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1.
Genes (Basel) ; 12(4)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918852

RESUMO

Steroid metabolism is a fundamental process in the porcine testis to provide testosterone but also estrogens and androstenone, which are essential for the physiology of the boar. This study concerns boars at an early stage of puberty. Using a RT-qPCR approach, we showed that the transcriptional activities of several genes providing key enzymes involved in this metabolism (such as CYP11A1) are correlated. Surprisingly, HSD17B3, a key gene for testosterone production, was absent from this group. An additional weighted gene co-expression network analysis was performed on two large sets of mRNA-seq to identify co-expression modules. Of these modules, two containing either CYP11A1 or HSD17B3 were further analyzed. This comprehensive correlation meta-analysis identified a group of 85 genes with CYP11A1 as hub gene, but did not allow the characterization of a robust correlation network around HSD17B3. As the CYP11A1-group includes most of the genes involved in steroid synthesis pathways (including LHCGR encoding for the LH receptor), it may control the synthesis of most of the testicular steroids. The independent expression of HSD17B3 probably allows part of the production of testosterone to escape this control. This CYP11A1-group contained also INSL3 and AGT genes encoding a peptide hormone and an angiotensin peptide precursor, respectively.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Redes Reguladoras de Genes , Transdução de Sinais , Testículo/metabolismo , Testosterona/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Masculino , Suínos
2.
Reprod Toxicol ; 101: 63-73, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33675932

RESUMO

We have reported sub-fertility in F1 progeny rats with gestational exposure to hexavalent chromium [Cr(VI)], which had disrupted Sertoli cell (SC) structure and function, and decreased testosterone (T). However, the underlying mechanism for reduced T remains to be understood. We tested the hypothesis "transient prenatal exposure to Cr(VI) affects testicular steroidogenesis by altering hormone receptors and steroidogenic enzyme proteins in Leydig cells (LCs)." Pregnant Wistar rats were given drinking water containing 50, 100, and 200 mg/L potassium dichromate during gestational days 9-14, encompassing fetal differentiation window of the testis from the bipotential gonad. F1 male rats were euthanized on postnatal day 60 (peripubertal rats with adult-type LCs alone). Results showed that prenatal exposure to Cr(VI): (i) increased accumulation of Cr(III) in the testis of F1 rats; (ii) increased serum levels of luteinizing and follicle stimulating hormones (LH and FSH), and 17ß estradiol, and decreased prolactin and T; (iii) decreased steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A1, 3ß- and 17ß-hydroxysteroid dehydrogenases, cytochrome P450 aromatase and 5α reductase proteins, (iv) decreased specific activities of 3ß and 17ß hydroxysteroid dehydrogenases; (v) decreased receptors of LH, androgen and estrogen in LCs; (vi) decreased 5α reductase and receptor proteins of FSH, androgen, and estrogen in SCs. The current study concludes that prenatal exposure to Cr(VI) disrupts testicular steroidogenesis in F1 progeny by repressing hormone receptors and key proteins of the steroidogenic pathway in LCs and SCs.


Assuntos
Carcinógenos Ambientais/toxicidade , Cromo/toxicidade , Dicromato de Potássio/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Testículo/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Colestenona 5 alfa-Redutase/metabolismo , Cromo/sangue , Feminino , Hormônios/sangue , Masculino , Troca Materno-Fetal , Dicromato de Potássio/sangue , Gravidez , Ratos Wistar , Receptores do LH/metabolismo , Receptores da Prolactina/metabolismo , Receptores de Esteroides/metabolismo , Testículo/metabolismo , Testículo/patologia
3.
J Steroid Biochem Mol Biol ; 210: 105846, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33609690

RESUMO

A new androsterone derivative bearing a 16ß-picolyl group (compound 5; FCO-586-119) was synthetized in four steps from the lead compound 1 (RM-532-105). We measured its inhibitory activity on 17ß-HSD3 using microsomal fraction of rat testes as well as transfected LNCaP[17ß-HSD3] cells. We then assessed its metabolic stability as well as its cytotoxic effect against a panel of cancer cell lines. The addition of a picolyl moiety at C-16 of RM-532-105 steroid core improves the 17ß-HSD3 inhibitory activity in the microsomal fraction of rat testes, but not in whole LNCaP[17ß-HSD3] cells. Interestingly, this structural modification enhances 3-fold the metabolic stability in conjunction with a significant cytotoxic effect against pancreatic, ovarian, breast, lung, and prostate cancer cells. Because the inhibitory activity data against 17ß-HSD3 suggested that both steroid derivatives are non-competitive inhibitors, we performed docking and molecular dynamics simulations using a homology model of this membrane-associated enzyme. The results of these simulations revealed that both RM-532-105 (1) and FCO-586-119 (5) can compete for the cofactor-binding site displaying better binding energy than NADP+.


