The expression of arginine-vasopressin (AVP) is regulated by estradiol and testosterone (T) in different neuronal populations by mechanisms that are not yet fully understood. Estrogen receptors (ERs) have been shown to participate in the regulation of AVP neurons by estradiol. In addition, there is evidence of the participation of ERβ in the regulation of AVP expression exerted by T via its metabolite <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) and its further conversion in the androgen metabolite and ERβ ligand 3β-diol. In this study we have explored the role of ERs in the regulation exerted by estradiol and T on AVP expression, using the human neuroblastoma cell line SH-SY5Y. Estradiol treatment increased AVP mRNA levels in SH-SY5Y cells in comparison with cells treated with vehicle. The stimulatory effect of estradiol on AVP expression was imitated by the ERα agonist 4,4',4',-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and blocked by the ER antagonist, ICI 182,780, and the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1hpyrazoledihydrochloride. In contrast, the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile reduced AVP expression, whereas the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol enhanced the action of estradiol on AVP expression. T increased AVP expression in SH-SY5Y cells by a mechanism that was dependent on aromatase but not on <em>5α</em>-reductase activity. The T effect was not affected by blocking the androgen receptor, was not imitated by the T metabolite <em>5α</em>-<em>DHT</em>, and was blocked by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1hpyrazoledihydrochloride. In contrast, <em>5α</em>-<em>DHT</em> had a similar effect as the ERβ agonists 2,3-bis(4-hydroxyphenyl)-propionitrile and 3β-diol, reducing AVP expression. These findings suggest that estradiol and T regulate AVP expression in SH-SY5Y cells through ERs, exerting a stimulatory action via ERα and an inhibitory action via ERβ.

OBJECTIVE

Testosterone replacement therapy improves bladder capacity in urinary tract dysfunction. There is no information, however, about the role of this steroid hormone on the muscle tension of the bladder outflow region. The current study investigated the mechanisms underlying the testosterone-induced action in the pig bladder neck.

METHODS

Urothelium-denuded bladder neck strips were mounted in myographs for isometric force recordings and for simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension. The relaxations to testosterone, the non-aromatizable metabolite 4,5α-dihydrotestosterone (DHT) and electrical field stimulation (EFS) were carried out on phenylephrine (PhE)-precontracted strips.

RESULTS

Testosterone and DHT evoked similar concentration-dependent relaxations only at very high pharmacological concentrations. The presence of the urothelium and the inhibition of intracellular androgenic receptor (AR), aromatase, 5α-reductase, nitric oxide (NO) synthase, guanylyl cyclase, cyclooxygenase (COX), large-, intermediate- and small-Ca(2+)-activated K(+) channels or ATP-dependent K(+) channels failed to modify the testosterone relaxations. Neuronal voltage-gated Ca(2+) (VOC) channels and voltage-gated K(+) (K(V)) channel blockers potentiated these responses. EFS evoked frequency-dependent relaxations, which were not changed by threshold concentrations of testosterone. In Ca(2+)-free potassium rich physiological saline solution, testosterone inhibited the contractions induced by CaCl(2) and the L-type VOC channel activator (±)-BAY K 8644. Relaxations elicited by testosterone were accompanied by simultaneous decreases in smooth muscle [Ca(2+)](i).

CONCLUSIONS

Testosterone produces relaxation of the pig urinary bladder neck through mechanisms independent of urothelium, AR, aromatase, 5α-reductase, NO synthase, guanylyl cyclase, COX and K(+) channels. Testosterone-induced relaxation is produced via the inhibition of the extracellular Ca(2+) entry through L-type VOC channels.

Androgens may provide protective effects in the vasculature under pathophysiological conditions. Our past studies have shown that dihydrotestosterone (<em>DHT</em>) decreases expression of cyclooxygenase-2 (COX-2) during cytokine, endotoxin, or hypoxic stimulation in human vascular smooth muscle cells, in an androgen receptor (AR)-independent fashion. Classically <em>DHT</em> is regarded as a pure AR agonist; however, it can be endogenously metabolized to <em>5α</em>-androstane-3β, 17β-diol (3β-diol), which has recently been shown to be a selective estrogen receptor (ERβ) agonist. Therefore, we hypothesized that <em>DHT</em>'s anti-inflammatory properties following cytokine stimulation are mediated through ERβ. Using primary human brain vascular smooth muscle cells (HBVSMC), we tested whether <em>DHT</em>'s effect on IL-1β induced COX-2 expression was mediated via AR or ERβ. The metabolism of <em>DHT</em> to 3β-diol is a viable pathway in HBVSMC since mRNA for enzymes necessary for the synthesis and metabolism of 3β-diol [3alpha-hydroxysteroid dehydrogenase (HSD), 3β-HSD, 17β-HSD, CYP7B1] was detected. In addition, the expression of AR, ERα, and ERβ mRNA was detected. When applied to HBVSMC, <em>DHT</em> (10nM; 18 h) attenuated IL-1β-induced increases in COX-2 protein expression. The AR antagonist bicalutamide did not block <em>DHT</em>'s ability to reduce COX-2. Both the non-selective estrogen receptor antagonist ICI 182,780 (1 μM) and the selective ERβ antagonist PHTPP (1 μM) inhibited the effect of <em>DHT</em>, suggesting that <em>DHT</em> actions are ERβ-mediated. In HBVSMC and in rat mesenteric arteries, 3β-diol, similar to <em>DHT</em>, reduced cytokine-induced COX-2 levels. In conclusion, <em>DHT</em> appears to be protective against the progression of vascular inflammation through metabolism to 3β-diol and activation of ERβ.

