11-oxygenated Androgens Research Articles

Intracrine activation of 11-oxygenated androgens by AKR1C3 modulates lipid metabolism in human female adipose tissue

Intracrine activation of 11-oxygenated androgens by AKR1C3 modulates lipid metabolism in human female adipose tissue

Measurement of Salivary Adrenal-Specific Androgens as Biomarkers of Therapy Control in 21-Hydroxylase Deficiency.

Monitoring of hormonal control represents a key part of the management of congenital adrenal hyperplasia (CAH). Monitoring strategies remain suboptimal because they rely on frequent blood tests and are not specific for adrenal-derived hormones. Recent evidence suggests the crucial role of adrenal-specific 11-oxygenated-C19 androgens in the pathogenesis of CAH. To establish a correlation between plasma and salivary adrenal-specific androgens in CAH as a noninvasive monitoring strategy. This prospective cross-sectional study recruited patients between 2015 and 2018. Multicenter study including 13 tertiary centers in the United Kingdom. Seventy-eight children with CAH and 62 matched healthy controls. Using liquid chromatography-tandem mass spectrometry, plasma and salivary concentrations of five steroids were measured: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT). The correlation between plasma and salivary steroids was analyzed to assess their use in clinical practice. Strong correlations between plasma and salivary steroid concentrations in patients with CAH were detected: 17OHP (rs = 0.871; P < 0.001), A4 (rs = 0.931; P < 0.001), T (rs = 0.867; P < 0.001), 11OH4A (rs = 0.876; P < 0.001), and 11KT (rs = 0.944; P < 0.001). These results were consistent for patient subgroups based on sex and age. Analysis of patient subgroups based on 17OHP concentrations established clear correlations between plasma and salivary concentrations of the adrenal-specific androgen 11KT. The current study identified tight correlations between plasma and saliva for the adrenal-derived 11-oxygenated C19 androgen 11KT, as well as 17OHP and A4, which are widely used for monitoring treatment in CAH. This combination of steroid hormones will serve as an improved noninvasive salivary test for disease monitoring in patients with CAH.

A liquid chromatography-tandem mass spectrometry assay for the profiling of classical and 11-oxygenated androgens in saliva.

Background Classical and 11-oxygenated androgens both contribute to the androgen pool. Regular monitoring of the androgen status is required in disorders of steroidogenesis, and multiplexing of androgens improves the diagnostic ability of an assay. Due to the cheap non-invasive collection, saliva is advantageous when multiple samples are required. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers sensitive, simultaneous quantification of steroids with short run times. Here, we have developed an LC-MS/MS assay for the simultaneous measurement of 17-hydroxyprogesterone, androstenedione, testosterone, 11β-hydroxyandrostenedione and 11-ketotestosterone in saliva. Methods Samples (300 μL unstimulated whole saliva) were prepared by supported liquid extraction with dichloromethane and were reconstituted in 40% methanol. After online solid phase extraction with C18 cartridges, liquid chromatography was performed on a C8 column using a water/methanol gradient containing 0.1% formic acid and 2 mmol/L ammonium acetate. A Waters TQ-S mass spectrometer was used for quantification. Results Total run time was 6.4 min. For all analytes, recovery was between 89% and 109%, ion suppression between 86% and 105%. Intra- and inter-assay comparisons showed a coefficient of variation &lt;10% and the bias between measured and nominal concentration varied between –8% and 10%. Interference with a large set of natural and synthetic steroids was excluded. The assay was applied for the measurement of the androgen profile in healthy men ( n = 17) and women ( n = 10) which confirmed the sensitivity of the assay to be appropriate. Conclusion We present a novel LC-MS/MS assay for the comprehensive profiling of classical and 11-oxygenated androgens with potential for routine clinical application.

