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A comprehensive atlas of sex steroid distribution in multiple tissues is currently lacking, and how circulating and tissue sex steroid levels correlate remains unknown. Here, we adapted and validated a gas chromatography tandem mass spectrometry method for simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), androstenedione, progesterone (Prog), estradiol, and estrone in mouse tissues. We then mapped the sex steroid pattern in 10 different endocrine, reproductive, and major body compartment tissues and serum of gonadal intact and orchiectomized (ORX) male mice. In gonadal intact males, high levels of DHT were observed in reproductive tissues, but also in white adipose tissue (WAT). A major part of the total body reservoir of androgens (T and DHT) and Prog was found in WAT. Serum levels of androgens and Prog were strongly correlated with corresponding levels in the brain while only modestly correlated with corresponding levels in WAT. After orchiectomy, the levels of the active androgens T and DHT decreased markedly while Prog levels in male reproductive tissues increased slightly. In ORX mice, Prog was by far the most abundant sex steroid, and, again, WAT constituted the major reservoir of Prog in the body. In conclusion, we present a comprehensive atlas of tissue and serum concentrations of sex hormones in male mice, revealing novel insights in sex steroid distribution. Brain sex steroid levels are well reflected by serum levels and WAT constitutes a large reservoir of sex steroids in male mice. In addition, Prog is the most abundant sex hormone in ORX mice., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Introduction: The two major androgens in humans are testosterone (T) and dihydrotestosterone (DHT). DHT is produced via the classical, backdoor, and alternative steroidogenic pathways. In addition, recent studies have identified C11-oxy C19 steroids as novel human androgens. Although the placenta is known to be involved in steroid metabolism, androgen levels in full-term placentas have poorly been investigated., Subjects and Methods: Ten placentas of healthy full-term neonates (five males and five females) were examined. We quantified progesterone, androstenedione (A4), T, allopregnanolone, androsterone, and estradiol, as well as four C11-oxy androgens (11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketoandrostenedione (11KA4), and 11-ketotestosterone (11KT)), using liquid chromatography-tandem mass spectrometry., Results: In all samples, levels of the ten steroids were above the detection limit. Progesterone was by far most abundant, while levels of T and androsterone were relatively low. Levels of 11KT and 11KA4 were higher than those of T and A4, respectively. There were no differences in steroid levels between male and female samples., Discussion: This study demonstrates that full-term placentas contain several steroids in the classical, backdoor, and alternative pathways. Placentas are likely to function as the supplier of progesterone to other steroidogenic tissues. More importantly, we found that placentas comprise relatively large amounts of 11KA4 and 11KT, which may be produced through steroid transfer from the adrenal gland or from the maternal circulation. These results indicate that the placenta participates in a feto-maternal multi-organ network for androgen biosynthesis.

Background: Higher endogenous testosterone levels are associated with reduced chronic disease risk and mortality. Since the mid-20th century, there have been significant changes in dietary patterns, and men's testosterone levels have declined in western countries. Cross-sectional studies show inconsistent associations between fat intake and testosterone in men., Methods: Studies eligible for inclusion were intervention studies, with minimal confounding variables, comparing the effect of low-fat vs high-fat diets on men's sex hormones. 9 databases were searched from their inception to October 2020, yielding 6 eligible studies, with a total of 206 participants. Random effects meta-analyses were performed using Cochrane's Review Manager software. Cochrane's risk of bias tool was used for quality assessment., Results: There were significant decreases in sex hormones on low-fat vs high-fat diets. Standardised mean differences with 95 % confidence intervals (CI) for outcomes were: total testosterone [-0.38 (95 % CI -0.75 to -0.01) P = 0.04]; free testosterone [-0.37 (95 % CI -0.63 to -0.11) P = 0.005]; urinary testosterone [-0.38 (CI 95 % -0.66 to -0.09) P = 0.009]; and dihydrotestosterone [-0.3 (CI 95 % -0.56 to -0.03) P = 0.03]. There were no significant differences for luteinising hormone or sex hormone binding globulin. Subgroup analysis for total testosterone, European and North American men, showed a stronger effect [-0.52 (95 % CI -0.75 to -0.3) P < 0.001]., Conclusions: Low-fat diets appear to decrease testosterone levels in men, but further randomised controlled trials are needed to confirm this effect. Men with European ancestry may experience a greater decrease in testosterone, in response to a low-fat diet., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The androgens testosterone and dihydrotestosterone (DHT) are essential for a variety of systemic functions in mature males. Alteration of these hormones results in late-onset hypogonadism (LOH) and benign prostate hyperplasia (BPH). The fruit bodies of fungi of the genus Cordyceps have been regarded as folk medicine or health food with tonic and antifatigue effects. The extract from the fruit body of Cordyceps militaris parasitizing Samia cynthia ricini (CM) was evaluated as a novel-candidate natural product for ameliorating male andropause symptoms. To explore the effects of CM on LOH and BPH, CM was applied to rat models and cultured testicular cells and prostate cells. The concentrations of androgens in the serum and culture media were determined by ELISA. Expression of steroidogenic enzymes and androgen-related genes was evaluated by qPCR, and prostatic cell proliferation was assessed with the cell-viability assay. CM maintained the serum levels of testosterone and DHT, but inhibited testosterone-induced prostate hypertrophy. CM also increased the secretion of testosterone and DHT by primary testicular cells, with no changes in the mRNA expression of steroidogenic enzymes, but decreased the growth of prostatic cell lines. Our data suggest that CM could improve both LOH and BPH in males.

Cranberry powder (CR) is reported to be effective against lower urinary tract symptoms (LUTS) and recurrent urinary tract infections. Benign prostatic hyperplasia (BPH) in men older than 50 years is a common cause of LUTS. Here, we attempted to evaluate if CR is also effective for treating BPH using a BPH-induced rat model, which was orally administered CR. Male Sprague-Dawley rats weighing 200-250 g were randomly divided into the following six groups ( n  = 9): noncastration group; castration group; BPH group; BPH and cranberry for 8-week (CR8W) group; BPH and cranberry for 4-week (CR4W) group; and BPH and saw palmetto group (saw palmetto). Compared with the BPH group, the CR8W group showed a significant decrease in prostate weight (by 33%), dihydrotestosterone (DHT) levels (by 18% in serum and 28% in prostate), 5-alpha reductase levels (18% reduction of type 1 and 35% of type 2), and histological changes. These results indicate that CR could attenuate BPH by inhibiting 5-alpha reductase and by reducing other biomarkers such as prostate weight and DHT levels. Thus, CR may be an effective candidate for the development of a functional food for BPH treatment. IACUC (USW-IACUC-R-2015-004).

Background: Age-dependent alterations of hormonal states have been considered to be involved in age related decline of cognitive abilities. Most of the studies in animal models are based on hormonal substitution in adrenal- and/or gonadectomized rodents or infusion of steroid hormones in intact rats. Moreover, the manipulations have been done timely, closely related to test procedures, thus reflecting short-term hormonal mechanisms in the regulation of learning and memory. Here we studied whether more general states of steroid and thyroid hormone profiles, independent from acute experiences, may possibly reflect long-term learning capacity. A large cohort of aged (17-18 months) intact male rats were tested in a spatial hole-board learning task and a subset of inferior and superior learners was included into the analysis. Young male adult rats (16 weeks of age) were also tested. Four to 8 weeks after testing blood plasma samples were taken and hormone concentrations of a variety of steroid hormones were measured by gas chromatography-tandem mass spectrometry or radioimmunoassay (17β-estradiol, thyroid hormones)., Results: Aged good learners were similar to young rats in the behavioral task. Aged poor learners but not good learners showed higher levels of triiodothyronine (T3) as compared to young rats. Aged good learners had higher levels of thyroid stimulating hormone (TSH) than aged poor learning and young rats. Both aged good and poor learners showed significantly reduced levels of testosterone (T), 4-androstenedione (4A), androstanediol-3α,17β (AD), dihydrotestosterone (DHT), 17-hydroxyprogesterone (17OHP), higher levels of progesterone (Prog) and similar levels of 17β-estradiol (E2) as compared to young rats. The learning, but not the memory indices of all rats were significantly and positively correlated with levels of dihydrotestosterone, androstanediol-3α,17β and thyroxine (T4), when the impacts of age and cognitive division were eliminated by partial correlation analyses., Conclusion: The correlation of hormone concentrations of individuals with individual behavior revealed a possible specific role of these androgen and thyroid hormones in a state of general preparedness to learn.

