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1.
Gene ; 747: 144672, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32305634

RESUMO

Brain and muscle Arnt-like protein-1 (BMAL1) is a clock gene that plays an important role in hormone secretion and apoptosis, but its effect on Leydig cells is unidentified. Here the role of BMAL1 in apoptosis and testosterone secretion in TM3 Leydig cell line were investigated by inhibiting its expression using small interfering RNA (siRNA). Results showed that BMAL1 knockdown promoted the apoptosis of Leydig cells and expression of (BCL2 associated X) BAX mRNA and protein, and reduced the expression of (B-cell lymphoma-2) BCL-2 mRNA and protein. BMAL1 inhibition resulted in decreased testosterone secretion and reduced expression of key genes during hormone synthesis, specifically steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). In addition, BMAL1 knockdown reduced the expression of phosphorylated p85 and AKT as confirmed by western blot. In conclusion, BMAL1 may affect testosterone secretion and apoptosis in mouse Leydig cells through regulation of the PI3K/AKT signaling pathway.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Apoptose , Técnicas de Silenciamento de Genes , Testosterona/metabolismo , Fatores de Transcrição ARNTL/genética , Animais , Apoptose/genética , Linhagem Celular , Regulação da Expressão Gênica , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Testosterona/biossíntese , Transcrição Genética
2.
Artigo em Inglês | MEDLINE | ID: mdl-32178576

RESUMO

The objective of present study was to investigate in vitro protective potential of resveratrol in TM3 Leydig cells with induced oxidative stress using hydrogen peroxide (H2O2). Leydig cells experiencing oxidative stress exhibit reduced activities in androgens production, and become hypofunctional with age, which is also related to growing oxidative stress, while resveratrol has received growing attention as a cytoprotective agent. TM3 mouse Leydig cells were cultivated during 24 h in the presence of resveratrol (5, 10, 25, 50 and 100 µM) alone, or in combination with H2O2 (300/600 µM) to induce oxidative stress. Mitochondrial activity was evaluated using MTT test, triple assay was used in order to assess cell viability parameters, intracellular generation of superoxide was determined by the nitroblue-tetrazolium assay, and quantification of steroid hormones was performed by the enzyme- linked immunosorbent assay. Resveratrol alone treatment led to the most significantly improved values of all tested parameters in the cells of experimental group with addition of 10 µM of resveratrol in comparison to the control group. In the case of cells with induced oxidative stress (300 µM H2O2) resveratrol administration resulted in significantly increased (P < 0.05) metabolic activity, as well as cell membrane integrity at concentration 10 µM. Significantly improved (P < 0.001) lysosomal activity showed cells treated with 5 and 10 µM of resveratrol, and the level of both measured hormones was significantly higher (P < 0.05) in cells supplemented with 10 µM of resveratrol. Significant decline of superoxide radical production was observed in all experimental groups in comparison to the control exposed to H2O2 alone. With respect to cells exposed to higher concentration of H2O2 (600 µM), results showed positive effect of resveratrol only in biosynthesis of both androgens with significant increased values in experimental group treated with 5 µM (P < 0.05) and 10 µM (P < 0.01) of resveratrol, in addition, in the case of testosterone we recorded significant higher (P < 0.05) values in cells with addition of 25 and 50 µM resveratrol when compared to H2O2 control. More specific and systematic research focused especially on androgen biosynthesis is necessary related to the biological activity of resveratrol in male reproductive system due to inconsistent results of studies.


Assuntos
Peróxido de Hidrogênio/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Resveratrol/farmacologia , Animais , Antioxidantes/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Superóxidos/metabolismo , Testosterona/biossíntese
3.
Gen Comp Endocrinol ; 288: 113371, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31857076

RESUMO

Mammalian bombesin-related peptide, neuromedin B (NMB) action is mediated by its receptor (NMBR), and NMB/NMBR system plays a major role in regulating hormone secretions, reproduction and cell growth. Here we report the functions of NMB in regulating steroidogenesis (testosterone synthesis), cell viability and apoptosis. The primary rabbit Leydig cells were employed as the paradigm for this research. We initially confirmed that NMBR is distributed in Leydig cells of rabbit testis, and a certain dose of NMB could increase the secretion of testosterone in primary cultured rabbit Leydig cells. Subsequently, the accumulated NMBR, StAR, CYP11A1, 3ß-HSD and PKC protein could be induced by a certain dose of NMB in Leydig cells. Moreover, we found that NMB could decrease the cell viability, and decreased the expression of PCNA protein in Leydig cells; meanwhile, except for 100 nM, other doses of NMB could suppress the cell apoptosis, and regulate Caspase-3 protein expression in Leydig cells, respectively. These results identify that NMB may be a key factor in regulating testosterone synthesis through taking part in NMBR/PKC/steroidogenesis signaling pathway, as well as the cell viability and proliferation in rabbit Leydig cells.


