Is idiopathic hirsutism idiopathic?

BACKGROUND: Hirsutism is the excessive growth of terminal hair in a male pattern in a female. In most hirsute women, hirsutism is caused by increased androgens. However, not all women with hirsutism actually show elevated levels of circulating androgens with standard laboratory tests, in which case we speak of idiopathic hirsutism (IH). OBJECTIVES: The aim of this paper is to investigate whether there are biochemical markers that can be used to unravel the cause in IH. METHODS: An electronic search through the PubMed database was conducted to find studies describing potential biomarkers for IH. RESULTS: The majority of included studies claimed an increased 5α-reductase (5α-RD) activity in women with IH by means of increased DHT metabolite levels. Studies investigating abnormalities of the androgen receptor (AR) and serum levels of indirect markers showed no significant differences. CONCLUSIONS: Our literature search showed that polymorphisms of the AR as well as indirect markers seem to be nonspecific, but that the dihydrotestosterone-reduced metabolite 5α-androstane-3α,17ß-diol glucuronide is markedly enhanced in women with IH, suggesting an increased 5α-RD activity in these women. Further studies need to be performed to determine the clinical usefulness of 3α-diol G as a biomarker for IH.

Formation of 5α-dihydrotestosterone from 5α-androstane-3α,17ß-diol in prostate cancer LAPC-4 cells - Identifying inhibitors of non-classical pathways producing the most potent androgen.

5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17ß-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4-14C]-3α-diol from [4-14C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC50 = 1.2-14.1 µM). The most potent inhibitor 25 (IC50 = 1.2 µM) possesses a 4-(4-CF3-3-CH3O-benzyl)piperazinyl methyl side chain at C3ß and 17ß-OH/17α-CCH functionalities at C17 of a 5α-androstane core.

Identification and functional characterization of microRNAs in rat Leydig cells during development from the progenitor to the adult stage.

The aim of the present study was to identify microRNAs (miRNAs) that regulate the proliferation and differentiation of Leydig cells (LCs) of rat. Three small RNA libraries derived from progenitor LCs (PLCs), immature LCs (ILCs) and adult LCs (ALCs) were analyzed by microarrays. In total, 68 differentially expressed miRNAs (DEMs) were identified. Based on the trend of DEM expression from PLCs to ALCs, primary LCs were transfected with miRNA mimics or inhibitors. Five miRNAs (miR-30a-5p, miR-3585-5p, miR-212-3p, miR-369-5p and miR-434-3p) promoted PLC proliferation, and 3 miRNAs (miR-17-5p, miR-532-3p and miR-329-3p) activated caspase-3, which triggered LC apoptosis. For steroidogenesis, 18 miRNAs could elevate or inhibit androsterone release at the PLC stage. Eleven and 9 miRNAs inhibited the production of 5α-androstane-3α,17ß-diol in ILCs and testosterone in ALCs, respectively. miR-17-5p, miR-29a-3p and miR-299a-5p decreased androgen production by LCs at all developmental stages. Furthermore, the miR-299a-5p-mediated decrease in androgen production by the LC lineage was primarily achieved by downregulating the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1). These findings provide insights into the regulatory roles of miRNAs during the postnatal development of LCs and suggest potential strategies for the treatment of steroid-related disorders.

DHRS7 (SDR34C1) - A new player in the regulation of androgen receptor function by inactivation of 5α-dihydrotestosterone?

DHRS7 (SDR34C1) has been associated with potential tumor suppressor effects in prostate cancer; however, its function remains largely unknown. Recent experiments using purified recombinant human DHRS7 suggested several potential substrates, including the steroids cortisone and Δ4-androstene-3,17-dione (androstenedione). However, the substrate and cofactor concentrations used in these experiments were very high and the physiological relevance of these observations needed to be further investigated. In the present study, recombinant human DHRS7 was expressed in intact HEK-293 cells in order to investigate whether glucocorticoids and androgens serve as substrates at sub-micromolar concentrations and at physiological cofactor concentrations. Furthermore, the membrane topology of DHRS7 was revisited using redox-sensitive green-fluorescent protein fusions in living cells. The results revealed that (1) cortisone is a substrate of DHRS7; however, it is not reduced to cortisol but to 20ß-dihydrocortisone, (2) androstenedione is not a relevant substrate of DHRS7, (3) DHRS7 catalyzes the oxoreduction of 5α-dihydrotestosterone (5αDHT) to 3α-androstanediol (3αAdiol), with a suppressive effect on androgen receptor (AR) transcriptional activity, and (4) DHRS7 is anchored in the endoplasmic reticulum membrane with a cytoplasmic orientation. Together, the results show that DHRS7 is a cytoplasmic oriented enzyme exhibiting 3α/20ß-hydroxysteroid dehydrogenase activity, with a possible role in the modulation of AR function. Further research needs to address the physiological relevance of DHRS7 in the inactivation of 5αDHT and AR regulation.

