of E2 oxidation seen in both LNCaP and DU1<em>4</em>5 cells. This is the first report on the expression of four different 17betaHSD types in human prostate cancer cells. It ought to be emphasized that for the first time, analysis of different 17betaHSD activities in either intact or fractionated cells harmonizes with the expression of relevant mRNAs species.

The inhibitory activity of aminoglutethimide, <em>4</em>-hydroxy<em>androstenedione</em>, 7 alpha-(<em>4</em>'-amino)phenylthio<em>androstenedione</em>, and cyanoketone (2 alpha-cyano-<em>4</em>,<em>4</em>,17 alpha-trimethyl-17 beta-hydroxy-5-androstene-3-one) on androgen aromatization by human mammary tumors was examined. <em>Androstenedione</em> and dehydroepiandrosterone were incubated with mammary tumor homogenates in the presence of a reduced nicotinamide adenine dinucleotide phosphate-generating system with or without inhibitor. All four compounds were found to be equally effective in inhibiting estrogen synthesis from dehydroepiandrosterone, but only aminoglutethimide, <em>4</em>-hydroxy<em>androstenedione</em>, and 7 alpha-(<em>4</em>'-amino)phenylthio<em>androstenedione</em> were capable of inhibiting <em>androstenedione</em> aromatization. Inhibitions of androgen aromatization ranging between 81 and 97% were found and were essentially similar for both estrogen receptor-negative and estrogen receptor-positive tumors. Kinetic analysis showed 7 alpha-(<em>4</em>'-amino)phenylthio<em>androstenedione</em> to be the most effective inhibitor with apparent Ki of 0.03<em>4</em> microM, followed by aminoglutethimide (Ki 0.26 microM) and <em>4</em>-hy droxy<em>androstenedione</em> (Ki 01.<em>4</em>7 microM) using <em>androstenedione</em> as a substrate. These results are discussed in relation to the significance of estrogen synthesis in mammary tumors.

We have measured microsomal steroid aromatase activity in the fetal component of ovine placental cotyledons collected from pregnant ewes between 12<em>4</em> days and 127 days of gestation. Aromatase activity was determined by quantifying the [3H]water by-product when [1 beta-3H(N)] <em>androstenedione</em> was used as substrate. The mean microsomal aromatase activity (+/- SD) was 5.7 +/- 2.2 pmol.min-1.mg protein-1 (n = 12) and was 9% of the aromatase activity of human placental microsomes [mean (+/- SD) of 66.1 +/- 25.0 pmol.min-1.mg protein-1 (n = 7)]. The apparent Km for ovine placental aromatase for <em>androstenedione</em>, at pH 7.<em>4</em> and 37 degrees C, was 50 nM while the Vmax was 20.6 pmol.min-1.mg protein-1. The respective concentrations effecting 50% inhibition of ovine placental aromatase activity (the I50) for econazole, <em>4</em>-hydroxy<em>androstenedione</em>, imazalil, miconazole, ketoconazole and aminoglutethimide were 0.03, 0.05, 0.15, 0.50, 5.0 and 5.5 microM. The order of relative potencies were similar to those obtained for human placental aromatase. Ketoconazole and aminoglutethimide were approx 10 times more potent inhibitors of the sheep enzyme relative to the human. Aromatase activity was not confined to the microsomal fraction of ovine placental tissue but was distributed throughout all the particulate subcellular fractions. The proportionally high activity of the tissue homogenate (1.75 pmol.min-1.mg protein-1) is suggestive that in the last third of pregnancy, aromatase is not rate limiting with regard to placental estrogen production. It would appear, therefore, that the major factor regulating placental estrogen synthesis in ovine pregnancy is the availability of substrate.

BACKGROUND

Adrenal hyperandrogenism affects approximately 25% of polycystic ovary syndrome (PCOS) patients but its relation to obesity is not totally understood.

OBJECTIVE

This study aimed to assess dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) levels in relation to body mass index (BMI) in PCOS.

METHODS

This was a prospective observational study at an institutional practice at an obstetrics/gynecology hospital.

METHODS

The study included 136 PCOS patients, 20-35 years old, and <em>4</em>2 age-matched control women. The participants were classified with the BMI cutoff value of 27 kg/m(2) as follows: 1) high-BMI PCOS patients; 2) low-BMI PCOS patients; 3) high-BMI control women; and <em>4</em>) low-BMI control women. The data were reanalyzed with the BMI cutoff value of 30 kg/m(2) to corroborate the findings in obese and nonobese patients.

METHODS

Blood samples were taken and LH, FSH, insulin, T, androstenedione (A<em>4</em>), DHEA, DHEAS, and glucose levels were determined. Homeostatic model assessment was calculated. Pelvic and abdominal ultrasound for ovarian morphology and adipose tissue, respectively, were performed.