Assuntos
Apoptose/efeitos dos fármacos , Hormônios Esteroides Gonadais/biossíntese , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/fisiologia , Neurocinina B/análogos & derivados , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Lipogênese/efeitos dos fármacos , Lipogênese/fisiologia , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Neurocinina B/farmacologia , Coelhos , Receptores da Bombesina/metabolismo , Testosterona/biossíntese , Testosterona/metabolismo
4.
Food Chem Toxicol ; 135: 111057, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31846720

RESUMO

Prenatal nicotine exposure (PNE) may lead to offspring's testicular dysplasia. Here, we confirmed the intergenerational effect of PNE on testosterone synthetic function and explored its epigenetic programming mechanism. Pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg.d) from gestational day 9-20. Some dams were anesthetized to obtain fetal rats, the rest were allowed to spontaneous labor to generate F1 and F2 generation. In utero, PNE impaired testicular development and testosterone production. Meanwhile, the expression of steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were decreased both in F1 and F2 generations. Furthermore, PNE enhanced the expression of fetal testicular nicotinic acetylcholine receptors (nAChRs) and histone deacetylase 4 (HDAC4), while obviously weakened histone 3 lysine 9 acetylation (H3K9ac) level of StAR/3ß-HSD promoter from GD20 to postnatal week 12 and even in F2 generation. In vitro, nicotine increased nAChRs and HDAC4 expression, and decreased the StAR/3ß-HSD H3K9ac level and expression, as well as the testosterone production in Leydig cells. Antagonism of nAChRs and inhibition of HDAC4 reversed the aforementioned changes. In conclusion, PNE programmed testicular low steroidogenesis and its heritability in male offspring rats. The underlying mechanism was associated to the low-level programming of StAR/3ß-HSD H3K9ac via nAChR/HDAC4.


Assuntos
Epigênese Genética/efeitos dos fármacos , Histona Desacetilases/metabolismo , Comportamento Materno , Nicotina/administração & dosagem , Receptores Nicotínicos/metabolismo , Testículo/efeitos dos fármacos , Testosterona/biossíntese , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Nicotina/farmacologia , Gravidez , Ratos , Ratos Wistar , Testículo/embriologia , Testículo/metabolismo
5.
Anim Genet ; 50(6): 705-711, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31476086

RESUMO

The genetic background of disorders of sex development (DSD) in dogs with a normal male sex chromosome set (78,XY) is poorly described. In this study, we present for the first time, an analysis of six genes of the testosterone pathway, encoding enzymes (CYP17A1, HSD3B2, HSD17B3, SRD5A2) and transcription factors (NR5A1, AR). The entire coding sequence and flanking regions of the introns, 5'-UTR and 3'-UTR were analyzed in five DSD dogs (78,XY, SRY-positive) with ambiguous external genitalia and in 15 control dogs. A homozygous deletion of 2 bp in exon 2 of HSD17B3 (hydroxysteroid 17-beta dehydrogenase 3) was found in a Dachshund dog with enlarged clitoris, vulva and abdominal gonads and decreased serum testosterone level. In silico analysis revealed that this deleterious variant causes truncation of the encoded polypeptide (from 306 to 65 amino acids) and deprivation of the active site of the encoded enzyme. Genotyping of 23 control Dachshund dogs showed a normal homozygous genotype. Thus, we assumed that the 2-bp deletion is the causative variant. Moreover, 24 SNPs (four in CYP17A1, three in HSD3B2, six in HSD17B3, five in SRD5A2, one in AR and five in NR5A1), two intronic indels (one in HSD3B2 and one in SRD5A2) and two microsatellite polymorphisms in exon 1 of AR were found. Six SNPs appeared to be novel. No association with DSD phenotype was observed. Identification of the first case of DSD in domestic animals caused by a deleterious variant of a gene involved in testosterone synthesis showed that these genes are important candidates in such studies.


