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Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells. Show less

The Claisen condensations of 3β-acetoxypregn-5-en-20-one (1) and 3β-acetoxypregna-5,16-diene (7) with dimethyl oxalate are known to lead to 3β-hydroxy-21-methoxalylpregn-5-en-20-one (2) and 3β-hydroxy-21-methoxalylpregna-5,16-dien-20-one (8), respectively. The reactions of 2 with p-substituted phenylhydrazines afford pyrazol-5-yl derivatives (5) as main, and 3-yl regioisomers (4) as minor products. The corresponding reactions of 16-ene analogue 8 afford only pyrazol-5-yl regioisomer 9. Oppenauer oxidation of the pyrazolyl compounds yields the corresponding Δ(4)-3-ketosteroids. We investigated the antiandrogenic effects of new methoxycarbonylpyrazolyl compounds through determination of their in vitro inhibition of the activities of rat testicular C17,20-lyase, Δ(5)-3β-hydroxysteroid dehydrogenase (Δ(5)-3β-HSD) and 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3). A Δ(5)-3β-hydroxy compound in the D-ring-saturated androst-5-ene series bearing an unsubstituted phenyl group on the pyrazolyl heterocycle (5a) proved to be a potent inhibitor of Δ(5)-3β-HSD. The 4-methoxyphenyl derivative (5e) and the 3-oxo counterpart (6a) of 5a also displayed substantial inhibition. The other tested compounds exerted only weak inhibitory action against the enzymes investigated. The newly synthetized compounds were evaluated in vitro by means of MTT assays for antiproliferative activity against Hela (cervical carcinoma), A431 (skin epidermoid carcinoma) and MCF7 (breast adenocarcinoma) cells. In all four groups (3β-hydroxy- and 3-ketosteroids with saturated or unsaturated ring D), the most potent analogs contain a 4-tolyl or 4-methoxyphenyl group. Compound 5d exhibited substantial antiproliferative action against the three cell lines investigated, whereas 9d inhibited the growth of Hela cells markedly. The most noteworthy inhibition was exerted by 6a against A431 cells. Show less

Estrogens have important roles in the pathogenesis of endometrial cancer. They can have carcinogenic effects through stimulation of cell proliferation or formation of DNA-damaging species. To characterize model cell lines of endometrial cancer, we determined the expression profiles of the estrogen receptors (ERs) ESR1, ESR2 and GPER, and 23 estrogen biosynthetic and metabolic genes, and investigated estrogen biosynthesis in the control HIEEC cell line and the Ishikawa and HEC-1A EC cell lines. HIEEC and Ishikawa expressed all ERs to different extents, while HEC-1A cells lacked expression of ESR1. Considering the estrogen biosynthetic and metabolic enzymes, these cells showed statistically significant different gene expression profiles for SULT2B1, HSD3B2, CYP19A1, AKR1C3, HSD17B1, HSD17B7, HSD17B12, CYP1B1, CYP3A5, COMT, SULT1A1, GSTP1 and NQO2. In these cells, E2 was formed from E1S and E1, while androstenedione was not converted to estrogens. HIEEC and Ishikawa had similar profiles of androstenedione and E1 metabolism, but hydrolysis of E1S to E1 was weaker in Ishikawa cells. HEC-1A cells were less efficient for activation of E1 into the potent E2, but metabolized androstenedione to other androgenic metabolites better than HIEEC and Ishikawa cells. This study reveals that HIEEC, Ishikawa, and HEC-1A cells can all form estrogens only via the sulfatase pathway. HIEEC, Ishikawa, and HEC-1A cells expressed all the major genes in the production of hydroxyestrogens and estrogen quinones, and in their conjugation. Significantly higher CYP1B1 mRNA levels in Ishikawa cells compared to HEC-1A cells, together with lack of UGT2B7 expression, indicate that Ishikawa cells can accumulate more toxic estrogen-3,4-quinones than HEC-1A cells, as also for HIEEC cells. This study provides further characterization of HIEEC, Ishikawa, and HEC-1A cells, and shows that they differ greatly in expression of the genes investigated and in their capacity for E2 formation, and thus they represent different in vitro models. Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3 or HSD17B3) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T), by stereoselectively reducing the C17 ketone of 4-androstene-3,17-dione (4-dione), with NADPH as cofactor. Since T plays an important role in androgen-sensitive diseases, this enzyme is thus an interesting therapeutic target. In an attempt to design compounds to lower the level of T, we synthesized androsterone derivatives substituted at position 3 as inhibitors of 17β-HSD3, and selected one of the most potent compounds for additional studies. In an enzymatic assay in homogenized and whole HEK-293 cells overexpressing 17β-HSD3, the inhibitor RM-532-105 efficiently inhibited the conversion of natural substrate 4-dione (50nM) into T with an IC50 of 26nM and 5nM, respectively. Moreover, the inhibitor RM-532-105 (10mg/kg) reached a plasma concentration of 250ng/mL at 7h (AUC 24h: 3485ngh/mL) after subcutaneous (s.c.) injection in the rat. In order to mimic the human situation in which 4-dione is converted to T in the testis, we used intact rats. Treatment for 7 days with 17β-HSD3 inhibitor RM-532-105 by s.c. injection or oral gavage exerted no effect on the testis, prostate and seminal vesicle weight and no modification in the levels of plasma steroids. However, after this treatment, the concentration of inhibitor in plasma increased depending on the dose. We thereafter determined the concentration of inhibitor in the testis and we discovered that the compound was slightly present. In fact, at 10mg/kg, the inhibitor RM-532-105 seems to have difficulty penetrating inside the testis and was found to be concentrated in the testicular capsule, and therefore unable to inhibit the 17β-HSD3 located inside the testis. However, with a higher dose of 50mg/kg injected s.c. in rats, RM-532-105 significantly decreased the level of T and dihydrotestosterone measured in plasma at 2h. Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is a key enzyme involved in the biosynthesis of testosterone and dihydrotestosterone. These hormones are known to stimulate androgen-dependent prostate cancer. In order to generate effective inhibitors of androgen biosynthesis without androgenic effect, we synthesized a new family of 3-spiromorpholinone and 3-spirocarbamate androsterone derivatives bearing diversified hydrophobic groups. We also tested their inhibitory activity in a microsomal fraction of 17β-HSD3-containing rat testes, and their androgenic effect on androgen-sensitive LAPC-4 cells. From our first structure-activity relationship (SAR) study, we noted that compound 7e inhibited 17β-HSD3 (77% at 0.1 μM) compared to our reference compound RM-532-105 (76% at 0.1 μM), but exhibited a residual androgenic effect. A library of 7e analogue compounds was next synthesized in order to generate compounds with reduced androgenic activity. In this new SAR study, the sulfonamide compound 7e21 and the carboxamide compound 7e22 inhibited 17β-HSD3 (IC50 = 28 and 88 nM, respectively). These two compounds were not androgenic and not cytotoxic even at the highest concentration tested, but their inhibitory activity decreased in intact LNCaP cells overexpressing 17β-HSD3 (LNCaP[17β-HSD3]). Structural modifications of these two lead compounds could however be tested to produce a second generation of 17β-HSD3 inhibitors. Show less