RESULTS

Obese PCOS patients presented significantly more insulin resistance than nonobese PCOS patients. The LH levels and LH/FSH ratio were significantly higher in low-BMI than in high-BMI PCOS patients. The A<em>4</em> and DHEAS levels were significantly higher in nonobese than in obese PCOS patients. A significant correlation between LH and A<em>4</em> in nonobese PCOS patients was observed. The frequency of hyperandrogenism by increased A<em>4</em>, and DHEA along with DHEAS was significantly higher in low-BMI PCOS patients compared with high-BMI PCOS patients. Some findings observed with the BMI cutoff value of 27 kg/m(2) changed with the cutoff value of 30 kg/m(2).

CONCLUSIONS

Low BMI more so than high BMI is associated with increased LH, high A<em>4</em>, DHEA, and DHEAS levels in PCOS patients. The BMI cutoff value of 27 kg/m(2) classified better than 30 kg/m(2) for hormonal and metabolic characteristics.

The multi-tyrosine kinase inhibitor sunitinib is used in the treatment of several solid tumors. Animal experiments pointed to an adrenotoxic effect of sunitinib. Therefore, we evaluated the expression of key targets of sunitinib in human adrenocortical carcinoma (ACC) tumor samples and investigated its in vitro effects in ACC cell lines. We carried out immunohistochemistry for vascular endothelial growth factor (VEGF) and its receptor (VEGF-R2) in 157 ACC samples and nine normal adrenal glands. VEGF and VEGF-R2 protein were expressed in 72 and 99% of ACC samples, respectively. Using NCI-H295 and SW13 ACC cell lines, we investigated the effects of sunitinib on cell proliferation. Sunitinib reduced dose-dependently cell viability of both NCI-H295 and SW13 cells (SW13: 0.1 μM 96 ± 7%, 1 μM 90 ± 9%*, 5 μM 62 ± 6%*, controls 100 ± 9%; *p < 0.05). To determine sunitinib effects on steroidogenesis, we measured steroid hormones in cell culture supernatant by gas chromatography-mass spectrometry. We observed a pronounced decrease of cortisol secretion (1 μM 90.1 ± 1.5%*, 5 μM 57.2 ± 0.3%*, controls 100 ± 2.<em>4</em>%) and a concomitant increase in the DHEA/<em>4</em>-<em>androstenedione</em> and 17-hydroxypregnenolone/17-hydroxyprogesterone ratios, indicating specific inhibition of 3β-hydroxysteroid dehydrogenase (HSD3B2). In yeast microsomes transformed with HSD3B2, no direct inhibition of HSD3B2 by sunitinib was detected. Sunitinib induced down-regulation of HSD3B2 mRNA and protein in ACC cell lines (mRNA: 1 μM <em>4</em><em>4</em> ± 16%*; 5 μM 22 ± 2%*; 10 μM 19 ± <em>4</em>%*; protein: 1 μM 82 ± 8%; 5 μM 63 ± 8%*; 10 μM 55 ± 9%*). CYP11B1 was down-regulated at mRNA but not at protein level and CYP11A1 remained unchanged. In conclusion, target molecules of sunitinib are expressed in the vast majority of ACC samples. Sunitinib exhibits anti-proliferative effects in vitro, and appears to specifically block adrenal steroidogenesis by down-regulation of HSD3B2, rendering it a promising option for treatment of ACC.

To evaluate the effect of growth hormone on testicular response to human chorionic gonadotropins (hCG) in vivo in humans, we selected patients with combined deficits of GH and gonadotropins who were in substitution treatment with both GH (from the time of diagnosis) and gonadotropins (from the time of induction of puberty). Testicular response to gonadotropin therapy was then evaluated during and six months after the cessation of GH treatment. Blood samples were collected before and 2, <em>4</em> and 6 days after hCG administration. hCG responses were calculated and expressed as the areas under the response curve. We studied four hypogonadotropic patients (aged 18-19 years) with associated GH deficiency. Their gonadotropin treatment consisted of hCG 1,500 IU every six days, and FSH 75 IU every three days. The GH therapy replacement consisted of <em>4</em> IU thrice weekly. Testosterone, <em>androstenedione</em>, 17 alpha-hydroxyprogesterone and estradiol were measured. In all subjects the testosterone area during GH treatment was significantly higher compared to the testosterone area obtained without GH administration (2993 +/- 1091 vs 2310 +/- 751; M +/- SD; p < 0.0<em>4</em>). The <em>androstenedione</em> area followed a similar pattern (708 +/- 377 vs <em>4</em>02 +/- 2<em>4</em>8; M +/- SD; p < 0.05). The 17 alpha-hydroxyprogesterone area, on the contrary, was significantly higher during GH withdrawal (5<em>4</em>2 +/- 307 vs 235 +/- 190; M +/- SD; p < 0.05). As far as the estradiol area is concerned, no significant differences were found (22,860 +/- 10,082 vs 25,697 +/- 13,6<em>4</em>0; M +/- SD). In conclusion, GH administration seems to improve testosterone production induced by human chorionic gonadotropins.(ABSTRACT TRUNCATED AT 250 WORDS)