Assuntos
Transtornos do Desenvolvimento Sexual/veterinária , Doenças do Cão/genética , Testosterona/biossíntese , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Códon de Terminação , Transtornos do Desenvolvimento Sexual/genética , Transtornos do Desenvolvimento Sexual/patologia , Cães , Feminino , Deleção de Genes , Genitália/patologia , Masculino , Testosterona/sangue
6.
Molecules ; 24(14)2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315257

RESUMO

As a result of the findings of scientists working on the biosynthesis and metabolism of steroids in the plant and animal kingdoms over the past five decades, it has become apparent that those compounds that naturally occur in animals can also be found as natural constituents of plants and vice versa, i.e., they have essentially the same fate in the majority of living organisms. This review summarizes the current state of knowledge on the occurrence of animal steroid hormones in the plant kingdom, particularly focusing on progesterone, testosterone, androstadienedione (boldione), androstenedione, and estrogens.


Assuntos
Fitosteróis/metabolismo , Plantas/metabolismo , Esteroides/biossíntese , Androstadienos/metabolismo , Androstenodiona/biossíntese , Animais , Vias Biossintéticas , Estrogênios/biossíntese , Progesterona/biossíntese , Testosterona/biossíntese
7.
Toxicol Lett ; 314: 53-62, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31319113

RESUMO

Benzyl butyl phthalate (BBP) is a widely used plasticizer and has raised public health concerns. Here, we report the effects of BBP on the testis development during rat puberty. BBP (0, 10, 100 or 1000 mg/kg) was gavaged to 35-day-old male Sprague Dawley rats for 21 days. The serum testosterone levels, Leydig cell number, the expressions of Leydig and Sertoli cell genes and proteins were measured. The in vitro effects on steroidogenesis and gene expression in immature Leydig cells were observed. BBP significantly increased serum testosterone level at 10 mg/kg but lowered its level at 1000 mg/kg without affecting serum luteinizing hormone and follicle-stimulating hormone levels. BBP increased Leydig cell number at all doses but inhibited steroidogenic capacity per Leydig cell at 1000 mg/kg. BBP significantly increased the ratio of phosphos-AKT2 (pAKT2)/AKT2, and phosphos-ERK1/2 (pERK1/2)/ERK1/2 in the testis. Mono-benzyl phthalate (the metabolite of BBP) inhibited steroidogenesis but BBP did not affect androgen production in immature Leydig cells in vitro. In conclusion, BBP non-linearly regulates Leydig cell development by increasing Leydig cell number but inhibiting steroidogenesis.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Plastificantes/toxicidade , Desenvolvimento Sexual/efeitos dos fármacos , Testosterona/biossíntese , Fatores Etários , Animais , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Testosterona/sangue
8.
Andrologia ; 51(9): e13372, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31347712

RESUMO

The aim of this investigation was to evaluate changes in testosterone and some of the functional and regulatory molecules of testis such as P450scc, steroidogenic acute regulatory protein (StAR), tumour necrosis factor-α (TNF-α), interleukin-1α (IL-1α), interleukin-1ß (IL-1ß) and nerve growth factor (NGF) following exposure to 900 MHz radio frequency (RF). Thirty adult male Sprague Dawley rats (190 ± 20 g BW) were randomly classified in three equal groups, control (sham, without any exposure), short-time exposure (2 hr) (STE) and long-time exposure (4 hr) (LTE). The exposure was performed for 30 consecutive days. The testosterone level in both exposed groups was significantly less than control (p < .05). Level of TNF-α in both exposed groups was significantly greater than control (p < .05). IL-1α and NGF levels in LTE were significantly higher than the STE and control groups (p < .05). Level of IL-1ß in LTE was significantly higher than control (p < .05). Expression of both P450scc and StAR mRNA was significantly down-regulated in both exposed groups compared to control (p < .05). Our results showed that RFW can affect testis and reproductive function through changes in factors, which are important during steroidogenesis, and also through changes in inflammatory factors, which regulate Leydig cell functions.