Dihydrotestosterone synthesis pathways from inactive androgen 5α-androstane-3ß,17ß-diol in prostate cancer cells: Inhibition of intratumoural 3ß-hydroxysteroid dehydrogenase activities by abiraterone.

Intratumoural dihydrotestosterone (DHT) synthesis could be an explanation for castration resistance in prostate cancer (PC). By using liquid chromatography-mass spectrometry, we evaluated the intratumoral DHT synthesis from 5α-androstane-3ß,17ß-diol (3ß-diol), which is inactive androgen metabolized from DHT. 3ß-diol had biochemical potential to be converted to DHT via three metabolic pathways and could stimulate PC cell growth. Especially, 3ß-diol was not only converted back to upstream androgens such as dehydroepiandrosterone (DHEA) or Δ5-androstenediol but also converted directly to DHT which is the main pathway from 3ß-diol to DHT. Abiraterone had a significant influence on the metabolism of DHEA, epiandrosterone and 3ß-diol, by the inhibition of the intratumoural 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activities which is one of key catalysts in androgen metabolic pathway. The direct-conversion of 3ß-diol to DHT was catalysed by 3ß-HSD and abiraterone could inhibit this activity of 3ß-HSD. These results suggest that PC had a mechanism of intratumoural androgen metabolism to return inactive androgen to active androgen and intratumoural DHT synthesis from 3ß-diol is important as one of the mechanisms of castration resistance in PC. Additionally, the inhibition of intratumoural 3ß-HSD activity could be a new approach to castration-resistant prostate cancer treatment.

Elevated expression of the Sertoli cell androgen receptor disrupts male fertility.

Recently, we created a unique gain-of-function mouse model with Sertoli cell-specific transgenic androgen receptor expression (TgSCAR) showing that SCAR activity controls the synchronized postnatal development of somatic Sertoli and Leydig cells and meiotic-postmeiotic germ cells. Moderate TgSCAR (TgSCAR(m)) expression reduced testis size but had no effect on male fertility. Here, we reveal that higher TgSCAR expression (TgSCAR(H)) causes male infertility. Higher SCAR activity, shown by upregulated AR-dependent transcripts (Rhox5, Spinw1), resulted in smaller adult TgSCAR(H) testes (50% of normal) despite normal or elevated circulating and intratesticular testosterone levels. Unlike fertile TgSCAR(m) males, testes of adult TgSCAR(H) males exhibited focal regions of interstitial hypertrophy featuring immature adult Leydig cells and higher intratesticular dihydrotestosterone and 5α-androstane 3α,17ß-diol levels that are normally associated with pubertal development. Mature TgSCAR(H) testes also exhibited markedly reduced Sertoli cell numbers (70%), although meiotic and postmeiotic germ cell/Sertoli cell ratios were twofold higher than normal, suggesting that elevated TgSCAR activity supports excessive spermatogenic development. Concurrent with the higher germ cell load of TgSCAR(H) Sertoli cells were increased levels of apoptotic germ cells in TgSCAR(H) relative to TgSCAR(m) testes. In addition, TgSCAR(H) testes displayed unique morphological degeneration that featured accumulated cellular and spermatozoa clusters in dilated channels of rete testes, consistent with reduced epididymal sperm numbers. Our findings reveal for the first time that excessive Sertoli cell AR activity in mature testes can reach a level that disturbs Sertoli/germ cell homeostasis, impacts focal Leydig cell function, reduces sperm output, and disrupts male fertility.

Androgen activity, ischaemic heart disease and risk factors among men in NHANES III.