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria such as Escherichia coli causes uterine inflammation and leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS on steroid production in bovine theca cells at different stages of follicular development. Theca cells isolated from pre- and post-selection follicles (PRFs, <8.5 mm in diameter, and POFs, >8.5 mm in diameter, respectively) of bovine ovaries were exposed to LPS under luteinizing hormone (LH) conditions, estradiol (E2) conditions or both conditions in vitro. Bovine theca cells expressed the LPS receptor gene complex: Toll-like receptor <em>4</em> (TLR<em>4</em>), CD1<em>4</em> and MD2. LPS suppressed progesterone (P<em>4</em>) and <em>androstenedione</em> (A<em>4</em>) production with downregulation of steroidogenic enzyme transcripts when theca cells were stimulated with LH. By contrast, LPS did not affect P<em>4</em> or A<em>4</em> production when theca cells were stimulated with E2. P<em>4</em> and A<em>4</em> production in theca cells from PRFs was suppressed by LPS as early as at <em>4</em>8 h of culture, whereas the effect of LPS on theca cells from POFs was observed at 96 h of culture. The results demonstrate that LPS inhibits steroid production in theca cells under LH conditions. Moreover, theca cells from POFs showed a slower response to LPS compared with that of theca cells from PRFs, which might imply a distinct effect of LPS on follicles at different developmental stages. These findings suggest a possible mechanism of ovarian dysfunction and subsequent infertility in cows with endometritis.

BACKGROUND

Cytochrome P<em>4</em>50 monooxygenases--able to regio- and stereoselectively hydroxylate non-activated carbon atoms--are important enzymes for the synthesis of valuable intermediates in the production of steroid hormones in the pharmaceutical industry. However, up to now only a few bacterial enzymes able to hydroxylate steroids have been reported. CYP15<em>4</em>C5 from Nocardia farcinica IFM 10152, a bacterial P<em>4</em>50 monooxygenase, was previously shown to convert testosterone to 16α-hydroxytestosterone. Since the hydroxylation at 16α-position is of special interest for the pharmaceutical industry, we have studied this enzyme in more detail to investigate its activity and selectivity in bioconversions of further steroids.

RESULTS

CYP15<em>4</em>C5 was coexpressed in Escherichia coli together with putidaredoxin and putidaredoxin reductase from Pseudomonas putida as redox partners for electron transfer and applied in bioconversions of various pregnanes and androstanes [pregnenolone (1), dehydroepiandrosterone (2), progesterone (3), <em>androstenedione</em> (<em>4</em>), testosterone (5) and nandrolone (6)]. Structure elucidation of the formed products revealed an exclusive regio- and stereoselectivity of CYP15<em>4</em>C5, always yielding the corresponding 16α-hydroxylated steroids. Application of whole cells expressing the three components, P<em>4</em>50, Pdx and PdR, in steroid biotransformations resulted in significantly higher conversions and total turnover numbers (TTN) compared to reactions using cell-free extracts. Additionally, considerably higher substrate loads (up to 15 mM) were tolerated by the whole-cell system. Furthermore, turnover numbers (TON) were determined for the six different steroids using whole cells. Thus, testosterone was found to be the worst substrate with a TON of only 0.8 μmol substrate consumed min-1 μmol(-1) CYP15<em>4</em>C5, while progesterone and pregnenolone were converted the fastest resulting in TON of 3.3 μmol substrate consumed min(-1) μmol(-1) CYP15<em>4</em>C5.

CONCLUSIONS

CYP15<em>4</em>C5 from N. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of different steroids as well as its efficient expression in E. coli at high yields. Using this enzyme, 16α-hydroxylated steroids, which are important precursors for the synthesis of high value steroidal drugs in the pharmaceutical industry, can be selectively produced on preparative scale with TTN (μmol substrate consumed μmol(-1) CYP15<em>4</em>C5) exceeding 2000.

BACKGROUND

Large cell calcifying Sertoli cell tumors (LCCSCT) present in isolation or, especially in children, in association with Carney Complex (CNC) or Peutz-Jeghers Syndrome (PJS). These tumors overexpress aromatase (CYP19A1), which leads to increased conversion of delta-<em>4</em>-<em>androstenedione</em> to estrone and testosterone to estradiol. Prepubertal boys may present with growth acceleration, advanced bone age, and gynecomastia.

OBJECTIVE

To investigate the outcomes of aromatase inhibitor therapy (AIT) in prepubertal boys with LCCSCTs.

METHODS

Case series of a very rare tumor and chart review of cases treated at other institutions.

METHODS

Tertiary care and referral center.