Prostate cancer (PCa) is the second most common type of male malignancy worldwide. The transcription factor zinc finger E‑box binding homeobox 1 (ZEB1) is associated with epithelial‑mesenchymal transition and is also involved in regulation of androgen receptor (AR) expression, the main ligands of which are testosterone and dihydrotestosterone (DHT). These androgens are synthesized through the steroidogenic pathway within the prostate, and their synthesis is altered in PCa. The present study aimed to determine the ZEB1‑induced alterations in androgen synthesis and AR expression in the DU145 PCa cell line. Reverse transcription‑quantitative polymerase chain reaction, western blotting and immunocytochemistry were used to determine the mRNA and protein expression levels, and cellular localization of steroidogenic pathway enzymes in the DU145 cell line in response to ZEB1 silencing. Furthermore, the concentrations of testosterone and DHT were detected in cell culture medium using ELISA. ZEB1‑silenced cells exhibited an increase in testosterone and DHT production, an increase in AR expression and an alteration in the steroidogenic pathway. In particular, steroidogenic acute regulatory protein and 5α‑reductase 2 expression levels were decreased, whereas cytochrome P450 family 17 subfamily A member 1, 5α‑reductase 1, aldo‑keto reductase family 1 member D1 and aldo‑keto reductase family 1 member C2 expression levels were increased. In conclusion, the present study provided novel information regarding the regulation of intratumoral androgen production in PCa, which is relevant for the progression of the disease to a castration‑resistant form.

Research Question: Can IVF outcomes be predicted from the steroid profile generated by liquid chromatography-mass spectrometry (LC-MS/MS) from follicular fluid collected from a single dominant follicle and serum after ovarian stimulation., Design: Prospective observational cohort study in which serum and follicular fluid were collected from women and used to generate steroid profiles by LC-MS/MS. A total of 93 consecutive women enrolled for IVF treatment were recruited at the Fertility Unit, Royal Prince Alfred Women and Babies Hospital, Sydney between September 2014 and July 2015. Baseline and serum levels at oocyte retrieval, as well as follicular fluid samples from the largest single antral follicle, were collected. All samples underwent steroid analysis within a single batch to measure progesterone (P4), oestradiol (E2), oestrone (E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), and 3 α, 5α androstanediol (3α-diol) and 3β, 5α androstanediol (3β-diol)., Results: P4, E2, E1, A4, T, DHEA and A4 were detectable in all baseline serum levels, at oocyte retrieval and in follicular fluid samples, whereas DHT, 3α-diol and 3β-diol were only detectable in a minority of samples. The most consistent predictor of pre-transfer (number of follicles >14mm in diameter, oocytes retrieved or fertilized, day-5 blastocysts) outcomes was baseline serum anti-Müllerian hormone. In follicular fluid, E2 was a negative predictor of the number of oocytes retrieved and the number of day-5 blastocysts but no follicular fluid steroids predicted pregnancy outcome., Conclusions: None of the nine steroids measured in follicular fluid predicted pregnancy outcome in women undergoing IVF., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

In this study, proton affinitive derivatization using picolinic acid and its analogs (3- and 6-methylpicolinic acid and 5-butylpicolinic acid) with proton affinitive moieties was performed for the highly sensitive determination of testosterone (T) and 5α-dihydrotestosterone (DHT) in saliva by LC-ESI-MS/MS. T and DHT were converted to their corresponding picolinate esters and their chromatographic behavior was investigated with a reversed phase column. The picolinate ester of each steroid exhibited a clear single peak and elution occurred in the following order: picolinate, 3/6-methylpicolinate, and 5-butylpicolinate. Estimation and understanding of the separation and retention time of each picolinate ester was made simple using the develop method. Although the peaks of picolinate and 3/6-methylpicolinate esters were suppressed by interference from the saliva background (matrix effect), the 5-butylpicolinate esters were only marginally affected.

Objectives: To develop a high-throughput screening system to measure the conversion of testosterone to dihydrotestosterone (DHT) in cultured human prostate cancer cells using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS)., Results: After optimizing the cell reaction system, this method demonstrated a screening capability of 103 samples, including 78 single compounds and 25 extracts, in less than 12 h without manual sample preparation. Consequently, fucoxanthin, phenethyl caffeate, and Curcuma longa L. extract were validated as bioactive chemicals that inhibited DHT production in cultured DU145 cells. In addition, naringenin boosted DHT production in DU145 cells., Conclusion: The method can facilitate the discovery of bioactive chemicals that modulate the DHT production, and four phytochemicals are potential candidates of nutraceuticals to adjust DHT levels in male hormonal dysfunction.

Background: Acorus gramineus has been reported to exhibit various pharmacological effects including inhibition of cholesterol synthesis, enhancement of lipid metabolism, prevention of dementia and inhibition of mast cell growth. According to the Chinese compendium of materia media, it has been reported that Acorus spp. is effective for sedation, dementia prevention as well as diuretic effect. In addition, it showed more than equivalent activity compared to furosoemide, a drug known to be effective in diuretic action in animal model study. However, their effectiveness against benign prostatic hyperplasia (BPH) of Acorus gramineus has not been reported. This study was designed to evaluate the effect of Acorus gramineus root hot water extract (AG) against BPH in vivo., Methods: Male rats, 10 weeks of age and weighing 405 g ± 10 g, were used for this study. Biomarkers were evaluated including prostate weight, prostate weight ratio, hormonal changes, 5-α reductase type II androgen receptor (AR) of the prostate gland and anti-oxidant activation factors related to BPH. These biomarkers were measured in vivo test., Results: AG showed significant effect at the 250 and 500 mg/kg/day in rats. Groups treated with AG displayed significantly lower levels of prostate gland weight (0.79 g) compared to the BPH induced group (1.19 g). Also, dihydrotestosterone (DHT) level was decreased from 61.8 to 100% and androgen receptor expression level was decreased from 111 to 658%. Any hematological toxicity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level wasn't observed., Conclusion: This study indicated that AG was effective for reducing BPH symptoms., Trial Registration: Not applicable.

The presence of the vertebrate steroids, testosterone (T) and 17β-estradiol in mollusks is often cited as evidence that they are involved in the control of their reproduction. In this paper, we show that a likely source of T in at least one species, the common mussel (Mytilus spp.), is from uptake from water. When mussels were exposed to waterborne tritiated T ([ 3 H]-T) in a closed container, the radioactivity decreased rapidly and exponentially until, by 24h, approximately 35% remained in the water. The rate of uptake of radiolabel could not be saturated by concentrations as high as 16.5μgL -1 (mean measured) of non-radiolabeled T, showing that the animals have a very high capacity for uptake of T. At least 30% of the applied radioactivity could be extracted from the tissues of the animals with organic solvents and most of this (26% of the total applied radioactivity) was in the fatty acid ester fraction. Following alkaline hydrolysis, reverse phase HPLC and TLC, this fraction was shown to consist predominantly of 5α-dihydrotestosterone and 5α-androstane-3β,17β-diol, while T was a minor component. These steroids were definitively identified in the fatty acid ester fraction by mass spectrometry. Overall, less than 5% of the [ 3 H]-T applied to the system remained untransformed at the end of exposure. After ten days of depuration there was no reduction in the total amount of radioactivity in the tissues, nor any changes in the ratio of the metabolites in the ester fraction. These findings show that any association between T presence and reproductive status or sex is confounded by their significant capacity for uptake, and that T undergoes extensive metabolism in mussels in vivo and therefore may not be representative of the androgenic burden of the animals. Consequently, measurements of T in mussel tissue offer little utility as an indicator of reproductive status or sex., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

The use of manure as an agricultural amendment is increasing the release of steroid hormones into the environment. Most research in this field has focused on estrogenic phenomena, with less attention paid to androgenic substances. The present study assessed androgenic activity in broiler manure using in vitro approaches based on cells stably transfected with androgen receptor. Leaching experiments were also performed to observe whether endocrine disruptors present in manure pass through a soil column and potentially reach groundwater. In parallel, an analytical chemistry method was used to determine the contribution of the most important natural androgens to androgenicity. Samplings were performed at 4 farms in 2 seasons. All but 2 samples showed androgen activity. In leakage experiments, however, no androgenic activity was detectable in leachates or in soils after leaching. According to the analytical results, androgenicity can be attributed mainly (but not completely) to androstenedione, and dihydrotestosterone. Similarly to the bioassays, chemical analysis did not reveal the presence of any androgen in leachates or soils. These results point to a rapid degradation of the substances responsible for androgenic activity in soils under the experimental conditions of the present study. However, the long-term effects associated with the constant and intensive application of manure to agricultural land require further attention. Environ Toxicol Chem 2017;36:1746-1754. © 2016 SETAC., (© 2016 SETAC.)