AIM: Observationally, low serum testosterone among men is associated with cardiovascular diseases and its risk factors, but it is unclear whether raising endogenous androgens would be protective. To clarify the role of androgens, the association of two different androgen biomarkers (serum testosterone and androstanediol glucuronide) with cardiovascular disease risk factors and mortality was examined in a nationally representative sample of US men. RESEARCH DESIGN AND METHODS: Multivariable linear and proportion hazards regression were used to examine the adjusted associations of serum testosterone and androstanediol glucuronide with cardiovascular disease risk factors and death from major cardiovascular diseases in 1460 men from NHANES III phase 1 (1988-1991) followed-up through 2006. RESULTS: Serum testosterone and androstanediol glucuronide were weakly correlated (0·13). Serum testosterone was associated with healthier values of most cardiovascular disease risk factors but not with death from ischaemic heart disease or stroke, adjusted for age, education, race/ethnicity, smoking and alcohol use. Similarly adjusted, androstanediol glucuronide was associated with unhealthier values of some cardiovascular risk factors and death from ischaemic heart disease (hazard ratio 1·16, 95% confidence interval 1·003-1·33 per standard deviation). CONCLUSIONS: Androgen biomarkers had inconsistent associations with cardiovascular disease risk factors and ischaemic heart disease. Androstanediol glucuronide, rather than serum testosterone, had associations with cardiovascular disease risk factors more similar to those seen in randomized controlled trials of testosterone therapy, with corresponding implications for raising androgens.

Neurosteroid interactions with synaptic and extrasynaptic GABA(A) receptors: regulation of subunit plasticity, phasic and tonic inhibition, and neuronal network excitability.

RATIONALE: Neurosteroids are steroids synthesized within the brain with rapid effects on neuronal excitability. Allopregnanolone, allotetrahydrodeoxycorticosterone, and androstanediol are three widely explored prototype endogenous neurosteroids. They have very different targets and functions compared to conventional steroid hormones. Neuronal γ-aminobutyric acid (GABA) type A (GABA(A)) receptors are one of the prime molecular targets of neurosteroids. OBJECTIVE: This review provides a critical appraisal of recent advances in the pharmacology of endogenous neurosteroids that interact with GABA(A) receptors in the brain. Neurosteroids possess distinct, characteristic effects on the membrane potential and current conductance of the neuron, mainly via potentiation of GABA(A) receptors at low concentrations and direct activation of receptor chloride channel at higher concentrations. The GABA(A) receptor mediates two types of inhibition, now characterized as synaptic (phasic) and extrasynaptic (tonic) inhibition. Synaptic release of GABA results in the activation of low-affinity γ2-containing synaptic receptors, while high-affinity δ-containing extrasynaptic receptors are persistently activated by the ambient GABA present in the extracellular fluid. Neurosteroids are potent positive allosteric modulators of synaptic and extrasynaptic GABA(A) receptors and therefore enhance both phasic and tonic inhibition. Tonic inhibition is specifically more sensitive to neurosteroids. The resulting tonic conductance generates a form of shunting inhibition that controls neuronal network excitability, seizure susceptibility, and behavior. CONCLUSION: The growing understanding of the mechanisms of neurosteroid regulation of the structure and function of the synaptic and extrasynaptic GABA(A) receptors provides many opportunities to create improved therapies for sleep, anxiety, stress, epilepsy, and other neuropsychiatric conditions.

Androgen metabolite-dependent growth of hormone receptor-positive breast cancer as a possible aromatase inhibitor-resistance mechanism.

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 (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3ß,17ß-diol (3ß-diol) generated from DHT by 3ß-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 3ß-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 DHT into 3ß-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.

Spectrophotometric method for the assay of steroid 5α-reductase activity of rat liver and prostate microsomes.

A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17ß-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR.

The androgen metabolite, 5α-androstane-3ß,17ß-diol (3ß-diol), activates the oxytocin promoter through an estrogen receptor-ß pathway.