MON-203 Local Activation of 11-Oxygenated Androgens by AKR1C3 Is the Predominant Source of Androgens in Human Female Adipose Tissue

Polycystic ovary syndrome (PCOS) affects 10% of women and is a lifelong metabolic disorder, with increased risk of type 2 diabetes, fatty liver and cardiovascular disease. Androgen excess is an important driver of metabolic risk. Excess androgen activation in adipose tissue from PCOS women, through conversion of androstenedione (A4) to testosterone (T) by the enzyme AKR1C3, has been shown to drive insulin resistance and lipotoxicity, with insulin upregulating AKR1C3 further. Recent work has shown that 11-oxygenated androgens including 11-ketotestosterone (11KT), which binds and activates the androgen receptor with similar potency to T, constitute the majority of circulating androgens in PCOS, and that AKR1C3 can activate 11-ketoandrostenedione (11KA4) to 11KT with even higher efficiency than A4 to T. This raises the possibility that 11KT is more relevant than T in mediating excess androgen effects in PCOS. Here, we examined the contributions of classic and 11-oxygenated androgen pathways to intra-adipose androgen activation and the functional significance of the involved enzymes. To this end, we performed ex vivo primary adipose tissue incubations with corresponding subcutaneous (sc) and omental (om) adipose tissue obtained from eight women undergoing bariatric surgery (age range 32-59 years; BMI range 44-57). Incubations carried out with 100nM A4 and 11KA4, respectively, revealed significantly higher AKR1C3-mediated conversion of 11KA4 to 11KT than conversion of A4 to T, both in sc and om tissue. We observed a significant reduction of both 11KT and T generation after the addition of a selective AKR1C3 inhibitor to the primary tissue incubations. Conversely, co-incubation with a selective HSD11B1 inhibitor significantly enhanced the activation of 11KA4 to 11KT by decreasing the inactivation of 11KA4 to 11β-hydroxyandrostenedione; HSD11B1 inhibition had no impact on classic androgen pathway activation of A4 to T. Supernatants and tissues are currently undergoing investigation by mass spectrometry and NMR spectroscopy to determine the differential impact of T and 11KT generation on the adipose-specific global metabolome and lipidome. We conclude that local activation of 11-oxygenated androgens is the predominant source of androgen exposure in adipose tissue. AKR1C3 inhibition presents a promising tool to reduce androgen burden in PCOS, with effective reduction in both classic and 11-oxygenated pathway androgen biosynthesis in adipose tissue.

SAT-345 11-oxygenated Adrenal Androgens Are Produced In Several Mammalian Species

Background: The term “adrenal androgen” is often used to describe the adrenal-produced androgen precursors, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). The human adrenal, however, also secretes 11-oxygenated androgens that include 11-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT), of which 11OHT and 11KT are bioactive. These clinically important steroids have been implicated in diseases of androgen excess, including premature adrenarche, congenital adrenal hyperplasia and polycystic ovary syndrome. While the production of these steroids is established in humans, their presence in other mammalian species has not been examined. Objectives: The goal of this study was to characterize circulating 11-oxyandrogens across species and identify possible animal models to study the regulation and functions of these steroids. Methods: To eliminate possible confounding contributions of testicular androgens, serum was obtained from a range of adult female animals, including birds, rodents, domestic animals and primates (15 species, n=5 each). Sera concentrations of DHEA, DHEAS, 11OHA4, 11KA4, 11OHT and 11KT were analyzed by liquid chromatography-tandem mass spectrometry. Results: As previously demonstrated, significant amounts of circulating DHEA and DHEAS were found only in primates. 11-oxyandrogens also circulated at similar concentrations in all the primates studied (rhesus monkeys, baboons, chimpanzees and humans) of which 11OHA4 was observed to be the most abundant circulating steroid. As expected, neither rats or mice had circulating 11-oxyandrogens, likely because they lack adrenal 17α-hydroxylase/17,20-lyase expression. Low levels of 11-oxyandrogens were detected in dogs, cows, sheep and horses. Interestingly, pigs and guinea pigs had significant circulating levels of 11-oxyandrogens. Circulating levels of 11OHA4 and 11KA4 in pigs (3.2 ± 0.8 nM; 0.4 ± 0.1 nM respectively) and guinea pigs (7.0 ± 0.2 nM; 1.2 ± 0.1 nM) were similar or higher than humans (3.4 ± 0.6 nM; 0.4 ± 0.1 nM). Serum concentrations of 11OHT in guinea pigs were lower (0.1 ± 0.02 nM) as compared to humans (0.3 ± 0.04 nM) and pigs (0.5 ± 0.2 nM). Conversely, circulating 11KT levels were most abundant in humans (0.7 ± 0.2 nM), followed by guinea pigs (0.4 ± 0.1 nM) and pigs (0.2 ± 0.03 nM). Conclusions: 11-oxygenated androgens are produced in multiple primate species. In addition, guinea pigs and pigs have circulating concentrations of 11-oxyandrogens similar to those seen in primates, and could be considered as appropriate animal models to define the physiology of the 11-oxyandrogens.