Several studies have demonstrated that the presence of stressors during pregnancy induces adverse effects on the neuroendocrine system of the offspring later in life. In the present work, we investigated the effects of early programming on the male reproductive system, employing a prenatal stress (PS) paradigm. This study found that when pregnant dams were placed in a plastic restrainer three times a day during the last week of pregnancy, the offspring showed reduced anogenital distance and delayed testicular descent. Serum luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels were decreased at postnatal day (PND) 28 and testosterone was decreased at PND 75. Increased testosterone plus dihydrotestosterone (T + <em>DHT</em>) concentrations correlated with increased testicular <em>5α</em> Reductase-1 (<em>5α</em>R-1) mRNA expression at PND 28. Moreover, PS accelerated spermatogenesis at PND 35 and 60, and increased mean seminiferous tubule diameter in pubertal offspring and reduced Leydig cell number was observed at PND 35 and 60. PS offspring had increased androgen receptor (AR) mRNA level at PND 28, and at PND 35 had increased the numbers of Sertoli cells immunopositive for AR. Overall, the results confirm that stress during gestation can induce long-term effects on the male offspring reproductive system. Of particular interest is the pre-pubertal imbalance of circulating hormones that probably trigger accelerated testicular development, followed by an increase in total androgens and a decrease in testosterone concentration during adulthood. Exposure to an unfavourable intrauterine environment might prepare for harsh external conditions by triggering early puberty, increasing reproductive potential.

Androgen ablation is the standard of care prescribed to patients with advanced or metastatic prostate cancer (PCa) to slow down disease progression. Unfortunately, a majority of PCa patients under androgen ablation progress to castration-resistant prostate cancer (CRPC). Several mechanisms including alternative intra-prostatic androgen production and androgen-independent androgen receptor (AR) activation have been proposed for CRPC progression. Aldo-keto reductase family 1 member C3 (AKR1C3), a multi-functional steroid metabolizing enzyme, is specifically expressed in the cytoplasm of PCa cells; and positive immunoreactivity of the type A γ-aminobutyric acid receptor (GABAAR), an ionotropic receptor and ligand-gated ion channel, is detected on the membrane of PCa cells. We studied a total of 72 radical prostatectomy cases by immunohistochemistry, and identified that 21 cases exhibited positive immunoreactivities for both AKR1C3 and GABAAR. In the dual positive cancer cases, AKR1C3 and GABAAR subunit α1 were either expressed in the same cells or in neighboring cells. Among several possible substrates, AKR1C3 reduces <em>5α</em>-dihydrotesterone (<em>DHT</em>) to form <em>5α</em>-androstane-3α, 17β-diol (3α-diol). 3α-diol is a neurosteroid that acts as a positive allosteric modulator of the GABAAR in the central nervous system (CNS). We examined the hypothesis that 3α-diol-regulated pathological effects in the prostate are GABAAR-dependent, but are independent of the AR. In GABAAR-positive, AR-negative human PCa PC-3 cells, 3α-diol significantly stimulated cell growth in culture and the in ovo chorioallantoic membrane (CAM) xenograft model. 3α-diol also up-regulated expression of the epidermal growth factor (EGF) family of growth factors and activation of EGF receptor (EGFR) and Src as measured by quantitative polymerase chain reaction and immunoblotting, respectively. Inclusion of GABAAR antagonists reversed 3α-diol-stimulated tumor cell growth, expression of EGF family members, and activation of EGFR and Src to the level observed in untreated cells. Results from the present study suggest that 3α-diol may act as an alternative intra-prostatic neurosteroid that activates AR-independent PCa progression. The involvement of AKR1C3-mediated steroid metabolisms in modulating GABAAR activation and promoting PCa progression requires continued studies.

Although chronic hyperandrogenism suppresses antral follicular development, a phenomenon often observed in polycystic ovarian syndrome (PCOS), whether and how deregulation of androgen receptor (AR) signaling is involved, is not well understood. In the present study, we examined the role of ring finger protein 6 (RNF6) in AR ubiquitination and the possible dysregulation in the expression and actions of growth differentiation factor 9 (GDF9) and kit-ligand (Kitlg) in a chronic androgenized PCOS rat model. <em>5α</em>-dihydrotestosterone (<em>DHT</em>) treatment in vivo inhibited antral follicle growth, a response mediated through increased RNF6 content, suppressed K63- but increased K48-linked AR ubiquitination as well as the mRNA expression and content of soluble KIT-L (sKitlg) and content of GDF9. These androgenic responses were attenuated by gonadotropin treatment in vivo. Growth of antral follicles from <em>DHT</em>-treated rats in vitro was significantly slower when compared to those of control but was significantly enhanced by exogenous GDF9, suggesting the <em>DHT</em>-induced antral follicular growth arrest is in part the results of GDF9 suppression. Our findings indicate how hyperandrogenism modulates RNF6 content and subsequently AR ubiquitination, resulting in antral follicle growth arrest in a chronically androgenized PCOS rat model.