Testosterone has been shown to suppress the acute stress-induced activation of the hypothalamic-pituitary-adrenal axis; however, the mechanisms underlying this response remain unclear. The hypothalamic-pituitary-adrenal axis is regulated by a neuroendocrine subpopulation of medial parvocellular neurons in the paraventricular nucleus of the hypothalamus (PVN). These neurons are devoid of androgen receptors (ARs). Therefore, a possibility is that the PVN target neurons respond to a metabolite in the testosterone catabolic pathway via an AR-independent mechanism. The dihydrotestosterone metabolite, 5α-androstane-3ß,17ß-diol (3ß-diol), binds and activates estrogen receptor-ß (ER-ß), the predominant ER in the PVN. In the PVN, ER-ß is coexpressed with oxytocin (OT). Therefore, we tested the hypothesis that 3ß-diol regulates OT expression through ER-ß activation. Treatment of ovariectomized rats with estradiol benzoate or 3ß-diol for 4 days increased OT mRNA selectively in the midcaudal, but not rostral PVN compared with vehicle-treated controls. 3ß-Diol treatment also increased OT mRNA in the hypothalamic N38 cell line in vitro. The functional interactions between 3ß-diol and ER-ß with the human OT promoter were examined using an OT promoter-luciferase reporter construct (OT-luc). In a dose-dependent manner, 3ß-diol treatment increased OT-luc activity when cells were cotransfected with ER-ß, but not ER-α. The 3ß-diol-induced OT-luc activity was reduced by deletion of the promoter region containing the composite hormone response element (cHRE). Point mutations of the cHRE also prevented OT-luc activation by 3ß-diol. These results indicate that 3ß-diol induces OT promoter activity via ER-ß-cHRE interactions.

Androgen deprivation promotes intratumoral synthesis of dihydrotestosterone from androgen metabolites in prostate cancer.

Intratumoral synthesis of dihydrotestosterone (DHT) from precursors cannot completely explain the castration resistance of prostate cancer. We showed that DHT was intratumorally synthesized from the inactive androgen metabolites 5α-androstane-3α/ß,17ß-diol (3α/ß-diol) in prostate cancer cells via different pathways in a concentration-dependent manner. Additionally, long-term culture in androgen-deprived media increased transcriptomic expression of 17ß-hydroxysteroid dehydrogenase type 6 (HSD17B6), a key enzyme of oxidative 3α-HSD that catalyzes the conversion of 3α-diol to DHT in prostate cancer cells. Correspondingly, the score for HSD17B6 in tissues of 42 prostate cancer patients undergoing androgen deprivation therapy (ADT) was about 2-fold higher than that in tissues of 100 untreated individuals. In men receiving ADT, patients showing biochemical progression had a higher HSD17B6 score than those without progression. These results suggested that 3α/ß-diol also represent potential precursors of DHT, and the back conversion of DHT from androgen derivatives can be a promising target for combination hormone therapy.

Interaction of Androst-5-ene-3ß,17ß-diol and 5α-androstane-3ß,17ß-diol with estrogen and androgen receptors: a combined binding and cell study.

Androst-5-ene-3ß,17ß-diol (ADIOL) and 5α-androstane-3ß,17ß-diol (3ß-DIOL), metabolites of dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), 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 3ß-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 3ß-DIOL inhibited the estrogen-stimulated BC cell growth. This inhibition was counteracted by anti-androgens, which were unable to affect the ADIOL and 3ß-DIOL stimulatory effects in E2-free medium. On the other hand, in the presence of tamoxifen, ADIOL and 3ß-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 3ß-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>3ß-DIOL>testosterone (T)>DHT for ER and DHT>T>3ß-DIOL>ADIOL>E1>E2 for AR. The relative binding affinities of ADIOL, 3ß-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'.

Anabolic potential of bone mineral in human periosteal fibroblasts using steroid markers of healing.

A deproteinized natural cancellous bone mineral (B) was studied in a cell culture model for its anabolic potential using two radiolabelled steroid substrates, 14C-testosterone (14C-T) and 14C-4-androstenedione (14C-4-A) independently; in the presence or absence of the anti-androgen finasteride (F) and minocycline (M). Culture medium was assayed for the biologically active metabolite 5 alpha-dihydrotestosterone (DHT) a marker of regenerative potential and wound healing. Confluent monolayer cultures of human periosteal fibroblasts were incubated in Eagle's minimum essential medium with each of the substrates 14C-T and 14C-4-A. Incubations were performed with previously established optimal concentrations of B5 (milligrams/ml), M25 (µg/ml) and F5 (µg/ml) alone and in combination (n=6) for 24h. The eluent was solvent extracted with ethyl acetate (2 ml x 2) and subjected to TLC in a benzene/acetone solvent system (4:1 v/v) for separation of metabolites; they were quantified using a radioisotope scanner. The yield of DHT was increased over controls in response to B and M with both substrates 14C-T and 14C-4-A by 1.7, 1.8-fold and 1.7, 1.6-fold respectively (n=6; p<0.001; one way ANOVA). Combined incubations of B and M resulted in similar yields. F inhibited DHT yields with both radiolabelled substrates by 2-3-fold (n=6; p<0.001) which was overcome by a combined incubation of F+B to values similar to those of controls (p<0.01). Documented pro-anabolic effects of minocycline were applicable as a standard for confirmation of responses to B. Significant increases in yields of DHT in response to B and M with both substrates indicate their anabolic potential in periosteal fibroblasts with implications for wound healing.