SAT-LB002 Steroid Metabolome Analysis Reveals 11-Ketotestosterone as the Most Abundant Androgen in Castration-Resistant Prostate Cancer Patients on Second-Line Therapies

Introduction Metastatic castration-resistant prostate cancer (CRPC) remains a clinical challenge despite antitumoral therapies such as taxane chemotherapeutics, enzalutamide, and abiraterone acetate. Clinical studies have highlighted the benefits of these therapies on overall and progression-free survival, but the changes these treatments impose on the steroid metabolome have not yet been fully mapped. In this study, we investigated the steroid metabolomes of CRPC patients on second-line therapies before and during treatment and after clinical progression was observed. Method Plasma samples from 30 patients from the ongoing CIRCUS trial were selected. Patients were included if progression on androgen-deprivation therapy was confirmed and patients were scheduled to receive additional secondary treatment with antiandrogens (enzalutamide (n=10) or apalutamide (n=1)), abiraterone + prednisone (n=2), docetaxel + prednisone (DP, n=10) or cabazitaxel + prednisone (CP, n=14). Seven patients completed two treatments during their enrollment in CIRCUS. Following liquid-liquid extraction of steroids from plasma, multi-steroid profiling by liquid chromatography-tandem mass spectrometry was carried out targeting 16 steroid hormones from classic and 11-oxygenated androgen pathways as well as mineralocorticoid and glucocorticoid biosynthetic pathways. Results Accurate quantification was achieved for all targeted steroids with the exception of dihydrotestosterone and androstenedione. The most abundant active androgen in CRPC patients was 11-ketotestosterone (11KT) with a median concentration of 0.46 nM (range 0.03 - 2.39 nM), whereas the median testosterone concentration was 0.14 nM (range 0.03 - 0.76 nM). 11KT and its precursors were suppressed by CP treatment. Analysis of all patients who started treatment with glucocorticoids revealed a potent suppression of 11KT by 88.5% (range 17.9 - 97.5%) and testosterone by 70.1% (range 24.1 - 95%), as well as suppression of 11-oxygenated androgen precursor steroids. Increased 11KT levels were observed in the DP-treated patients at the time of progressive disease. Discussion In this study, we present in-depth characterization of the previously unexplored steroid metabolomes in CRPC patients revealing 11KT as the most abundant androgen. 11KT has been shown to bind and activate the AR with similar potency to testosterone and our findings suggest that the 11-oxygenated androgens may be the dominant AR agonists in the CRPC setting. Routine quantification of testosterone alone may therefore underestimate the androgenic potential in CRPC patients. Suppression of adrenal androgens, including 11KT, was achieved by glucocorticoid treatment, which may explain the beneficial effects of glucocorticoids in CRPC patients. Funding This project was funded by the Daniel den Hoed foundation. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Circulating 11-oxygenated androgens across species