The influence of medroxyprogesterone acetate (MPA) and various other steroid hormones on the lateral diffusion of the fluorescent lipid probe 1-acyl-2-(N-4-nitrobenzo-2-oxa-1.3-diazolyl)-aminocarpropylphos phatidylcholine (NBD-PC) in the plasma membrane of intact mammary cancer cells (CAMA-1 cell line) has been studied by fluorescence recovery after photobleaching technique. Incubation of cells with MPA in serum free medium at ambient temperature for 1 hr led to a significant decrease in the lateral diffusion coefficient of NBD-PC. MPA induced this change over a limited concentration range with 10(-7)-10(-5) mol/l near-maximal or maximal effects, and 10(-8) mol/l exhibiting no effect. Exposure of the CAMA-1 cells to 10(-7) mol/l MPA in undiluted serum induced a significant effect following 1.5 hr of treatment with no increase in effectiveness up to 4 hr of incubation. As compared to MPA, the other steroids tested were less effective or ineffective. The influence on lateral lipid mobility diminished as follows: MPA greater than progesterone greater than <em>5a</em>-<em>DHT</em> approximately 17 beta-estradiol greater than dexamethasone, and roughly seems to parallel their lipid solubility as estimated by partition coefficients in an n-octanol-water system. Any involvement of classical steroid hormone receptors in the mechanism of membrane action could be excluded. Nongenomic steroid effects on the plasma membrane are assumed. As the structure and function of biomembranes are modulated by lipid-bilayer fluidity and membranes crucially participate in nearly all aspects of cell biology, it is concluded that direct interactions of MPA with membranes potentially play a role in the antitumor activity of the compound.

BACKGROUND

Affected by steroid <em>5α</em>-reductase type 2 deficiency (<em>5α</em>-RD2), 46, XY individuals present divergent phenotypes characterized by undervirilization of male external genitalia. To identify the disorder, mutational analysis of <em>5α</em>-reductase type 2 gene (SRD5A2) is a reliable approach. The genotype-phenotype relationship has not been elucidated.

OBJECTIVE

To improve the diagnosis and expand the knowledge of the disease, we collected and analysed relevant data of clinical diagnosis, biological investigation and molecular determination in 45 children with the SRD5A2 gene mutations from South China in our centre.

METHODS

We studied a cohort of 45 Chinese children with SRD5A2 gene mutations.

RESULTS

Isolated microphallus (35·6%) and microphallus associated with various degrees of hypospadias (55·6%) were frequent phenotype. Female external genitalia with clitoromegaly (8·9%) were rare. 16 of 18 (88·9%) cases had hCG-stimulated T/DHT ratio above 10. In 45 patients, we identified 15 different mutations. Five have never been described: p.His90ThrfsX31, p.Gly21Arg, p.Gly149Asp, p.Arg145Leu and p.Gly66Arg. The p.Arg227Gln mutation was detected in 41 (91·1%) patients. The p.Leu89Val polymorphism was found in 38 (84·4%) patients. Homozygous mutations were presented in 16 (35·6%) patients, compound heterozygous mutations in 20 (44·4%) patients, compound heterozygous mutations alone with the p.Leu89Val polymorphism in nine (20·0%) patients. Exons 1 and 4 were most affected, and the number of mutant alleles per exon was 78·1% and 12·2%, respectively.

CONCLUSIONS

The study demonstrated a wide spectrum of phenotypes, biological profiles and genotypes in the children with <em>5α</em>-RD2 from South China. The heterozygous mutation p.Arg227Gln is presumably a hot spot mutation and suggests a founder effect in the population of South China that may explain a moderate phenotype among our patients. Our report provides new insights into the molecular mechanism of <em>5α</em>-RD2 and help to the diagnosis and treatment of this disease.

Breast cancer (BC) is among the most prevalent type of malignancy affecting females worldwide. BC is classified into different types according to the status of the expression of receptors such as estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR). Androgen receptor (AR) appears to be a promising therapeutic target of breast cancer. Binding of <em>5α</em>-dihydrotestosterone (<em>DHT</em>) to AR controls the expression of microRNA (miRNA) molecules in BC, consequently, affecting protein expression. One of these proteins is the transmembrane glycoprotein cluster of differentiation 44 (CD44). Remarkably, CD44 is a common marker of cancer stem cells in BC. It functions as a co-receptor for a broad diversity of extracellular matrix ligands. Several ligands, primarily hyaluronic acid, can interact with CD44 and mediate its functions. CD44 promotes a variety of functions independently or in cooperation with other cell-surface receptors through activation of varied signaling pathways like Rho GTPases, Ras-MAPK, and PI3K/AKT pathways to regulate cell adhesion, migration, survival, invasion, and epithelial-mesenchymal transition. In this review, we present the relations between AR, miRNA, and CD44 and their roles in BC.