Assuntos
17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Androsterona/química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , 17-Hidroxiesteroide Desidrogenases/química , 17-Hidroxiesteroide Desidrogenases/metabolismo , Androstanos/química , Androsterona/análogos & derivados , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estabilidade de Medicamentos , Inibidores Enzimáticos/síntese química , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Ratos Sprague-Dawley , Sulfonamidas/química
4.
J Steroid Biochem Mol Biol ; 209: 105849, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33610799

RESUMO

OBJECTIVE: Although the ovaries produce the majority of estrogens in women before menopause, estrogen is also synthesized in peripheral tissues such as adipose tissue (AT). The typical female AT distribution, concentrated in subcutaneous and femoro-gluteal regions, is estrogen-mediated, but the significance of estrogen synthesis in AT of premenopausal women is poorly understood. DESIGN AND METHODS: Serum and subcutaneous and visceral AT homogenates from 28 premenopausal women undergoing non-malignant surgery were analyzed for estrone, estradiol, and serum estrone sulfate (E1S) concentrations with liquid chromatography-tandem mass spectrometry. Isotopic precursors were used to measure enzyme activities of estrone-producing steroid sulfatase and estradiol-producing 17ß-hydroxysteroid dehydrogenases (17ß-HSD). Messenger RNA (mRNA) expression levels of genes for estrogen-metabolizing enzymes were analyzed using real-time reverse transcription quantitative polymerase chain reaction. RESULTS: While estradiol was the predominant circulating active estrogen, estrone dominated in AT, with a higher concentration in visceral than subcutaneous AT (median, 2657 vs 1459 pmol/kg; P = 0.002). Both AT depots converted circulating E1S to estrone, and estrone to estradiol. Median levels of estrone were five to ten times higher in subcutaneous and visceral AT than in serum (P < 0.001) and the estradiol level in visceral AT was 1.3 times higher than in serum (P < 0.005). The local estrone concentration in visceral AT correlated positively with mRNA expression of estrone-producing enzyme aromatase (r = 0.65, P = 0.003). Waist circumference correlated positively with increased estradiol production in subcutaneous AT (r = 0.60, P = 0.039). CONCLUSIONS: Premenopausal AT demonstrated high estrogenic enzyme activity and considerable local estrogen concentrations. This may be a factor promoting female-typical AT distribution in premenopausal women.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Aromatase/metabolismo , Estrogênios/metabolismo , Gordura Intra-Abdominal/metabolismo , Pré-Menopausa , Gordura Subcutânea/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , Adulto , Aromatase/genética , Feminino , Humanos , Pessoa de Meia-Idade
5.
J Cell Physiol ; 236(9): 6706-6725, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33598947

RESUMO

Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0-5 IU/ml) and 22R-OH-cholesterol (0-30 µM) addition stimulated testosterone synthesis in primary goat LCs in a dose-dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real-time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis-related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1-Luc circadian oscillations were clearly observed in dexamethasone-treated goat LCs transfected with the pLV6-Bmal1-Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis-related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability.


Assuntos
Fatores de Transcrição ARNTL/genética , Relógios Circadianos/genética , Regulação da Expressão Gênica , Cabras/genética , Células Intersticiais do Testículo/metabolismo , Fosfoproteínas/genética , Testosterona/biossíntese , Transcrição Genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Relógios Circadianos/efeitos dos fármacos , Dexametasona/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Cabras/sangue , Humanos , Hidroxicolesteróis/metabolismo , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Modelos Biológicos , Testosterona/sangue , Transcrição Genética/efeitos dos fármacos
6.
Reprod Toxicol ; 101: 50-62, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33548410

