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1.
Life Sci ; 246: 117431, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061868

RESUMO

Melatonin is an endogenous indoleamine hormone involved in various physiological processes. However, the mechanism of melatonin in mediating Leydig cells function has not been fully explained. In this study, we investigated the mechanism through which melatonin inhibits apoptosis in mouse Leydig cells by activating the retinoic acid-related orphan nuclear receptor (ROR) α/p53 signaling pathway. We confirmed the expression and localization of RORα in mouse Leydig cells using immunofluorescence. After treatment with 10 ng/mL melatonin for 36 h, RORα mRNA and protein levels were significantly increased (P < 0.01). TUNEL and flow cytometry showed that melatonin significantly decreased the TUNEL-positive cell ratio and apoptosis rate (P < 0.05). Moreover, melatonin decreased BAX expression and increased BCL-2 expression (P < 0.05). However, the RORα inhibitor SR1001 reversed the inhibitory effects of melatonin on apoptosis (P < 0.05). Additionally, analysis of p53 expression showed that melatonin inhibited p53 mRNA and protein expression (P < 0.05), whereas SR1001 reversed these effects. p53 reversed the anti-apoptotic process involving RORα-mediated melatonin in mouse Leydig cells. Collectively, our findings suggested that melatonin inhibited apoptosis via the RORα/p53 pathway.


Assuntos
Apoptose/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Melatonina/farmacologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos
2.
Environ Pollut ; 256: 113421, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31677866

RESUMO

Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant that has been used as an additive for fat- or oil-containing foods. The exposure index value increases with extended usage of the chemical. Further, estimated total amount of BHT could exceed standard regulation, considering dietary intake or another exposure. Although BHT may induce side effects in reproductive systems, adequate research had not yet been performed to confirm them. In this study, we investigated the effects of BHT on mouse Leydig cells (TM3), which are components of testis. Our results indicated that BHT suppressed cellular proliferation and induced cell cycle arrest in TM3 cells. Moreover, BHT hampered cytosolic and mitochondrial calcium homeostasis in TM3 cells. Furthermore, BHT treatment led to endoplasmic reticulum (ER) stress and DNA fragmentation, simultaneously stimulating intrinsic apoptosis signal transduction. To elucidate the mode of action of BHT on Leydig cells, we performed western blot analysis and confirmed the activation of the PI3K/AKT and MAPK pathways. Collectively, our results demonstrated that BHT has toxic effects on mouse Leydig cells via induction of calcium dysregulation and ER-mitochondria dysfunction.


Assuntos
Hidroxitolueno Butilado/toxicidade , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Poluentes Ambientais/toxicidade , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Fenóis/toxicidade , Fosfatidilinositol 3-Quinases , Testículo/metabolismo , Testes de Toxicidade
3.
Chemosphere ; 241: 125036, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31606569

RESUMO

Dimethoate is an organophosphate pesticide. It is widely used in agriculture. However, whether it blocks pubertal development of Leydig cells remains unknown. In the current study, we exposed male Sprague Dawley rats with 7.5 and 15 mg kg-1 dimethoate from postnatal day 35-56. We also exposed Leydig cells isolated from 35-day-old rats for 3 h. Dimethoate reduced serum testosterone levels at 7.5 and 15 mg kg-1 but increased serum luteinizing hormone and follicle stimulating hormone levels at 15 mg kg-1. Dimethoate did not influence Leydig cell number but reduced Leydig cell size and down-regulated Star, Cyp11a1, and Hsd3b1 in Leydig cells as well as their protein expression. Dimethoate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.05 µM. It significantly inhibited luteinizing hormone and 8Br-cAMP stimulated androgen outputs at 50 µM. It significantly inhibited 22R-hydroxycholesterol and progesterone-mediated androgen outputs at 50 µM. Further study demonstrated that dimethoate also down-regulated the expression of Star, Cyp11a1, and Hsd3b1 at 5 or 50 µM in vitro. Dimethoate did not directly inhibit rat testicular steroidogenic enzyme activities at 50 µM. In conclusion, dimethoate targets Star, Cyp11a1, and Hsd3b1 transcription, thus blocking Leydig cell differentiation during puberty.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Dimetoato/farmacologia , Células Intersticiais do Testículo/citologia , Puberdade , Androgênios/metabolismo , Animais , Inseticidas/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Complexos Multienzimáticos/genética , Fosfoproteínas/genética , Progesterona Redutase/genética , Ratos , Ratos Sprague-Dawley , Esteroide Isomerases/genética , Testosterona/sangue , Transcrição Genética
4.
Environ Pollut ; 256: 112957, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31672375