MAMLD1 has been implicated in testicular function in both human and mouse fetuses. Although three patients with MAMLD1 mutations were reported to have hypergonadotropic hypogonadism in their teens, the functional significance of MAMLD1 in the postnatal testis remains unclear. Here, we analyzed the phenotype of Mamld1 knockout (KO) male mice at reproductive ages. The reproductive organs of KO male mice were morphologically unremarkable, except for relatively small testes. Seminiferous tubule size and number of proliferating spermatogonia/spermatocytes were reduced in the KO testis. Daily sperm production of KO mice was mildly attenuated, whereas total sperm counts in epididymal semen remained normal. Sperm motility and morphology, as well as androgen levels in serum and testicular tissues and the number of pups born from cross-mated wildtype (WT) female mice, were comparable between WT and KO male mice. These results indicate that MAMLD1 contributes to the maintenance of postnatal testicular growth and daily sperm production but is dispensable for androgen biosynthesis and fertility. MAMLD1 likely plays supporting roles in multiple and continuous steps of male reproduction., Competing Interests: The authors declare no conflict of interest.

Regular resistance exercise induces skeletal muscle hypertrophy and improvement of glycemic control in type 2 diabetes patients. Administration of dehydroepiandrosterone (DHEA), a sex steroid hormone precursor, increases 5α-dihydrotestosterone (DHT) synthesis and is associated with improvements in fasting blood glucose level and skeletal muscle hypertrophy. Therefore, the aim of this study was to investigate whether increase in muscle DHT levels, induced by chronic resistance exercise, can contribute to skeletal muscle hypertrophy and concomitant improvement of muscular glucose metabolism in type 2 diabetic rats. Male 20-week-old type 2 diabetic rats (OLETF) were randomly divided into 3 groups: sedentary control, resistance training (3 times a week on alternate days for 8 weeks), or resistance training with continuous infusion of a 5α-reductase inhibitor (n = 8 each group). Age-matched, healthy nondiabetic Long-Evans Tokushima Otsuka (LETO) rats (n = 8) were used as controls. The results indicated that OLETF rats showed significant decrease in muscular DHEA, free testosterone, DHT levels, and protein expression of steroidogenic enzymes, with loss of skeletal muscle mass and hyperglycemia, compared to that of LETO rats. However, 8-week resistance training in OLETF rats significantly increased the levels of muscle sex steroid hormones and protein expression of steroidogenic enzymes with a concomitant increase in skeletal muscle mass, improved fasting glucose level, and insulin sensitivity index. Moreover, resistance training accelerated glucose transporter-4 (GLUT-4) translocation and protein kinase B and C-ζ/λ phosphorylation. Administering the 5α-reductase inhibitor in resistance-trained OLETF rats resulted in suppression of the exercise-induced effects on skeletal muscle mass, fasting glucose level, insulin sensitivity index, and GLUT-4 signaling, with a decline in muscular DHT levels. These findings suggest that resistance training-induced elevation of muscular DHT levels may contribute to improvement of hyperglycemia and skeletal muscle hypertrophy in type 2 diabetic rats., Competing Interests: The authors have declared that no competing interests exist.

Secretions of chloride (Cl(-))- and bicarbonate (HCO3(-))-rich fluid by the seminal vesicles could involve cystic fibrosis transmembrane regulator (CFTR), which activity can be stimulated by cAMP generated from the reaction involving adenylate cyclase (AC). In this study, we investigated levels of CFTR, AC, and cAMP in the seminal vesicles under testosterone influence. Orchidectomized adult male rats received 7-day treatment with 125 or 250 μg/kg/day of testosterone with or without flutamide or finasteride. At the end of the treatment, animals were sacrificed and seminal vesicles were harvested for analyses of CFTR and AC protein expression level by Western blotting. Distribution of CFTR and AC in seminal vesicles was observed by immunohistochemistry. Levels of cAMP and dihydrotestosterone in seminal vesicle homogenates were measured by ELISA. Cystic fibrosis transmembrane regulator, AC, and cAMP levels increased with increasing doses of testosterone (P < 0.05 compared to nontreated orchidectomized rats). Cystic fibrosis transmembrane regulator and AC were expressed at the apical membrane of the epithelium lining the seminal vesicle lumen with higher expression levels observed in testosterone-treated rats than in non-treated orchidectomized rats (P < 0.05). The inhibitory effects of flutamide or finasteride on these parameters were greater in 250 μg/kg/day testosterone-treated rats than their effects in 125 μg/kg/day testosterone-treated rats. Higher dihydrotestosterone levels were observed in seminal vesicle homogenates after treatment with 250 μg/kg/day than with 125 μg/kg/day of testosterone (P < 0.05). Increased levels of CFTR, AC, and cAMP in seminal vesicles might contribute toward an increase in Cl(-) and HCO3(-) concentrations in the seminal fluid as reported under testosterone influence., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: The effects on scalp and serum dihydrotestosterone (DHT) of different doses of a novel topical solution of 0.25% finasteride (P-3074), a type 2 5α-reductase, were investigated in men with androgenetic alopecia., Methods: Two randomized, parallel-group studies were conducted. Study I: 18 men received 1 mL (2.275 mg) P-3074, applied to the scalp once a day (o.d.) or twice a day (b.i.d), or 1 mg oral tablet o.d. for 1 week. Study II: 32 men received P-3074 at the dose of 100 (0.2275 mg), 200 (0.455 mg), 300 (0.6285 mg), or 400 (0.91 mg) μL or the vehicle o.d. for 1 week. Scalp and serum DHT and serum testosterone were evaluated at baseline and treatment end., Results: Change from baseline in scalp DHT was -70% for P-3074 o.d. and approx. -50% for P-3074 b.i.d. and the tablet. Serum DHT decreased by 60 - 70%. The doses of 100 and 200 μL P-3074 resulted in a -47/-52% scalp DHT reduction, similar to the 300 and 400 μL doses (i.e., -37/-54%). A -5.6% inhibition was observed for the vehicle. Serum DHT was reduced by only -24/-26% with 100 and 200 μL P-3074 and by -44/-48% with 300 and 400 μL P-3074. No relevant changes occurred for serum testosterone., Conclusions: The novel finasteride 0.25% solution applied o.d. at the doses of 100 and 200 μL results in an appropriate inhibition of scalp DHT potentially minimizing the untoward sexual side-effects linked to a systemic DHT reduction.

Accurate measurement of sex steroid concentrations in rodent serum is essential to evaluate mouse and rat models for sex steroid-related disorders. The aim of the present study was to develop a sensitive and specific gas chromatography-tandem mass spectrometry (GC-MS/MS) method to assess a comprehensive sex steroid profile in rodent serum. A major effort was invested in reaching an exceptionally high sensitivity for measuring serum estradiol concentrations. We established a GC-MS/MS assay with a lower limit of detection for estradiol, estrone, T, DHT, progesterone, androstenedione, and dehydroepiandrosterone of 0.3, 0.5, 4.0, 1.6, 8, 4.0, and 50 pg/mL, respectively, whereas the corresponding values for the lower limit of quantification were 0.5, 0.5, 8, 2.5, 74, 12, and 400 pg/mL, respectively. Calibration curves were linear, intra- and interassay coefficients of variation were low, and accuracy was excellent for all analytes. The established assay was used to accurately measure a comprehensive sex steroid profile in female rats and mice according to estrous cycle phase. In addition, we characterized the impact of age, sex, gonadectomy, and estradiol treatment on serum concentrations of these sex hormones in mice. In conclusion, we have established a highly sensitive and specific GC-MS/MS method to assess a comprehensive sex steroid profile in rodent serum in a single run. This GC-MS/MS assay has, to the best of our knowledge, the best detectability reported for estradiol. Our method therefore represents an ideal tool to characterize sex steroid metabolism in a variety of sex steroid-related rodent models and in human samples with low estradiol levels.