RESUMO

Evidences have shown that alterations in testicular dehydrogenase and ionic-ATPase activities have important implications in spermatogenesis and sperm capacitation, a penultimate biochemical change required for fertilization. Previous studies have revealed that taurine and coenzyme-Q10 (COQ-10), which are synergistic testicle-active bioflavonoids, with proven gonadotropin-enhancing properties reduce testicular damage in rats. Hence, this study investigated the effects of taurine and COQ-10 or their combination alone, and in the preventive and reversal of chlorpromazine-induced inhibition of testicular dehydrogenase enzymes, electrogenic pumps, sperm capacitation and acrosomal-reaction in male Wister rats. In the drug-treatment alone or preventive-protocol, rats received oral treatment of saline (10 mL/kg), taurine (150 mg/kg/day), COQ-10 (10 mg/kg/day) or both alone repeatedly for 56 days, or in combination with chlorpromazine (30 mg/kg/p.o./day) from days 29-56. In the reversal-protocol, the animals received chlorpromazine for 56 days prior to saline, taurine, COQ-10 or the combination from days 29-56. Thereafter, spermatogenesis (sperm count, viability, motility and morphology), testicular dehydrogenase [3beta-hydroxysteroid dehydrogenase (3ß-HSD), 17beta-hydroxysteroid dehydrogenase (17ß-HSD), glucose-6-phosphate dehydrogenase (G6PDH), lactate dehydrogenase-X (LDH-X)], ATPase (Na+/K+, Ca2+, Mg2+, H+) activities, sperm capacitation and acrosomal reaction were evaluated. Taurine and COQ-10 or their combination increased spermatogenesis, testicular 3ß-HSD, 17ß-HSD, G6PDH and LDH-X enzymes of naïve and chlorpromazine-treated rats. Both taurine and COQ-10 increased Na+/K+, Ca2+, Mg2+ and H+-ATPase activities. Also, taurine and COQ-10 or their combination prevented and reversed chlorpromazine-induced inhibition of sperm capacitation and acrosomal-reaction. The study showed that taurine and COQ-10 prevent and reverse chlorpromazine-induced inhibition of spermatogenesis, epididymal sperm capacitation and acrosomal reaction in rats through increased testicular dehydrogenases and electrogenic pump activities.


Assuntos
Antipsicóticos/toxicidade , Clorpromazina/toxicidade , Coenzimas/uso terapêutico , Flavonoides/uso terapêutico , Substâncias Protetoras/uso terapêutico , Taurina/uso terapêutico , Testículo/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Coenzimas/farmacologia , Sinergismo Farmacológico , Flavonoides/farmacologia , Glucosefosfato Desidrogenase/metabolismo , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Substâncias Protetoras/farmacologia , Ratos , Ratos Wistar , Contagem de Espermatozoides , Motilidade Espermática , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Taurina/farmacologia , Testículo/metabolismo
7.
J Ethnopharmacol ; 267: 113540, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33152430

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Quite a few plants are in use to treat female infertility and associated problems. Availing the cues from traditional knowledge, phytochemical studies and ethnopharmacological evidences, the aphrodisiac plant Ficus religiosa (F. religiosa) is widely in use to cure infertility in women. For instance, the juice of leaf and aerial root of F. religiosa is reported to normalize the dysregulated menstrual cycle in women. Besides, it is believed that regular circumambulation of F. religiosa during the early hours of the morning helps women in alleviating infertility which could be attributed to the potential phytovolatiles released from F. religiosa. However, the evidences for therapeutic potential of F. religiosa in treating female infertility are arbitrary and mostly anecdotal. AIM OF THE STUDY: The present study was aimed at examining if extracts of fresh and/or dry leaf of F. religiosa would cure polycystic ovary syndrome (PCOS) in the rat model. METHODS: Rats were divided into seven groups; control (Group I), PCOS-induced (P.O, Letrozole -1 mg/kg BW for 21 days) and untreated (Group II), PCOS-induced and treated with the leaf extracts of F. religiosa (Groups III-VI), and, PCOS-induced and treated with pioglitazone (Group VII). The estrous intervals, body and organ weights (ovary and uterus), and serum hormones (testosterone, luteinizing hormone [LH], estrogen, and progesterone) were measured, and the expression of Cyp19a1 (aromatase), and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) were assessed in the experimental rats. The levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and antioxidants (MDA, GSH, GPx, SOD, and CAT) were also quantified. Besides, the putative volatile compounds in the esterified leaf extracts were identified using Gas Chromatography-Mass Spectrometry (GC-MS). RESULTS: Letrozole treatment induced irregular estrous and altered weight of organs and hormonal milieu, which were reverted to normal in leaf extracts-treated PCOS-induced rats. Remarkably, fresh leaf treatment up-regulated Cyp19a1and PPAR-γ and increased the levels of 3ß-HSD and 17ß-HSD. We found 3-acetoxy-3-hydroxy-propionic acid in fresh and dry leaf extracts, which we attribute to efficacy of the extracts in alleviating PCOS. CONCLUSION: Put together, our findings suggest the leaves of F. religiosa as potential in alleviating PCOS, mainly due to the presence of putative volatile molecules. Further screening of the leaves of F. religiosa is recommended to identify other key molecules and to develop a systematic therapeutic intervention for PCOS.


