With aging, abnormal benign growth of the prostate results in benign prostate hyperplasia (BPH) with concomitant lower urinary tract symptoms (LUTS). Because the prostate is an androgen target tissue, and transforms testosterone into <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>), a potent androgen, via <em>5α</em>-reductase (<em>5α</em>-R) activity, inhibiting this key metabolic reaction was identified as a target for drug development to treat symptoms of BPH. Two drugs, namely finasteride and dutasteride were developed as specific <em>5α</em>-reductase inhibitors (<em>5α</em>-RIs) and were approved by the U.S. Food and Drug Administration for the treatment of BPH symptoms. These agents have proven useful in the reducing urinary retention and minimizing surgical intervention in patients with BPH symptoms and considerable literature exists describing the benefits of these agents. In this review we highlight the adverse side effects of <em>5α</em>-RIs on sexual function, high grade prostate cancer incidence, central nervous system function and on depression. <em>5α</em>-Rs isoforms (types 1-3) are widely distributed in many tissues including the central nervous system and inhibition of these enzymes results in blockade of synthesis of several key hormones and neuro-active steroids leading to a host of adverse effects, including loss of or reduced libido, erectile dysfunction, orgasmic dysfunction, increased high Gleason grade prostate cancer, observed heart failure and cardiovascular events in clinical trials, and depression. Considerable evidence exists from preclinical and clinical studies, which point to significant and serious adverse effects of <em>5α</em>-RIs, finasteride and dutasteride, on sexual health, vascular health, psychological health and the overall quality of life. Physicians need to be aware of such potential adverse effects and communicate such information to their patients prior to commencing <em>5α</em>-RIs therapy.

Sex steroids are important regulators for longitudinal growth, bone mass accrual, and sexual dimorphism of the skeleton. 17β-Estradiol regulates proliferation and differentiation of female chondrocytes via a membrane-associated signaling pathway in addition to its estrogen receptor (ER) mediated effects. In contrast, testosterone does not elicit a similar membrane response, either in male or female cells. Whereas female rat growth plate chondrocytes convert testosterone to 17β-estradiol, male chondrocytes produce <em>5α</em>-dihydrotestosterone (<em>DHT</em>). Previously <em>DHT</em> was found to mediate sex-specific effects of testosterone in male cells, but it is not known if a membrane-signaling pathway is involved. In this study, we hypothesized that <em>DHT</em> can induce sex-specific rapid membrane effects similar to other steroid hormones. Confluent cultures of chondrocytes isolated from resting zones of growth plates of both male and female rats were treated with 10(-10)-10(-7)M testosterone or <em>DHT</em> for 3, 9, 90 and 270min and protein kinase C (PKC) and phospholipase A2 (PLA2) activities were measured. To examine potential signaling pathways involved in PKC activation, male chondrocytes were treated with 10(-7)M <em>DHT</em> for 9min in the presence or absence of the phospholipase C (PLC) inhibitor U73122, the secretory PLA2 inhibitor quinacrine or the cytosolic PLA2 inhibitor AACOCF3; the Gαi inhibitor pertussis toxin (PTX) or Gαs activator cholera toxin (CTX), and the general G-protein inhibitor GDPβS; thapsigargin, an inhibitor of a Ca-ATPase pump in the endoplasmic reticulum; verapamil and nifedipine, inhibitors of specific L type Ca2+ channels on the cell membrane; and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR); as well as the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide. <em>DHT</em> induced a dose-dependent increase in PKC and PLA2 activity in male cells with the highest increase at 10(-7)M <em>DHT</em> (p<0.05), whereas testosterone had no effect. PKC activity was augmented at 9 and 90 min, and then decreased to baseline at 270min. Neither testosterone nor <em>DHT</em> affected PKC in female cells. U73122, quinacrine, and AACOCF3 inhibited <em>DHT</em>-induced activation of PKC. <em>DHT</em> treatment for 9 min had no effect in [(3)H]-thymidine incorporation in quiescent confluent cultures but caused a dose dependent increase in alkaline phosphatase specific activity. Inhibition of PLC reduced the response of to <em>DHT</em> in a dose dependent manner, indicating that PLC is involved. In conclusion, our study indicates that <em>DHT</em>, but not testosterone, has sex-specific rapid membrane effects in male growth plate chondrocytes involving PLC and PLA2-mediated PKC signaling pathways. Together with previous observations showing that male cells convert testosterone to <em>DHT</em>, these results suggest that <em>DHT</em> might act in the membrane through an autocrine/paracrine mechanism.