Increased levels of testosterone (T2 ), dihydrotestosterone (DHT) and estradiol (E2 ) in children may be responsible for their early/delayed puberty and obesity conditions. Therefore, multimode sensors based on carbon matrices, such as graphite, graphene, fullerene C60 and multiwall carbon nanotubes modified with maltodextrin, were designed to assess reliably T2 , DHT and E2 in children saliva. The modes used for the assay of hormones were stochastic mode (for qualitative and quantitative determination of hormones) and differential pulse voltammetry mode (for quantitative determination of hormones). The advantage of this type of sensors, for hormone analysis, is their possibility to reach low concentration levels- are placed for children saliva under the detection limit of standard methods (e.g. ELISA used for the determination of these hormones in saliva). This made the multimode sensors an excellent tool for clinical analysis and especially for determination of substances of clinical importance in saliva samples. The proposed method is fast and simple, and no sampling of saliva is required., (Copyright © 2014 John Wiley & Sons, Ltd.)

The androgens testosterone (T) and dihydrotestosterone (DHT) play a key role in the function and integrity of prostate tissue, but are also implicated in prostate cancer and benign prostatic hyperplasia (BPH). The reduction of androgen levels can be achieved by the inhibition of 3-oxo-5α-steroid-4-dehydrogenase (5α-reductase), which is responsible for the irreversible conversion of T into its more active metabolite DHT. In fact, the use of 5α-reductase inhibitors (RIs), like finasteride, can be a valuable strategy for the treatment of BPH and in chemoprevention of prostate tumors. In this work a new method based on a dispersive liquid-liquid microextraction (DLLME) procedure, followed by gas chromatography-mass spectrometry (GC-MS), to evaluate the 5α-reductase activity, by measuring the conversion percentage of T into DHT was optimized and validated. Enzymatic assays were carried out in human prostate microsomes, using T as substrate. T and DHT were extracted by the developed DLLME technique and quantified, after silylation, by GC-MS. Variables affecting the extraction efficiency and derivatization of T and DHT were evaluated. The optimized method showed good linearity (with correlation coefficients over 0.9994 for T and 0.9995 for DHT), good recoveries (higher than 80%), and good intra- and inter-day precision (below 13%, 3 levels, n=6). The detection limits for T and DHT were 0.5 nM and the limits of quantification were 5 nM. The new GC-MS method is a good alternative to the already described methods, to evaluate 5α-reductase activity, since it avoids the use of radioactive compounds and corresponds to a fast and sensitive methodology with a good extraction efficiency, accuracy and high recovery. As this method allows the evaluation of 5α-reductase activity, also permits the study of inhibitory efficacy of new molecules as potential RIs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Physiologically relevant steroid 5α-reductase (SRD5A) activity that is essential for dihydrotestosterone (DHT) biosynthesis in human castration-resistant prostate cancer (CRPC) has not been fully characterized yet. In this study to ascertain the potential SRD5A activity, we cultured two human CRPC cell lines, C4-2 and C4-2AT6, with the steroid precursor: ¹³C-[2,3,4]-androstenedione (13C-Adione), and analyzed the sequential biosynthesis of ¹³C-[2,3,4]-testosterone (13C-T) and ¹³C-[2,3,4]-DHT (13C-DHT) by liquid chromatography/mass spectrometry (LC/MS/MS). The 13C-DHT/13C-T concentration ratio detected by LC/MS/MS in C4-2AT6 cells appeared to reflect the SRD5A activity. The ratio in C4-2AT6 was significantly lower than that in C4-2. An increased concentration of DHT did not have a positive effect on cell proliferation, rather it exhibited inhibitory effects. 5α-reductase inhibitors did not have any inhibitory effect at clinically achievable concentrations. These results indicate that CRPC cells may have an unknown regulation system to protect themselves from an androgenic suppressive effect mediated by SRD5A activity.

Prostate cancer is the product of dysregulated homeostasis within the aging prostate. Supplementation with selenium in the form of selenized yeast (Se-yeast) significantly reduced prostate cancer incidence in the Nutritional Prevention of Cancer Trial. Conversely, the Selenium and Vitamin E Cancer Prevention Trial (SELECT) showed no such cancer-protective advantage using selenomethionine (SeMet). The possibility that SeMet and Se-yeast are not equipotent in promoting homeostasis and cancer risk reduction in the aging prostate has not been adequately investigated; no direct comparison has ever been reported in man or animals. Here, we analyzed data on prostatic responses to SeMet or Se-yeast from a controlled feeding trial of 49 elderly beagle dogs-the only non-human species to frequently develop prostate cancer during aging-randomized to one of five groups: control; low-dose SeMet, low-dose Se-yeast (3 μg/kg); high-dose SeMet, high-dose Se-yeast (6 μg/kg). After seven months of supplementation, we found no significant selenium form-dependent differences in toenail or intraprostatic selenium concentration. Next, we determined whether SeMet or Se-yeast acts with different potency on six markers of prostatic homeostasis that likely contribute to prostate cancer risk reduction-intraprostatic dihydrotestosterone (DHT), testosterone (T), DHT:T, and epithelial cell DNA damage, proliferation, and apoptosis. By analyzing dogs supplemented with SeMet or Se-yeast that achieved equivalent intraprostatic selenium concentration after supplementation, we showed no significant differences in potency of either selenium form on any of the six parameters over three different ranges of target tissue selenium concentration. Our findings, which represent the first direct comparison of SeMet and Se-yeast on a suite of readouts in the aging prostate that reflect flux through multiple gene networks, do not further support the notion that the null results of SELECT are attributable to differences in prostatic consequences achievable through daily supplementation with SeMet, rather than Se-yeast.

Androgens are key mediators of prostate development and function, a role that extends to the development of prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In prostate, DHT is the major androgen and reduction and glucuronidation are the major metabolic pathways for DHT elimination. A streamlined method for quantitation of dihydrotestosterone (DHT), 5α-androstan-3α,17β-diol (3α-diol), and 3α-diol glucuronide (diol-gluc) was established and validated for use with archived prostate tissue specimens to facilitate examination of the roles of the underlying metabolism. This involved a sequential 70/30 hexane/ethyl acetate (hex/EtOAc) extraction of steroids, followed by an ethyl acetate extraction for diol-gluc. Derivatization of the hex/EtOAc fraction with2-fluoro-1-methylpyridinium p-toluene-4-sulfonate (FMP) was used to enhance sensitivity for hydroxyl steroids and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized for analysis of both fractions. The method was validated with calibration standards followed by recovery assessment from spiked samples of BPH and normal prostate. Lower limits of quantitation (LLOQ) were 50 pg/g, 20 pg/g and 100 pg/g for DHT, 3α-diol and diol-gluc, respectively for extracts from 50mg equivalents of tissue. Prepared samples were stable for up to three weeks at 4 °C and 37 °C. The method provides excellent sensitivity and selectivity for determination of tissue levels of DHT, 3α-diol, and diol-gluc. Furthermore, this protocol can easily be extended to other hydroxyl steroids, is relatively straightforward to perform and is an effective tool for assessing steroid levels in archived clinical prostate samples., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

In order to assess whether intratesticular hormone content may be helpful for prediction of successful conception in men with fertility problems, five sex steroids, testosterone, dihydrotestosterone, androstenedione, estradiol and, for the first time epitestosterone, were measured in testicular tissue obtained by surgical retrieval from total 84 men. The group consisted of non-obstructive azoospermic men, aged 21-67 years who attended the centre for in vitro fertilization. Steroids after ether extraction and solvent partition were separated by high performance liquid chromatography and then measured by specific radioimmunoassays. The values varied considerably with means ± S.D. 2.43±2.47, 0.27±0.24, 0.080±0.13, 0.071±0.089 and 0.31±0.27 for testosterone, dihydrotestosterone, androstenedione, estradiol and epitestosterone, respectively.