Assuntos
Aromatase/metabolismo , Ficus , Hormônios Esteroides Gonadais/biossíntese , Ovário/efeitos dos fármacos , PPAR gama/metabolismo , Extratos Vegetais/farmacologia , Síndrome do Ovário Policístico/tratamento farmacológico , 17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Aromatase/genética , Modelos Animais de Doenças , Feminino , Ficus/química , Ovário/enzimologia , PPAR gama/genética , Extratos Vegetais/isolamento & purificação , Folhas de Planta , Síndrome do Ovário Policístico/enzimologia , Síndrome do Ovário Policístico/genética , Ratos Wistar , Transdução de Sinais , Regulação para Cima
8.
ChemMedChem ; 16(1): 259-291, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33151004

RESUMO

17ß-Hydroxysteroid dehydrogenases catalyse interconversion at the C17 position between oxidized and reduced forms of steroidal nuclear receptor ligands. The type 1 enzyme, expressed in malignant cells, catalyses reduction of the less-active estrone to estradiol, and inhibitors have therapeutic potential in estrogen-dependent diseases such as breast and ovarian cancers and in endometriosis. Synthetic decoration of the nonsteroidal N-phenyl-1,2,3,4-tetrahydroisoquinoline (THIQ) template was pursued by using Pomeranz-Fritsch-Bobbitt, Pictet-Spengler and Bischler-Napieralski approaches to explore the viability of this scaffold as a steroid mimic. Derivatives were evaluated biologically in vitro as type 1 enzyme inhibitors in a bacterial cell homogenate as source of recombinant protein. Structure-activity relationships are discussed. THIQs possessing a 6-hydroxy group, lipophilic substitutions at the 1- or 4-positions in combination with N-4'-chlorophenyl substitution were most favourable for activity. Of these, one compound had an IC50 of ca. 350 nM as a racemate, testifying to the applicability of this novel approach.


Assuntos
17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Desenho de Fármacos , Tetra-Hidroisoquinolinas/química , 17-Hidroxiesteroide Desidrogenases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Humanos , Concentração Inibidora 50 , Conformação Molecular , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/metabolismo
9.
Chem Pharm Bull (Tokyo) ; 69(1): 52-58, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33087639

RESUMO

17ß Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is the key enzyme in the biosynthesis of testosterone, which is an attractive therapeutic target for prostate cancer (PCa). H10, a novel curcumin analogue, was identified as a potential 17ß-HSD3 inhibitor. The pharmacokinetic study of H10 in rats were performed by intraperitoneal (i.p.), intravenous (i.v.) and oral (p.o.) administration. In addition, the inhibitory effects of H10 against liver CYP3A4 were investigated in vitro using human liver microsomes (HLMs). The acute and chronic toxicological characteristics were characterized using single-dose and 30 d administration. All the mice were alive after i.p. H10 with dose of no more than 100 mg/kg which are nearly the maximum solubility in acute toxicity test. The pharmacokinetic characteristics of H10 fitted with linear dynamics model after single dose. Furthermore, H10 could bioaccumulate in testis, which was the target organ of 17ß-HSD3 inhibitor. H10 distributed highest in spleen, and then in liver both after single and multiple i.p. administration. Moreover, H10 showed weak inhibition towards liver CYP3A4, and did not cause significant changes in aspartate transaminase (AST) and alanine transaminase (ALT) levels after treated with H10 for continuously 30 d. Taken together, these preclinical characteristics laid the foundation for further clinical studies of H10.


Assuntos
17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Curcumina/farmacologia , Citocromo P-450 CYP3A/metabolismo , Inibidores Enzimáticos/farmacologia , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Curcumina/administração & dosagem , Curcumina/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
10.
Reprod Domest Anim ; 56(1): 74-82, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33111336

RESUMO

The oestrogens have been highly implicated in the fertility of female animals. It is widely known that the oestrogens are primarily synthetized by the ovarian granulosa cells (GCs), and the final and essential step of this process is to catalyse the oestrone to the more active oestradiol by the protein coded by hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1) gene. However, the molecular mechanism regarding the transcription of HSD17B1 remains to be fully elucidated in ovarian GCs. In this study, the 5'-deletion, luciferase assay and chromatin immunoprecipitation (ChIP) were utilized to explore the molecular regulation of transcription of HSD17B1 with the porcine ovarian GCs as the cellular model. After the deletions with -2105 to -1754 bp, -1753 to -1429 bp, -1430 to -1081 bp and -1082 to -730 bp, the relative luciferase activity of HSD17B1 promoter did not change significantly, but the deletion of -731 to -332 bp significantly increased the relative luciferase activity of HSD17B1 promoter, and an insertion (GTTT) that might raise the transcription of HSD17B1 was identified at -401 bp of HSD17B1. These findings suggested the region from -731 to +38 bp was the core promoter of HSD17B1, and the region between -731 to -332 bp might be a silence element for HSD17B1. Furthermore, the forkhead box A2 (FoxA2) directly bound at -412 to -401 bp to negatively but p53 bound at -383 to -374 bp to positively regulate the transcription and translation of HSD17B1 in ovarian GCs. These findings will improve our understanding on HSD17B1-mediated oestrogens and provide useful information for further investigations into fertility of females.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Transcrição Genética , Proteína Supressora de Tumor p53/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Feminino , Regulação Enzimológica da Expressão Gênica , Células da Granulosa , Regiões Promotoras Genéticas , Sus scrofa
11.
Chem Biol Interact ; 336: 109271, 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33002461