Vocal control nuclei in songbirds display seasonal changes in volume that are regulated by testosterone (T) and its androgenic (<em>5α</em>-dihydrotestosterone; <em>DHT</em>) or estrogenic (17β-estradiol; E(2)) metabolites. In male canaries, T regulates expression of the microtubule-associated protein doublecortin (DCX), a marker of neurogenesis. We examined the effect of T and its two metabolites alone or in combination on DCX expression in adult female canaries. Treatment with T or with <em>DHT</em>+E(2) increased HVC volume and neuron numbers as well as the total numbers of fusiform (migrating) and round (differentiating) DCX neurons in the nucleus but generally not in adjacent areas. <em>DHT</em> or E(2) alone did not increase these measures but increased the density of fusiform DCX cells per section. Similar results were observed in area X, although some effects did not reach significance, presumably because plasticity in X is mediated transsynaptically and follows HVC changes with some delay. There was no effect of any treatment on the total number of neurons in area X, and no change in DCX cell densities was detected in the lateral magnocellular nucleus of the anterior nidopallium, nor in other parts of the nidopallium. <em>DHT</em> and E(2) by themselves thus increase density of DCX cells migrating through HVC but are not sufficient in isolation to induce the recruitment of these newborn neurons in the nucleus. These effects are generally not observed in the rest of the nidopallium, implying that steroids only act on the attraction and recruitment of new neurons in HVC without having any major effects on their production at the ventricle wall.

Exposure to phthalates in utero alters fetal rat testis gene expression and testosterone production, but much remains to be done to understand the mechanisms underlying the direct action of phthalate within the fetal testis. We aimed to investigate the direct mechanisms of action of mono-(2-ethylhexyl) phthalate (MEHP) on the rat fetal testis, focusing on Leydig cell steroidogenesis in particular. We used an in vitro system based on the culture for three days, with or without MEHP, of rat fetal testes obtained at 14.5 days post-coitum.Exposure to MEHP led to a dose-dependent decrease in testosterone production. Moreover, the production of 5 alpha-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) (-68%) and androstenedione (-54%) was also inhibited by 10 µM MEHP, whereas 17 alpha-hydroxyprogesterone (17α-OHP) production was found to increase (+41%). Testosterone synthesis was rescued by the addition of androstenedione but not by any of the other precursors used. Thus, the hormone data suggested that steroidogenesis was blocked at the level of the 17,20 lyase activity of the P450c17 enzyme (CYP17), converting 17α-OHP to androstenedione. The subsequent gene expression and protein levels supported this hypothesis. In addition to Cyp17a1, microarray analysis showed that several other genes important for testes development were affected by MEHP. These genes included those encoding insulin-like factor 3 (INSL3), which is involved in controlling testicular descent, and Inha, which encodes the alpha subunit of inhibin B.These findings indicate that under in vitro conditions known to support normal differentiation of the fetal rat testis, the exposure to MEHP directly inhibits several important Leydig cell factors involved in testis function and that the Cyp17a1 gene is a specific target to MEHP explaining the MEHP-induced suppression of steroidogenesis observed.

BACKGROUND

Currently available <em>5α</em>-reductase inhibitors are not completely effective for treatment of benign prostate enlargement, prevention of prostate cancer (CaP), or treatment of advanced castration-recurrent (CR) CaP. We tested the hypothesis that a novel <em>5α</em>-reductase, <em>5α</em>-reductase-3, contributes to residual androgen metabolism, especially in CR-CaP.

METHODS

A new protein with potential <em>5α</em>-reducing activity was expressed in CHO-K1 cellsandTOP10 E. coli for characterization. Protein lysates and total mRNA were isolated from preclinical and clinical tissues. Androgen metabolism was assessed using androgen precursors and thin layer chromatography or liquid chromatography tandem mass spectrometry.

RESULTS

The relative mRNA expression for the three <em>5α</em>-reductase enzymes in clinical samples of CR-CaP was <em>5α</em>-reductase-3 ≫ <em>5α</em>-reductase-1> <em>5α</em>-reductase-2. Recombinant <em>5α</em>-reductase-3 protein incubations converted testosterone, 4-androstene-3,17-dione (androstenedione) and 4-pregnene-3,20-dione (progesterone) to dihydrotestosterone, <em>5α</em>-androstan-3,17-dione, and <em>5α</em>-pregnan-3,20-dione, respectively. <em>5α</em>-Reduced androgen metabolites were measurable in lysates from androgen-stimulated (AS) CWR22 and CR-CWR22 tumors and clinical specimens of AS-CaP and CR-CaP pre-incubated with dutasteride (a bi-specific inhibitor of <em>5α</em>-reductase-1 and 2).

CONCLUSIONS

Human prostate tissues contain a third <em>5α</em>-reductase that was inhibited poorly by dutasteride at high androgen substrate concentration in vitro, and it may promote DHT formation in vivo, through alternative androgen metabolism pathways when testosterone levels are low.

BACKGROUND

<em>5α</em>-Reductase 1 and 2 (SRD5A1, SRD5A2) inactivate cortisol to <em>5α</em>-dihydrocortisol in addition to their role in the generation of <em>DHT</em>. Dutasteride (dual SRD5A1 and SRD5A2 inhibitor) and finasteride (selective SRD5A2 inhibitor) are commonly prescribed, but their potential metabolic effects have only recently been identified.

OBJECTIVE

Our objective was to provide a detailed assessment of the metabolic effects of SRD5A inhibition and in particular the impact on hepatic lipid metabolism.