Assuntos
Exposição Ambiental/efeitos adversos , Células Intersticiais do Testículo/efeitos da radiação , Ondas de Rádio/efeitos adversos , Reprodução/efeitos da radiação , Animais , Telefone Celular , Enzima de Clivagem da Cadeia Lateral do Colesterol/análise , Enzima de Clivagem da Cadeia Lateral do Colesterol/biossíntese , Relação Dose-Resposta à Radiação , Regulação para Baixo , Células Intersticiais do Testículo/metabolismo , Masculino , Modelos Animais , Fosfoproteínas/análise , Fosfoproteínas/biossíntese , Ratos , Ratos Sprague-Dawley , Testosterona/análise , Testosterona/biossíntese , Fatores de Tempo
9.
Int J Nanomedicine ; 14: 4601-4611, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31296989

RESUMO

Introduction: The ratio of Ce3+/Ce4+ in their structure confers unique functions on cerium oxide nanoparticles (CeO2NPs) containing rare earth elements in scavenging free radicals and protecting against oxidative damage. The potential of CeO2NPs to protect testosterone synthesis in primary mouse Leydig cells during exposure to 1,800 MHz radiofrequency (RF) radiation was examined in vitro. Methods: Leydig cells were treated with different concentrations of CeO2NPs to identify the optimum concentration for cell proliferation. The cells were pretreated with the optimum dose of CeO2NPs for 24 hrs and then exposed to 1,800 MHz RF at a power density of 200.27 µW/cm2 (specific absorption rate (SAR), 0.116 W/kg) for 1 hr, 2 hrs, or 4 hrs. The medium was used to measure the testosterone concentration. The cells were collected to determine the antioxidant indices (catalase [CAT], malondialdehyde [MDA], and total antioxidant capacity [T-AOC]), and the mRNA expression of the testosterone synthase genes (Star, Cyp11a1, and Hsd-3ß) and clock genes (Clock, Bmal1, and Rorα). Results: Our preliminary result showed that 128 µg/mL CeO2NPs was the optimum dose for cell proliferation. Cells exposed to RF alone showed reduced levels of testosterone, T-AOC, and CAT activities, increased MDA content, and the downregulated genes expression of Star, Cyp11a1, Hsd-3ß, Clock, Bmal1, and Rorα. Pretreatment of the cells with 128 µg/mL CeO2NPs for 24 hrs followed by RF exposure significantly increased testosterone synthesis, upregulated the expression of the testosterone synthase and clock genes, and increased the resistance to oxidative damage in Leydig cells compared with those in cells exposed to RF alone. Conclusion: Exposure to 1,800 MHz RF had adverse effects on testosterone synthesis, antioxidant levels, and clock gene expression in primary Leydig cells. Pretreatment with CeO2NPs prevented the adverse effects on testosterone synthesis induced by RF exposure by regulating their antioxidant capacity and clock gene expression in vitro. Further studies of the mechanism underlying the protective function of CeO2NPs against RF in the male reproductive system are required.


Assuntos
Antioxidantes/farmacologia , Cério/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Ondas de Rádio/efeitos adversos , Testosterona/biossíntese , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Cério/química , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Intersticiais do Testículo/fisiologia , Masculino , Malondialdeído/metabolismo , Camundongos Endogâmicos C57BL , Nanopartículas/química
10.
Theriogenology ; 134: 98-103, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31158736

RESUMO

The testosterone levels decreased by T-2 toxin in mouse Leydig cells were reported previously. It is not known, however, whether l-arginine improves the situation and what's the mechanism. Leydig cells were isolated and cultured with control, 10 nM T-2 toxin, 0.25, 0.5 or 1.0 mM l-arginine, and 10 nM T-2 toxin supplemented with 0.25, 0.5 or 1.0 mM l-arginine for 24 h. Cells and supernatants were collected to detect the mRNA expression and activities of P450scc (cholesterol side-chain cleavage enzyme), 3ß-HSD-1 (3ß-hydroxysteroid dehydrogenase/isomerase-1) and StAR (steroidogenic acute regulatory protein). Results revealed that l-arginine increased the testosterone levels declined by T-2 toxin and up-regulated the activities and mRNA expression of P450scc, 3ß-HSD-1 and StAR down-regulated by T-2 toxin in Leydig cells. Therefore, we concluded that l-arginine ameliorated the testosterone levels decreased by T-2 Toxin via regulating the mRNA expression and activities of P450scc, 3ß-HSD-1 and StAR in mouse Leydig cells.