RESUMO

3,17ß-Hydroxysteroid dehydrogenase in Comamonas testosteroni (C. testosteroni) is a key enzyme involved in the degradation of steroid compounds. Recently, we found that LuxR is a negative regulator in the expression of the 3,17ß-HSD gene. In the present work, we cultured wild-type and LuxR knock-out mutants of C. testosteroni with inducers such as testosterone, estradiol, progesterone or estrone. HPLC analysis showed that the degradation activities towards testosterone, estradiol, progesterone, and estrone by C.T.-LuxR-KO1 were increased by 7.1%, 9.7%, 11.9% and 3.1%, respectively compared to the wild-type strain. Protein conformation of LuxR was predicted by Phyre 2 Server software, where the N-terminal 86(Ile), 116(Ile), 118(Met) and 149(Phe) residues form a testosterone binding hydrophobic pore, while the C-terminus forms the DNA binding site (HTH). Further, luxr point mutant plasmids were prepared by PCR and co-transformed with pUC3.2-4 into E. coli HB101. ELISA was used to determine 3,17ß-HSD expression after testosterone induction. Compared to wild-type luxr, 3,17ß-HSD expression in mutants of I86T, I116T, M118T and F149S were decreased. The result indicates that testosterone lost its capability to bind to LuxR after the four amino acid residues had been exchanged. No significant changes of 3,17ß-HSD expression were found in K354I and Y356 N mutants compared to wild-type luxr, which indicates that these two amino acid residues in LuxR might relate to DNA binding. Native LuxR protein was prepared from inclusion bodies using sodium lauroylsarcosinate. Molecular interaction experiments showed that LuxR protein binds to a nucleotide sequence which locates 87 bp upstream of the ßhsd promoter. Our results revealed that steroid induction of 3,17ß-HSD in C. testosteroni in fact appears to be a de-repression, where testosterone prevents the LuxR regulator protein binding to the 3,17ß-HSD promoter domain.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Comamonas testosteroni/enzimologia , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Comamonas testosteroni/citologia , Comamonas testosteroni/crescimento & desenvolvimento , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/deficiência , Transativadores/química , Transativadores/deficiência
12.
Biomolecules ; 10(11)2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33198360

RESUMO

Steroid hormone levels are associated with estrous behavior, which affects timely mating and reproductive efficiency in pigs. 17ß-hydroxysteroid dehydrogenase type 14 (HSD17B14) modulates steroid synthesis and metabolism. To identify the functional single nucleotide polymorphisms (SNPs) in the porcine HSD17B14 gene, ear tissues from Large White and Mi gilts were collected to extract genomic DNA. Variable lengths of truncated promoter of HSD17B14 gene were used to determine the promoter activity by a dual luciferase reporter system. The vector HSD17B14Phe or HSD17B14Val was transfected into porcine granulosa cells (GCs). The core promoter region was identified between -72bp and -218bp. Six of seven SNPs had significant differences of allele frequency between Large White and Mi gilts. The plasmids with the wild genotype AA of rs329427898 maintained a smaller fraction of promoter activity compared with the plasmids with the mutant genotype GG, while the plasmids with wild the genotype TT of rs319864566 had a greater promoter activity than the plasmids with the mutant genotype CC. A missense mutation (Phe73Val) caused changes in the structural dynamics and function of the HSD17B14 protein. The highly expressed HSD17B14Val degraded less estradiol into estrone, while the relatively lowly expressed HSD17B14Phe degraded more estradiol into estrone, suggesting the protein activity of HSD17B14Phe was greater than that of HSD17B14Val. Moreover, the HSD17B14Phe group has a greater apoptosis rate of porcine GCs. The HSD17B14 gene could been used as a candidate molecular marker for estrus behavior in pigs.