RESUMO

Parabens are class of preservatives used in vast majority of commercial products, and a potential Endocrine Disrupting Chemical (EDC). The present study was undertaken to delineate the effects of n-butylparaben on F1 male progeny exposed maternally through gestation and lactation via subcutaneous route. The F0 dams were given subcutaneous injections of n-butylparaben from gestation day (GD) 6 to postnatal day (PND) 21 with doses of 10, 100, 1000 mg/kg Bw/day in corn oil. The F1 male rats were monitored for pubertal development and sexual maturation; these were sacrificed on PND 30, 45 and 75. On PND 75, these F1 male rats were subjected for fertility assessment with unexposed female rats. A delayed testicular descent at 100 and 1000 mg/kg Bw dose and delayed preputial separation at 10 mg/kg Bw dose was observed in exposed F1 male rats. Decreased sperm count, motility and Daily Sperm Production was observed at 100 mg/kg Bw dose at PND 75. Interestingly, the sperm transit time in the epididymis was accelerated at this dose. Significant perturbed testicular expression of steroid receptors (ERα and ß, AR), INSL3 and StAR genes with increased T and LH levels indicates direct effect on spermatogenesis and steroidogenesis. These F1 generation adult rats were sub-fertile with increased (%) pre- and post-implantation loss at 100 and 1000 mg/kg Bw/day dose. This is the first report on n-butylparaben highlighting the involvement of testicular leydig cells with accelerated sperm transit time leading to reduced fertility in the maternally exposed F1 male rats through estrogenic/anti-androgenic action.


Assuntos
Disruptores Endócrinos/toxicidade , Fertilidade/efeitos dos fármacos , Parabenos/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Receptores de Esteroides/metabolismo , Espermatogênese/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lactação , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Ratos , Receptores de Esteroides/genética , Maturidade Sexual/efeitos dos fármacos , Contagem de Espermatozoides , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/embriologia , Testículo/crescimento & desenvolvimento
5.
Environ Pollut ; 255(Pt 2): 113316, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31610511

RESUMO

Paraquat is a fast and non-selective herbicide that is widely used in crop cultivation and conservation tillage systems. Animal experiments have shown that paraquat decreases sperm quality and testicular organ coefficient, but its effects on the development of Leydig cells remain unclear. The objective of the current study was to investigate the effects of paraquat exposure on the Leydig cell development in rats during puberty. Twenty-eight male 35-day-old Sprague-Dawley rats were divided into 4 groups: 0, 0.5, 2.0, and 8 mg kg-1 d-1 paraquat. Paraquat was gavaged for 10 d. Adult Leydig cells were isolated and treated with paraquat for 24 h. Paraquat in vivo significantly decreased body and testis weights at 8 mg kg-1 and lowered serum testosterone levels at 2 and 8 mg kg-1 without affecting the levels of serum luteinizing hormone and follicle-stimulating hormone. Paraquat did not alter Leydig cell number and PCNA labeling index. Real-time PCR showed that paraquat down-regulated the expression of Lhcgr, Scarb1, Cyp11a1, Cyp17a1, and Hsd17b3 genes and their proteins at 2 or 8 mg kg-1, while it up-regulated the expression of Srd5a1 at 8 mg kg-1. Paraquat increased ROS and decreased testosterone production by Leydig cells at 1 and 10 µM after in vitro 24-h exposure. Vitamin E (40 µg/ml) reversed paraquat-induced ROS and suppression of testosterone synthesis in vitro. In conclusion, paraquat directly delays Leydig cell differentiation to block testosterone synthesis via down-regulating the expression of critical testosterone synthesis-related genes and up-regulating the expression of testosterone metabolic enzyme (Srd5a1) gene and possibly via increasing ROS production.


Assuntos
Herbicidas/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Paraquat/toxicidade , Animais , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo , Hormônio Foliculoestimulante/sangue , Herbicidas/metabolismo , Hormônio Luteinizante/sangue , Masculino , Ratos , Ratos Sprague-Dawley , Maturidade Sexual , Esteroide 17-alfa-Hidroxilase/metabolismo , Testículo/efeitos dos fármacos , Testosterona/sangue , Regulação para Cima
6.
Chem Biol Interact ; 312: 108817, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499053