The androgen precursors, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) are produced in high amounts by the adrenal cortex primarily in humans and a few other primates. The human adrenal also secretes 11-oxygenated androgens (11-oxyandrogens), including 11β-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11β-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT), of which 11OHT and 11KT are bioactive androgens. The 11-oxyandrogens, particularly 11KT, have been recognized as biologically important testicular androgens in teleost fishes for decades, but their physiological contribution in humans has only recently been established. Beyond fish and humans, however, the presence of 11-oxyandrogens in other species has not been investigated. This study provides a comprehensive analysis of a set of C19 steroids, including the traditional androgens and 11-oxyandrogens, across 18 animal species. As previously shown, serum DHEA and DHEAS were much higher in primates than all other species. Circulating 11-oxyandrogens, especially 11KT, were observed in notable amounts in male, but not in female trout, consistent with gonadal origin in fish. The circulating concentrations of 11-oxyandrogens ranged from 0.1 to 10 nM in pigs, guinea pigs and in all the primates studied (rhesus macaque, baboon, chimpanzee and human) but not in rats or mice, and 11OHA4 was consistently the most abundant. In contrast to fish, serum 11KT concentrations were similar in male and female primates for each species, despite significantly higher circulating testosterone in males, suggesting that 11KT production in these species is not testis-dependent and primarily originates from adrenal-derived 11-oxyandrogen precursors.

Abstract #425 Development and Validation of a Liquid Chromatography Tandem Mass Spectrometry Bioanalytical Method for the 11-Oxygenated Androgens 11-Ketotestosterone, 11-Hydroxytestosterone and 11-Hydroxyandrostenedione in Serum or Plasma

Abstract #425 Development and Validation of a Liquid Chromatography Tandem Mass Spectrometry Bioanalytical Method for the 11-Oxygenated Androgens 11-Ketotestosterone, 11-Hydroxytestosterone and 11-Hydroxyandrostenedione in Serum or Plasma

Exogenous Testosterone Does Not Influence 11-Oxygenated C19 Steroid Concentrations in Healthy Postmenopausal Women.

Context11β-Hydroxyandrostenedione (11OHA4), 11β-hydroxytestosterone (11OHT), and their respective peripheral derivatives, 11-ketoandrostenedione (11KA4) and 11-ketotesosterone (11KT), have been implicated in androgen-related physiopathology. Little is known of these steroids in postmenopausal women or whether exogenous testosterone therapy influences their levels.ObjectiveThe impact of exogenous testosterone on serum levels of 11-oxygenated steroids was determined in healthy postmenopausal women.Participants and MethodsLevels of 19-carbon (C19) steroids were measured by liquid chromatography–tandem mass spectrometry in serum obtained at baseline and at 12 and 26 weeks from 73 healthy postmenopausal women, aged 55 to 65 years, who participated in a randomized, double-blind, placebo-controlled clinical trial assessing the effects of transdermal testosterone on cognitive performance.ResultsOf the 11-oxygenated androgens, 11OHA4 was the most abundant (median, 6.46 nmol/L; range, 1.51 to 23.82 nmol/L), with concentrations several fold greater than its precursor androstenedione (median, 1.38 nmol/L; range, 0.52 to 2.92 nmol/L). Baseline median (range) testosterone and 11KT levels were similar [0.56 (0.23 to 1.48) nmol/L; 0.85 (0.25 to 2.86) nmol/L, respectively). 11OHT was closely correlated with 11KT (Spearman rank correlation coefficient, 0.79; P < 0.001) and 11OHA4 correlated with 11KA4 (Spearman rank correlation coefficient, 0.73; P < 0.001). Testosterone therapy resulted in an increase in serum testosterone level, whereas all 11-oxygenated androgens remained unchanged throughout the 26 weeks of treatment.ConclusionAfter menopause, the adrenal production of 11-oxygenated derivatives of androstenedione and testosterone contributes importantly to the total circulating androgen pool. Exogenous testosterone does not influence the circulating levels 11-oxygenated C19 steroids.