Assuntos
17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Estro/metabolismo , Polimorfismo de Nucleotídeo Único , Suínos/metabolismo , Animais , Estradiol/metabolismo , Estrogênios/metabolismo , Estrona/metabolismo , Feminino , Genótipo , Células da Granulosa/metabolismo , Regiões Promotoras Genéticas , Suínos/genética
13.
J Steroid Biochem Mol Biol ; 204: 105750, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32920127

RESUMO

Ghrelin is a 28-amino acid peptide hormone that regulates ovarian steroid hormone synthesis; however, there is limited evidence regarding the regulation of this pathway by ghrelin in mice ovary. Thus, we aimed to investigate whether central ghrelin action plays a role in murine reproductive health by inhibiting steroid synthesis. Further, we sought to examine the mechanism of central ghrelin action in ovarian steroid hormone synthesis. After the administration of intracerebroventricular ghrelin (1 nmol), we found reduced serum concentrations of oestradiol and progesterone and reduced secretion of follicle-stimulating hormone and luteinising hormone. Although ghrelin reduced 3ß-hydroxysteroid dehydrogenase mRNA and protein levels in the hypothalamus, it did not affect the expression of steroidogenic acute regulatory protein and cytochrome P450 17A1. In the ovary, central ghrelin regulation indirectly inhibited the mRNA and protein levels of steroidogenic acute regulatory protein, cytochrome P450 17A1, and 3ß-hydroxysteroid dehydrogenase. Moreover, no changes were observed in the expression of proliferating cell nuclear antigen and phosphorylation of extracellular signal-regulated kinase. We hypothesised that central ghrelin regulation suppressed serum oestradiol and progesterone levels by indirectly inhibiting the expression of steroidogenic acute regulatory protein, cytochrome P450 17A1, and 3ß-hydroxysteroid dehydrogenase in the ovary. In this regulation, the suppressed secretion of the follicle-stimulating hormone and luteinising hormone in the pituitary by ghrelin could be involved. Furthermore, hypothalamic 3ß-hydroxysteroid dehydrogenase expression is reduced by ghrelin injection.


Assuntos
Grelina/metabolismo , Hormônios/sangue , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Feminino , Hipotálamo/metabolismo , Injeções Intraventriculares , Camundongos Endogâmicos C57BL , Ovário/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reprodução , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo
14.
Toxicology ; 444: 152577, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32898603

RESUMO

Trimethyltin (TMT) is widely used in industry and agriculture. The present study aims to clarify the effects of in vitro TMT exposure on androgen biosynthesis and metabolism in immature Leydig cells (ILCs), and to unveil the underlying mechanism. It was found that 1-10µM TMT decreased ILC androgen productions under basal conditions. TMT at 10µM decreased luteinizing hormone (LH) or 8-Br-cAMP (8BR)-stimulated androgen productions from ILCs. TMT at 10µM decreased 22R-hydroxycholesterol (22R) and androstenedione (D4)-mediated androgen productions from ILCs. TMT at 0.1-10µM down-regulated the mRNA or protein expression levels of STAR, CYP11A1, 17ß-HSD3, or NR5A1. TMT at 10µM directly inhibited the enzyme activities of CYP11A1 and 17ß-HSD3. In conclusion, the present study demonstrated that in vitro TMT exposure decreased ILC function of androgen production, via exerting negative effects on the mRNA/protein expression levels, or enzyme activities of STAR, CYP11A1, 17ß-HSD3, or NR5A1.


Assuntos
Androgênios/biossíntese , Células Intersticiais do Testículo/efeitos dos fármacos , Compostos de Trimetilestanho/toxicidade , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Células Intersticiais do Testículo/metabolismo , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos Sprague-Dawley , Fator Esteroidogênico 1/genética , Fator Esteroidogênico 1/metabolismo
15.
Eur Rev Med Pharmacol Sci ; 24(17): 8997-9007, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32964989

RESUMO

OBJECTIVE: The authors performed a systematic review and meta-analysis to investigate the role of rs72613567 within hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) in liver diseases. MATERIALS AND METHODS: Relevant studies on the effects of HSD17B13 rs72613567 on liver diseases were found using the PubMed, Web of Science, and Embase databases, up to March 2020. The keywords "HSD17B13", "polymorphism", "variant" and "rs72613567" were used. Odds ratios (OR) and 95% confidence interval (CI) were extracted or estimated from each eligible study. A random-effects model was applied to pool results. RESULTS: We included a large population for the assessment of any liver disease (n=564702), cirrhosis (n=559834), and hepatocellular carcinoma (HCC) (n=183179), respectively. The results demonstrated that the TA allele of HSD17B13 rs72613567 could provide substantial protection from these disorders (any liver diseases: pooled OR=0.73, 95% CI=0.61-0.87; liver cirrhosis: pooled OR=0.81, 95% CI=0.76-0.88; HCC: pooled OR=0.64, 95% CI=0.53-0.77). In addition, four studies were summarized based on the histological features of nonalcoholic fatty liver disease (NAFLD). HSD17B13 rs72613567 showed a tendency towards decreased inflammation, reduced fibrosis, and milder disease severity in NAFLD. CONCLUSIONS: Our study highlights that HSD17B13 rs72613567 is an important protective factor in multiple categories of liver diseases.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Hepatopatias/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , Progressão da Doença , Humanos , Hepatopatias/patologia , Hepatopatia Gordurosa não Alcoólica/patologia
16.
J Lipid Res ; 61(11): 1400-1409, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32973038