Osteoarthritis (OA) is a multi-factorial disease leading progressively to loss of articular cartilage and subsequently to loss of joint function. While hypertrophy of chondrocytes is a physiological process implicated in the longitudinal growth of long bones, hypertrophy-like alterations in chondrocytes play a major role in OA. We performed a quantitative proteomic analysis in osteoarthritic and normal chondrocytes followed by functional analyses to investigate proteome changes and molecular pathways involved in OA pathogenesis. Chondrocytes were isolated from articular cartilage of ten patients with primary OA undergoing knee replacement surgery and six normal donors undergoing fracture repair surgery without history of joint disease and no OA clinical manifestations. We analyzed the proteome of chondrocytes using high resolution mass spectrometry and quantified it by label-free quantification and western blot analysis. We also used WebGestalt, a web-based enrichment tool for the functional annotation and pathway analysis of the differentially synthesized proteins, using the Wikipathways database. ClueGO, a Cytoscape plug-in, is also used to compare groups of proteins and to visualize the functionally organized Gene Ontology (GO) terms and pathways in the form of dynamical network structures. The proteomic analysis led to the identification of a total of ~2400 proteins. 269 of them showed differential synthesis levels between the two groups. Using functional annotation, we found that proteins belonging to pathways associated with regulation of the actin cytoskeleton, EGF/EGFR, TGF-β, MAPK signaling, integrin-mediated cell adhesion, and lipid metabolism were significantly enriched in the OA samples (p ≤10(-5)). We also observed that the proteins GSTP1, PLS3, MYOF, HSD17B12, PRDX2, APCS, PLA2G2A SERPINH1/HSP47 and MVP, show distinct synthesis levels, characteristic for OA or control chondrocytes. In this study we compared the quantitative changes in proteins synthesized in osteoarthritic compared to normal chondrocytes. We identified several pathways and proteins to be associated with OA chondrocytes. This study provides evidence for further testing on the molecular mechanism of the disease and also propose proteins as candidate markers of OA chondrocyte phenotype. Show less