METHODS

We conducted a randomized study in 12 healthy male volunteers with detailed metabolic phenotyping performed before and after a 3-week treatment with finasteride (5 mg od) or dutasteride (0.5 mg od). Hepatic magnetic resonance spectroscopy (MRS) and two-step hyperinsulinemic euglycemic clamps incorporating stable isotopes with concomitant adipose tissue microdialysis were used to evaluate carbohydrate and lipid flux. Analysis of the serum metabolome was performed using ultra-HPLC-mass spectrometry.

METHODS

The study was performed in the Wellcome Trust Clinical Research Facility, Queen Elizabeth Hospital, Birmingham, United Kingdom.

METHODS

Incorporation of hepatic lipid was measured with MRS.

RESULTS

Dutasteride, not finasteride, increased hepatic insulin resistance. Intrahepatic lipid increased on MRS after dutasteride treatment and was associated with increased rates of de novo lipogenesis. Adipose tissue lipid mobilization was decreased by dutasteride. Analysis of the serum metabolome demonstrated that in the fasted state, dutasteride had a significant effect on lipid metabolism.

CONCLUSIONS

Dual-SRD5A inhibition with dutasteride is associated with increased intrahepatic lipid accumulation.

Prostate cancer is a commonly diagnosed cancer in men, and dietary chemoprevention by pomegranate (Punica granatum) extracts has shown noticeable benefits. In this study, we investigated the growth inhibitory, antiandrogenic, and pro-apoptotic effects of 13 pure compounds found in the pomegranate in androgen-dependent LNCaP human prostate cancer cells. Cells deprived of steroid hormones were exposed to increasing concentrations (1-100 μM) of pomegranate compounds in the presence of 0.1 nM dihydrotestosterone (<em>DHT</em>), and the inhibition of cell growth was measured by WST-1 colorimetric assay after a 4 day exposure. Four compounds, epigallocatechin gallate (EGCG), delphinidin chloride, kaempferol, and punicic acid, were found to inhibit <em>DHT</em>-stimulated cell growth at concentrations of 10 μM and above. These four pomegranate compounds inhibited <em>DHT</em>-stimulated androgen receptor nuclear accumulation and the expression of the androgen receptor-dependent genes prostate specific antigen and steroid <em>5α</em>-reductase type 1 at concentrations ≥10 μM. We determined the possible contribution of apoptosis to the observed decrease in cell growth and found that three compounds, EGCG, kaempferol, and, in particular, punicic acid, induced DNA fragmentation after a 24 h treatment, at concentrations in the 10-100 μM range. Punicic acid, an important fatty acid in pomegranate seeds, was further found to induce intrinsic apoptosis via a caspase-dependent pathway. In conclusion, punicic acid, the main constituent of pomegranate seed (70-80%), exhibited potent growth inhibitory activities in androgen-dependent LNCaP cells, which appear to be mediated by both antiandrogenic and pro-apoptotic mechanisms.

Recent evidence indicates that the estrogen receptor-α-negative, androgen receptor (AR)-positive molecular apocrine subtype of breast cancer is driven by AR signaling. The MDA-MB-453 cell line is the prototypical model of this breast cancer subtype; its proliferation is stimulated by androgens such as <em>5α</em>-dihydrotestosterone (<em>DHT</em>) but inhibited by the progestin medroxyprogesterone acetate (MPA) via AR-mediated mechanisms. We report here that the AR gene in MDA-MB-453 cells contains a G-T transversion in exon 7, resulting in a receptor variant with a glutamine to histidine substitution at amino acid 865 (Q865H) in the ligand binding domain. Compared with wild-type AR, the Q865H variant exhibited reduced sensitivity to <em>DHT</em> and MPA in transactivation assays in MDA-MB-453 and PC-3 cells but did not respond to non-androgenic ligands or receptor antagonists. Ligand binding, molecular modeling, mammalian two-hybrid and immunoblot assays revealed effects of the Q865H mutation on ligand dissociation, AR intramolecular interactions, and receptor stability. Microarray expression profiling demonstrated that <em>DHT</em> and MPA regulate distinct transcriptional programs in MDA-MB-453 cells. Gene Set Enrichment Analysis revealed that <em>DHT</em>- but not MPA-regulated genes were associated with estrogen-responsive transcriptomes from MCF-7 cells and the Wnt signaling pathway. These findings suggest that the divergent proliferative responses of MDA-MB-453 cells to <em>DHT</em> and MPA result from the different genetic programs elicited by these two ligands through the AR-Q865H variant. This work highlights the necessity to characterize additional models of molecular apocrine breast cancer to determine the precise role of AR signaling in this breast cancer subtype.