A method was developed for the simultaneous determination and identification of 12 steroid hormone residues in pig tissues, including stanolone, aldosterone, boldenone, danazol, metandienone, methyltestosterone, nadrolone, norethindrone, progesterone, stanozolol, testosterone and testosterone propionate, using liquid chromatography-tandem triple-quadrupole linear ion trap mass spectrometry(LC-MS/MS). Homogenized pig tissue samples were purified with a Waters MCX solid phase extraction column after enzymatic hydrolysis by beta-glucuronidase, then separated on a Venusil MP C18 column (100 mm x 2, 1 mm, 3 microm) using gradient elution with the mobile phases of acetonitrile and water with 0.1% (v/v) formic acid. A multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information dependent acquisition (IDA) experiment. The compound identification was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The results showed that the limits of detection (LODs, S/N = 3) were in the range of 0.2 - 0.5 microg/kg for the steroid hormones, and with a good linearity (r > 0.99) ranged from 0.5 to 100.0 microg/L. The average recoveries (n = 6) of the 12 steroid hormones spiked in pig tissue samples at 5.0 microg/kg ranged from 72.0% to 98.1% with the relative standard deviations (RSDs) between 3.1% and 12.5%. The method was applied for the qualitative and quantitative determination of steroid hormone residues in pig tissues with sensitive and accurate characteristics.

A method of steroid profiling, including androgens, progestins, corticoids and sterols, was developed to evaluate the concentrations of steroids as well as the activities of the enzymes responsible for steroidogenesis in hair by gas chromatography/mass spectrometry. The extraction efficiencies of steroids from the hair matrix were improved by ultrasonication for 1 h at 50 °C. The overall recoveries ranged from 71 to 132%, with a limit of quantification for all analytes ranging from 1 to 50 ng/g. The devised method was used to identify the metabolic changes for both male-pattern baldness (MPB) and the drug efficiency of dutasteride, which inhibits 5α-reductase. Increased dihydrotestosterone levels and the dihydrotestosterone/testosterone (DHT/T) ratio, which is responsible for the 5α-reductase activity, were observed in the MPB patients. A dutasteride treatment resulted in decreases in the DHT and 5α-androstanedione concentrations and DHT/T ratio in the hair samples. Hair steroid profiling reflects the sebaceous status in the scalp and may be useful for monitoring the metabolic responses to both the disease and drug actions., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objective: To determine whether trilostane or ketotrilostane is more potent in dogs and determine the trilostane and ketotrilostane concentrations that inhibit adrenal gland cortisol, corticosterone, and aldosterone secretion by 50%., Sample: 24 adrenal glands from 18 mixed-breed dogs., Procedures: Adrenal gland tissues were sliced, placed in tissue culture, and stimulated with 100 pg of ACTH/mL alone or with 5 concentrations of trilostane or ketotrilostane. Trials were performed independently 4 times. In each trial, 6 samples (1 for each time point) were collected for each of the 5 concentrations of trilostane and ketotrilostane tested as well as a single negative control samples. At the end of 0, 1, 2, 3, 5, and 7 hours, tubes were harvested and media and tissue slices were assayed for cortisol, corticosterone, aldosterone, and potassium concentrations. Data were analyzed via pharmacodynamic modeling. One adrenal slice exposed to each concentration of trilostane or ketotrilostane was submitted for histologic examination to assess tissue viability., Results: Ketotrilostane was 4.9 and 2.4 times as potent in inhibiting cortisol and corticosterone secretion, respectively, as its parent compound trilostane. For trilostane and ketotrilostane, the concentrations that inhibited secretion of cortisol or corticosterone secretion by 50% were 480 and 98.4 ng/mL, respectively, and 95.0 and 39.6 ng/mL, respectively., Conclusions and Clinical Relevance: Ketotrilostane was more potent than trilostane with respect to inhibition of cortisol and corticosterone secretion. The data should be useful in developing future studies to evaluate in vivo serum concentrations of trilostane and ketotrilostane for efficacy in the treatment of hyperadrenocorticism.

The mechanism accounting for the development of castration-resistant prostate cancer (CRPC) remains unclear. Studies in CRPC tissues suggest that, after androgen deprivation therapy (ADT), the adrenal androgens may be an important source of testosterone (T) and 5-alpha dihydrotestosterone (DHT) in CRPC tissues. To clarify the role of adrenal androgens in the prostatic tissues (prostatic tissue adrenal androgens) during ADT, we developed a high sensitive and specific quantification method for the levels of androgens in prostatic tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human prostatic tissues were purified using mixed-mode reversed-phase, strong anion exchange Oasis cartridges (Oasis MAX). Analysis of steroids was performed using LC-MS/MS after picolinic acid derivatization. The validation tests showed that our method of quantitative analysis was precise and sensitive enough for the quantification of dehydroepiandrosterone (DHEA), androstenedione, androstenediol, T, and DHT in the prostatic tissue. The levels of adrenal androgens in prostate cancer tissues after ADT were similar to those in untreated PCa. Especially, DHEA was the most existing androgen precursor in PCa tissues after ADT. The levels of DHEA were high in PCa tissues, irrespective of ADT. We assumed that DHEA played a significant role in the synthesis of T and DHT in PCa tissues after ADT., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In rats with a DHT deficiency induced by finasteride, morphological changes in the seminiferous epithelium were observed. The structural alterations were manifested by the premature germ cells sloughing into the lumen of seminiferous tubules. The etiology of this disorder could be connected with intercellular junctions disintegration. We showed in the immunohistochemical study the changes in expression of some proteins building tight and adherens junctions. The depression of N-cadherin, β-catenin and occludin immunoexpressions could be the reason for the release of immature germ cells from the seminiferous epithelium. However, the observed increase of the immunohistochemical reaction intensity of vinculin, one of the cadherin/catenin complex regulators, could be insufficient to maintain the proper function of adherens junctions. The hormonal imbalance appears to influence the pattern of expression of junctional proteins in the seminiferous epithelium. It could lead to untimely germ cells sloughing, and ultimately could impair fertility.

Bone may contain an intraskeletal reservoir of sex steroids that is capable of producing biological effects. The purposes of these experiments were to 1) establish and validate methods to extract and measure intraskeletal sex hormones, 2) compare serum and intraskeletal sex hormone abundance, and 3) determine the impact of testosterone-enanthate administration and orchiectomy on intraskeletal sex hormone concentrations. Tibiae from male F344 rats were crushed, suspended in an aqueous buffer, disrupted mechanically and sonically, extracted with organic solvents, dried, and reconstituted in assay buffer appropriate for measurement of testosterone, dihydrotestosterone, and estradiol by immunoassay. Prior to extraction, bone homogenate was spiked with [³H]testosterone, [³H]dihydrotestosterone, or [³H]estradiol, and >80% of each ³H-labeled sex hormone was recovered. Extracted bone samples were also assayed with and without known amounts of unlabeled sex hormones, and >97% of the expected hormone concentrations were measured. Administration of testosterone-enanthate increased intraskeletal testosterone 11-fold and intraskeletal dihydrotestosterone by 82% without altering intraskeletal estradiol (P < 0.01). Conversely, orchiectomy did not alter intraskeletal testosterone or estradiol but increased intraskeletal dihydrotestosterone by 39% (P < 0.05). In intact rats, intraskeletal testosterone and dihydrotestosterone were directionally higher than in serum, whereas intraskeletal estradiol was directionally lower than serum. Serum androgens were positively correlated with intraskeletal androgens (r = 0.74-0.96, P < 0.001); however, neither serum nor intraskeletal androgens nor serum estradiol were correlated with intraskeletal estradiol. We report the validation of a novel method for measuring intraskeletal sex hormones. Our findings demonstrate that the intraskeletal sex steroid reservoirs are modifiable and only partially influenced by circulating sex hormones.