METHODS

Six boys, five with PJS and one with CNC, were referred to the National Institutes of Health for treatment of LCCSCT. All patients had gynecomastia, testicular enlargement, and advanced bone ages, and were being treated by their referring physicians with AIT.

METHODS

Patients were treated for a total of 6-60 months on AIT.

METHODS

Height, breast tissue mass, and testicular size were all followed; physical examination, scrotal ultrasounds, and bone ages were obtained, and hormonal concentrations and tumor markers were measured.

RESULTS

Tumor markers were negative. All patients had decreases in breast tissue while on therapy. Height percentiles declined, and predicted adult height moved closer to midparental height as bone age advancement slowed. Testicular enlargement stabilized until entry into central puberty. Only one patient required unilateral orchiectomy.

CONCLUSIONS

Patients with LCCSCT benefit from AIT with reduction and/or elimination of gynecomastia and slowing of linear growth and bone age advancement. Further study of long-term outcomes and safety monitoring are needed but these preliminary data suggest that mammoplasty and/or orchiectomy may be foregone in light of the availability of medical therapy.

Nuclear receptor subfamily <em>4</em> group A member1 (NR<em>4</em>A1), an orphan nuclear receptor, is involved in the transcriptional regulation of thecal cell androgen biosynthesis and paracrine factor insulin-like 3 (INSL3) expression. Androgens are known to play an important regulatory role in ovarian follicle growth. Using a chronically androgenized rat model, a preantral follicle culture model and virus-mediated gene delivery, we examined the role and regulation of NR<em>4</em>A1 in the androgenic control of preantral follicular growth. In the present study, Ki67 staining was increased in preantral follicles on ovarian sections from 5α-dihydrotestosterone (DHT)-treated rats. Preantral follicles from DHT-treated rats cultured for <em>4</em> d exhibited increased growth and up-regulation of mRNA abundance of G(1)/S-specific cyclin-D2 (Ccnd2) and FSH receptor (Fshr). Similarly, DHT (1 μm) increased preantral follicular growth and Ccnd2 and Fshr mRNA abundance in vitro. The NR<em>4</em>A1 expression was high in theca cells and was down-regulated by DHT in vivo and in vitro. Forced expression of NR<em>4</em>A1 augmented preantral follicular growth, <em>androstenedione</em> production, and Insl3 expression in vitro. Inhibiting the action of androgen (with androgen receptor antagonist flutamide) or INSL3 (with INSL3 receptor antagonist INSL3 B-chain) reduced NR<em>4</em>A1-induced preantral follicular growth. Furthermore, NR<em>4</em>A1 overexpression enhanced DHT-induced preantral follicular growth, a response attenuated by inhibiting INSL3. In conclusion, DHT promotes preantral follicular growth and attenuates thecal NR<em>4</em>A1 expression in vivo and in vitro. Our findings are consistent with the notion that NR<em>4</em>A1 serves as an important point of negative feedback to minimize the excessive preantral follicle growth in hyperandrogenism.

Aromatase catalyzes the conversion of androgens to estrogens through a series of monooxygenations to achieve the 19-desmolation and aromatization of the neutral steroid ring-A structure. We have separated two forms of aromatase, a major (P2a) and a minor (P3) form, from human term placenta through solubilization and chromatography. Partially purified aromatase in each form was immunoaffinity chromatographed to give a single band (SDS-PAGE) cytochrome P-<em>4</em>50 of 55 kDa, utilizing a mouse monoclonal anti-human placental aromatase cytochrome P-<em>4</em>50 IgGi (MAb3-2C2) which is capable of suppressing placental aromatase activity. The purified cytochrome P-<em>4</em>50 showed specific aromatase activity of 25-30 nmol/min per mg with Km of 20-30 nM for <em>androstenedione</em> on reconstitution with NADPH-cyt P-<em>4</em>50 reductase and dilauroyl L-alpha-phosphatidylcholine. This one step represents a higher than 100-fold purification with maintenance of the same Km. The stability analysis showed a half-life of more than 5 yr for solubilized aromatase and 2 months for the aromatase cytochrome P-<em>4</em>50 on storage at -90 degrees C. Contrary to the recent claim that estrogen biosynthesis by reconstituted human placental cytochrome P-<em>4</em>50 is by trans-diaxial 1 alpha,2 beta-hydrogen elimination, all of our partially purified forms and reconstituted aromatase synthesized estrogens by cis-1 beta, 2 beta-hydrogen elimination. Use of purified aromatase and [19-3H3, <em>4</em>-1<em>4</em>C]<em>androstenedione</em> led us to discover a metabolic switching by aromatase to 2 beta-hydroxylation of androgen. Results of the MAb3-2C2 suppression of aromatase activity in different species and tissues including human, baboons, horses, cows, pigs and rats indicated the presence of various isozymes of aromatase.