RESUMO

Human genetic studies recently identified an association of SNPs in the 17-ß hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/ß-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Hepatopatias/tratamento farmacológico , 17-Hidroxiesteroide Desidrogenases/análise , 17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Células Cultivadas , Doença Crônica , Humanos , Hepatopatias/metabolismo , Hepatopatias/patologia , Bibliotecas de Moléculas Pequenas/farmacologia
17.
J Clin Endocrinol Metab ; 105(12)2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32865200

RESUMO

CONTEXT: Girls with premature adrenarche (PA) may have a higher risk of developing polycystic ovary syndrome (PCOS) and metabolic syndrome. The biological purpose of adrenarche is unknown and the role of novel biosynthetic pathways remains unclear. OBJECTIVE: To compare the urinary steroid metabolome and enzyme activities of girls with PA to age-matched control girls and to published steroid values of girls with normal adrenarche and of women with PCOS and their newborn daughters. DESIGN: Prospective observational study from 2009 to 2014. SETTING: Academic pediatric endocrinology referral center. PARTICIPANTS: Twenty-three girls with PA and 22 healthy, age-matched girls. MAIN OUTCOME MEASURES: Steroid metabolites in 24-hour urine samples, including 4 progesterones, 5 corticosterones, aldosterone, 13 androgens, 2 estrogens, 14 glucocorticoids, and enzyme activities represented by metabolite ratios. RESULTS: Girls with PA had a higher body mass index (mean standard deviation scores 0.9 vs -0.3, P = 0.013). Androgen excretion was higher in PA girls than in control girls (median 3257 nmol/24 hours vs 1627 nmol/24 hours, P < 0.001), in particular metabolites from alternate androgen pathways. The amount of progesterone, corticosterone, aldosterone, estrogen, and cortisol metabolites were similar between groups. Activities of 17ß-hydroxysteroid-dehydrogenase and of 17,20-lyase were higher in girls with PA. Activities of 3ß-hydroxysteroid-dehydrogenase, 21-hydroxylase, and 5α-reductase activity were not different between groups, in contrast to published results on girls with normal adrenarche or PCOS females. CONCLUSIONS: Metabolites and enzymes involved in alternate androgen pathways appear to be markers of PA. Prospective studies should assess whether steroid production in PA also differs from adrenarche at normal timing and persists into adulthood.


Assuntos
17-Hidroxiesteroide Desidrogenases/sangue , Adrenarca/sangue , Puberdade Precoce/sangue , Esteroide 17-alfa-Hidroxilase/sangue , 17-Hidroxiesteroide Desidrogenases/metabolismo , Adrenarca/metabolismo , Adrenarca/fisiologia , Androgênios/sangue , Androgênios/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Corticosterona/sangue , Corticosterona/metabolismo , Estrogênios/sangue , Estrogênios/metabolismo , Feminino , Humanos , Hidrocortisona/sangue , Hidrocortisona/metabolismo , Metaboloma , Puberdade Precoce/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Suíça , Regulação para Cima
18.
J Steroid Biochem Mol Biol ; 204: 105763, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32987128

RESUMO

Pubertal ovarian function might be dependent on the factors present in the pre-pubertal stages. Visfatin regulates ovarian steroidogenesis in adult. To date, no study has investigated the role of visfatin either in pre-pubertal or pubertal mice ovary. Thus, we investigated the role of visfatin in pre-pubertal mice ovary in relation to steroidogenesis and proliferation and apoptosis in vitro by inhibiting the endogenous visfatin by a specific inhibitor, FK866. Inhibition of visfatin increased the estrogen secretion and also up-regulated the expression of CYP11A1, 17ßHSD and CYP19A1 in mice ovary. Furthermore, active caspase3 was up-regulated along with the down-regulation of BAX and BCL2 in the pre-pubertal ovary after visfatin inhibition. The expression of GCNA, PCNA, and BrdU labeling was also decreased by FK866 treatment. These results suggest that visfatin inhibits steroidogenesis, increases proliferation, and suppresses apoptosis in the pre-pubertal mice ovary. So, visfatin is a new regulator of ovary function in pre-pubertal mice.