Owing to the heterogeneity in the clinical symptoms of polycystic ovary syndrome (PCOS), the early pathophysiological mechanisms of PCOS remain unclear. Clinical, experimental, and genetic evidence supports an interaction between genetic susceptibility and the influence of maternal environment in the pathogenesis of PCOS. To determine whether prenatal androgen exposure induced PCOS-related metabolic derangements during pubertal development, we administrated <em>5α</em>-dihydrotestosterone (<em>DHT</em>) in pregnant rats and observed their female offspring from postnatal 4 to 8 weeks. The prenatally androgenized (PNA) rats exhibited more numerous total follicles, cystic follicles, and atretic follicles than the controls. Fasting glucose, insulin, leptin levels, and homeostatic model assessment for insulin resistance were elevated in the PNA rats at the age of 5-8 weeks. Following intraperitoneal glucose tolerance tests, glucose and insulin levels did not differ between two groups; however, the PNA rats showed significantly higher 30- and 60-min glucose levels than the controls after insulin stimulation during 5-8 weeks. In addition, prenatal <em>DHT</em> treatment significantly decreased insulin-stimulated phosphorylation of AKT in the skeletal muscles of 6-week-old PNA rats. The abundance of IR substrate 1 (IRS1) and IRS2 was decreased in the skeletal muscles and liver after stimulation with insulin in the PNA group, whereas phosphorylation of insulin-signaling proteins was unaltered in the adipose tissue. These findings validate the contribution of prenatal androgen excess to metabolic derangements in pubertal female rats, and the impaired insulin signaling through IRS and AKT may result in the peripheral insulin resistance during pubertal development.

Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (<em>DHT</em>). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. <em>5α</em>-Androstane-3β,17β-diol (3β-diol) generated from <em>DHT</em> by 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3β-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of <em>DHT</em> into 3β-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen metabolite-induced ER activation and growth mechanisms. Androgen metabolite-dependent growth of breast cancer cells may therefore play a role in AI-resistance.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 (Cyp)-derived arachidonic acid metabolite that has been shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation, and promote hypertension. We examined if 20-HETE contributes to microvascular remodeling in hypertension. In Sprague-Dawley rats, administration of the 20-HETE biosynthesis inhibitor HET0016 or the 20-HETE antagonist N-20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented <em>5α</em>-dihydrotestosterone (<em>DHT</em>)-induced increases in blood pressure as well as abrogated <em>DHT</em>-induced increases in the media-to-lumen ratio (M/L), media thickness, and collagen IV deposition in renal interlobar arteries. Reserpine prevented blood pressure elevation in <em>DHT</em>-treated rats but did not affect microvascular remodeling (M/L, media thickness, and collagen deposition); under these conditions, treatment with the 20-HETE antagonist attenuated microvascular remodeling, suggesting that 20-HETE contributes to <em>DHT</em>-induced vascular remodeling independent of blood pressure elevation. In Cyp4a14(-/-) mice, which display androgen-driven and 20-HETE-dependent hypertension, treatment with the 20-HETE antagonist abolished remodeling of renal resistance arteries measured as media thickness (24 ± 1 vs. 15 ± 1 μm) and M/L (0.29 ± 0.03 vs. 0.17 ± 0.01). Moreover, in Cyp4a12 transgenic mice in which the expression of Cyp4a12-20-HETE synthase is driven by a tetracycline-sensitive promoter, treatment with doxycycline resulted in blood pressure elevation (140 ± 4 vs. 92 ± 5 mmHg) and a significant increase in remodeling of renal resistance arteries (media thickness: 23 ± 1 vs. 16 ± 1 μm; M/L: 0.39 ± 0.04 vs. 0.23 ± 0.02); these increases were abrogated by cotreatment with 20-HEDE. This study demonstrated that 20-HETE is a key regulator of microvascular remodeling in hypertension; its effect is independent of blood pressure elevation and androgen levels.

Human aldo-keto reductase family 1 member C3 (AKR1C3) was initially identified as a critical enzyme in reducing <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) to <em>5α</em>-androstane-3α,17β-diol (3α-diol) and oxidizing 3α-diol to androsterone. Based on these enzymatic activities, AKR1C3 was originally named type 2 3α-hydroxysteroid dehydrogenase (HSD)/type 5 17β-HSD. Additionally, AKR1C3 was demonstrated to be capable of metabolizing other steroids including estrogen and progesterone. Subsequently, AKR1C3 was shown to possess 11-ketoprostaglandin reductase activity in metabolizing prostaglandins and dihydrodiol dehydrogenase x (DDx) activity in metabolizing xenobiotics. Tissue distribution of AKR1C3 has been detected in both sex hormone-dependent organs such as the testis, breast, endometrium, and prostate as well as sex hormone-independent organs including the kidney and urothelium. Although prominent expression of AKR1C isozymes has been reported in human non-small cell lung carcinoma (NSCLC), the expression of AKR1C3 in small cell carcinoma of the lung has not been described. Also, the expression of AKR1C3 in normal lung has not been described. In this study, we demonstrated strong AKR1C3 immunoreactivity in bronchial epithelium but not in bronchial glands or alveolar pneumocytes. Strong AKR1C3 immunoreactivity was also demonstrated in columnar epithelium but only weak immunoreactivity in squamous epithelium of the gastrointestinal junction. Although AKR1C3 immunoreactivity was absent in small cell carcinoma of the lung, positive AKR1C3 immunoreactivity was extensively present in both adenocarcinoma and squamous cell carcinoma arising from the lung and the gastroesophageal junction. AKR1C3 may serve as an adjunct marker for differentiating small cell carcinoma from NSCLC. However, roles of AKR1C3 in adenocarcinoma, squamous cell carcinoma, and small cell carcinoma pathogenesis require further studies.