The reductive 17β-hydroxysteroid dehydrogenases which catalyze the last step in estrogen activation for estrogen dependent breast cancer cells were studied. Their biological function and the effects of their knockdown for cancer cell proliferation were demonstrated. The multidisciplinary study involves enzyme catalysis, sex-hormone and cell cycle regulation, as well as cell proliferation in breast cancer cells. Reductive 17β-HSD1, -7 and -12 were studied in the main breast cancer epithelial cells MCF-7 and T47D. Modification of estradiol and 5α-dihydrotestosterone concentrations was monitored by ELISA assay while corresponding cell viability measured by MTT assay. Cell cycle was determined by flow cytometry. Dual activity of estradiol activation and 5α-dihydrotestosterone reduction by 17β-HSD1 and -7 was critical for breast cancer cell (T47D and MCF-7) viability. Cell viability was decreased by 35.8% ± 1.6% in T47D cells after simultaneously knocking down 17β-HSD1 and -7. MCF-7 cell viability was decreased by 29.3% ± 4.2% using a combination of siRNAs and inhibitors. By knocking down 17β-HSD7, we have provided the first demonstration of the significant role of this enzyme in the stimulation of breast cancer cell viability as a result of its high activity on androgen reduction with positive feedback on estradiol production. A further decrease in cell viability was not observed with additional knockdown of 17β-HSD12 after 17β-HSD1 and 7. Breast cancer cell cycle progression was impeded to enter the S phase from G0-G1 after knocking down 17β-HSD1 and -7. In summary, this is the first demonstration that the dual activity in estrone activation and 5α-dihydrotestosterone reduction are the functional basis of reductive 17β-HSDs in breast cancer cells. 17β-HSD1 and -7 are principal reductive 17β-HSDs and major players in the viability of estrogen-dependent breast cancer cells. Combined targeting of these enzymes may be potential for molecular therapy of such cancer. Show less

Deficiency of 17β-hydroxysteroid dehydrogenase type3 (17β-HSD3) isoenzyme which catalyzes the synthesis of testosterone from Δ4-androstenedione, is the cause of 46, XY disorders of sex development (DSD). 17β-HSD3 deficiency is a rare autosomal recessive disorder, which is caused by mutations in the HSD17B gene found on chromosome 9q22. Up to now, almost 33 mutations in the HSD17B3 gene have been reported. Here, we report a patient with a novel mutation in HSD17B3 gene leading to 17β-HSD3 deficiency. The patient was admitted because of primary amenorrhea and signs of virilization at puberty. The chromosome analysis showed a 46, XY karyotype. Hormonal evaluation revealed a high Δ4-androstenedione level with a low serum testosterone/androstenedione (T/A) ratio. Sequence analysis of HSD17B3 gene revealed the presence of a homozygous missense mutation in exon 11 resulting in a premature stop codon (p.Y287). Gonadectomy was performed after the molecular diagnosis and estrogen replacement therapy was initiated. With this report, we emphasize that 17β-HSD3 deficiency should be considered in virilized female patients at puberty if the T/A ratio is less than 0.8, and the molecular analysis should be performed for the precise diagnosis and genetic counseling. Show less

17-β-Hydroxysteroid dehydrogenase type 3 (17βHSD-3) is present almost exclusively in the testes, and converts androstenedione (A) to testosterone (T). 17βHSD-3 deficiency is rare. The diagnosis can be missed in early childhood as the clinical presentation may be subtle. The most frequent presentation of 17 HSD-3 deficiency is a 46,XY individual with female external genitalia, labial fusion and a blind ending vagina, with or without clitoromegaly. A low testosterone/androstenedion (T/A) ratio is suggestive of 17βHSD-3 deficiency, and such diagnosis can be confirmed with molecular genetic studies. A 12-day newborn was referred to our hospital because of palpable gonads in the labia majora. On physical examination, the baby had female external genitalia and palpable gonads in the labia majora. T/A ratio was 0.26 and the diagnosis was 17βHSD-3 deficiency, which was confirmed by the evidence of compound heterozygousity novel frameshift mutations in exon 9 and 10 of HSD17B3 gene. Show less

We report two newborns with female external genitalia and bilateral inguinal swelling who were diagnosed with 17β-hydroxysteroid dehydrogenase type 3 deficiency, a rare cause of 46,XY disorder of sexual development. The first case had normal clitoral size and vaginal and urethral openings, palpable gonads in the inguinal region, low testosterone, and low levels of basal and GNRH-stimulated gonadotropin. The second case had similar external genitalia, low testosterone but borderline basal and normal stimulated gonadotropin levels. Low testosterone/androstenedione ratios (0.22 and 0.24, respectively; normal, >0.8) after human chorionic gonadotropin stimulation indicated 17β-hydroxysteroid dehydrogenase type 3 deficiency. HSD17B3 sequencing revealed a homozygous novel mutation (c.464A>C, p.H155P) in exon 6 in the first case and homozygous c.239G>A (p.R80Q) in exon 3 in the second. Show less