The principle steroidal androgens are testosterone and its metabolite <em>5α</em>-dihydrotestosterone (<em>DHT</em>), which is converted from testosterone by the enzyme <em>5α</em>-reductase. Through the classic pathway with androgens crossing the plasma membrane and binding to the androgen receptor (AR) or via mechanisms independent of the ligand-dependent transactivation function of nuclear receptors, testosterone induces genomic and non-genomic effects respectively. AR is widely distributed in several tissues, including vascular endothelial and smooth muscle cells. Androgens are essential for many developmental and physiological processes, especially in male reproductive tissues. It is now clear that androgens have multiple actions besides sex differentiation and sexual maturation and that many physiological systems are influenced by androgens, including regulation of cardiovascular function [nitric oxide (NO) release, Ca2+ mobilization, vascular apoptosis, hypertrophy, calcification, senescence and reactive oxygen species (ROS) generation]. This review focuses on evidence indicating that interplay between genomic and non-genomic actions of testosterone may influence cardiovascular function.

UNASSIGNED

Avian mothers can potentially alter the phenotypes of their offspring by varying the concentration of steroid hormones in their eggs. We explored variation in androstenedione (A4), testosterone (T), <em>5α</em>-dihydrotestosterone (<em>DHT</em>), 17β-estradiol (E2), and corticosterone (CORT) in the yolks of 12 free-living great tit Parus major clutches. We analyzed variation and covariation in greater detail than previous studies, using models for variation with laying sequence that take into account variable clutch size and comparing correlations between pairs of hormones at the within- and between-clutch levels. We also investigated relationships between hormone levels and various environmental, life history, and parental traits. For three of the five steroids, we found no significant correlates, but based on individual statistical tests (a) <em>DHT</em> varied between clutches with male age (1 year old vs older); (b) <em>DHT</em> and CORT were negatively correlated within clutches with the average temperature on the day (<em>DHT</em> and CORT) or 3 days (<em>DHT</em> only) preceding laying; and (c) <em>DHT</em> in the last egg of the clutch relative to the clutch mean was positively correlated with the interval between clutch completion and the onset of incubation (incubation delay). Relationships with ambient temperature and incubation delay have not previously been reported for any yolk hormone in birds. Intriguingly, the three relationships for <em>DHT</em> are consistent with more <em>DHT</em> being transferred to eggs in situations that could be more energetically challenging for the female. More research is needed to determine the generality of the patterns we found and to understand their functional significance.

UNASSIGNED

The yolks of birds' eggs contain steroid hormones produced by the mother which can affect the development and behavior of the resultant chicks. We analyzed five steroid hormones in the yolks of wild great tits and show for the first time that yolk hormone levels are related to ambient temperature in the day(s) just before laying and, in the last-laid egg, with the day it is laid relative to the onset of incubation, and that the concentrations of pairs of yolk hormones can vary with each other in a different way between and within clutches. These results contribute insights into the ways in which yolk hormones may adaptively modify the chicks or may reflect physiological processes occurring in the mother.

Androgens are steroid hormones with pleotropic and diverse biochemical and physiological functions, and androgen deficiency exerts a negative impact on human health. Testosterone (T) either directly or via its transformation into the more potent metabolite <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) or via aromatization into estradiol (E2) modulates important biochemical signaling pathways of human physiology and plays a critical role in the growth and/or maintenance of functions in a host of tissues and organs. T and <em>5α</em>-<em>DHT</em> play an important role in regulating physiology of the muscle, adipose tissue, liver, bone, and central nervous system, as well as reproductive and sexual functions. Thus, androgen deficiency (also referred to as hypogonadism) is a well-recognized medical condition and if remained untreated will have a negative impact on human health and quality of life.In this chapter, we have summarized the negative impact of T deficiency (TD) on a host of physiological functions including reduced lean body mass (LBM), increased fat mass (FM), increased insulin resistance (IR), metabolic syndrome (MetS) and adiposity, reduced bone mineral density (BMD), anemia, sexual dysfunction, and reduced quality of life and increased mortality. In addition, we discuss another critical aspect of unrecognized form of androgen deficiency resulting from inhibition of <em>5α</em>-reductases with drugs, such as finasteride and dutasteride, to block transformation of T into <em>5α</em>-<em>DHT</em> in the course of treatment of benign prostatic hyperplasia (BPH) and male pattern hair loss, also known as androgenetic alopecia (AGA). The negative impact of drugs that inhibit transformation of T to <em>5α</em>-<em>DHT</em> by <em>5α</em>-reductases on metabolic function is manifested in fat accumulation in the liver, which may predispose to nonalcoholic fatty liver disease (NAFLD). Also, inhibition of <em>5α</em>-<em>DHT</em> formation increases glucose synthesis and reduces glucose disposal potentially contributing to hyperglycemia, IR, and elevated activities of liver function enzymes concomitant with reduction in circulating T levels, worsening erectile dysfunction (ED), and reduced quality of life.Although we have attempted to summarize the current literature pertaining to this critical topic "androgen deficiency" and its impact on men's health and quality of life, there remain many gaps in the knowledge regarding the biochemical pathways that are involved in the pathophysiology of androgen deficiency. We wish to clearly state that there are areas of controversies, including whether age-related androgen deficiency (functional hypogonadism) merits treatment and whether T therapy provided real proven benefits. Finally, considerable debate exists with respect to the potential and purported cardiovascular (CV) risks of treating TD with exogenous T. For brevity sake, we will not discuss in detail the benefits of T therapy in men with TD since this topic is comprehensively covered by Dr. F. Saad's chapter in this book, entitled "Testosterone Therapy and Glucose Homeostasis in Men with Testosterone Deficiency (Hypogonadism)."We have made a concerted effort to address the controversy of T therapy in men with TD in the discussion. However, we wish to acknowledge that these issues will remain a matter of debate for some time to come. Only with advances in fundamental basic science and clinical research, some of these controversial issues may be laid to rest. Nevertheless, we believe that there is considerable body of credible evidence to suggest that T therapy of men with TD is safe and effective and provides a host of health benefits and therefore merits considerations in men with TD, irrespective of the underlying cause or etiology. An additional aspect of androgen deficiency is the drug-induced reduction in <em>5α</em>-<em>DHT</em> levels by the use of <em>5α</em>-reductase inhibitors. We also believe that physicians prescribing <em>5α</em>-reductase inhibitors (i.e., finasteride or dutasteride) for relief of BPH symptoms or treatment of hair loss should engage their patients in a productive discussion regarding the potential adverse side effects of these medications on their overall health and quality of life.