11-Oxygenated androgen precursors are the preferred substrates for aldo-keto reductase 1C3 (AKR1C3): Implications for castration resistant prostate cancer

The progression of castration resistant prostate cancer (CRPC) is driven by the intratumoral conversion of adrenal androgen precursors to potent androgens. The expression of aldo-keto reductase 1C3 (AKR1C3), which catalyses the reduction of weak androgens to more potent androgens, is significantly increased in CRPC tumours. The oxidation of androgens to their inactive form is catalysed by 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2), but little attention is given to the expression levels of this enzyme. In this study, we show that the 11-oxygenated androgen precursors of adrenal origin are the preferred substrate for AKR1C3. In particular we show that the enzymatic efficiency of AKR1C3 is 8- and 24-fold greater for 11-ketoandrostenedione than for the classic substrates androstenedione and 5α-androstanedione, respectively. Using three independent experimental systems and a computational model we subsequently show that increased ratios of AKR1C3:17βHSD2 significantly favours the flux through the 11-oxygenated androgen pathway as compared to the classical or 5α-androstanedione pathways. Our findings reveal that the flux through the classical and 5α-androstanedione pathways are limited by the low catalytic efficiently of AKR1C3 towards classical androgens combined with the high catalytic efficiency of 17βHSD2, and that the expression of the oxidative enzyme therefore plays a vital role in determining the steady state concentration of active androgens. Using microarray data from prostate tissue we confirm that the AKR1C3:17βHSD2 ratio is significantly increased in patients undergoing androgen deprivation therapy as compared to benign tissue, and further increased in patients with CRPC. Taken together this study therefore demonstrates that the ratio of AKR1C3:17βHSD2 is more important than AKR1C3 expression alone in determining intratumoral androgen levels and that 11-oxygenated androgens may play a bigger role in CRPC than previously anticipated.

Androgen excess is due to elevated 11-oxygenated androgens in treated children with congenital adrenal hyperplasia

Adrenal androgen excess is the hallmark of classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Recently, 11-oxygenated C19 steroids, a class of highly active adrenal-derived androgens, have been described in patients with CAH.The aim of our study was to elucidate the significance of 11-oxygenated androgens in children with CAH.We retrospectively analysed 190 daily urinary excretion rates of glucocorticoid-, 17α-hydroxyprogesterone (17OHP)-, and androgen metabolites determined by gas chromatography-mass spectrometry of 99 children aged 3.0–10.9 years with classic CAH on hydrocortisone and fludrocortisone treatment. Daily urinary steroid metabolite excretions were transformed into z-scores using references of healthy children. Androgen metabolite z-scores were separately calculated for androsterone (AN), the major urinary metabolite of androstenedione (A4), testosterone and 5α-dihydrotestosterone, for urinary metabolites of dehydroepiandrosterone (DHEA), and for 11β-hydroxyandrosterone (11OHAN), the major urinary metabolite of adrenal-derived 11-oxygenated androgens. Multivariate regression analysis was applied to analyse the precursors of 11OHAN synthesis.11OHAN, cortisol-, and 17OHP metabolite z-scores were elevated in treated children with CAH, whereas AN- and DHEA metabolite z-scores were normalized or suppressed. Multivariate regression analysis revealed that 11OHAN excretion was strongest associated with 21-deoxycortisol (β = 0.379; P =.0006), followed by A4 (β = 0.280; P = .0008)) and 17OHP (β = 0.243; P = .04) metabolite excretion.Androgen excess in treated children with CAH is solely due to elevated 11-oxygenated androgens that derive in addition to the known conversion from A4 also by direct conversion from 21-deoxycortisol. 11-Oxygenated androgens may represent better biomarkers of adrenal androgen status and treatment response than conventional androgens.

Histological and transcriptomic effects of 17\u03b1-methyltestosterone on zebrafish gonad development

BackgroundSex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation.ResultsAt a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf).ConclusionOur study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish.

Clinical significance of 11-oxygenated androgens.