Androgen deprivation therapy is the most common treatment option for advanced prostate cancer. Almost all prostate cancers recur during androgen deprivation therapy, and new evidence suggests that androgen receptor activation persists despite castrate levels of circulating androgens. Quantitation of tissue levels of androgens is critical to understanding the mechanism of recurrence of prostate cancer during androgen deprivation therapy. A liquid chromatography atmospheric pressure photoionization tandem mass spectrometric method was developed for quantitation of tissue levels of androgens. Quantitation of the saturated keto-steroids dihydrotestosterone and 5-alpha-androstanedione required detection of a novel parent ion, [M + 15](+). The nature of this parent ion was explored, and the method was applied to prostate tissue and cell culture with comparison to results achieved using electrospray ionization.

Introduction: The 5-alpha-reductase inhibitor finasteride is used for the treatment of androgenic alopecia, benign prostate hyperplasia and prostate cancer. Besides inhibiting the conversion of testosterone to the biologically more active 5alpha-dihydrotestosterone, it also inhibits the production of neurosteroids. Decreased neurosteroid levels are postulated to be involved in the pathophysiology of psychiatric disorders such as depression. As neurosteroids metabolized by 5-alpha-reductase influence neural plasticity, we investigated whether finasteride treatment alters adult hippocampal neurogenesis, implicated in the pathophysiology of depression., Methods: Male C57BL/6N mice were treated subchronically (7 days) with finasteride or vehicle. Adult neurogenesis was assessed at two different time points after treatment (day 1; day 35) using immunohistochemistry., Results: Finasteride treatment led to a significant decrease in brain 5alpha-dihydrotestosterone levels and induced a reversible reduction in the number of newborn cells and young neurons in the hippocampus. 35 days after the last finasteride injection, neurogenesis had returned to normal., Discussion: These data indicate that inhibition of 5-alpha-reductase activity by finasteride treatment influences neuronal plasticity on a structural level. These changes might contribute to the pathophysiology of depressive episodes observed after finasteride treatment., (Copyright Georg Thieme Verlag KG Stuttgart New York.)

Androgens and estrogens are known regulators of fetal and postnatal lung development, but their levels in the developing lung have never been determined. We present here, for the first time, a gas chromatography-mass spectrometry (GC/MS) quantification of dihydrotestosterone, testosterone, androstenedione, and estradiol in canalicular, saccular, and alveolar stage lungs of both sexes. Testosterone, androstenedione, and estradiol were observed in all the analyzed lung samples from gestation day (GD) 16.5 to postnatal day (PN) 30, totalizing 383 individual mice. Levels of these three steroids decreased between birth and PN 5. In contrast, dihydrotestosterone was detected only in male samples on GD 19.5, PN 0, and PN 30. A significant sex difference was observed for testosterone and androstenedione but not for estradiol. Steroid levels were also determined in skinned hind legs for comparison. Three-way analysis of variance revealed that tissue (lung or leg) had a significant effect on testosterone levels for both sexes, but not on androstenedione and estradiol levels. Low but significant testosterone and androstenedione levels were observed in all the females and in prepubertal male samples. These levels must be sufficient to induce androgen receptor activation, as suggested by our recent report showing the presence of androgen receptor in the nucleus of several lung cells in corresponding developmental ages and sexes.

Background: Various hormone refractory prostate cancer cell models have been established with androgen depletion and have helped to clarify the mechanism for the transition into androgen-depletion independent status. However, the mechanism of bicalutamide resistance remains unclear because few cell models have been generated., Methods: We generated a bicalutamide-resistant subline, LNCaP-BC2, from LNCaP after prolonged treatment with bicalutamide. Androgen and/or bicalutamide responsiveness for proliferation and prostate-specific antigen (PSA) secretion were examined in vitro and in vivo. Testosterone and dihydrotestosterone (DHT) levels in xenografted tumors were analyzed by liquid chromatography-tandem mass spectrometry. Androgen receptor (AR) gene mutation and amplification and AR and pAR(210) expression were determined., Results: LNCaP-BC2 did not grow in an androgen-depleted medium and proliferation was stimulated in a tenfold lower concentration of androgen than that of LNCaP. LNCaP-BC2 grew in castrated male mice, and the DHT level in grafted LNCaP-BC2 tumors was 7.7-fold lower than in LNCaP tumors. Bicalutamide stimulated LNCaP-BC2 proliferation and PSA secretion in vitro and the antitumor activity of bicalutamide against LNCaP-BC2 was weaker than that of LNCaP in vivo. Additional AR mutation and AR gene amplification were not detected in LNCaP-BC2, but AR and pAR(210) expression and PSA secretion in LNCaP-BC2 were higher than in LNCaP., Conclusions: Bicalutamide-resistant LNCaP-BC2 exhibited AR overexpression and hypersensitivity to low levels of androgen. Our data suggests that AR overexpression is a significant mechanism of bicalutamide resistance similar to resistance from chronic androgen depletion. In addition, pAR(210) overexpression could be a potential mechanism for hypersensitivity to low androgen in LNCaP-BC2.

Sex steroids are essential for spermatogenesis; however, normal intratesticular concentrations of these hormones in man have not been extensively studied. To improve our understanding of intratesticular hormone concentrations, we performed bilateral testicular aspirations in a group of normal men, determined sex steroid concentrations within each testis, and compared these levels to serum hormone concentrations. Ten healthy human subjects aged 20-49 underwent bilateral testicular aspirations. Intratesticular hormone concentrations of testosterone, dihydrotestosterone (DHT), and estradiol were measured using liquid chromatography-tandem mass spectrometry. Intratesticular testosterone concentrations ranged from 119 to 1251 ng/mL, with a mean of 635 +/- 368 ng/mL. Intratesticular estradiol ranged from 0.41 to 3.9 ng/mL, with a mean of 2.4 +/- 1.3 ng/mL. Intratesticular DHT ranged from 1.1 to 7.9 ng/mL, with a mean of 3.5 +/- 3.2 ng/mL. Intratesticular testosterone and estradiol concentrations correlated highly with serum luteinizing hormone (LH; r = 0.87 and r = 0.70 respectively, P < .01). Intratesticular testosterone correlated highly with serum testosterone. Moreover, a significant correlation between the right and left testes was observed for testosterone (r = 0.82, P = .003), but not for estradiol or DHT. Intratesticular hormone concentrations can be safely assessed by testicular aspiration. Intratesticular testosterone and estradiol correlate highly with serum LH concentrations, and variation in serum LH accounts for most of the variation in intratesticular testosterone among men. In addition, intratesticular testosterone is highly correlated between testes in a given individual. Direct measurement of intratesticular testosterone will improve our understanding of the relationship between intratesticular sex steroids and spermatogenesis, and may have implications for the development of male hormonal contraception.

Background: Human prostate cancer cells are routinely maintained in media supplemented with 10% Fetal Calf Serum (FCS) to provide androgen. In the present study, total and free testosterone levels in 10%FCS supplemented tissue culture media were determined and compared to levels in intact and castrated human males. Dextran-coated charcoal stripped FCS (i.e., DC-FCS) is often used instead of FCS to minimize the level of androgen provided in 10% serum supplemented media. Therefore, total and free testosterone levels in 10%DC-FCS containing media were likewise determined., Methods: Total testosterone, free testosterone, and total dihydrotestosterone (DHT) were determined on RPMI-1640 media supplemented with either 10%FCS or 10%DC-FCS by ELISA assays before and after exposure to LNCaP human prostate cancer cells in culture. The growth and PSA secretion by these cells was also determined., Results: Ten percentage FCS supplemented media contains a castrate level of testosterone. However, even with this castrate starting level of testosterone, LNCaP cells concentrate and metabolize the testosterone to produce a physiologic (i.e., 10 nM) level of intracellular DHT which optimally stimulates the growth of these cells in vitro., Conclusions: The present studies document that prostate cancer cells auto-regulate their androgen metabolism so that an optimal level of DHT for growth is maintained during both up and down fluctuations in the supply of testosterone. These results have significant implications for whether exogenous androgen should be added to the 10%FCS supplemented media to grow prostate cancer cells from intact versus castrated patients., (Copyright 2009 Wiley-Liss, Inc.)