Assuntos
Arginina/farmacologia , Células Intersticiais do Testículo/metabolismo , Substâncias Protetoras/farmacologia , Toxina T-2/toxicidade , Testosterona/biossíntese , Animais , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Masculino , Camundongos , RNA Mensageiro/metabolismo
11.
Food Funct ; 10(7): 4113-4123, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31233037

RESUMO

This study was motivated by clinical observations that dysmetabolic iron overload syndrome (DIOS) and an androgen deficiency are common features observed in obese adult men; however, the molecular mechanism underlying the effects of DIOS on androgen deficiency remains to be elucidated. We established a DIOS animal model by feeding Sprague-Dawley rats an iron/fat-enriched diet (50% fat plus 0.25, 1, or 2 g ferric iron per kg diet) for 12 weeks to induce iron dysfunction (indicated by decreased tissue iron efflux) in obese rats. Obese rats fed an iron/fat-enriched diet showed decreased levels of testicular total Testosterone (T) and iron exporter ferroportin but increased levels of testicular iron and hepcidin, and these effects were more evident with a >1 g ferric iron per kg diet. A western blot analysis showed that an iron/fat-enriched diet triggered testicular endoplasmic reticular (ER) stress but decreased mitochondrion biogenesis proteins (PGC1α and TFAM) and T-converting proteins (StAR, CYP11A, and 17ß-HSD). TUNEL staining showed that >1 g ferric iron induced apoptosis mainly in germ cells and Leydig's cells. Uncontrolled testicular iron efflux may cause mitochondrial-ER dysfunction and affect T biosynthesis. Future study targeting the testicular hepcidin-ferroportin axis may offer a therapeutic tool to alleviate testicular iron retention and mitochondrial-ER stress in Leydig's cells.


Assuntos
Transporte Biológico , Ferro/metabolismo , Obesidade/metabolismo , Testosterona/biossíntese , Animais , Apoptose , Proteínas de Transporte de Cátions/metabolismo , Dieta Hiperlipídica/efeitos adversos , Hepcidinas/metabolismo , Sobrecarga de Ferro , Células Intersticiais do Testículo , Masculino , Mitocôndrias , Modelos Animais , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos , Testículo/metabolismo , Testículo/patologia
12.
Andrologia ; 51(8): e13323, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134680

RESUMO

High-fat diets (HFDs) are detrimental to steroidogenesis and male fertility. This study aimed to investigate the protective effects of melatonin (MT) treatment on testicular dysfunction in mice fed with HFD. C57BL/6J male mice were randomly divided into three groups: CTRL, HFD and HFD + MT. MT treatment mitigated the increase in body weight and adipose tissue in HFD-fed mice. Serum levels of sex hormones were improved upon MT supplementation, and the expression of the testosterone synthesis proteins, StAR and P450scc was rescued as well. MT treatment significantly up-regulated the expression of SIRT1, SOD2, and GPx4 and down-regulated the expression of GRP78 and CHOP, indicating an attenuation of oxidative stress (OS) and endoplasmic reticulum (ER) stress. In TM3 cells, MT treatment protected against H2 O2 -induced steroidogenic collapse by improving mitochondrial function and attenuating OS and ER stress. These results indicate that MT treatment can improve steroidogenesis in mice fed with HFD and may have therapeutic value in the treatment of obesity-associated hypogonadism.


Assuntos
Hipogonadismo/tratamento farmacológico , Células Intersticiais do Testículo/efeitos dos fármacos , Melatonina/administração & dosagem , Obesidade/complicações , Testosterona/biossíntese , Animais , Linhagem Celular , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Hipogonadismo/etiologia , Hipogonadismo/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Obesidade/etiologia , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/metabolismo , Resultado do Tratamento
13.
Environ Toxicol ; 34(8): 968-978, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31077554

RESUMO

The aim of this study was to investigate the protective effects of Nano-Se against Ni-induced testosterone synthesis disorder in rats and determine the underlying protective mechanism. Sprague-Dawley rats were co-treated with Ni (5.0 mg/kg, i.p.) and Nano-Se (0.5, 1.0, and 2.0 mg/kg, oral gavage) for 14 days after which various endpoints were evaluated. The Ni-induced abnormal pathological changes and elevated 8-OHdG levels in the testes were attenuated by Nano-Se administration. Importantly, decreased serum testosterone levels in the Ni-treated rats were significantly restored by Nano-Se treatment, particularly at 1.0 and 2.0 mg/kg. Furthermore, the mRNA and protein levels of testosterone synthetase were increased by Nano-Se compared to the Ni group, whereas phosphorylated protein expression levels of mitogen-activated protein kinase (MAPK) pathways were suppressed by Nano-Se administration in the Ni-treated rats. Overall, the results suggest that Nano-Se may ameliorate the Ni-induced testosterone synthesis disturbance via the inhibition of ERK1/2, p38, and JNK MAPK pathways.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Níquel/toxicidade , Selênio/farmacologia , Testosterona/biossíntese , Animais , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Nanopartículas , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia , Testosterona/sangue , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Toxicol Lett ; 308: 56-64, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30935992