Previous studies have shown a role of d-aspartic acid (d-Asp) in testicular steroidogenesis. Here, we evaluated the effects of d-Asp on androgen production and on expression levels of mRNAs encoding specific steroidogenic key molecules. d-Asp was endogenously present in adult rat testis and its content paralleled to serum luteinizing hormone (LH) and, local and circulating <em>androstenedione</em> and testosterone levels. In vivod-Asp administration induced serum LH release, causing an indirect increase of <em>androstenedione</em> and testosterone levels by enhancing steroidogenic acute regulatory protein (StAR), cytochrome P<em>4</em>50 cholesterol side-chain cleavage enzyme (P<em>4</em>50scc) and 3β-hydroxysteroid dehydrogenase/D5-D<em>4</em> isomerases (3β-HSD) mRNA levels. The direct endocrine role of d-Asp was evaluated using cultured immature Leydig cells (ILCs) obtained from 35days old rats. Cytoplasm and nucleus of ILCs localized d-Asp, while StAR marked the cytoplasm only. After 12h from d-Asp in vitro administration, ILCs resulted intensely d-Asp stained, and StaR protein level, evaluated by Western blotting, significantly increased. After 2<em>4</em>h, significant <em>androstenedione</em> and testosterone syntheses were induced. At molecular level, d-Asp administration significantly increased StAR, P<em>4</em>50scc and 3β-HSD mRNAs at 2, <em>4</em> and 12h, respectively. The temporal shift on relative mRNA expression levels indicated that d-Asp exerted its physiological role through sequential gene cascade activation of those molecules implicated in the synthesis of androgens. Conclusively, our findings demonstrated that d-Asp is a local messenger in testis and give a contribution in understanding the complexity of local endocrine regulation as well as the molecular events leading the acquisition to a steroidogenic competence by ILCs.

The oxidative effect of nicotine was investigated using androgen biomarkers of redox status and wound healing in fibroblasts; using the antioxidant glutathione for confirmation of responses. Cultures of human gingival (HGF) and periosteal fibroblasts (HPF) were incubated with substrates 1<em>4</em>C-testosterone/1<em>4</em>C-<em>4</em>-<em>androstenedione</em> in the presence or absence of serial concentrations of nicotine (N(100-500)), glutathione (G(1-5)) and their combinations, in medium. At 2<em>4</em> h the medium was solvent extracted for metabolites, separated by TLC and quantified using radioisotope scanning. Nicotine caused significant inhibition in yields of the physiologically active metabolite 5α-dihydrotestosterone (DHT) in HGF and HPF, overcome to varying degrees by the anti-oxidant glutathione (n = 6; p<0.01, one way ANOVA); this is suggestive of moderation of an oxidative mechanism induced by nicotine. Down-regulation of 5α-reductase activity by nicotine resulting in reduced yields of DHT was overcome by glutathione. Overcoming oxidative stress in a redox environment is applicable to treatment outcome.

BACKGROUND

Insulin resistance and hyperandrogenism are the cardinal features of polycystic ovary syndrome (PCOS). Women with insulin receptor (INSR) mutations develop severe hyperandrogenism secondary to hyperinsulinaemia. We hypothesised that insulin might drive adipose testosterone generation from androstenedione through aldoketoredutase type 3 (AKR1C3) in women with insulin resistance. Here we assessed the effect of insulin on AKR1C3 activity in vivo and in vitro.

METHODS

Ten women with PCOS, ten healthy controls, and three women with INSR mutations underwent dehydroepiandrosterone (DHEA) challenge; serum androgens were measured every 30 min for 4 h after ingestion of 100 mg DHEA. Additionally, paired subcutaneous and omental fat samples were obtained during abdominal surgery from 38 women. AKR1C3 expression was measured by real-time PCR. Serum steroids and cultured cell media androgens were measured with liquid chromatography tandem mass spectrometry.

RESULTS

Women with PCOS had higher androstenedione concentrations than did controls and women with INSR mutations (p=0·01 and p=0·005, respectively). However, area under the curve for testosterone was higher in women with INSR mutations after DHEA than in women with PCOS and controls (874·2 [SE 242] vs 425 [136] and 375·2 [109], p<0·001 for both). AKR1C3 mRNA expression was significantly higher in subcutaneous than in omental adipose tissue (p=0·004). AKR1C3 expression correlated positively with body-mass index in subcutaneous fat (Spearman correlation=0·51, p=0·006). Insulin significantly increased AKR1C3 expression in differentiated subcutaneous adipocytes (p=0·04). Incubation with insulin significantly increased testosterone generation from androstenedione in cultured subcutaneous cell media compared with controls (p<0·001).

CONCLUSIONS

We have found in-vivo and in-vitro evidence of modulation of AKR1C3 activity by insulin in PCOS and in women with INSR mutations. Insulin seems to drive adipose androgen generation by increasing AKR1C3 activity in female subcutaneous adipose tissue. Selective AKR1C3 inhibition might offer a novel therapeutic target to reduce androgen burden and improve metabolic phenotype in PCOS.