Androst-5-ene-3β,17β-diol (ADIOL) and <em>5α</em>-androstane-3β,17β-diol (3β-DIOL), metabolites of dehydroepiandrosterone (DHEA) and dihydrotestosterone (<em>DHT</em>), respectively, are known to possess estrogenic properties. To better understand their hormonal action and roles in the proliferation of breast cancer (BC) cells, we studied their binding to sex-hormone receptors in estrogen receptor (ER)-positive (ZR-75-1 and T-47D) and ER-negative (MDA-MB-231) human BC cells. The results demonstrated that estradiol (E2), ADIOL and 3β-DIOL stimulated the proliferation of ZR-75-1 and T-47D cells, but had no effect on ER-negative cells. In the presence of estradiol, ADIOL and 3β-DIOL inhibited the estrogen-stimulated BC cell growth. This inhibition was counteracted by anti-androgens, which were unable to affect the ADIOL and 3β-DIOL stimulatory effects in E2-free medium. On the other hand, in the presence of tamoxifen, ADIOL and 3β-DIOL showed an additional anti-proliferative activity on hormone-sensitive BC cells compared with tamoxifen treatment alone. These results are similar to previous reports obtained using MCF-7 cells, which confirmed that ADIOL and 3β-DIOL stimulated estrogen-dependent BC cell growth via ERs, but inhibited growth via androgen receptors (ARs). Several steroids bind to both ER and AR in a different preference and degree, i.e. E2>estrone (E1>>ADIOL>3β-DIOL>testosterone (T>><em>DHT</em> for ER and <em>DHT</em>)T>3β-DIOL>ADIOL>E1>E2 for AR. The relative binding affinities of ADIOL, 3β-DIOL, and E2 corresponded well to their respective potential in stimulating cell proliferation of ZR-75-1 and T-47D cells in our results. The intrinsic relationship between cell proliferation effects and binding affinities for receptors of several steroids was revealed here by a combined binding and cell study. This article is part of a Special Issue entitled 'Synthesis and biological testing of steroid derivatives as inhibitors'.

Prostate cancer (PCa) is the 2nd leading cause of cancer-related death among men in the United States and its progression is tightly associated with the androgen/androgen receptor (AR) signals. Men castrated before puberty (eunuchs) or men with inherited deficiency of type II <em>5α</em>-reductase (with failure to convert testosterone to the more potent dihydrotestosterone) (<em>DHT</em>) do not develop PCa. To date, androgen deprivation therapy (ADT) with anti-androgen treatments to reduce or prevent androgens from binding to the AR remains the main therapeutic option for advanced PCa since its discovery by Huggins and Hodges in 1941. Multiple strategies related to surgical/chemical castration with combinations of various anti-androgens, including Cyproterone Acetate, Flutamide, Nilutamide, Bicalutamide (Casodex) and Enzalutamide, as well as some androgen synthesis blockers, including Abiraterone, have been used to control PCa progression. However, patients on ADT with anti-androgen treatment eventually develop resistance, which might be accompanied with the unwanted side effects of enhanced metastasis. New therapeutic approaches via directly targeting the AR with ASC-J9®, Cisplatin, EPI-001, Niclosamide, and VPC compounds as well as silencing AR with siRNAs or non-coding RNAs have been developed to further suppress PCa at the castration resistant stages.

BACKGROUND

<em>5α</em> steroid reductase deficiency (<em>5α</em>SRD) is an autosomal recessive enzymatic deficiency and mutations in the <em>5α</em> steroid reductase type 2 gene (SRD5A2) result in male pseudohermaphrodism caused by decreased dihydrotestosterone (<em>DHT</em>) synthesis.