RESUMO

Although it is well acknowledged that the anti-androgenic phthalate diesters can be readily hydrolysed into their monoester counterparts, their metabolites' toxicology remains obscure. Herein, we tested the hypothesis that hydrolysis of one of the two ester bonds can mediate phthalate diesters' potential endocrine effects in MLTC-1 Leydig cells, in line with their ability to disrupt androgen secretion in humans. Five diesters (DMP, DEP, DBP, DBzP and DEHP) and five monoesters (MMP, MEP, MBP, MBzP and MEHP) phthalates as mixtures or individually were applied to cell lines to investigate differences in phthalates' hydrolysis associated with varying side-chain structures and steroidogenic effects. Short-chain diesters DMP, DEP and DBP are more readily hydrolysed compared to the long-chain DEHP, while aromatic alkyl chain DBzP cannot be metabolized completely in vitro. When the hydrolysis processes are interrupted, the diester phthalates' steroidogenic effects can be influenced via regulating related steroidogenic pathway genes. With 10-100 µM treatment exposures, androgenic effects were observed only with DMP or DEP but not for MMP or MEP; while the phthalate diesters DBP, DBzP or DEHP generally exhibited more complex steroidogenic effects than their corresponding monoester counterparts (i.e., biphasic androgen and anti-androgen effects for diesters but monotonic androgen effects for monoesters were observed). DBP elicited hydrolysis-related steroidogenic modulation, in which the anti-androgenic effects of diester DBP reversed into the androgenic effects of monoester MBP at 100 µM. Phthalate metabolites appear to exert different effects at an endocrine level compared to parent compounds, and deeper insights into how the hydrolytic process is related to this alternating toxicity would improve our understanding of a risk assessment for these widespread contaminants in male reproduction.


Assuntos
Androstenodiona/biossíntese , Disruptores Endócrinos/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Testosterona/biossíntese , Animais , Carboxilesterase/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Meios de Cultura , Relação Dose-Resposta a Droga , Disruptores Endócrinos/química , Hidrólise , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Ácidos Ftálicos/química
15.
Steroids ; 146: 79-91, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30951760

RESUMO

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold ('cut-off') concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatography tandem mass spectrometry (LC-MS/MS) and LC high resolution mass spectrometry (LC-HRMS). Samples were analysed both underivatised and derivatised to increase the certainty of identification. In addition to previously reported endogenous steroids, analysis of non-spiked testicular tissue samples demonstrated the presence of boldenone and boldienone at trace levels in the equine testis. Incubation of homogenates with deuterium or carbon isotope labelled testosterone and androstenedione resulted in the matching stable isotope analogues of boldenone and boldienone being formed. Additionally, deuterium and carbon labelled 2-hydroxyandrostenedione was detected, raising the possibility that this steroid is a biosynthetic intermediate. In conclusion, boldenone and boldienone are naturally present in the equine testis, with the biosynthesis of these steroids arising from the conversion of testosterone and androstenedione. However, additional work employing larger numbers of animals, further enzyme kinetic experiments and pure reference standards for 2-OH androstenedione isomers would be required to better characterize the pathways involved in these transformations.


Assuntos
Testículo/metabolismo , Testosterona/análogos & derivados , Animais , Cavalos , Masculino , Testosterona/biossíntese , Testosterona/química , Testosterona/metabolismo
16.
Dokl Biochem Biophys ; 484(1): 78-81, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012020

RESUMO

Abstract-It was shown that the thienopyrimidine derivative TP03, a low-molecular-weight agonist of the luteinizing hormone receptor (LHR), during the treatment of male rats for 7 days steadily increased the production of testosterone (T), whose elevated level was retained for 7 days, and increased the expression of the gene for LHR, which indicates the maintenance of the sensitivity of Leydig cells to gonadotropins. At the same time, the steroidogenic effect of human chorionic gonadotropin (hCG), which significantly increased the T level on the first day of administration, was further weakened, which was accompanied by a decrease in the expression of the gene for LHR in the testes, indicating the development of resistance of Leydig cells to hCG. Along with this, in the case of hCG administration, a compensatory increase in the expression of genes of the steroidogenic enzymes, such as cytochrome P450scc and dehydrogenase 3ß-HSD, was shown in the testes, while in the case of TP03 administration this effect was absent.