Skeletal muscle can synthesize testosterone and <em>5α</em>-dihydrotestosterone (<em>DHT</em>) from dehydroepiandrosterone (DHEA) via steroidogenic enzymes in vitro, but hormone levels and steroidogenic enzyme expression decline with aging. Resistance exercise has been shown to increase in plasma sex steroid hormone levels. However, it remains unclear whether resistance training can restore impaired steroidogenic enzyme expressions in older individuals. Six young and 13 older men were recruited, and muscle biopsies were taken from the vastus lateralis at basal state. The same group of older subjects underwent resistance training involving knee extension and flexion exercises for 12 wk, and post-training biopsies were performed 4-5 d after the last exercise session. Muscular sex steroid hormone levels and sex steroidgenesis-related enzyme expressions were significantly lower in older subjects than younger ones at baseline, but 12 wk of resistance training significantly restored hormone levels (DHEA: 432±26 at baseline, 682±31 pg/μg protein, <em>DHT</em>: 6.2±0.9 at baseline, 9.8±1.4 pg/μg protein). Furthermore, the steroidogenesis-related enzymes such as 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, and <em>5α</em>-reductase expressions were significantly restored by resistance training. We conclude progressive resistance training restores age-related declines in sex steroidogenic enzyme and muscle sex steroid hormone levels in older men.

The purpose of this study was to determine the effects of di(n-butyl) phthalate (DBP) administration on male reproductive organ development in F1 Sprague-Dawley rats following in utero exposure. During gestation days (GD) 10-19, pregnant rats were administered daily, orally, DBP at 250, 500, or 700 mg/kg or flutamide (1, 12.5, or 25 mg/kg/d) as a positive control. The male offspring were sacrificed at 31 d of age. DBP and flutamide dose-dependently significantly increased the incidence of hypospadias and cryptorchidism in F1 male offspring. The weights of testes and accessory sex organs (epididymides, seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles (LABC), and Cowper's glands) were significantly reduced in DBP-treated animals. Furthermore, cauda agenesis of epididymides and ventral prostate atrophy were observed in high-dose 700-mg/kg DBP males. Anogenital distance (AGD) and levels of dihydrotestosterone (<em>DHT</em>) and testosterone were significantly decreased in the DBP (700 mg/kg/d)-treated groups. In particular, the expression of androgen receptor (AR) and <em>5α</em>-reductase type 2 in the proximal penis was markedly depressed following administration of DBP (700 mg/kg/d) or flutamide (25 mg/kg/d). The expression of sonic hedgehog (Shh) in the urethral epithelium of the proximal penis was significantly less in the DBP (700 mg/kg/d)- or flutamide (25 mg/kg/d)-treated groups. In addition, DBP dose-dependently significantly increased the expression of estrogen receptor (ER α) in the undescended testis. Data demonstrated that in utero exposure to DBP produced several abnormal responses in male reproductive organs, and these effects may be due to disruption of the stage-specific expression of genes related to androgen-dependent organs development.

BACKGROUND

Polycystic ovarian syndrome (PCOS) is a spectrum of heterogeneous disorders of reproduction and metabolism in women with potential systemic sequel such as diabetes and obesity. Although, PCOS is believed to be caused by genetic abnormalities, the genetic background that can be associated with PCOS phenotypes remains unclear due to the complexity of the trait. In this study, we used a rat model which exhibits reproductive and metabolic abnormalities similar to the human PCOS to unravel the molecular mechanisms underlining this complex syndrome.

METHODS

Female Sprague-Dawley rats were randomly assigned to <em>DHT</em> and control (CTL) groups. Rats in the <em>DHT</em> group were implanted with a silicone capsule continuous-releasing 83 μg <em>5α</em>-dihydrotestosterone (<em>DHT</em>) per day for 12 weeks to mimic the hyperandrogenic state in women with PCOS. The animals were euthanized at 15 weeks of age and the pairs of ovaries were excised and the ovarian cortex tissues were used for gene expression analysis. Total RNA was from the ovarian cortex was amplified, labeled and hybridized to the Affymetrix GeneChip® Rat Genome 230 2.0 Array. A linear model system for microarray data analysis was used to identify genes affected in <em>DHT</em> treated rat ovaries and the molecular pathway of those genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis tool.