The androgen metabolite, 5α-androstane-3ß,17ß-diol, decreases cytokine-induced cyclooxygenase-2, vascular cell adhesion molecule-1 expression, and P-glycoprotein expression in male human brain microvascular endothelial cells.

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 (DHT), decreases inflammatory markers in vascular smooth muscle cells, independent of androgen receptor (AR) stimulation. The principal metabolite of DHT, 5α-androstane-3ß,17ß-diol (3ß-diol), activates estrogen receptor (ER)ß and similarly decreases inflammatory markers in vascular cells. Therefore, we tested the hypothesis that either DHT or 3ß-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 ERß and steroid metabolizing enzymes necessary for DHT conversion to 3ß-diol were detected in male HBMECs demonstrating that the enzymes and receptors for production of and responsiveness to 3ß-diol are present. Western analysis showed that 3ß-diol reduced COX-2 and Pgp expression; the effect on Pgp was inhibited by the ER antagonist, ICI-182,780. IL-1ß-caused an increase in COX-2 and VCAM-1 that was reduced by either DHT or 3ß-diol. 3ß-diol also decreased cytokine-induced Pgp expression. ICI-182,780 blocked the effect of 3ß-diol on COX-2 and VCAM-1, but not Pgp expression. Therefore, in cytokine-stimulated male HBMECs, the effect of 3ß-diol on proinflammatory mediator expression is ER dependent, whereas its effect on Pgp expression is ER independent. These studies suggest a novel role of 3ß-diol in regulating blood-brain barrier function and support the concept that 3ß-diol can be protective against proinflammatory mediator stimulation.

The ERß ligand 5α-androstane, 3ß,17ß-diol (3ß-diol) regulates hypothalamic oxytocin (Oxt) gene expression.

The endocrine component of the stress response is regulated by glucocorticoids and sex steroids. Testosterone down-regulates hypothalamic-pituitary-adrenal (HPA) axis activity; however, the mechanisms by which it does so are poorly understood. A candidate testosterone target is the oxytocin gene (Oxt), given that it too inhibits HPA activity. Within the paraventricular nucleus of the hypothalamus, oxytocinergic neurons involved in regulating the stress response do not express androgen receptors but do express estrogen receptor-ß (ERß), which binds the dihydrotestosterone metabolite 3ß,17ß-diol (3ß-diol). Testosterone regulation of the HPA axis thus appears to involve the conversion to the ERß-selective ligand 5α-androstane, 3ß-diol. To study mechanisms by which 3ß-diol could regulate Oxt expression, we used a hypothalamic neuronal cell line derived from embryonic mice that expresses Oxt constitutively and compared 3ß-diol with estradiol (E2) effects. E2 and 3ß-diol elicited a phasic response in Oxt mRNA levels. In the presence of either ligand, Oxt mRNA levels were increased for at least 60 min and returned to baseline by 2 h. ERß occupancy preceded an increase in Oxt mRNA levels in the presence of 3ß-diol but not E2. In tandem with ERß occupancy, 3ß-diol increased occupancy of the Oxt promoter by cAMP response element-binding protein and steroid receptor coactivator-1 at 30 min. At the same time, 3ß-diol led to the increased acetylation of histone H4 but not H3. Taken together, the data suggest that in the presence of 3ß-diol, ERß associates with cAMP response element-binding protein and steroid receptor coactivator-1 to form a functional complex that drives Oxt gene expression.

Activation of the androgen receptor by intratumoral bioconversion of androstanediol to dihydrotestosterone in prostate cancer.

The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report, we demonstrate the extent of AR transactivation in the presence of 5α-androstane-3α,17ß-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17ß-hydroxysteroid dehydrogenase 6 (17ß-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17ß-HSD6 in prostate-derived cell lines and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor-bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis.

Transformation of a series of saturated isomeric steroidal diols by Aspergillus tamarii KITA reveals a precise stereochemical requirement for entrance into the lactonization pathway.