Assuntos
3-Hidroxiesteroide Desidrogenases/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Células Intersticiais do Testículo/metabolismo , Receptores do LH/agonistas , Testosterona/biossíntese , Tienopiridinas/farmacologia , Animais , Gonadotropina Coriônica/farmacologia , Células Intersticiais do Testículo/citologia , Masculino , Ratos
17.
Artigo em Inglês | MEDLINE | ID: mdl-30925854

RESUMO

The aim of our in vitro study was to assess the potential effect of 4-octylphenol (4-OP) on the basal and human chorionic gonadotropin (hCG)-stimulated cholesterol levels and biosynthesis of steroid hormones in cultured mouse Leydig cells. In addition, we evaluated the intracellular superoxide production following 4-OP treatment. Isolated mouse Leydig cells were cultured in the presence or absence of 1 IU/mL (hCG) with the addition of 0.04; 0.2; 1.0; 2.5 and 5.0 µg/mL 4-OP during 44 h. The level of cholesterol was determined from the culture medium using photometry. Quantification of steroid hormones was performed by the enzyme linked immunosorbent assay and intracellular generation of superoxide radicals was assessed by the nitroblue-tetrazolium assay. Administered concentrations of 4-OP (0.04-5.0 µg/mL) did not affect basal and hCG-stimulated cholesterol level significantly. However, basal DHEA secretion was increased significantly (P < 0.001) in the highest experimental dose (5 µg/mL) of 4-OP. By hCG-stimulated DHEA secretion, a significant (P < 0.001) decrease was recorded at 5.0 µg/mL 4-OP in comparison to the control group. The stimulatory effect of 4-OP was also confirmed in androstenedione secretion, when 2.5 and 5.0 µg/mL increased hormone secretion significantly (P˂0.05; P˂0.001). Exposure to experimental concentrations (0.04 to 5.0 µg/mL) of tested chemical reduced hCG-stimulated androstenedione formation, but not significantly. Measurements of basal testosterone production have shown significant (P˂0.01; P˂0.001) increase at 1.0; 2.5 and 5.0 µg/mL of 4-OP. Furthermore, 44 h treatment by 4-OP (1.0-5.0 µg/mL) caused significant (P˂0.01; P˂0.001) intracellular accumulation of superoxide radicals in exposed cells. A considerably more detailed and systematic research is required for a better understanding of risks associated with male reproductive system in humans and wildlife.


Assuntos
Androgênios/metabolismo , Gonadotropina Coriônica/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Fenóis/farmacologia , Superóxidos/metabolismo , Androstenodiona/metabolismo , Animais , Células Cultivadas , Humanos , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Testosterona/biossíntese
18.
Cell Mol Biol Lett ; 24: 21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30915128

RESUMO

Melatonin receptors MT1 and MT2 (genes officially named MTNR1A and MTNR1B, respectively) play crucial roles in melatonin-mediated regulation of circadian rhythms, the immune system, and control of reproduction in seasonally breeding animals. In this study, immunolocalization assay showed that MT1 and MT2 are highly expressed in Leydig cell membrane. To understand the biological function of melatonin receptors in hCG-induced testosterone synthesis, we generated melatonin receptor knockdown cells using specific siRNA and performed testosterone detection after hCG treatment. We found that knockdown of melatonin receptors, especially MTNR1A, led to an obvious decrease (> 60%) of testosterone level. Our further study revealed that knockdown of melatonin receptors repressed expression, at both the mRNA level and the protein level, of key steroidogenic genes, such as p450scc, p450c17 and StAR, which are essential for testosterone synthesis. hCG triggered endoplasmic reticulum (ER) stress to regulate steroidogenic genes' expression and apoptosis. To further investigate the potential roles of melatonin receptors in hCG-induced regulation of ER stress and apoptosis, we examined expression of some crucial ER stress markers, including Grp78, Chop, ATF4, Xbp1, and IRE1. We found that inhibition of melatonin receptors increased hCG-induced expression of Grp78, Chop and ATF4, but not Xbp1 and IRE1, suggesting that hCG may modulate IRE1 signaling pathways in a melatonin receptor-dependent manner. In addition, our further data showed that knockdown of MTNR1A and MTNR1B promoted hCG-induced expression of apoptosis markers, including p53, caspase-3 and Bcl-2. These results suggested that the melatonin receptors MTNR1A and MTNR1B are essential to repress hCG-induced ER stress and cell apoptosis. Our studies demonstrated that the mammalian melatonin receptors MT1 and MT2 are involved in testosterone synthesis via mediating multiple cell pathways.