17-β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) is an important enzyme involved in the final steps of androgen synthesis and is required for the development of normal male external genitalia. 46,XY individuals with deficiency of this enzyme present a wide clinical spectrum from a female appearance of the external genitalia through ambiguous genitalia to a predominantly male genitalia with micropenis or hypospadias. This paper reports a one-year-old 46,XY patient with 17β-HSD3 deficiency who presented with female external genitalia and bilaterally palpable gonads in the inguinal region. The low T/Δ4 ratio after human chorionic gonadotropin (hCG) stimulation suggested 17β-HSD3 deficiency. A homozygous mutation, c.761₇₆₂delAG, was determined at the intron 9/exon 10 splice site of the HSD17B3 gene. To the best of our knowledge, this mutation has not been reported thus far, but its localization and type would imply a complete disruption of the 17β-HSD3 which may explain the phenotype of our patient. Show less

Synthesis of three clinical candidates for medicines, a human urate transporter-1 inhibitor, an arginine vasopressin antagonist, and a 17β-hydroxysteroid dehydrogenase type-3 inhibitor, is described. These compounds were synthesized via construction of their 3,4-dihydro-2H-benzo[b][1,4]oxazine, dibenzodiazepine, and dibenzazocine skeletons, respectively, using the reductive ring-expansion reaction of the corresponding bicyclic or tricyclic oximes with diisobutylaluminum hydride. Show less

We determined the functional role of the Sertoli cell glucocorticoid receptor (GR) in vivo using a transgenic Cre-loxP approach to conditionally disrupt GR expression. Sertoli cell GR knockout (SCGRKO) was shown by absent Sertoli cell-specific GR immunolocalization and reduced levels of glucocorticoid-responsive Stc1 and Tsc22d3 mRNA in SCGRKO relative to control testes. Adult SCGRKO testes exhibited distinct morphological changes, including reduced seminiferous tubular lumen formation, decreased total Sertoli cell numbers, and parallel reductions in meiotic spermatocyte and postmeiotic spermatid numbers. Conversely, tubular diameter was increased and testis size was normal in SCGRKO males. Decreased serum FSH and testicular Fshr mRNA levels were consistent with reduced Sertoli cell number. Adult SCGRKO testes also displayed atypical germ cells and interstitial focal accumulations of hypertrophic lipid-laden, immature-like Leydig cells. Circulating LH, and testicular Lhr mRNA, testosterone, dihydrotestosterone, and 3α/3β-diol levels were all reduced in mature SCGRKO mice, whereas serum testosterone and dihydrotestosterone levels remained normal. Moreover, Sertoli cell GR disruption caused differential changes to steroidogenic enzyme transcripts, with down-regulated testicular Cyp11a1 contrasting with up-regulated Hsd17b3 expression. Reduced SCGRKO testicular expression of Hsd11b2, encoding an enzyme for corticosterone inactivation, supports a dynamic coupling between Hsd11b and androgen production. Our novel SCGRKO model has revealed that Sertoli cell-mediated GR actions support normal testicular function. Sertoli cell GR is required to maintain normal testicular Sertoli/germ cell numbers and circulating gonadotropin levels, as well as optimal Leydig cell maturation and steroidogenesis, providing new insight into gluocorticoid-mediated impact on male reproduction. Show less

The genetic etiology of sporadic neuroblastoma is still largely obscure. In a genome-wide association study, we identified single-nucleotide polymorphisms (SNP) associated with neuroblastoma at the CASC15, BARD1, LMO1, DUSP12, HSD17B12, HACE1, and LIN28B gene loci, but these explain only a small fraction of neuroblastoma heritability. Other neuroblastoma susceptibility genes are likely hidden among signals discarded by the multiple testing corrections. In this study, we evaluated eight additional genes selected as candidates for further study based on proven involvement in neuroblastoma differentiation. SNPs at these candidate genes were tested for association with disease susceptibility in 2,101 cases and 4,202 controls, with the associations found replicated in an independent cohort of 459 cases and 809 controls. Replicated associations were further studied for cis-effect using gene expression, transient overexpression, silencing, and cellular differentiation assays. The neurofilament gene NEFL harbored three SNPs associated with neuroblastoma (rs11994014: Pcombined = 0.0050; OR, 0.88; rs2979704: Pcombined = 0.0072; OR, 0.87; rs1059111: Pcombined = 0.0049; OR, 0.86). The protective allele of rs1059111 correlated with increased NEFL expression. Biologic investigations showed that ectopic overexpression of NEFL inhibited cell growth specifically in neuroblastoma cells carrying the protective allele. NEFL overexpression also enhanced differentiation and impaired the proliferation and anchorage-independent growth of cells with protective allele and basal NEFL expression, while impairing invasiveness and proliferation of cells homozygous for the risk genotype. Clinically, high levels of NEFL expression in primary neuroblastoma specimens were associated with better overall survival (P = 0.03; HR, 0.68). Our results show that common variants of NEFL influence neuroblastoma susceptibility and they establish that NEFL expression influences disease initiation and progression. Show less