RESUMO

Aconitine might have reproductive toxicity and the effects of aconitine on androgen synthesis in Leydig cells remain unclear. Here, we explore how aconitine affects androgen synthesis and metabolism in rat immature Leydig cells in vitro. Immature Leydig cells were isolated from 35-day-old male Sprague Dawley rats and cultured with 0-50 µM aconitine for 3 h in combination with LH, 8Br-cAMP, 22R-hydroxycholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively. Medium androgens were measured. The levels of Leydig cell mRNAs, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14, were measured by qPCR. ROS and apoptosis were determined after 24-h aconitine treatment. Aconitine inhibited basal androgen production in Leydig cells at 0.05 µM and the higher concentrations. Aconitine blocked pregnenolone, progesterone, and androstenedione mediated androgen outputs without affecting 22R-hydroxycholesterol-mediated androgen production at 5 µM. Aconitine also inhibited LH and 8Br-cAMP stimulated androgen outputs at 5 µM. Further investigation showed that aconitine blocked androgen synthesis via down-regulating the expression of Scarb1, Hsd3b1, Cyp17a1, and Hsd17b3. At 50 µM, aconitine also induced ROS generation and increased apoptotic rate of Leydig cells. Aconitine lowered serum testosterone levels at 1.5 mg/kg after 7 days of oral exposure from postnatal day 35. In conclusion, aconitine inhibits androgen synthesis.


Assuntos
Aconitina/farmacologia , Androgênios/metabolismo , Regulação para Baixo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Testosterona/sangue , Testosterona/farmacologia
7.
Reprod Biol Endocrinol ; 17(1): 71, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31472681

RESUMO

BACKGROUND: Palmitic acid (PA) is a common saturated fatty acid that induces apoptosis in various types of cells, including testicular Leydig cells. There is evidence suggesting that PA is increased in patients with obesity and that PA-induced cell apoptosis may play an important role in obesity-related male infertility. Curcumin, a natural polyphenol, has been reported to exert cytoprotective effects in various cell types. However, the cytoprotective effect of curcumin against PA-induced apoptosis in Leydig cells remains unknown. Therefore, the current study was performed to investigate the protective effects of curcumin in response to PA-induced toxicity and apoptosis in murine Leydig tumor cell line 1 (MLTC-1) cells and explore the mechanism underlying its anti-apoptotic action. METHODS: MLTC-1 cells were cultured in Roswell Park Institute-1640 medium and divided into five groups. First four groups were treated with 50-400 µM PA, 400 µM PA + 5-40 µM curcumin, 400 µM PA + 500 nM 4-phenylbutyric acid (4-PBA, an endoplasmic reticulum (ER) stress inhibitor), and 500 nM thapsigargin (TG, an ER stress inducer) + 20 µM curcumin, respectively, followed by incubation for 24 h. Effects of PA and/or curcumin on viability, apoptosis, and ER stress in MLTC-1 cells were then determined by cell proliferation assay, flow cytometry, and western blot analysis. The fifth group of MLTC-1 cells was exposed to 400 µM of PA and 5 IU/mL of human chorionic gonadotropin (hCG) for 24 h in the absence and presence of curcumin, followed by measurement of testosterone levels in cell-culture supernatants by enzyme-linked immunosorbent assay (ELISA). Rats fed a high-fat diet (HFD) were treated with or without curcumin for 4 weeks, and the testosterone levels were detected by ELISA. RESULTS: Exposure to 100-400 µM PA reduced cell viability, activated caspase 3, and enhanced the expression levels of the apoptosis-related protein BCL-2-associated X protein (BAX) and ER stress markers glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in MLTC-1 cells. Treating cells with 500 nM 4-PBA significantly attenuated PA-induced cytotoxicity through inhibition of ER stress. Curcumin (20 µM) significantly suppressed PA- or TG-induced decrease in cell viability, caspase 3 activity, and the expression levels of BAX, CHOP, and GRP78. In addition, treating MLTC-1 cells with 20 µM curcumin effectively restored testosterone levels, which were reduced in response to PA exposure. Similarly, curcumin treatment ameliorated the HFD-induced decrease in serum testosterone level in vivo. CONCLUSIONS: The present study suggests that PA induces apoptosis via ER stress and curcumin ameliorates PA-induced apoptosis by inhibiting ER stress in MLTC-1 cells. This study suggests the application of curcumin as a potential therapeutic agent for the treatment of obesity-related male infertility.