Four isomers of 5α-androstan-3,17-diol have been transformed by the filamentous fungus Aspergillus tamarii, an organism which has the ability to convert progesterone to testololactone in high yield through an endogenous four step enzymatic pathway. The only diol handled within the lactonization pathway was 5α-androstan-3α,17ß-diol which, uniquely underwent oxidation of the 17ß-alcohol to the 17-ketone prior to its Baeyer-Villiger oxidation and the subsequent production of 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one. This demonstrated highly specific stereochemical requirements of the 17ß-hydroxysteroid dehydrogenase for oxidation of this specific steroidal diol to occur. In contrast, the other three diols were transformed within the hydroxylation pathway resulting in functionalization at C-11ß. Only 5α-androstan-3ß,17α-diol could bind to the hydroxylase in multiple binding modes undergoing monohydroxylation in 6ß and 7ß positions. Evidence from this study has indicated that hydroxylation of saturated steroidal lactones may occur following binding of ring-D in its open form in which an α-alcohol is generated with close spatial parity to the C-17α hydroxyl position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR and DEPT analysis and further characterized using infra-red, elemental analysis and accurate mass measurement.

The testosterone metabolite 3α-diol enhances female rat sexual motivation when infused in the nucleus accumbens shell.

AIM: The purpose of this study was to provide a quantitative assessment of female rat sexual behaviors after acute exposure to the A-ring reduced testosterone metabolite, androstanediol (3α-Diol), through the nucleus accumbens (NA) shell. MAIN OUTCOME MEASURES: Quantitative analyses of female rat sexual behaviors and assessment of protein levels for the enzyme glutamic acid decarboxylase isoform 67 (GAD67) and gephyrin, a protein that participates in the clustering of GABA-A receptors in postsynaptic cells, were accomplished. METHODS: Female rats were ovariectomized and primed with estrogen and progesterone to induce sexual behaviors. Females received a 3α-Diol infusion via guided cannula that aimed to the NA shell five minutes prior to a sexual encounter with a stud male. The following parameters were videotaped and measured in a frame by frame analysis: lordosis quotient (LQ), Lordosis rating (LR), frequency and duration of proceptive behaviors (hopping/darting and ear wiggling). Levels of GAD67 and gephyrin were obtained by Western blot analysis two or twenty-four hours after the sexual encounter. RESULTS: Acute exposure to 3α-Diol in the NA shell enhanced LR, ear wiggling, and hopping/darting but not LQ. Some of these behavioral effects were counteracted by co-infusion of 3α-Diol plus the GABAA-receptor antagonist GABAzine. A transient reduction of GAD67 levels in the NA shell was detected. CONCLUSIONS: The testosterone metabolite 3α-Diol enhances sexual proceptivity, but not receptivity, when infused into the NA shell directly. The GABAergic system may participate in the androgen-mediated enhancement of female rat sexual motivation.

Studies on neurosteroids XXVI. Fluoxetine-evoked changes in rat brain and serum levels of neuroactive androgen, 5 alpha-androstane-3 alpha,17 beta-diol.

It is well known that fluoxetine (Fluox), a selective serotonin reuptake inhibitor, increases the brain content of allopregnanolone (AP), a potent neuroactive steroid that positively modulates the action of gamma-aminobutyric acid (GABA) at the GABA type A receptors, but the influence of Fluox on the brain and serum levels of a neuroactive androgen, 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), is poorly understood. In this study, we examined the Fluox-evoked changes in the 3alpha,5alpha-Adiol levels and compared the level changes of 3alpha,5alpha-Adiol with those of AP. The brain and serum 3alpha,5alpha-Adiol and AP levels were determined using previously developed LC-MS/MS. The ratio of the brain 3alpha,5alpha-Adiol to the serum 3alpha,5alpha-Adiol concentrations (B/S value) was significantly elevated in the rats intraperitoneally administered Fluox (10 mg/kg). Although the magnitude of the Fluox-evoked level change in 3alpha,5alpha-Adiol was much lower than that in AP, this study demonstrated that the 3alpha,5alpha-Adiol content is also influenced by Fluox. The most probable cause for the increase in the B/S value by the Fluox treatment is the activation of the 3alpha-hydroxysteroid dehydrogenase enzyme followed by the promotion of the de novo biosynthesis of 3alpha,5alpha-Adiol in the brain.