Assuntos
Gonadotropina Coriônica/farmacologia , Deleção de Genes , Células Intersticiais do Testículo/metabolismo , Receptor MT1 de Melatonina/metabolismo , Testosterona/metabolismo , Animais , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Masculino , Camundongos , RNA Interferente Pequeno/metabolismo , Esteroides/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testosterona/biossíntese
19.
Bioprocess Biosyst Eng ; 42(6): 933-940, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30848360

RESUMO

Boldenone (BD) is an important steroid hormone drug which is the derivative of testosterone. In this study, an ordered biotransformation method was proposed employing Arthrobacter simplex and recombinant Pichia pastoris with 17ß-hydroxysteroid dehydrogenase from Saccharomyces cerevisiae to produce BD from androst-4-ene-3,17-dione (AD) efficiently. To lower the oxidation towards BD in A. simplex, the transformation was conducted sequentially by C1,2 dehydrogenation in A. simplex and 17ß-carbonyl reduction in recombinant P. pastoris GS115. Moreover, the reaction system was inactivated before recombinant P. pastoris GS115 was added to further inhibit the oxidation of BD by A. simplex, and the productivity of BD was improved 10.6% compared with the control. Furthermore, by optimizing the conditions of transformation from AD to BD, 4.2 g/L BD was generated with 83% productivity from 5.0 g/L AD, which was the highest productivity reported by biological method. This study offers a promising method to produce BD by ordered biotransformation system, which can also be used to manufacture other steroidal compounds that are difficult to acquire directly.


Assuntos
Androstenodiona/metabolismo , Arthrobacter/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , Pichia/metabolismo , Testosterona/análogos & derivados , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Arthrobacter/genética , Biotransformação , Microrganismos Geneticamente Modificados/genética , Pichia/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Testosterona/biossíntese
20.
Int J Mol Sci ; 20(3)2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717178

RESUMO

Cadmium (Cd) is harmful for humans and animals, especially for the reproductive system. However, the mechanism of its toxicity has not been elucidated, and how to alleviate its toxicity is very important. This study aimed to explore the role and mechanism of action of sulforaphane (SFN) in protecting mouse Leydigs (TM3) cells from cadmium (Cd)-induced damage. The half-maximal inhibitory concentration (IC50) of Cd and the safe doses of SFN were determined using a methyl thiazolyl tetrazolium (MTT) assay. The testosterone secretion from TM3 cells was measured using the enzyme-linked immunosorbent assay. The intracellular oxidative stress was evaluated using corresponding kits. The cell apoptosis was detected using flow cytometry. The mRNA expression of genes associated with NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling was detected using reverse transcription⁻polymerase chain reaction, including Nrf2, heme oxygenase I (HO-1), glutathione peroxidase (GSH-Px), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS). The protein expression of Nrf2, GSH-Px, HO-1, γ-GCS, and NQO1 was detected using Western blot analysis. The results showed that the IC50 of Cd to TM3 cells was 51.4 µmol/L. SFN reduced the release of lactate dehydrogenase from Cd-exposed cells. Cd + SFN 2.5 treatment significantly elevated testosterone concentration compared with the Cd group (p < 0.05). SFN significantly increased total superoxide dismutase (T-SOD) and GSH-Px activity and GSH content in Cd-treated cells (p < 0.05; p < 0.01), inhibited the production of malondialdehyde or reactive oxygen species caused by Cd (p < 0.05; p < 0.01), and reduced the apoptotic rate of Cd-induced TM3 cells (p < 0.01). SFN upregulated the mRNA expression of Nrf2, GSH-Px, HO-1, NQO1, and γ-GCS in Cd-treated cells, indicating the protective effect of SFN against Cd-induced oxidative stress or cell apoptosis by activating the Nrf2/ARE signaling pathway.


Assuntos
Antioxidantes/farmacologia , Cloreto de Cádmio/antagonistas & inibidores , Isotiocianatos/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Elementos de Resposta Antioxidante/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Testosterona/biossíntese
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