Assuntos
Nicotinamida Fosforribosiltransferase/metabolismo , Ovário/metabolismo , 17-Hidroxiesteroide Desidrogenases/metabolismo , Acrilamidas/farmacologia , Animais , Apoptose , Aromatase/metabolismo , Proliferação de Células , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Feminino , Camundongos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Ovário/efeitos dos fármacos , Piperidinas/farmacologia , Maturidade Sexual
19.
J Clin Endocrinol Metab ; 105(10)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32750115

RESUMO

CONTEXT: The levels of adrenal androgens are increased through the action of steroidogenic enzymes with morphological changes in the adrenal zona reticularis. OBJECTIVE: We investigated longitudinal changes in androgen levels and steroidogenic enzyme activities during early childhood. DESIGN AND PARTICIPANTS: From a prospective children's cohort, the Environment and Development of Children cohort, 114 boys and 86 girls with available blood samples from ages 2, 4, and 6 years were included. OUTCOME MEASUREMENTS: Serum concentrations of adrenal androgens using liquid chromatography-tandem mass spectrometry and steroidogenic enzyme activity calculated by the precursor/product ratio. RESULTS: During ages 2 to 4 years, 17,20-lyase and dehydroepiandrosterone (DHEA) sulfotransferase activities increased (P < 0.01 for both in boys). During ages 4 to 6 years, 17,20-lyase activity persistently increased, but 3ß-hydroxysteroid dehydrogenase (HSD) and 17ß-HSD activities decreased (P < 0.01 for all). Serum DHEA sulfate (DHEA-S) levels persistently increased from 2, 4, to 6 years, and DHEA, 17-hydroxyprogesterone, and androstenedione levels increased during ages 4 to 6 years (P < 0.01 for all). Serum DHEA-S levels during early childhood were associated with body mass index z-scores (P = 0.001 in only boys). CONCLUSION: This study supports in vivo human evidence of increased 17,20-lyase and DHEA sulfotransferase activities and decreased 3ß-HSD activity during early childhood.


Assuntos
3-Hidroxiesteroide Desidrogenases/sangue , Adrenarca/sangue , Androgênios/sangue , Esteroide 17-alfa-Hidroxilase/sangue , Sulfotransferases/sangue , 17-Hidroxiesteroide Desidrogenases/sangue , 17-Hidroxiesteroide Desidrogenases/metabolismo , 17-alfa-Hidroxiprogesterona/sangue , 17-alfa-Hidroxiprogesterona/metabolismo , 3-Hidroxiesteroide Desidrogenases/metabolismo , Adrenarca/metabolismo , Androgênios/metabolismo , Androstenodiona/sangue , Androstenodiona/metabolismo , Criança , Pré-Escolar , Sulfato de Desidroepiandrosterona/sangue , Sulfato de Desidroepiandrosterona/metabolismo , Feminino , Seguimentos , Humanos , Estudos Longitudinais , Masculino , Estudos Prospectivos , Esteroide 17-alfa-Hidroxilase/metabolismo , Sulfotransferases/metabolismo , Zona Reticular/metabolismo
20.
Biomolecules ; 10(9)2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32825572

RESUMO

Progressive mitochondrial dysfunction due to the accumulation of amyloid beta (Aß) peptide within the mitochondrial matrix represents one of the key characteristics of Alzheimer's disease (AD) and appears already in its early stages. Inside the mitochondria, Aß interacts with a number of biomolecules, including cyclophilin D (cypD) and 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10), and affects their physiological functions. However, despite intensive ongoing research, the exact mechanisms through which Aß impairs mitochondrial functions remain to be explained. In this work, we studied the interactions of Aß with cypD and 17ß-HSD10 in vitro using the surface plasmon resonance (SPR) method and determined the kinetic parameters (association and dissociation rates) of these interactions. This is the first work which determines all these parameters under the same conditions, thus, enabling direct comparison of relative affinities of Aß to its mitochondrial binding partners. Moreover, we used the determined characteristics of the individual interactions to simulate the concurrent interactions of Aß with cypD and 17ß-HSD10 in different model situations associated with the progression of AD. This study not only advances the understanding of Aß-induced processes in mitochondria during AD, but it also provides a new perspective on research into complex multi-interaction biomolecular processes in general.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ciclofilina D/metabolismo , Proteínas Mitocondriais/metabolismo , 17-Hidroxiesteroide Desidrogenases/química , Peptídeos beta-Amiloides/química , Técnicas Biossensoriais , Ciclofilina D/química , Humanos , Proteínas Mitocondriais/química , Ressonância de Plasmônio de Superfície
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