RESULTS

A total of 573 gene transcripts, including CPA1, CDH1, INSL3, AMH, ALDH1B1, INHBA, CYP17A1, RBP4, GAS6, GAS7 and GATA4, were activated while 430 others including HSD17B7, HSD3B6, STAR, HMGCS1, HMGCR, CYP51, CYP11A1 and CYP19A1 were repressed in DHT-treated ovaries. Functional annotation of the dysregulated genes revealed that biosynthesis and metabolism of steroids, cholesterol and lipids to be the most top functions enriched by the repressed genes. However, cell differentiation/proliferation, transcriptional regulation, neurogenesis, cell adhesion and blood vessel development processes were enriched by activated genes.

CONCLUSIONS

The dysregulation of genes associated with biosynthesis and metabolism of steroids, cholesterol and lipids, cell differentiation/proliferation in DHT- treated ovaries could be a molecular clue for abnormal steroidogenesis, estrous cycle irregularity, abnormal folliculogenesis, anovulation and lipid metabolism in PCOS patients.

The enzyme <em>5α</em>-reductase, which converts testosterone to dihydrotestosterone (<em>DHT</em>), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of <em>5α</em>-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of <em>5α</em>-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of <em>5α</em>-reductase isoenzymes in a cell type-specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of <em>5α</em>-reductase isoenzymes may confer response or resistance to <em>5α</em>-reductase inhibitors and thus may have importance in prostate cancer prevention.

Activation of the hypothalamo-pituitary-adrenal (HPA) axis is a basic reaction of animals to environmental perturbations that threaten homeostasis. These responses are ultimately regulated by neurons residing within the paraventricular nucleus (PVN) of the hypothalamus. Within the PVN, corticotrophin-releasing hormone (CRH), vasopressin (AVP), and oxytocin (OT) expressing neurons are critical as they can regulate both neuroendocrine and autonomic responses. Estradiol (E2) and testosterone (T) are well known reproductive hormones; however, they have also been shown to modulate stress reactivity. In rodent models, evidence shows that under some conditions E2 enhances stress activated adrenocorticotropic hormone (ACTH) and corticosterone secretion. In contrast, T decreases the gain of the HPA axis. The modulatory role of testosterone was originally thought to be via 5 alpha reduction to the potent androgen dihydrotestosterone (<em>DHT</em>) and its subsequent binding to the androgen receptor, whereas E2 effects were thought to be mediated by estrogen receptors alpha (ERalpha) and beta (ERbeta). However, <em>DHT</em> has been shown to be metabolized to the ERbeta agonist, <em>5α</em>- androstane 3β, 17β Diol (3β-Diol). The actions of 3β-Diol on the HPA axis are mediated by ERbeta which inhibits the PVN response to stressors. In gonadectomized rats, ERbeta agonists reduce CORT and ACTH responses to restraint stress, an effect that is also present in wild-type but not ERbeta-knockout mice. The neurobiological mechanisms underlying the ability of ERbeta to alter HPA reactivity are not currently known. CRH, AVP, and OT have all been shown to be regulated by estradiol and recent studies indicate an important role of ERbeta in these regulatory processes. Moreover, activation of the CRH and AVP promoters has been shown to occur by 3β-Diol binding to ERbeta and this is thought to occur through alternate pathways of gene regulation. Based on available data, a novel and important role of 3β-Diol in the regulation of the HPA axis is suggested.

BACKGROUND

Normal and pathologic growth of the prostate is dependent on the synthesis of dihydrotestosterone (<em>DHT</em>) from testosterone by <em>5α</em>-reductase. Finasteride is a selective inhibitor of <em>5α</em>-reductase 2, one isozyme of <em>5α</em>-reductase found in abundance in the human prostate. The objective of this study was to investigate the effects of finasteride on androgen receptor expression and tissue morphology in human benign prostatic hyperplasia specimens.

METHODS

Patients undergoing transurethral resection of the prostate and either treated or not treated with finasteride between 2004 and 2010 at the University of Wisconsin-Hospital were retrospectively identified using an institutional database. Prostate specimens from each patient were triple-stained for androgen receptor, prostate-specific antigen, and basal marker cytokeratin 5. Morphometric analysis was performed using the multispectral imaging, and results were compared between groups of finasteride treated and non-treated patients.

RESULTS

Epithelial androgen receptor but not stromal androgen receptor expression was significantly lower in patients treated with finasteride than in non-treated patients. Androgen receptor-regulated prostate-specific antigen was not significantly decreased in finasteride-treated patients. Significant luminal epithelial atrophy and basal cell hyperplasia were prevalent in finasteride treated patients. Epithelial androgen receptor expression was highly correlated to the level of luminal epithelial atrophy.

CONCLUSIONS

In this study, finasteride decreased the expression of epithelial androgen receptor in a tissue specific manner. The correlation between epithelial androgen receptor and the extent of luminal epithelial atrophy suggests that epithelial androgen receptor may be directly regulating the atrophic effects observed with finasteride treatment.