Background: Investigating the mechanisms of human primordial germ cell (PGC) and gamete development are important for understanding the causes of infertility and effects of environmental chemicals on reproductive development. However, there are practical and ethical difficulties associated with obtaining human tissue in early development. The aim of this study was to investigate whether human embryonic stem cell-hESC-generated germ cells could provide an in vitro model of gamete development., Method: Human ESCs were differentiated as embryoid bodies (EBs) in vitro. Gene and protein marker expression profiles of EBs in different periods of culture were analysed by quantitative polymerase chain reaction (Q-PCR) and immunolocalization to monitor germ cell development. Secretion of hormones involved in germ cell maturation was measured, to detect the existence of a germ cell niche within EBs., Results: Q-PCR revealed gene expression profiles consistent with PGC formation and germ cell development. A small population of post-meiotic spermatid cells were identified using sperm-specific antibodies (Protamine 1 and 1.97). Although gene expression profiles characteristic of oocyte development and follicle-like structures were detected, a committed oocyte with extra-cellular zona pellucida was not recognized with zona pellucida-specific monoclonal antibody., Conclusions: hESCs can form PGCs and post-meiotic spermatids in vitro, however, there remains doubt about oocyte development. Levels of steroid hormones produced by EBs were significant when compared with known values for a similar quantity of human testis, suggesting that hESC may intrinsically create a favourable hormonal niche for spermatogenesis.

We developed highly sensitive detection of testosterone (T) and 5alpha-dihydrotestosterone (DHT) by liquid chromatography-electrospray ionization tandem mass spectrometry using high proton affinitive derivatization of 17beta-hydroxyl group of T and DHT with picolinic acid, mobile phase consisting of MeCN-MeOH-H(2)O-formic acid and conventional octadecylsilica (ODS) column. Purification of the derivatives was carried out using solid-phase extraction with ODS cartridge. By this method, T and DHT were determined simultaneously with limits of quantification (LOQs) of 1 pg/0.2 ml in serum, and T and DHT with LOQs of 0.5 pg and 1 pg/3mg in prostate tissue, respectively, under acceptable assay performance (intra-assay and inter-assay accuracy and precision). The present method provides reliable and reproducible results for quantification of T and DHT in small volumes of serum and prostate samples for diagnosis in prostatic disorders and male climacteric.

Treatments for advanced prostate cancer (CaP) typically involve androgen deprivation therapy. However, most patients eventually develop castration-resistant CaP (CRPC) for which highly effective therapies are limited. We explored the efficacy of a novel agent, HE3235, in inhibiting growth of CRPC in preclinical models. Castrated male mice were implanted subcutaneously with LuCaP35V CaP xenografts in the presence and absence of 5'-androstenediol (AED) and treated with HE3235. To investigate the effect of HE3235 on CaP tumor in the bone, castrated mice were injected intratibially with C4-2B CaP cells and treated with HE3235. Serum prostate-specific antigen (PSA) levels, tumor volume, immunohistochemistry, gene expression, and levels of intratumoral androgens were analyzed. HE3235 significantly prolonged the tumor doubling time of LuCaP35V, decreased androgen receptor expression, and lowered levels of intratumoral testosterone by approximately 89% and dihydrotestosterone by approximately 63% in both the presence and the absence of AED. HE3235 inhibited tumor growth in the bone environment. Weights of tumored tibiae of HE3235-treated animals were lower than those of control (P = .031), and normalized PSA levels were also significantly decreased at the end of study by HE3235 treatment (P = .0076). HE3235 inhibits the growth of subcutaneous CRPC as well as CRPC in the bone environment. Our data show that HE3235 exhibits a wide range of effects, including alteration of androgen receptor signaling and reductions in levels of intratumoral androgens. Our results support ongoing clinical investigations into the effectiveness of HE3235 in the setting of CRPC and warrants further studies into the mechanisms behind the effects of HE3235.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to extract and quantify the androgen concentration in the rat prostate. This method introduced a novel 96-well plate format for the extraction and derivatization of testosterone (T) and dihydrotestosterone (DHT) from rat prostatic tissue that greatly simplified the sample preparation procedure. Due to the difficulty to obtain reproducible specimens with non-detectable level of androgen, a matrix-free standard solution was used for method validation. Both T and DHT calibration curves were linear over the calibration range (12.5-2500 pg) with correlation coefficient values greater than 0.9900. The intra-day and inter-day accuracy, reported as %bias, and precision, reported as %CV, of T and DHT were within +/-10%. The lower limit of detection (LLOD) and lower limits of quantification (LLOQ) for both T and DHT were determined to be 5 and 12.5 pg. The validation results demonstrated the selectivity, sensitivity, accuracy, precision, linearity and ruggedness of the method, as well as the suitability of the method for simultaneous detection of T and DHT in rat prostatic tissues. The validated method was successfully applied to determine the physiological T and DHT level in rat prostatic tissues. Similarly to the serum concentration profile pattern, T and DHT intraprostatic levels peaked 2 h after lights-on and decreased after lights-off with DHT level approximately 4-fold greater than T.

Wastewater effluents are complex mixtures containing a variety of anthropogenic compounds, many of which are known endocrine disruptors. In order to characterize the developmental and behavioral effects of such a complex mixture, northern leopard frogs, Rana pipiens, were exposed to a range of concentrations (0%, 10%, 50%, 100%) of municipal wastewater effluent from the egg stage through metamorphosis. The estrogenic activity of the effluent was quantified by the calculation of an estradiol (E2) equivalency (EEQ) factor and was determined to be equivalent to 1.724+/-2.103ng/L E2. Individuals from the 50% and 100% wastewater treatments took significantly longer to reach metamorphosis than individuals in the 0% and 10% treatments. An increased incidence of male testicular oocytes was observed in the 50% and 100% treatments when compared to the control treatment. Morphological changes in the thyroid glands of 100% wastewater-treated individuals were also noted. No effects of wastewater exposure on growth, sex ratio, swim speed, startle response, or female gonadal development were observed. These results suggest that municipal wastewater effluent can alter the timing of the metamorphic process and impact male sexual development in R. pipiens.

Objective: To investigate the relationship between antimüllerian hormone (AMH) and steroidogenesis in follicles of normoandrogenic ovulatory women undergoing follicle-stimulating hormone (FSH) therapy for in vitro fertilization/embryo transfer (IVF-ET)., Design: Prospective cohort., Setting: Institutional/private practice., Patient(s): 26 normoandrogenic ovulatory women. All women received gonadotropin-releasing hormone (GnRH) analog and ovarian stimulation for IVF-ET., Intervention(s): Follicle fluid was aspirated at oocyte retrieval from the first follicle of each ovary., Main Outcome Measure(s): Follicle fluid was assayed for AMH, estradiol (E(2)), progesterone, androstenedione, testosterone, dihydrotestosterone, insulin, and FSH., Result(s): Intrafollicular AMH levels positively and negatively correlated with E(2) and FSH concentrations in follicles, respectively, causing a positive relationship between follicle fluid AMH levels and E(2)/FSH ratios as a measure of follicle sensitivity to FSH. A positive relationship also existed in follicles between AMH levels and E(2)/androgen ratios as a marker of aromatase activity., Conclusion(s): The AMH levels in follicles of IVF patients positively correlate with follicle sensitivity to FSH.

Saccharomyces cerevisiae bioluminescent bioreporter assays were developed previously to assess a chemical's estrogenic or androgenic disrupting potential. S. cerevisiae BLYES, S. cerevisiae BLYAS, S. cerevisiae BLYR, were used to assess their reproducibility and utility in screening 68, 69, and 71 chemicals for estrogenic, androgenic, and toxic effects, respectively. EC(50) values were 6.3 +/- 2.4 x 10(-10)M (n = 18) and 1.1 +/- 0.5 x 10(-8)M (n = 13) for BLYES and BLYAS, using 17beta-estradiol and 5alpha-dihydrotestosterone over concentration ranges of 2.5 x 10(-12) through 1.0 x 10(-6)M, respectively. Based on analysis of replicate standard curves and comparison to background controls, a set of quantitative rules have been formulated to interpret data and determine if a chemical is potentially hormonally active, toxic, both, or neither. The results demonstrated that these assays are applicable for Tier I chemical screening in Environmental Protection Agency's Endocrine Disruptor Screening and Testing Program as well as for monitoring endocrine-disrupting activity of unknown chemicals in water.