BACKGROUND

Wellcome Trust.

Perfluorinated chemicals (PFCs) disrupt cholesterol homeostasis. All steroid hormones are derived from cholesterol, and steroid hormones such as glucocorticoids and androgenic hormones mediate several vital physiologic functions. However, the in utero effects of PFCs exposure on the homeostasis of these steroid hormones are not well understood in humans.

We examined the relationship between prenatal exposure to perfluorooctane sulfonate (PFOS)/perfluorooctanoate (PFOA) and cord blood levels of glucocorticoid and androgenic hormones.

We conducted a hospital-based birth cohort study between July 2002 and October 2005 in Sapporo, Japan (n = 514). In total, 185 mother-infant pairs were included in the present study. Prenatal PFOS and PFOA levels in maternal serum samples were measured using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Cord blood levels of glucocorticoid (cortisol and cortisone) and androgenic hormones [dehydroepiandrosterone (DHEA) and androstenedione] were also measured in the same way.

We found a dose-response relationship of prenatal PFOS, but not PFOA, exposure with glucocorticoid levels after adjusting for potential confounders. Cortisol and cortisone concentrations were -23.98-ng/mL (95% CI: -0.47.12, -11.99; p for trend = 0.006) and -63.21-ng/mL (95% CI: -132.56, -26.72; p for trend < 0.001) lower, respectively, in infants with prenatal PFOS exposure in the fourth quartile compared with those in the first quartile. The highest quartile of prenatal PFOS exposure was positively associated with a 1.33-ng/mL higher DHEA level compared with the lowest quartile (95% CI: 0.17, 1.82; p for trend = 0.017), whereas PFOA showed a negative association with DHEA levels (quartile 4 vs. quartile 1: -1.23 ng/mL, 95% CI: -1.72, -0.25; p for trend = 0.004). We observed no significant association between PFCs and androstenedione levels.

Our results indicate that prenatal exposure to PFCs is significantly associated with glucocorticoid and DHEA levels in cord blood. Citation: Goudarzi H, Araki A, Itoh S, Sasaki S, Miyashita C, Mitsui T, Nakazawa H, Nonomura K, Kishi R. 2017. The association of prenatal exposure to perfluorinated chemicals with glucocorticoid and androgenic hormones in cord blood samples: the Hokkaido Study. Environ Health Perspect 125:111-118; http://dx.doi.org/10.1289/EHP142.

OBJECTIVE

To evaluate the clinical and endocrine responses of ferrets with adrenocortical disease (ACD) to treatment with a slow-release implant of deslorelin acetate.

METHODS

15 ferrets with ACD.

METHODS

Ferrets were treated SC with a single slow-release, 3-mg implant of deslorelin acetate. Plasma estradiol, androstenedione, and 17-hydroxyprogesterone concentrations were measured before and after treatment and at relapse of clinical signs; at that time, the adrenal glands were grossly or ultrasonographically measured and affected glands that were surgically removed were examined histologically.

RESULTS

Compared with findings before deslorelin treatment, vulvar swelling, pruritus, sexual behaviors, and aggression were significantly decreased or eliminated within 14 days of implantation; hair regrowth was evident 4 to 6 weeks after treatment. Within 1 month of treatment, plasma hormone concentrations significantly decreased and remained decreased until clinical relapse. Mean time to recurrence of clinical signs was 13.7 +/- 3.5 months (range, 8.5 to 20.5 months). In 5 ferrets, large palpable tumors developed within 2 months of clinical relapse; 3 of these ferrets were euthanatized because of adrenal gland tumor metastasis to the liver or tumor necrosis.

CONCLUSIONS

In ferrets with ACD, a slow-release deslorelin implant appears promising as a treatment to temporarily eliminate clinical signs and decrease plasma steroid hormone concentrations. Deslorelin may not decrease adrenal tumor growth in some treated ferrets. Deslorelin implants may be useful in the long-term management of hormone-induced sequelae in ferrets with ACD and in treatment of animals that are considered at surgical or anesthetic risk.