OBJECTIVE

Endurance training improves skeletal muscular function including energy metabolism and structure. Sex steroid hormones partly contribute to the exercise-induced muscular adaptations. Recently, we demonstrated that skeletal muscle contains steroidogenic converting enzymes to synthesize sex steroid hormones and an acute endurance exercise activates local steroidogenesis in skeletal muscle. However, whether chronic endurance training leads to enhanced steroidogenesis in skeletal muscle is unknown. Here, we examined changes in steroidogenic enzymes and sex steroid hormones in the skeletal muscle after chronic endurance exercise training.

METHODS

Eleven male rats were divided into two groups: sedentary (n = 6) and trained (n = 5). Endurance training was performed on a treadmill (30 m·min(-1), 30 min) for 5 d·wk(-1) for 12 wk. The posttraining harvesting was performed 48 h after the last exercise training.

RESULTS

The mRNA expressions of 3β-HSD, aromatase cytochrome P450, and <em>5α</em>-reductase in the skeletal muscle of trained rats were significantly higher than those of sedentary rats (P < 0.05). The protein expressions of aromatase cytochrome P450 and <em>5α</em>-reductase in the skeletal muscle of trained rats were also significantly higher than those of sedentary rats (P < 0.05). The muscular dihydrotestosterone (<em>DHT</em>) concentrations in the skeletal muscle of trained rats were significantly higher than those of sedentary rats (P < 0.01), but there was no change in dehydroepiandrosterone, total testosterone, free testosterone, and estradiol. Furthermore, muscle weight corrected for body weight of trained rats was moderately correlated with the level of muscular <em>DHT</em> concentration in trained rats (r = 0.41, P < 0.05).

CONCLUSIONS

Endurance exercise training enhances the muscular <em>DHT</em> concentration through <em>5α</em>-reductase in the skeletal muscle of rats, suggesting that local bioactive androgen metabolism may participate in exercise training-induced skeletal muscular adaptation.

We have previously established a cytochrome P450 4F2 (CYP4F2) transgenic mouse model. The present study elucidated the molecular foundation of hypertension by androgen-induction in this model. The renal expression of CYP4F2 in transgenic mice was highly expressed and strongly induced with <em>5α</em>-dihydrotestosterone (<em>DHT</em>) treatment determined by Western blot. <em>DHT</em> also increased the renal arachidonic acid ω-hydroxylation and urinary 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (P<0.01), and furthermore elevated the systolic blood pressure by 10 and 22 mm Hg (P<0.05) in female and castrated male transgenic mice, respectively. HET0016 completely eliminated the androgen-induced effects (P<0.01). Endogenous Cyp4a ω-hydroxylases, evaluated by real-time quantitative PCR, were significantly suppressed in transgenic mice (P<0.05). Importantly, transgenic mice with increased 20-HETE showed decreased epoxyeicosatrienoic acids (EETs) and increased dihydroxyeicosatetraenoic acids determined by liquid chromatography-tandem mass spectrometry, contributing to significantly raised ratio of 20-HETE/EETs in the urine and kidney homogenate (P<0.01). These data demonstrate that the androgen aggravated hypertension possibly through an altered ratio of 20-HETE/EETs in CYP4F2 transgenic mice.

<em>5α</em>-reductases convert testosterone to dihydrotestosterone (<em>DHT</em>). There are two <em>5α</em>-reductase isozymes, type 1 and type 2 in humans and animals. Mutations in type 2 isozyme with decreased enzymatic activity cause male pseudohermaphroditism. The affected 46XY individuals have high normal or elevated plasma testosterone levels with low normal or decreased <em>DHT</em> levels, resulting in an elevated testosterone/<em>DHT</em> ratios. They are born with ambiguous external genitalia and normal Wolffian differentiation. Their prostate is small and rudimentary, and plasma levels of prostate specific antigen (PSA) are low or undetectable in adulthood. Prostate cancer and benign prostate hyperplasia (BPH) have never been reported in these patients. Similar defects in prostate development are observed in animals with either <em>5α</em>-reductase-2 or <em>5α</em>-reductase-2 plus <em>5α</em>-reductase-1 gene knockout, and in animals treated with specific <em>5α</em>-reductase inhibitor. <em>5α</em>-reductase isozymes are expressed in multiple tissues, and the predominant isozyme in human prostate is <em>5α</em>-reductase-2. The expression of <em>5α</em>-reductase-2 gene in prostate cells is regulated by various factors. A high dietary fat intake, a risk factor of prostate cancer, induces prostate <em>5α</em>-reductase-2 gene expression and subsequently stimulates prostate growth, which is blocked by genistein, a phytoestrogen. Inhibition of <em>5α</em>-reductase activity by medication is used in the treatment of BPH and male-pattern baldness, while its use in prostate cancer prevention is still controversial although it can decrease the incidence of prostate cancer. The analyses of <em>5α</em>-reductases in humans and animals highlight the differences between testosterone and <em>DHT</em>, and the significance of <em>DHT</em> in male sexual differentiation and prostate physiology and pathophysiology.