The adrenal gland is considered a source of weak androgens, such as dehydroepiandrosterone, dehydroepiandrosterone sulfate, and androstenedione. Emerging evidence proposes a set of 11-oxygenated 19-carbon (11oxC19) adrenal-derived steroids as clinically important androgens. Such steroids include 11β-hydroxyandrostenedione, 11-ketoandrostenedione, 11β-hydroxytestosterone, and 11-ketotestosterone. The present review will discuss the synthesis, androgenic activity, and clinical implications of the 11oxC19 steroids. The clinical relevance of the 11oxC19 steroids resides in two key characteristics: the synthesis of all 11oxC19 originates predominantly in the adrenal cortex, and 11-ketotestosterone and its 5α-reduced metabolite, 11-ketodihydrotestosterone are potent agonists of the human androgen receptor, similar to the classic androgens testosterone and dihydrotestosterone, respectively. Recent studies have demonstrated higher than normal circulating levels of 11oxC19 steroids in patients with 21-hydroxylase deficiency and in polycystic ovary syndrome. The 11oxC19 steroids are also thought to contribute to castration-resistant prostate cancer progression. In addition, the 11oxC19 steroids might have clinical implications in adrenarche and postmenopausal women. Future prospective studies are needed to establish the clinical utility of the 11oxC19 steroids for individualized patient care. Preliminary data suggest that these biomarkers hold promise to improve the evaluation and management of androgen excess disorders.

11-Oxygenated Androgens Are Biomarkers of Adrenal Volume and Testicular Adrenal Rest Tumors in 21-Hydroxylase Deficiency

Context:Patients with 21-hydroxylase deficiency (21OHD) have long-term complications, resulting from poor disease control and/or glucocorticoid overtreatment. Lack of optimal biomarkers has made it challenging to tailor therapy and predict long-term outcomes.Objective:To identify biomarkers of disease control and long-term complications in 21OHD.Setting and Participants:Cross-sectional study of 114 patients (70 males), ages 2 to 67 years (median, 15 years), seen in a tertiary referral center.Methods:We correlated a mass-spectrometry panel of 23 steroids, obtained before first morning medication, with bone age advancement (children), adrenal volume (adults), testicular adrenal rest tumors (TART), hirsutism, menstrual disorders, and pituitary hormones.Results:Total adrenal volume correlated positively with 18 steroids, most prominently 21-deoxycortisol and four 11-oxygenated-C19 (11oxC19) steroids: 11β-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11ketoA4), 11β-hydroxytestosterone (11OHT), and 11-ketotestosterone (11ketoT) (r ≈ 0.7, P < 0.0001). Nine steroids were significantly higher (P ≤ 0.01) in males with TART compared with those without TART, including 11OHA4 (6.8-fold), 11OHT (4.9-fold), 11ketoT (3.6-fold), 11ketoA4 (3.3-fold), and pregnenolone sulfate (PregS; 4.8-fold). PregS (28.5-fold) and 17-hydroxypregnenolone sulfate (19-fold) levels were higher (P < 0.01) in postpubertal females with menstrual disorders. In males, testosterone levels correlated positively with all 11oxC19 steroids in Tanner stages 1 and 2 (r ≈ 0.7; P < 0.001) but negatively in Tanner stage 5 (r = −0.3 and P < 0.05 for 11ketoA4 and 11ketoT). In females, testosterone level correlated positively with all four 11oxC19 steroids across all Tanner stages (r ≈ 0.8; P < 0.0001).Conclusion:11oxC19 steroids and PregS might serve as clinically useful biomarkers of disease control and long-term complications in 21OHD.

11-Oxygenated C19 Steroids Are the Predominant Androgens in Polycystic Ovary Syndrome.