A complete renin-angiotensin system has been shown to be present in human placenta, but its physiological role is poorly known. To investigate the implication of this system in the regulation of steroid hormone secretion, we studied the effect of angiotensin-II on the release of estradiol and progesterone from human placental explants. Our experiments showed that angiotensin-II stimulated estradiol secretion from term placental explants in a dose- and time-dependent fashion, although progesterone release was unaffected. Estradiol release induced by angiotensin-II (0.2 mumol/L) was blocked by angiotensin AT1 receptor antagonist losartan in a dose-dependent manner, suggesting the involvement of the AT1 receptor subtype in the process. On the contrary, the angiotensin AT2 receptor antagonist PD123319 (1 mumol/L) or the angiotensin AT2 receptor agonist CGP<em>4</em>2112A (1 mumol/L) had no effect. Analysis of the amount of steroid hormones in the placental tissues incubated for 12 h showed that angiotensin-II increased estradiol production by 3<em>4</em>% compared with the unstimulated explants, whereas the total levels of the estrogen precursor <em>androstenedione</em> and testosterone were decreased by 30-<em>4</em>5% in the presence of the peptide, suggesting a stimulatory effect on the aromatization step. This hypothesis was reinforced by the absence of effect of angiotensin-II on both estradiol and testosterone concentrations in the placental explants pretreated with the aromatase inhibitor <em>4</em>-hydroxy<em>androstenedione</em> (25 mumol/L). Progesterone synthesis was not affected by angiotensin-II. The present study indicates that angiotensin-II induces the secretion of estradiol from human placenta through the angiotensin AT1 receptor subtype activation, and this effect seems to be linked to the stimulation of local androgen aromatization.

A sensitive ultra-performance liquid chromatography-electrospray tandem mass spectrometry method, combined with solid-phase extraction and silica cartridge cleanup, was established for nine androgens (<em>androstenedione</em>, 19-nor-<em>4</em>-androstene-3,17-diol, androsterone, epiandrosterone, testosterone, methyltestosterone, trenbolone, nandrolone, stanozolol) and nine progestogens (progesterone, 17alpha-hydroxyprogesterone, 21alpha-hydroxyprogesterone, 6alpha-methyl-hydroxyprogesterone, 17alpha,20beta-dihydroxy-<em>4</em>-pregnene-3-one, megestrol acetate, norethindrone, norgestrel, medroxyprogesterone acetate) in environmental waters. For the various water matrices considered, the overall method recoveries were from 78 to 100%, and no apparent signal suppression was found. The method detection limits for the eighteen analytes in the influent, effluent and surface water samples were 0.20-50, 0.0<em>4</em>-20 and 0.01-12 ng/L, respectively. This method was used to analyze the residual androgens and progestogens in the wastewater and surface water samples from Japan, and ten analytes (0.03 (medroxyprogesterone acetate)-1<em>4</em><em>4</em>1 ng/L (androsterone)) were detected in the wastewater samples, and four analytes (0.06 (progesterone)-0.<em>4</em>6 ng/L (<em>androstenedione</em>)) were detected in the surface water samples.

In young men, chronic ingestion of 100 mg <em>androstenedione</em> (ASD), three times per day, does not increase serum total testosterone but does increase serum estrogen and ASD concentrations. We investigated the effects of ASD ingestion in healthy 30- to 56-yr-old men. In a double-blind, randomly assigned manner, subjects consumed 100 mg ASD three times daily (n = 28), or placebo (n = 27) for 28 days. Serum ASD, dihydrotestosterone (DHT), free and total testosterone, estradiol, prostate-specific antigen (PSA), and lipid concentrations were measured at week 0 and each week throughout the supplementation period. Serum total testosterone and PSA concentrations did not change with supplementation. Elevated serum concentrations of ASD (300%), free testosterone (<em>4</em>5%), DHT (83%), and estradiol (68%) were observed during weeks 1-<em>4</em> in ASD (P < 0.05). There was no relationship between age and changes in serum ASD (r2 = 0.02<em>4</em>), free testosterone (r2 = 0.00), or estradiol (r2 = 0.029) concentrations with ASD, whereas the serum DHT response to ASD ingestion was related to age (r2 = 0.2<em>4</em><em>4</em>; P < 0.05). Serum concentrations of high-density lipoprotein cholesterol were decreased by 10% during the supplementation period (P < 0.05). These results suggest that the ingestion of 100 mg ASD, three times per day, does not increase serum total testosterone or PSA concentrations but does elicit increases in ASD, free testosterone, estradiol, and DHT and decreases serum high-density lipoprotein cholesterol concentrations.

Previously, we found that in addition to embryos, the uterine tissues may be a source of estradiol-17beta (E(2)) during early pregnancy in the pig. The aim of the present study was to determine whether porcine endometrium and myometrium secrete androgens - <em>androstenedione</em> (A(<em>4</em>)), testosterone (T) and estrone (E(1)) during early pregnancy and luteolysis (Days 1<em>4</em>-16) in pigs. Individual endometrial and myometrial slices (200 mg) were first pre-incubated (2<em>4</em> h) and then incubated (6 h, 37 degrees C in an atmosphere of 95% O(2) and 5% CO(2)) in the presence or absence of progesterone (P(<em>4</em>); 10(-5) M), oxytocin (OT; 10(-7) M) or OT plus P(<em>4</em>). Basal endometrial and myometrial secretion of A(<em>4</em>) and T did not differ between pregnant and cyclic gilts. Endometrial secretion of E(1) was higher in pregnant than cyclic gilts (p<0.05) while myometrial secretion of E(1) did not differ between the two groups of the examined pigs (p>0.05). Progesterone significantly increased A(<em>4</em>) and T secretion (p<0.001) by uterine tissues regardless of the reproductive status. In the presence of P(<em>4</em>), endometrial and myometrial secretion of E(1) was increased only during luteolysis (p<0.001). In both tissues, OT did not affect the examined steroid secretion and did not change the effect of P(<em>4</em>).