Changes in meat quality traits are strongly associated with alterations in postmortem metabolism which depend on genetic variations, especially nonsynonymous single nucleotide variations (nsSNVs) having critical effects on protein structure and function. To selectively identify metabolism-related nsSNVs, next-generation transcriptome sequencing (RNA-Seq) was carried out using RNAs from porcine liver, which contains a diverse range of metabolic enzymes. The multiplex SNV genotyping analysis showed that various metabolism-related genes had different nsSNV alleles. Moreover, many nsSNVs were significantly associated with multiple meat quality traits. Particularly, ch7:g.22112616A>G SNV was identified to create a single amino acid change (Thr/Ala) at the 145th residue of H1.3-like protein, very close to the putative 147th threonine phosphorylation site, suggesting that the nsSNV may affect multiple meat quality traits by affecting the epigenetic regulation of postmortem metabolism-related gene expression. Besides, one nonsynonymous variation, probably generated by gene duplication, led to a stop signal in porcine testicular carbonyl reductase (PTCR), resulting in a C-terminal (E281-A288) deletion. Molecular docking and energy minimization calculations indicated that the binding affinity of wild-type PTCR to <em>5α</em>-<em>DHT</em>, a C(21)-steroid, was superior to that of C-terminal-deleted PTCR or human carbonyl reductase, which was very consistent with experimental data, reported previously. Furthermore, P284 was identified as an important residue mediating the specific interaction between PTCR and <em>5α</em>-<em>DHT</em>, and phylogenetic analysis showed that P284 is an evolutionarily conserved residue among animal carbonyl reductases, which suggests that the C-terminal tails of these reductases may have evolved under evolutionary pressure to increase the substrate specificity for C(21)-steroids and facilitate metabolic adaptation. Altogether, our RNA-Seq revealed that selective nsSNVs were associated with meat quality traits that could be useful for successful marker-assisted selection in pigs and also represents a useful resource to enhance understanding of protein folding, substrate specificity, and the evolution of enzymes such as carbonyl reductase.

Modulation of sex steroid pre-receptor in adipose tissue is important for the development of metabolic diseases, but its roles in the pathogenesis of polycystic ovary syndrome (PCOS) has not been fully characterized. Herein we compared the expression of key sex steroid converting enzymes in the subcutaneous adipose tissue (SAT) between patients with PCOS and the matched controls. Most of the sex steroid converting enzymes were highly expressed in the SAT, except 17α-hydroxylase (CYP17A1). Compared with the controls, PCOS patients showed significantly higher levels of 3β-hydroxysteroid dehydrogenase1-2 (3β-HSD1-2), aldo-keto reductase 1C 1-3 (AKR1C1-3) and leptin, but lower level of P450 aromatase and <em>5α</em>-reductase 1. Interestingly, leptin was positively correlated to AKR1C2 expression and negatively to <em>5α</em>-reductase1 as well as peroxisome proliferator-activated receptor γ (PPARγ). In summary, the expression of enzymes synthesizing testosterone and enzymes inactivating <em>DHT</em> and progesterone was higher in SAT of PCOS patients compared to controls. Correlation analysis indicated that increased leptin expression may be negatively related to local <em>DHT</em> level. These data suggested that sex steroid converting enzymes expression was different in SAT of PCOS patients that might contribute to abnormal testosterone and leptin level of PCOS patients.

20-HETE is a potent inducer of endothelial ACE in vitro and administration of lisinopril or losartan attenuates blood pressure in models of 20-HETE-dependent hypertension. The present study was undertaken to further define the relationship between 20-HETE and the renin-angiotensin system in hypertension using an angiotensinogen-deficient mouse (Agt+/-). Treatment of male AGT+/- with <em>5α</em>-dihydrotestosterone (<em>DHT</em>) increased systolic BP from 102±2 to 125±3mmHg; in comparison, the same treatment raised BP in wild type (WT) from 110±2 to 138±2mmHg. <em>DHT</em> increased vascular 20-HETE levels in AGT+/- and WT from 1.5±0.7 and 2.1±0.6 to 13.0±2.0 and 15.8±4.0ng/mg, respectively. Concurrent treatment with the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented the increases in BP in both AGT+/- and WT mice. Administration of 20-HEDE at the peak of the <em>DHT</em>-induced BP increase (12 days) reduced BP to basal levels after 48h. Interestingly, basal levels of renal microvascular EETs were higher in AGT+/- compared to WT (55.2±9.7 vs 20.0±4.1ng/mg) and treatment of AGT+/- with <em>DHT</em> decreased the levels of EETs (28.4±5.1ng/mg). <em>DHT</em>-mediated changes in vascular EET level were not observed in WT mice. Vascular Cyp4a12 and ACE protein levels were increased in both AGT+/- and WT by 30-40% and decreased with concomitant administration of 20-HEDE. Lisinopril was as effective as 20-HEDE in preventing <em>DHT</em>-mediated increases in BP in both AGT+/- and WT mice. This study substantiates our previous findings that the RAS plays an important role in 20-HETE-mediated hypertension. It also proposes a novel interaction between 20-HETE and EETs.