The development of male internal and external genitalia in an XY fetus requires a complex interplay of many critical genes, enzymes, and cofactors. The enzyme 17β-hydroxysteroid-dehydrogenase type 3 (17βHSD3) is present almost exclusively in the testicles and converts Delta 4-androstenodione (Δ4) to testosterone. A deficiency in this enzyme is rare and is a frequently misdiagnosed autosomal recessive cause of 46,XY, disorder of sex development. The case report is of a 15-year-old adolescent, who was raised according to female gender. At puberty, the adolescent had a severe virilization and primary amenorrhea. The physical examination showed a male phenotype with micropenis and blind vagina. The Tanner stage was A3B1P4, nonpalpable gonads. The karyotype revealed 46,XY. The endocrinology study revealed: testosterone=2.38 ng/ml, Δ4>10.00 ng/ml, and low testosterone/Δ4 ratio=0.23. Magnetic resonance imaging of the abdominal-pelvic showed the presence of testicles in inguinal canal, seminal vesicle, prostate, micropenis, and absence of uterus and vagina. The genetic study confirmed the mutation p.Glu215Asp on HSD17B3 gene in homozygosity. The dilemma of sex reassignment was seriously considered when the diagnosis was made. During all procedures the patient was accompanied by a child psychiatrist/psychologist. The teenager desired to continue being a female, so gonadectomy was performed. Estrogen therapy and surgical procedure to change external genitalia was carried out. In this case, there was a severe virilization at puberty. It is speculated to be due to a partial activity of 17βHSD3 in the testicles and/or extratesticular ability to convert Δ4 to testosterone by 17βHSD5. Prenatal exposure of the brain to androgens has increasingly been put forward as a critical factor in gender identity development, but in this case the social factor was more important for the gender assignment. In this case, we highlight the late diagnosis, probably because the patient belongs to a poor family without proper primary medical care.We emphasize the psychological and social aspects in the sex assignment decision. Show less

In this study, we present a Sudanese 46,XY patient raised as a female and diagnosed at the age of 20 years with having 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency. She presented with primary amenorrhea, undeveloped breasts and a male pattern of secondary sexual characteristics. Examination of her external genitalia showed type IV genital circumcision. Steroid measurements both in urine and serum pointed to 17β-HSD3 deficiency. A novel homozygous splice-site mutation [c.524 + 2T>A] was detected in intron 7 of the HSD17B3 gene. In this patient, steroid concentration clearly supported both the clinical diagnosis of 17β-HSD3 deficiency and the functional relevance of the mutation. Interestingly, despite of the type IV genital circumcision, the patient expressed her interest in reassigning her sex from female to male. Show less

Forced vital capacity (FVC), a spirometric measure of pulmonary function, reflects lung volume and is used to diagnose and monitor lung diseases. We performed genome-wide association study meta-analysis of FVC in 52,253 individuals from 26 studies and followed up the top associations in 32,917 additional individuals of European ancestry. We found six new regions associated at genome-wide significance (P < 5 × 10(-8)) with FVC in or near EFEMP1, BMP6, MIR129-2-HSD17B12, PRDM11, WWOX and KCNJ2. Two loci previously associated with spirometric measures (GSTCD and PTCH1) were related to FVC. Newly implicated regions were followed up in samples from African-American, Korean, Chinese and Hispanic individuals. We detected transcripts for all six newly implicated genes in human lung tissue. The new loci may inform mechanisms involved in lung development and the pathogenesis of restrictive lung disease. Show less

Estrogens are well recognized to have beneficial effects on vulvovaginal atrophy because of menopause. The distribution of estrogen receptors and enzymes responsible for estradiol (E2) formation within the vagina may provide insight into how dehydroepiandrosterone, a precursor of both estrogens and androgens, improves vulvovaginal atrophy. The purpose of the study was to determine where the steroidogenic enzymes responsible for E2 formation as well as estrogen receptors are localized in vaginal specimens collected from cynomolgus monkeys (Macaca fascicularis), the closest model to the human. HSD3B1, HSD17B1, HSD17B5, HSD17B12, aromatase (CYP19A1), estrogen receptor (ER)-α, and ER-β were measured or localized by quantitative real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence. Estrogens were quantified by liquid chromatography/tandem mass spectrometry. All steroidogenic enzymes and estrogen receptors are localized mainly in the superficial layer of the stratified squamous epithelium, blood vessel walls, and muscle fibers of the vagina. Immunolabeling of HSD17B5 and HSD17B12 shows that these enzymes are uniformly distributed from the basal membrane to the superficial keratinized cells, whereas HSD3B1 and aromatase are particularly localized in the outer (external) portion of the epithelial layer. ER-α and ER-β are also distributed within the vaginal epithelium, with expression especially elevated at the basal membrane level. The enzymes responsible for E2 formation as well as ERs are expressed mainly in the superficial layer of the stratified epithelium as well as the muscle layer of the vagina. The present data provide morphologic and biochemical support for the role of local dehydroepiandrosterone transformation into estrogens in regulating epithelial cell maturation, pH, fluid secretion, smooth muscle activity, and blood flow regulation in the primate vagina. Show less

Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-)androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO), fluconazole (FLUC), flusilazole (FLUS), hexaconazole (HEXA), myconazole (MYC), penconazole (PEN), prochloraz (PRO), tebuconazole (TEBU), triadimefon (TRIA), and triticonazole (TRIT) were examined using murine Leydig (MA-10) cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T) secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC Show less

Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (αSma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and αSma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p < 0.001). Totally, the results have revealed that although washing is the only common technique for elimination of contaminant cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species. Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3 or HSD17B3) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T), by stereoselectively reducing the C17 ketone of 4-androstene-3,17-dione (4-dione), with NADPH as cofactor. Since T plays an important role in androgen-sensitive diseases, this enzyme is thus an interesting therapeutic target. In an attempt to design compounds to lower the level of T, we synthesized androsterone derivatives substituted at position 3 as inhibitors of 17β-HSD3, and selected one of the most potent compounds for additional studies. In an enzymatic assay in homogenized and whole HEK-293 cells overexpressing 17β-HSD3, the inhibitor RM-532-105 efficiently inhibited the conversion of natural substrate 4-dione (50nM) into T with an IC50 of 26nM and 5nM, respectively. Moreover, the inhibitor RM-532-105 (10mg/kg) reached a plasma concentration of 250ng/mL at 7h (AUC 24h: 3485ngh/mL) after subcutaneous (s.c.) injection in the rat. In order to mimic the human situation in which 4-dione is converted to T in the testis, we used intact rats. Treatment for 7 days with 17β-HSD3 inhibitor RM-532-105 by s.c. injection or oral gavage exerted no effect on the testis, prostate and seminal vesicle weight and no modification in the levels of plasma steroids. However, after this treatment, the concentration of inhibitor in plasma increased depending on the dose. We thereafter determined the concentration of inhibitor in the testis and we discovered that the compound was slightly present. In fact, at 10mg/kg, the inhibitor RM-532-105 seems to have difficulty penetrating inside the testis and was found to be concentrated in the testicular capsule, and therefore unable to inhibit the 17β-HSD3 located inside the testis. However, with a higher dose of 50mg/kg injected s.c. in rats, RM-532-105 significantly decreased the level of T and dihydrotestosterone measured in plasma at 2h. Show less

We examined whether variants in genes related to sex hormone biosynthesis and metabolism were associated with hypospadias in humans. We examined 332 relatively common tag single-nucleotide polymorphisms (tagSNPs) in 20 genes. Analyses included 633 cases (84 mild, 322 moderate, 212 severe and 15 undetermined severity) and 855 population-based non-malformed male controls born in California from 1990 to 2003. We used logistic regression models to estimate odds ratios (OR) and 95% confidence intervals (CI) for each SNP. Several of the 332 studied SNPs had p < 0.01: one in CYP3A4, four in HSD17B3, one in HSD3B1, two in STARD3, 10 in SRD5A2 and seven in STS. In addition, haplotype analyses gave several associations with p < 0.01. For HSD17B3, 14-SNP and 5-SNP blocks had ORs of 1.5 (95% CI 1.1, 2.0, p < 0.001) and 2.8 (95% CI 1.6, 4.8, p < 0.001) respectively. For SRD5A2, 9-SNP, 3-SNP and 8-SNP blocks had ORs of 1.7 (95% CI 1.3, 2.2, p < 0.001), 1.4 (95% CI 1.1, 1.8, p = 0.008) and 1.5 (95% CI 1.2, 1.9, p = 0.002) respectively. Our study indicates that several genes that contribute to sex hormone biosynthesis and metabolism are associated with hypospadias risk. Show less

Benign prostatic hyperplasia is still one of the most important problems of modern urology. We studied the gene expression of androgen metabolic enzymes involved in the synthesis of androgens directly in the target organs in 20 patients using real-time RP-PCR. A significant increase in the mRNA of HSD1 7B3 approximately 1,7 fold, SRD5A2 approximately 2 fold, HSD3B1 approximately 2 fold, STS approximately 2,3 fold u AR -2.5 fold and reduced HSD17B2 approximately 4 fold in samples of tumor tissue compared with adjacent tissues. Show less