CONCLUSIONS

1) porcine endometrium and myometrium was found to produce A(<em>4</em>), T and E(1) in vitro; 2) basal endometrial and myometrial production of A(<em>4</em>) and T did not differ between the examined reproductive periods; 3) the endometrium released more E(1) during early pregnancy than luteolysis; <em>4</em>) in the presence of substrate (P(<em>4</em>)), uterine tissues increased secretion of A(<em>4</em>) and T during early pregnancy and luteolysis; and 5) P(<em>4</em>) increased uterine production of E(1) only during luteolysis. These data demonstrated the presence of the active steroid pathway in porcine endometrium and myometrium which may serve as an alternative source of androgens and estrogens in pigs.

Interleukin 18 (IL-18) is a pleiotropic pro-inflammatory cytokine and is associated with arrested follicle development and anovulation which are the typical pathological changes of PCOS. Theca cells (TCs) have a key role in follicular growth and atresia. But whether IL-18 can directly affect ovarian TCs function is unknown. Therefore, the objective of this study was to determine the effect of IL-18 on proliferation and steroidogenesis of bovine TCs and to explore the biological effect of IL-18 on folliculogenesis. This work revealed that at 300-1000 pg/mL, IL-18 led to a time- and dose-dependently increase in cell proliferation (P < .05). IL-18 increased 17-hydroxyprogesterone (17OHP<em>4</em>) and <em>androstenedione</em> (A2) secretion with up-regulation of key steroidogenesis-related genes CYP11A1 and CYP17A1 (P < .05). Furthermore, our data demonstrated that the IL-18R protein is predominantly expressed in small-follicle (3-6 mm) TCs than large follicles (8-22 mm) by immunohistochemistry. We also found that the stimulation effects of IL-18 on TCs can be reversed with the addition of IL-18BP as early as at <em>4</em> hours of culture and reached the peak at 16 hours. We conclude that IL-18 appears to target TCs in bovine, and suggest an important role for this cytokine in ovarian function. Present findings further validate potential effects of IL-18 in the conditions associated with follicular dysplasia and excessive growth of ovarian TCs (such as PCOS). But additional research is needed to further understand the mechanism of action of IL-18 in theca cells as well as its precise role in folliculogenesis.

Keywords: IL‐18; bovine; polycystic ovary syndrome; proliferation; steroidogenesis; theca cells.

A series of thiol androgens were synthesized and investigated to characterize structural features important for the inhibition of aromatase. Analogues of <em>androstenedione</em> with thiol groups in either the 2 alpha-, 10 beta-, or 19-positions caused time-dependent inhibition of human placental aromatase. When their KI and kcat values were compared with those of <em>4</em>-hydroxyandrost-<em>4</em>-ene-3,17-dione (<em>4</em>-OHa) and 10 beta-propargylestr-<em>4</em>-ene-3,17-dione (PED), the thiol androgen 10 beta-mercaptoestr-<em>4</em>-ene-3,17-dione (10 beta-SHnorA) proved to be the most potent suicide substrate. However, 19-mercaptoandrost-<em>4</em>-ene-3,17-dione (19-SHA) was the best all-around inhibitor. All compounds except 19-SHA exhibited normal type I P-<em>4</em>50 difference spectra with partially purified/solubilized, human placental aromatase. The Ks values for the series of compounds compared qualitatively to the KI values determined from the time and concentration-dependent inhibition experiments. 19-SHA induced split Soret peaks at 380 and <em>4</em>7<em>4</em> nm, which suggested binding of the 19-thiolate directly to the ferric iron of aromatase. This binding could be displaced by aminoglutethimide but not by <em>androstenedione</em>. The inhibitory activity of 19-SHA may be explained by two independent mechanisms: (1) suicide inactivation of aromatase in the ferrous state; and (2) a direct "hyper-type II" binding to the remaining portion of the cytochrome in the ferric state. A free thiol group was necessary for the suicide inhibitory activity of 19-SHA; time-dependent inactivation of aromatase by 19-(acetylthio)androst-<em>4</em>-ene-3,17-dione (19-SAcA) and 19-xanthogenylandrost-<em>4</em>-ene-3,17-dione (19-XanA) could be prevented if the microsomes were preincubated with a carboxyesterase inhibitor. Aromatase previously inactivated by either thiol androgens,<em>4</em>-OHA, or PED could not be reactivated after incubation with the disulfide reducing agent dithiothreitol, which suggests that a disulfide bond may not be involved in aromatase inactivation by these inhibitors.