OBJECTIVE

To identify the specific mutations of the SRD5A2 gene in Cypriot patients with <em>5α</em>SRD.

METHODS

Five unrelated patients with 46,XY karyotype were examined. Four of them were born with ambiguous genitalia and 1 patient, who was raised as girl, presented with primary amenorrhea. The hCG test was informative (elevated testosterone/<em>DHT</em>) of <em>5α</em>SRD in 3 out of 4 subjects. Sequencing of the SRD5A2 gene was completed for all patients. Genomic DNA was also isolated from a total of 204 healthy unrelated Cypriot subjects. Screening for the IVS1-2A>G mutation was performed by using direct sequencing and restriction enzyme analysis.

RESULTS

The IVS1-2A>G was identified in homozygosity in 3 patients and in a compound heterozygote state in the other 2 patients, in combination with p.P181L and p.R171S in exon 3, respectively. The carrier frequency in the Cypriot population for the IVS1-2A>G mutation was estimated to be 0.98% or 2 in 204.

CONCLUSIONS

The same IVS1-2A>G mutation in the SRD5A2 gene seems to characterize all Cypriot patients with <em>5α</em>SRD diagnosed so far. Furthermore this relatively rare genetic defect, which has only been reported previously in a single case in the Eastern Mediterranean region, is very likely to be the result of a founder effect.

Ovarian steroids such as estrogen and progesterone have been reported to influence knee laxity. The effect of testosterone, however, remains unknown. This study investigated the effect of testosterone on the knee range of motion (ROM) and the molecular mechanisms that might involve changes in the expression of relaxin receptor isoforms, Rxfp1 and Rxfp2 in the patella tendon and lateral collateral ligament of the female rat knee. Ovariectomized adult female Wistar rats received three days treatment with peanut oil (control), testosterone (125 and 250 μg/kg) and testosterone (125 and 250 μg/kg) plus flutamide, an androgen receptor blocker or finasteride, a <em>5α</em>-reductase inhibitor. Duplicate groups received similar treatment however in the presence of relaxin (25 ng/kg). A day after the last drug injection, knee passive ROM was measured by using a digital miniature goniometer. Both tendon and ligament were harvested and then analysed for protein and mRNA expression for Rxfp1 and Rxfp2 respectively. Knee passive ROM, Rxfp1 and Rxfp2 expression were significantly reduced following treatment with testosterone. Flutamide or finasteride administration antagonized the testosterone effect. Concomitant administration of testosterone and relaxin did not result in a significant change in knee ROM as compared to testosterone only treatment; however this was significantly increased following flutamide or finasteride addition. Testosterone effect on knee passive ROM is likely mediated via dihydro-testosterone (<em>DHT</em>), and involves downregulation of Rxfp1 and Rxfp2 expression, which may provide the mechanism underlying testosterone-induced decrease in female knee laxity.

BACKGROUND

Finasteride, an inhibitor of <em>5α</em>-reductase (type II), lowers intraprostatic dihydrotestosterone (<em>DHT</em>), which is reflected in serum as reduced <em>5α</em>-androstane-3α,17β-diol glucuronide (3α-dG). It also modestly increases serum testosterone (T), estrone (E(1)), and estradiol (E(2)). In this altered hormonal milieu, it is unknown whether serum concentrations of these hormones are associated with prostate cancer risk.

METHODS

In this nested case-control study of men in the finasteride arm of the Prostate Cancer Prevention Trial, sex steroid hormones and sex hormone binding globulin were measured at baseline and approximately 3-year posttreatment in 553 prostate cancer cases and 694 controls.

RESULTS

Median posttreatment changes in concentrations of 3α-dG, T, E(1), and E(2) were -73.8%, +10.1%, +11.2%, and +7.5% (all P < 0.001), respectively. Neither the pre- nor posttreatment concentrations of 3α-dG, nor its change, were associated with risk. Pretreatment, high concentrations of E(1) and low concentrations of T were associated with increased cancer risk [OR; 95% confidence interval (CI) quartile 4 vs. 1: 1.38 (0.99-1.93) P(trend) = 0.03; 0.64 (0.43-0.93) P(trend) = 0.07, respectively]. Posttreatment, high concentrations of both E(1) and E(2) were associated with increased cancer risk [OR; 95% CI quartile 4 vs. 1: 1.54 (1.09-2.17) P(trend) = 0.03; 1.49 (1.07-2.07) P(trend) = 0.02, respectively].

CONCLUSIONS

Among finasteride-treated men, concentrations of 3α-dG were not associated with total or Gleason grades 2 to 6, 7 to 10, or 8 to 10 cancer. High serum estrogens may increase cancer risk when intraprostatic DHT is pharmacologically lowered.

CONCLUSIONS

Low posttreatment serum estrogens may identify men more likely to benefit from use of finasteride to prevent prostate cancer.