Objectives: Dutasteride exerts its beneficial effects on the prostate through suppression of intraprostatic dihydrotestosterone (DHT). The aim of this analysis was to assess the effects of the approved dose of dutasteride (0.5 mg/d), given for 2 weeks to 4 months, on the serum and intraprostatic DHT and testosterone levels in 3 randomized studies., Methods: Intraprostatic androgen levels were measured in benign prostatic tissue collected during transurethral resection of the prostate (benign prostatic hyperplasia studies, n = 256) or radical prostatectomy (prostate cancer study, n = 51), performed after 2 weeks, or 1, 3, or 4 months of treatment with dutasteride or with placebo or surgery alone. The serum androgen levels were assessed at the same points during treatment. Data from the control groups were pooled to provide 1 comparison group., Results: Dutasteride reduced the intraprostatic DHT levels by 83%, 90%, 92%, and 93% after 2 weeks and 1, 3, and 4 months of treatment, respectively, compared with placebo/surgery alone. Dutasteride reduced the serum DHT levels from baseline by 84% at 2 weeks and by approximately 90% at 1, 2, 3, and 4 months compared with a 5.2% increase in the control group. The decrease in DHT levels with dutasteride was accompanied by a reciprocal increase in the serum and intraprostatic testosterone levels. However, the intraprostatic testosterone levels in the dutasteride groups generally remained lower than the intraprostatic DHT levels in the control group., Conclusions: The results of our study have shown that dutasteride provides near-maximal suppression of both serum and intraprostatic DHT levels in men with benign prostatic hyperplasia or prostate cancer at all points assessed.

Maternal hormones are an excellent pathway for the mother to influence offspring development, and birds provide exceptional opportunities to study these hormone-mediated maternal effects. Two dominant hypotheses about the function of yolk androgens in avian eggs concern maternal manipulation of sibling competition and post hatching paternal care. In megapodes, however, neither sibling competition nor post hatching parental care exists. Eggs are incubated by external heat sources, and chicks dig themselves out of their underground nest and live independently of their parents and their siblings. In this first study on egg androgens of such a megapode, the Australian Brush-turkey Alectura lathami, we found nevertheless substantial amounts of maternal androgens. Since size of the incubation mound, incubation temperature, egg size and laying date greatly vary in this species, we analysed variation in testosterone (T), androstenedione (A4) and dihydrotestosterone (DHT) in relation to these factors. T concentrations were significantly higher in eggs from bigger mounds and laid at greater depth, which may compensate via anabolic effects for the longer duration and higher energetic requirements of chicks when digging themselves out. T concentrations were higher in smaller eggs, and both yolk A4 and T concentrations increased with laying date, perhaps as a compensatory measure, while DHT concentrations only varied across different mounds. These results indicate that maternal androgens may influence offspring development outside the contexts of sibling competition or parental care.

Greater offspring predation favors evolution of faster development among species. We hypothesized that greater offspring predation exerts selection on mothers to increase levels of anabolic androgens in egg yolks to achieve faster development. Here, we tested whether (1) concentrations of yolk androgens in passerine species were associated with offspring predation and (2) embryo and nestling development rates were associated with yolk androgen concentrations. We examined three androgens that increase in potency along the synthesis pathway: androstenedione (A(4)) to testosterone (T) to 5 alpha -dihydrotestosterone (5 alpha -DHT). Concentrations of none of these steroids were related to clutch size; only A(4) was allometrically related to egg volume. Species that experience greater predation showed higher yolk concentrations of T and 5 alpha -DHT. Higher concentrations of T and particularly 5 alpha -DHT were strongly correlated with faster development during the embryo period and less so during the nestling period. Development rates were most strongly correlated with 5 alpha -DHT, suggesting that potency increases along the androgen synthesis pathway and that effects are mediated by the androgen receptor pathway. These results are consistent with the hypothesis that selection for faster development by time-dependent offspring mortality may be achieved epigenetically by varying embryo exposure to maternal anabolic steroids.

Maternal effects occur when offspring phenotype is affected by environmental factors experienced by the mother and, in egg-laying species, are often mediated via egg resources. There is currently great interest among behavioural ecologists in maternally allocated yolk androgens, especially their relationship with offspring sex and development. Such studies need embryonic tissue for sexing, however, requiring eggs to be incubated (usually for 3 days). Therefore, there are concerns about whether the androgen concentrations assayed reflect those allocated by the mother. In addition, studies showing sex biases in maternal allocation of androgens could be confounded if male and female embryos uptake or metabolise androgens at different rates. We ran a series of experiments using zebra finch (Taeniopygia guttata) eggs to address these potential confounding factors. First we showed, using eggs naturally incubated for up to 5 days, that eggs containing embryos had lower yolk androgen concentrations than eggs that had failed to form embryos. We then tested various hypotheses for this difference using controlled incubation treatments. Our results suggested that (a) embryo development causes the yolk to become progressively more diluted with albumin; and (b) between 3 and 5 days of incubation embryos start uptaking or metabolising androgens. Crucially, we found no decline in yolk androgen concentration at 3 days incubation, and no evidence for sex-specific rates of uptake or metabolism of androgens. This strongly suggests that yolk androgen levels up to 3 days incubation do reflect those allocated by the mother, and that studies of sex biased maternal allocation of yolk androgens are not confounded by sex differences in embryo development.

Previous in vitro studies demonstrated that bioactive androgen 5alpha-dihydrotestosterone (DHT) exerted antiproliferative effects through an interaction with androgen receptor (AR) in breast carcinoma cells. However, AR status has not been examined in association with DHT concentration in breast carcinoma tissues, and significance of androgenic actions remains unclear in breast carcinomas. Therefore, in our study, we first examined intratumoral DHT concentrations in 38 breast carcinoma tissues using liquid chromatography/electrospray tandem mass spectrometry. Intratumoral DHT concentration was positively associated with 5alpha-reductase type 1 (5alphaRed1), and negatively correlated with aromatase. We then examined clinical significance of AR and 5alphaRed1 status in 115 breast carcinoma tissues by immunohistochemistry. Breast carcinomas positive for both AR and 5alphaRed1 were inversely associated with tumor size or Ki-67. These patients showed significant associations with a decreased risk of recurrence and improved prognosis for overall survival, and the AR / 5alphaRed1 status was demonstrated an independent prognostic factor. Moreover, we examined possible regulation of DHT production by aromatase in in vitro studies. DHT synthesis from androstenedione in MCF-7 cells was significantly inhibited by coculture with aromatase-positive stromal cells, which was significantly reversed by addition of aromatase inhibitor exemestane. These results suggest that intratumoral DHT concentration is mainly determined by 5alphaRed1 and aromatase in breast carcinoma tissues, and antiproliferative effect of DHT may primarily occur in the cases positive for both AR and 5alphaRed1. Aromatase inhibitors may be more effective in these patients, possibly due to increasing local DHT concentration with estrogen deprivation., ((c) 2006 Wiley-Liss, Inc.)

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the simultaneous quantitative analysis of dihydrotestosterone (DHT) and testosterone (T) from biological fluids has been developed. Commercially available deuterated analogues were used as internal standards. Steroids were extracted from serum or testicular fluid with hexane/ethyl acetate, evaporated to dryness, and treated with hydroxylamine to form their oxime derivatives. Upon chromatographic separation, the compounds were quantified using selected reaction monitoring (SRM). For T, the [M+H](+) ion at m/z 304 and the fragment ion at m/z 124 were used as the precursor and product ions. For DHT the ion cluster [M+H+ACN](+) at m/z 347 and the dissociated ion [M+H](+) at m/z 306 were used as the precursor and product ions, respectively. The limits of detectability on-column were in the sub-femtomole range for both compounds and the intra-day coefficient of variation (CV) for analysis from serum was less than 7% for both compounds. Given its high reproducibility, sensitivity, and relative simplicity, this assay should be of use in determining androgen levels in biospecimens, particularly in settings where sample quantity or steroid concentration are low., (Copyright (c) 2007 John Wiley & Sons, Ltd.)