Assuntos
Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Ácido Palmítico/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Fenilbutiratos/farmacologia , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Testículo/citologia
8.
Chem Biol Interact ; 312: 108792, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31491373

RESUMO

Cadmium (Cd) is an important toxic chemical due to its increasing levels in the environment and bioaccumulation in humans and animals. The present study was performed to evaluate the effects of long-term exposure to 1, 10, or 100 µg/L Cd in drinking water on the development, reproduction and neurotoxicity of offspring when administered to mice from parental puberty to postnatal 10 weeks in offspring. The development parameters measured in offspring included physical development, reflex ontogeny, body weight and body size. The reproductive indices measured consisted of anogenital distances (AGDs), estrous cycle, sperm quality, specific gene expression in Leydig or Sertoli cells, seminiferous epithelium cycle, sex hormone levels, histological morphology and apoptosis in testis or ovary, and the levels of oxidative stress. The determination of neurotoxicity included learning and memory ability, anxiety, and related serum indicators. In addition, blood lipid level, liver and kidney function were also determined by serum biochemical assays. The results showed that exposure to Cd in the present model had no adverse effects on development, but had some reproductive toxicity and neurotoxicity, including alteration of spermatogenic epithelial staging in testis and inducing anxiety in offspring. Furthermore, the levels of total protein, globulins, total bile acid and direct bilirubin were also significantly altered, especially in female offspring. The present study suggested that long-term exposure to low doses of Cd had adverse effects on the health of the next generation, and some harmful effects showed gender differences in offspring. The present study demonstrated that attention should be paid to Cd pollution in the environment, especially before pregnancy.


Assuntos
Cádmio/toxicidade , Reprodução/efeitos dos fármacos , Animais , Análise Química do Sangue , Feminino , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia
9.
Gen Comp Endocrinol ; 284: 113268, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31491376

RESUMO

CPFX is a highly effective antibiotic, but it has been reported to significantly impair both testicular function and structure in rats. In this study, we assessed reversal of CPFX-induced variation in mice testicular structure and testosterone synthesis by probiotic microbes in the infected model and normal model. We detected testicular weight, testicular structure and Leydig cell variables in numbers. We detected the levels of serum testosterone and steroidogenic enzymes, as well as DBC1, Sirt1, NF-κB, and related redox state and inflammatory response in the testes. The results showed that probiotic microbes had significantly elevated serum testosterone levels and steroidogenic enzymes, higher Sirt1, anti-oxidative enzymes and anti-inflammatory cytokine expression, and lower NF-κB, DBC1, oxidative damage, pro-inflammatory cytokine expression. The results suggest that the testis-protective, antiinflammatory and antioxidation effects of probiotics largely resulted from its ability to decrease oxidative stress and preserve antioxidant activity by stabilizing antioxidant defense systems, reducing oxidative damage and inflammatory response.


Assuntos
Ciprofloxacino/farmacologia , Probióticos/metabolismo , Testículo/metabolismo , Testículo/microbiologia , Testosterona/metabolismo , Animais , Antioxidantes/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citocinas/metabolismo , Epitélio/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Peso Molecular , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1/metabolismo , Testículo/efeitos dos fármacos
10.
Int J Mol Sci ; 20(16)2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430870

RESUMO

Zinc oxide nanoparticles (ZnO NPs) have shown adverse health impact on the human male reproductive system, with evidence of inducing apoptosis. However, whether or not ZnO NPs could promote autophagy, and the possible role of autophagy in the progress of apoptosis, remain unclear. In the current study, in vitro and in vivo toxicological responses of ZnO NPs were explored by using a mouse model and mouse Leydig cell line. It was found that intragastrical exposure of ZnO NPs to mice for 28 days at the concentrations of 100, 200, and 400 mg/kg/day disrupted the seminiferous epithelium of the testis and decreased the sperm density in the epididymis. Furthermore, serum testosterone levels were markedly reduced. The induction of apoptosis and autophagy in the testis tissues was disclosed by up-regulating the protein levels of cleaved Caspase-8, cleaved Caspase-3, Bax, LC3-II, Atg 5, and Beclin 1, accompanied by down-regulation of Bcl 2. In vitro tests showed that ZnO NPs could induce apoptosis and autophagy with the generation of oxidative stress. Specific inhibition of autophagy pathway significantly decreased the cell viability and up-regulated the apoptosis level in mouse Leydig TM3 cells. In summary, ZnO NPs can induce apoptosis and autophagy via oxidative stress, and autophagy might play a protective role in ZnO NPs-induced apoptosis of mouse Leydig cells.