N-acetyltransferase 1 (NAT1) is an enzyme that metabolizes carcinogens, which suggests a potential role in breast carcinogenesis. High NAT1 expression in breast tumors is associated with estrogen receptor α (ERα+) and the luminal subtype. We report that NAT1 mRNA transcript, protein, and enzyme activity were higher in human breast tumors with high expression of ERα/ESR1 compared with normal breast tissue. There was a strong correlation between NATb promoter and NAT1 protein expression/enzyme activity. High NAT1 expression in tumors was not the result of adipocytes, as evidenced by low perilipin (PLIN) expression. ESR1, NAT1, and XBP1 expression were associated in tumor biopsies. Direct regulation of NAT1 transcription by estradiol (E2) was investigated in ERα (+) MCF-7 and T47D breast cancer cells. E2 did not increase NAT1 transcript expression but increased progesterone receptor expression in a dose-dependent manner. Likewise, NAT1 transcript levels were not increased by dihydrotestosterone (<em>DHT</em>) or <em>5α</em>-androstane-3β, (3β-adiol) 17β-diol. Dithiothreitol increased levels of the activated, spliced XBP1 in ERα (+) MCF-7 and T47D breast cancer cells but did not affect NAT1 or ESR1 expression. We conclude that NAT1 expression is not directly regulated by E2, <em>DHT</em>, 3β-adiol, or dithiothreitol despite high NAT1 and ESR1 expression in luminal A breast cancer cells, suggesting that ESR1, XBP1, and NAT1 expression may share a common transcriptional network arising from the luminal epithelium associated with better survival in breast cancer. Clusters of high-expression genes, including NAT1, in breast tumors might serve as potential targets for novel therapeutic drug development.

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of <em>5α</em>-dihydrotestosterone (<em>DHT</em>)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled <em>DHT</em> binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased <em>DHT</em>-mediated transcriptional activity of the androgen receptor compared with <em>DHT</em> delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.

P-glycoprotein (Pgp), a multiple drug resistance transporter expressed by vascular endothelial cells, is a key component of the blood-brain barrier and has been shown to increase after inflammation. The nonaromatizable androgen, dihydrotestosterone (<em>DHT</em>), decreases inflammatory markers in vascular smooth muscle cells, independent of androgen receptor (AR) stimulation. The principal metabolite of <em>DHT</em>, <em>5α</em>-androstane-3β,17β-diol (3β-diol), activates estrogen receptor (ER)β and similarly decreases inflammatory markers in vascular cells. Therefore, we tested the hypothesis that either <em>DHT</em> or 3β-diol decrease cytokine-induced proinflammatory mediators, vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2), to regulate Pgp expression in male primary human brain microvascular endothelial cells (HBMECs). Using RT-qPCR, the mRNAs for AR, ERα, and ERβ and steroid metabolizing enzymes necessary for <em>DHT</em> conversion to 3β-diol were detected in male HBMECs demonstrating that the enzymes and receptors for production of and responsiveness to 3β-diol are present. Western analysis showed that 3β-diol reduced COX-2 and Pgp expression; the effect on Pgp was inhibited by the ER antagonist, ICI-182,780. IL-1β-caused an increase in COX-2 and VCAM-1 that was reduced by either <em>DHT</em> or 3β-diol. 3β-diol also decreased cytokine-induced Pgp expression. ICI-182,780 blocked the effect of 3β-diol on COX-2 and VCAM-1, but not Pgp expression. Therefore, in cytokine-stimulated male HBMECs, the effect of 3β-diol on proinflammatory mediator expression is ER dependent, whereas its effect on Pgp expression is ER independent. These studies suggest a novel role of 3β-diol in regulating blood-brain barrier function and support the concept that 3β-diol can be protective against proinflammatory mediator stimulation.

Stressors, during early life or adulthood, can alter steroid-sensitive behaviors, such as exploration, anxiety, and/or cognitive processes. We investigated if exposure to acute stressors in adulthood may alter behavioral and neuroendocrine responses of male rats that were exposed to gestational stress or not. We hypothesized that rats exposed to gestational and acute stress may show behavioral inhibition, increased corticosterone, and altered androgen levels in the hippocampus. Subjects were adult, male offspring of rat dams that were restrained daily on gestational days 14-20, or did not experience this manipulation. Immediately before testing, rats were restraint stressed for 20 min or not. During week 1, rats were tested in a battery of tasks, including the open field, elevated plus maze, social interaction, tailflick, pawlick, and defensive burying tasks. During week 2, rats were trained and tested 24 h later in the inhibitory avoidance task. Plasma corticosterone and androgen levels, and hippocampal androgen levels, were measured in all subjects. Gestational and acute restraint stress increased plasma levels of corticosterone, and reduced levels of testosterone's <em>5α</em>-reduced metabolites, dihydrotestosterone (<em>DHT</em>) and 3α-androstanediol (3α-diol), but not the aromatized metabolite, estradiol (E(2)), in plasma or the hippocampus. Gestational and acute restraint stress reduced central entries made in the open field, and latencies to enter the shock-associated side of the inhibitory avoidance chamber during testing. Gestational stress reduced time spent interacting with a conspecific. These data suggest that gestational and acute restraint stress can have actions to produce behavioral inhibition coincident with increased corticosterone and decreased <em>5α</em>-reduced androgens of adult male rats. Thus, gestational stress altered neural circuits involved in the neuroendocrine response to acute stress in early adulthood.