The rapid growth of the corpus luteum (CL) after ovulation is believed to be mainly due to an increase in the size of luteal cells (hypertrophy) rather than an increase in their number. However, the relationship between luteal growth and the proliferation of luteal steroidogenic cells (LSCs) is not fully understood. One goal of the present study was to determine whether LSCs proliferate during CL growth. A second goal was to determine whether luteinizing hormone (LH), which is known have roles in the proliferation and differentiation of follicular cells, also affects the proliferation of LSCs. Ki-67 (a cell proliferation marker) was expressed during the early, developing and mid luteal stages and some Ki-67-positive cells co-expressed HSD3B (a steroidogenic marker). DNA content in LSCs isolated from the developing CL increased much more rapidly (indicating rapid growth) than did DNA content in LSCs isolated from the mid CL. The cell cycle-progressive genes CCND2 (cyclin D2) and CCNE1 (cyclin E1) mRNA were expressed more strongly in the small luteal cells than in the large luteal cells. LH decreased the rate of increase of DNA in LSCs isolated from the mid luteal stage but not in LSCs from the developing stage. LH suppressed CCND2 expression in LSCs from the mid luteal stage but not from the developing luteal stage. Furthermore, LH receptor (LHCGR) mRNA expression was higher at the mid luteal stage than at the developing luteal stage. The overall results suggest that the growth of the bovine CL is due to not only hypertrophy of LSCs but also an increase in their number, and that the proliferative ability of luteal steroidogenic cells decreases between the developing and mid luteal stages. Show less

17-β-Hydroxysteroid dehydrogenase type 3 deficiency is a rare autosomal recessive cause of 46,XY disorder of sex development. Worldwide, about 30 mutations in the hydroxysteroid (17-beta) dehydrogenase 3 (HSD17B3) gene have been reported, involving all exons except exon 1. Herein, we investigated an Egyptian female with 46,XY karyotype and low testosterone/Δ4-androstenedione ratio. Genomic DNA was extracted from blood samples, and then, direct DNA sequencing of HSD17B3 gene was performed. The patient had a homozygous mutation c.198G>A in exon 1 resulting in a stop codon (p.W50X). The study presents the first mutation to be reported in exon 1 of the HSD17B3 gene. Show less

Deficiency of 17β-hydroxysteroid dehydrogenase type 3 (17βHSD3), an enzyme converting androstenedione (A) to testosterone (T), is a rare cause of autosomal recessive 46,XY disorder of sexual development (DSD). A 18-years phenotypically female patient from southern Italy presented with primary amenorrhea. She had deep voice, macrocephaly, enlarged and bulbous nasal tip, macrostomia, facial acne, breast asymmetry, hypoplasia of the first finger of right hand, proximal implant of the fifth metatarsus bilaterally as well as an increased muscle mass and hirsutism, with hair distribution on face, neck, chest, abdomen, pubic region and on upper and lower limbs. Genital exam showed thickened labra majora with absence of labra minora and a blind-ending pseudo-vagina with clitoris enlargement. Karyotype analysis showed a male genotype (46,XY). Hormonal evaluation showed decreased T (188 ng/dL-6.5 nmol/L) and increased A (10 ng/mL-34,96 nmol/L), considering male reference ranges, resulting in a decreased T/A ratio (0,186). MRI identified testicles in inguinal regions. Human Chorionic Gonadotropin test showed T/A ratio permanently under 0,8. These evidences were suggestive of a 46,XY DSD due to 17βHSD3 deficiency. An homozygous mutation (IVS3 -1 G>C or c.326-1G>C) of the 17βHSD3 gene was discovered. Psychologist identified a well determined female gender identity. It was decided to proceed with gonadectomy and vaginal enlargement by use of dilatators. The case described represents a new case of DSD due to 17βHSD3 deficiency. This patient, raised as a girl, is diagnosed in a very late stage. The identified mutation, previously reported only in Dutch and Brazilian population, is one of 27 presently known mutations of 17βHSD3 gene and is never reported in Italian population. Show less

The clinical presentations of 17β hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency, 5α-reductase type 2 deficiency, and complete androgen insensitivity syndrome can be similar. However, those with 17β-HSD3 deficiency and 5α-reductase type 2 deficiency will develop virilization and should undergo gonadectomy after genetic testing before the age of puberty if reared in the female sex. Two sisters were initially diagnosed with complete androgen insensitivity syndrome as young children after testes were discovered during hernia surgery. Virilization occurred in both sisters during puberty, and a diagnosis of 17β-HSD3 deficiency was suspected. Confirmatory diagnosis through gene sequencing identified a heterozygous mutation for both a known splicing mutation and a previously unreported amplification mutation of the HSD17B3 gene. Show less