Context:Androgen excess is a defining feature of polycystic ovary syndrome (PCOS), but the exact origin of hyperandrogenemia remains a matter of debate. Recent studies have highlighted the importance of the 11-oxygenated C19 steroid pathway to androgen metabolism in humans. In this study, we analyzed the contribution of 11-oxygenated androgens to androgen excess in women with PCOS.Methods:One hundred fourteen women with PCOS and 49 healthy control subjects underwent measurement of serum androgens by liquid chromatography-tandem mass spectrometry. Twenty-four–hour urinary androgen excretion was analyzed by gas chromatography-mass spectrometry. Fasting plasma insulin and glucose were measured for homeostatic model assessment of insulin resistance. Baseline demographic data, including body mass index, were recorded.Results:As expected, serum concentrations of the classic androgens testosterone (P < 0.001), androstenedione (P < 0.001), and dehydroepiandrosterone (P < 0.01) were significantly increased in PCOS. Mirroring this, serum 11-oxygenated androgens 11β-hydroxyandrostenedione, 11-ketoandrostenedione, 11β-hydroxytestosterone, and 11-ketotestosterone were significantly higher in PCOS than in control subjects, as was the urinary 11-oxygenated androgen metabolite 11β-hydroxyandrosterone. The proportionate contribution of 11-oxygenated to total serum androgens was significantly higher in patients with PCOS compared with control subjects [53.0% (interquartile range, 48.7 to 60.3) vs 44.0% (interquartile range, 32.9 to 54.9); P < 0.0001]. Obese (n = 51) and nonobese (n = 63) patients with PCOS had significantly increased 11-oxygenated androgens. Serum 11β-hydroxyandrostenedione and 11-ketoandrostenedione correlated significantly with markers of insulin resistance.Conclusions:We show that 11-oxygenated androgens represent the majority of circulating androgens in women with PCOS, with close correlation to markers of metabolic risk.

Presence of 11-ketotestosterone in pre-differentiated male gonads of Odontesthes bonariensis

The involvement of androgens during sex differentiation period was investigated in the pejerrey Odontesthes bonariensis, by classic biochemical studies and gonadal histology. We studied in particular whether the enzyme activities involved in 11-oxygenated androgen production were active in a gonadal/peritoneum complex (GPC) of very small larvae exposed to masculinizing temperatures previous to morphological sex differentiation (5weeks post-hatching). The GPC was incubated with 17-hydroxyprogesterone ((3)H-17P), and the presence of 11-KT as major metabolite in early gonads undergoing masculine pathway after temperature treatment exposure is reported. 11-KT was identified by thin-layer chromatography and high-pressure liquid chromatography. The present results show that 11-KT is produced at very early stages of testis development in pejerrey, being this androgen one of the main mediators of the masculinization induced by temperature treatment at the gonad level.

Molecular characterization of cyp11a1 and cyp11b1 and their gene expression profile in pejerrey ( Odontesthes bonariensis) during early gonadal development

Sex steroids are known to be involved in gonadal differentiation in fish, but whether androgens are early mediators of testis differentiation remains unclear. We studied the sex-related developmental variations in the gene expression of two key enzymes involved in steroids and androgen synthesis ( cyp11a1 and cyp11b1) in trunks and isolated gonads of pejerrey ( Odontesthes bonariensis) larvae during and after the sex determination period. Also, and in order to have a better characterization of this process we studied the expression of Sertoli ( dmrt1, amh, sox9) and Leydig ( nr5a1 or sf - 1) cell markers as well as a gene with higher expression in females ( cyp19a1a). No clear differences were observed in the expression of cyp11a1 and cyp11b1 during the temperature-sensitive window in the trunk of pejerrey larvae. Nevertheless, a clear increase of cyp11b1 was observed in isolated gonads taken from fish reared at the male producing temperature. In these gonads we also confirmed the trends of genes with higher expression in males ( dmrt1, amh) and females ( cyp19a1a) as previously described in larval trunks of pejerrey. Our results showed that the expression of cyp11b1 was positively associated with the morphological differentiation of the testis. Nevertheless the involvement of 11-oxygenated androgens during the temperature-sensitive window could not be clearly established.

Sex steroid dynamics during embryogenesis and sexual differentiation in Eurasian perch, Perca fluviatilis