BACKGROUND

Testosterone, a primary androgen in males, is converted into its most active form, dihydrotestosterone (<em>DHT</em>), by <em>5α</em>-reductase type 2 (encoded by the SRD5A2 gene) in the prostate. <em>DHT</em> is necessary for prostatic growth and has five times higher binding affinity than testosterone for androgen receptors. We hypothesized that polymorphic variations in the SRD5A2 gene may affect the risk of benign prostatic hyperplasia and prostate cancer.

METHODS

We analyzed SRD5A2 gene polymorphisms in 217 BPH patients, 192 PCa cases, and 171 controls. Genotyping was undertaken using direct DNA sequencing. Genotype data were compared between cases and controls using a Chi square statistical tool.

RESULTS

We found that the A49T locus was monomorphic with 'AA' genotype in all subjects. At V89L locus, the presence of 'VV' showed a marginally significant correlation with increased BPH risk (p=0.047). At the (TA)n locus, longer TA repeats were found to be protective against BPH (p=0.003). However, neither of these polymoprhisms correlated with the risk of PCa.

CONCLUSIONS

We conclude that A49T is monomorphic in the study population, VV marginally correlates with BPH risk, and longer (TA)n repeats are protective against BPH. None of these polymorphisms affect the risk of PCa.

In recent years, a growing number of human pharmaceuticals have been detected in the aquatic environment, generally at low concentrations (sub-ng/L-low μg/L). In most cases, these compounds are characterised by highly specific modes of action, and the evolutionary conservation of drug targets in wildlife species suggests the possibility that pharmaceuticals present in the environment may cause toxicological effects by acting through the same targets as they do in humans. Our research addressed the question of whether or not dutasteride, a pharmaceutical used to treat benign prostatic hyperplasia, may cause adverse effects in a teleost fish, the fathead minnow (Pimephales promelas), by inhibiting the activity of both isoforms of <em>5α</em>-reductase (<em>5α</em>R), the enzyme that converts testosterone into dihydrotestosterone (<em>DHT</em>). Mammalian pharmacological and toxicological information were used to guide the experimental design and the selection of relevant endpoints, according to the so-called "read-across approach", suggesting that dutasteride may affect male fertility and steroid hormone dynamics. Therefore, a 21-day reproduction study was conducted to determine the effects of dutasteride (10, 32 and 100 μg/L) on fish reproduction. Exposure to dutasteride significantly reduced fecundity of fish and affected several aspects of reproductive endocrine functions in both males and females. However, none of the observed adverse effects occurred at concentrations of exposure lower than 32 μg/L; this, together with the low volume of drug prescribed every year (10.34 kg in the UK in 2011), and the extremely low predicted environmental concentration (0.03 ng/L), suggest that, at present, the potential presence of dutasteride in the environment does not represent a threat to wild fish populations.

Efforts to establish sustainable and efficient aquaculture production of sablefish (Anoplopoma fimbria) have been constrained by delayed puberty in cultured females. This study integrates a series of experiments aimed at gaining an understanding of the reproductive physiology of puberty in female sablefish. We detected transcripts for the dopamine D2 receptor (drd2) in brain, pituitary and ovary of sablefish, and prepubertal females exhibited significantly elevated brain and pituitary drd2 expression relative to wild maturing females. Treatments with sustained-release cholesterol pellets containing testosterone (T) and the dopamine D2 receptor antagonist, metoclopramide (Met), stimulated expression of pituitary luteinizing hormone beta subunit (lhb) and follicle-stimulating hormone beta subunit (fshb), respectively, in prepubertal females, whereas a combination of T and gonadotropin-releasing hormone agonist (GnRHa) had a strong synergistic effect on lhb expression (2000-fold higher than control). Although T induced a significant increase in the maximum ovarian follicle volume, none of the treatments tested stimulated onset of vitellogenesis. Using liquid chromatography/tandem mass spectrometry, we demonstrated that Met stimulated production of T by previtellogenic ovarian follicles in vitro, whereas gonadotropin preparations enhanced 17α-hydroxyprogesterone, androstenedione (A4), T and 17β-estradiol (E2) production. Treatment with T increased production of A4, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, E2, 11-ketotestosterone, and <em>5α</em>-dihydrotestosterone (<em>DHT</em>). Interestingly, in the presence of high doses of T the previtellogenic ovary preferentially produced A4 and <em>DHT</em> over any other metabolite. Our data suggest the existence of dopamine inhibition of the reproductive axis in female sablefish. Treatments with Met and T elevated gonadotropin mRNAs in prepubertal females but failed to stimulate the transition into vitellogenic growth, suggesting a possible failure in pituitary gonadotropin protein synthesis/release. Previtellogenic ovarian follicles of sablefish are equipped to synthesize steroids, including those required for vitellogenic growth, and <em>DHT</em>, a steroid hormone whose role in reproduction of fishes remains unknown.