Several neuroblastoma (NB) susceptibility loci have been identified within LINC00340, BARD1, LMO1, DUSP12, HSD17B12, DDX4, IL31RA, HACE1 and LIN28B by genome-wide association (GWA) studies including European American individuals. To validate and comprehensively evaluate the impact of the identified NB variants on disease risk and phenotype, we analyzed 16 single nucleotide polymorphisms (SNPs) in an Italian population (370 cases and 809 controls). We assessed their regulatory activity on gene expression in lymphoblastoid (LCLs) and NB cell lines. We evaluated the cumulative effect of the independent loci on NB risk and high-risk phenotype development in Italian and European American (1627 cases and 2575 controls) populations. All NB susceptibility genes replicated in the Italian dataset except for DDX4 and IL31RA, and the most significant SNP was rs6435862 in BARD1 (P = 8.4 × 10(-15)). BARD1 showed an additional and independent SNP association (rs7585356). This variant influenced BARD1 mRNA expression in LCLs and NB cell lines. No evidence of epistasis among the NB-associated variants was detected, whereas a cumulative effect of risk variants on NB risk (European Americans: P (trend) = 6.9 × 10(-30), Italians: P (trend) = 8.55 × 10(13)) and development of high-risk phenotype (European Americans: P (trend) = 6.9 × 10(-13), Italians: P (trend) = 2.2 × 10(-1)) was observed in a dose-dependent manner. These results provide further evidence that the risk loci identified in GWA studies contribute to NB susceptibility in distinct populations and strengthen the role of BARD1 as major genetic contributor to NB risk. This study shows that even in the absence of interaction the combination of several low-penetrance alleles has potential to distinguish subgroups of patients at different risks of developing NB. Show less

Pediatric low grade gliomas are the most common central nervous system tumors and are still incurable among a subset of patients despite current treatment modalities. Steroid biosynthesis occurs in a wide variety of organs including the brain, to mediate an assortment of functions, including a proposed role in the growth of gliomas. Hence, targeting steroid biosynthesis and/or their signaling pathways, is anticipated as an effective approach for treating gliomas. In this study, we investigated whether our chemical library of steroid inhibitors can modulate the growth of pediatric low grade glioma cell lines (Res186, Res259, R286), and subsequently identified a potent inhibitor of 17β-hydroxysteroid dehydrogenase type 3, referred to as DK16, which functions by attenuating cell viability, proliferation, migration/invasion and anchorage independent growth and conversely induces apoptosis and cell cycle arrest in a dose and duration dependent manner. Further investigations into the mechanisms of how DK16 functions showed that this drug increased the BAX/BCL2 expression ratio, induced phosphatidylserine externalization, and mitochondrial membrane depolarizations culminating to the release and nuclear translocation of AIF. In addition, treatments of low grade glioma cell lines with DK16 increased the expression of pro-apoptotic mediators including CDK2 and CTSL1, and with the converse diminished expression of pro-survival and migratory/invasion genes like PRKCA, TERT, MAPK8, MMP1 and MMP2. Our findings collectively demonstrate the potent anti-neoplastic properties of DK16, a steroid biosynthesis inhibitor, on the growth of pediatric low grade gliomas. Show less

Prostate cancer is a heterogeneous genetic disease, and molecular methods for predicting prognosis in patients with aggressive form of the disease are urgently needed to better personalize treatment approaches. The objective was to identify host genetic variations in candidate steroidogenic genes affecting hormone levels and prostate cancer progression. The study examined two independent cohorts composed of 526 Caucasian men with organ-confined prostate cancer and 601 Taiwanese men on androgen-deprivation therapy. Caucasians were genotyped for 109 haplotype-tagging single-nucleotide polymorphisms (SNP) in CYP17A1, ESR1, CYP19A1, and HSD3B1, and their prognostic significance on disease progression was assessed using Kaplan-Meier survival curves and Cox regression models. Positive findings, including previously identified SRD5A1, SRD5A2, HSD17B2, HSD17B3, and HSD17B12 polymorphisms, were then explored in Taiwanese men (n = 32 SNPs). The influence of positive markers on the circulating hormonal levels was then appraised in Caucasians using specific and sensitive mass spectrometry-based methods. After adjusting for known risk factors, variants of CYP17A1 (rs6162), HSD17B2 (rs4243229 and rs7201637), and ESR1 (rs1062577) were associated with progressive disease in both cohorts. Indeed, the presence of these variations was significantly associated with progression in Caucasians (HR, 2.29-4.10; P = 0.0014-2 × 10(-7)) and survival in Taiwanese patients [HR = 3.74; 95% confidence interval (CI): 1.71-8.19, P = 0.009]. Remarkably, the CYP17A1 rs6162 polymorphism was linked to plasma dehydroepiandrosterone-sulfate (DHEA-S) levels (P = 0.03), HSD17B2 rs7201637 with levels of dihydrotestosterone (P = 0.03), and ESR1 rs1062577 with levels of estrone-S and androsterone-glucuronide (P ≤ 0.05). This study identifies, in different ethnic groups and at different disease stages, CYP17A1, HSD17B2, and ESR1 as attractive prognostic molecular markers of prostate cancer progression. Show less