It is widely accepted that sex steroid hormones play an important and a specific role during the process of sex differentiation in fish. In order to describe the role of the three main sex steroid hormones (testosterone—T, 17β-estradiol—E2 and 11keto-testosterone—11KT) during embryogenesis and sex differentiation in Eurasian perch, Perca fluviatilis, eggs, larvae and juveniles originating from two mixed-sex and two all-female progenies were regularly sampled from fertilization to hatching (D0) and from hatching to day 70 post-hatching (D70). Just after spawning, a significant amount of sex steroids [T (1634.2 pg g −1), E2 (554.4 pg g −1) and 11KT (1513.2 pg g −1)] was measured in non-fertilised eggs suggesting a maternal transmission of these steroids. From D2 to D70 post-hatching, E2 levels were significantly higher in mixed-sex progenies (median: 725.7 pg g −1) than in all-female progenies (156.2 pg g −1) and significantly increased after the onset of the histological differentiation of the gonad in both progenies (D35). Levels of 11KT were significantly higher in mixed-sex (median: 431.5 pg g −1) than in all-female progenies (below the limit of assay detection) and significantly increased at D35 in all-female progenies (median value: 343.2 pg g −1). Mean 11KT to E2 ratio was six-fold higher in mixed-sex progenies (1.35) than in all-female progenies (0.24). The data suggest that the 11-oxygenated androgen (11KT) plays a major role in the male differentiation process, and that sex differentiation in Eurasian perch is probably determined by the 11KT to E2 ratio.

A cDNA for European sea bass ( Dicentrachus labrax) 11β-hydroxylase: Gene expression during the thermosensitive period and gonadogenesis

Steroid P450 11β-hydroxylase, encoded by the CYP11B gene, is a key mitochondrial enzyme for the production of 11-oxygenated androgens, which have been shown to be potent masculinising steroids in several fish species. In this study we have isolated a CYP11B cDNA of 1903 base pairs from the testis of European sea bass ( Dicentrarchus labrax) encoding a predicted protein of 552 amino acids. The amino acid identities to other vertebrate 11β-hydroxylase proteins ranged from 66% to 72% to other fish; 45% to amphibian and 35–39% to mammalian. Southern blot indicated that a single CYP11B gene is present. Northern blot analysis detected two transcripts in testis and head kidney, one of the same size of the isolated clone and the other of 3.9 kb. Reverse transcriptase-polymerase chain reaction showed abundant mRNA expression only in testis and head kidney, being residual in a range of other tissues. Expression of CYP11B and CYP19A (which encodes for ovarian aromatase) was detected from at least 4 days post-hatching and did not appear to be affected by rearing temperature (15 and 20 °C) during the first 60 days, a period in which high temperatures promote masculinisation in European sea bass. Throughout, gonadogenesis (60–300 dph), a highly dimorphic pattern of CYP11B expression was consistent with a role of this gene in testicular development.

Effects of 4- tert-pentylphenol on the gene expression of P450 11β-hydroxylase in the gonad of medaka ( Oryzias latipes)

Alkylphenols including 4- tert-pentylphenol (4-PP) have been shown to alter sexual differentiation in fish due to their estrogenic properties. Medaka ( Oryzias latipes) is so sensitive to these substances because morphological sex reversal and testis-ova induction are well developed in the exposed males. However, little work has been done to characterize the molecular effects of estrogenic substances on the morphological and gonadal feminization in male fish. Cytochrome P450 11β-hydroxylase (P450 11β) is a key steroidogenic enzyme in production of 11-ketotestosterone which is the predominant androgen in male fish. In this study, we cloned a cDNA encoding medaka testicular P450 11β, and then investigated the gene expression of P450 11β in the testes of genetically male medaka exposed to 4-PP. The cDNA contains 1740 nucleotides that encode a protein of 543 amino acids, which shares 68.9% and 73.4% homology with testicular P450 11βs from Japanese eel ( Anguilla japonica) and rainbow trout ( Oncorhynchus mykiss), respectively. HeLa cells transfected with an expression vector containing the open reading frame of medaka P450 11β cDNA showed 11β-hydroxylating activity in the presence of exogenous testosterone. Analysis of tissue distribution by RT-PCR showed great abundance of P450 11β mRNA in testis. In the partial life-cycle exposure with 4-PP, morphologically sex-reversal was observed in XY medaka exposed to 4-PP concentrations of ≥ 238 μg/L. Furthermore, exposure to 4-PP completely inhibited P450 11β mRNA expression in the gonads of sex-reversed XY fish at 60-day posthatch. These results suggest that xeno-estrogen 4-PP may have inhibitory effects on the synthesis of testicular 11-oxygenated androgens through downregulation of P450 11β expression in the genetically male fish.