Assuntos
Autofagia/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Nanopartículas/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Óxido de Zinco/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos
11.
J Therm Biol ; 84: 1-7, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31466741

RESUMO

Heat can trigger testicular damage and impair fertility. Leydig cells produce testosterone in response to stimulation by luteinizing hormone (LH), which induces Ca2+ entry and K+ efflux through ion channels in their plasma membrane. Considering that mechanisms coordinating the Leydig cell responses to hyperthermic stress remain unclear; the present study analyzed the effects of heat stress (HS, 43°C, 15 min) and inhibition of Hsp90 on T-type calcium currents and voltage-dependent potassium currents (VKC) in mice Leydig cells. Results show that HS reduced the VKC steady state currents at +80 mV (45.3%) and maximum conductance (71.5%), as well as increased the activation time constant (31.7%) and the voltage for which half the channels are open (30%). Hsp90 inhibition did not change the VKC currents. T-type calcium currents were not affected by HS or Hsp90 inhibition. In conclusion, HS can slow the activation, reduce the currents and voltage dependence of the VKC, suggesting a possible role of these currents in the response to hyperthermic stress in Leydig cells.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Proteínas de Choque Térmico HSP90/fisiologia , Resposta ao Choque Térmico/fisiologia , Células Intersticiais do Testículo/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Animais , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Temperatura Alta , Lactamas Macrocíclicas/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos
12.
Molecules ; 24(17)2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31450679

RESUMO

The purpose of the present study is to examine the effects of melatonin on apoptosis and oxidative stress in mouse Leydig cells and to elucidate the mechanisms responsible for these effects. Our results indicated that 10 ng/mL of melatonin significantly promoted cell viability, the ratio of EdU-positive (5-Ethynyl-2'-deoxyuridine) cells, and increased the mRNA expression of proliferating cell nuclear antigen (PCNA), cyclin D1(CCND1), and cell division control protein 42 (CDC42) (p < 0.05). We also observed that melatonin inhibited apoptosis of mouse Leydig cells, accompanied with increased B-cell lymphoma-2 (BCL-2) and decreased BCL2 associated X (BAX) mRNA and protein expression. Moreover, addition of melatonin significantly decreased the reactive oxygen species (ROS) production and malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, while it increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels (p < 0.05). In addition, we also found that melatonin increased the expression of SIRT1 (Silent information regulator 1) (p < 0.05). To explore the role of SIRT1 signaling in melatonin-induced cells, mouse Leydig cells were pretreated with EX527, an inhibitor of SIRT1. The protective effects of melatonin on mouse Leydig cells were reversed by EX527, as shown by decreased cell proliferation and increased cell apoptosis and oxidative stress. In summary, our results demonstrated that melatonin inhibited apoptosis and oxidative stress of mouse Leydig cells through a SIRT1-dependent mechanism.


Assuntos
Apoptose/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Melatonina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos
13.
Drug Des Devel Ther ; 13: 2057-2066, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31296984

RESUMO

Background: Ginsenoside Rg3 has been reported to exert protection function on germ cells. However, the mechanisms by which Rg3 regulates apoptosis in mouse Leydig cells remain unclear. In addition, triptolide (TP) has been reported to induce infertility in male rats. Thus, this study aimed to investigate the protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells. Methods: CCK-8, immunofluorescence assay, Western blotting and flow cytometry were used to detect cell proliferation and cell apoptosis, respectively. In addition, the dual luciferase reporter system assay was used to detect the interaction between miR-26a and GSK3ß in MLTC-1 cells. Results: TP significantly inhibited the proliferation of MLTC-1 cells, while the inhibitory effect of TP was reversed by Rg3. In addition, TP markedly induced apoptosis in MLTC-1 cells via increasing the expressions of Bax, active caspase 3, Cyto c and active caspase 9, and decreasing the level of Bcl-2. However, Rg3 alleviated TP-induced apoptosis of MLTC-1 cells. Moreover, the level of miR-26a was obviously downregulated by Rg3 treatment. The protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells was abolished by miR-26a upregulation. Meanwhile, dual-luciferase assay showed GSK3ß was the direct target of miR-26a in MLTC-1 cells. Overexpression of miR-26a markedly decreased the level of GSK3ß. As expected, upregulation of miR-26a could abrogate the protective effects of Rg3 against TP-induced cytotoxicity via inhibiting the expression of GSK3ß. Conclusion: These results indicated that Rg3 could protect MLTC-1 against TP by downregulation of miR-26a. Therefore, Rg3 might serve as a potential agent for the treatment of male hypogonadism.


Assuntos
Antiespermatogênicos/antagonistas & inibidores , Diterpenos/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Ginsenosídeos/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , MicroRNAs/biossíntese , Fenantrenos/antagonistas & inibidores , Substâncias Protetoras/farmacologia , Animais , Antiespermatogênicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Diterpenos/farmacologia , Relação Dose-Resposta a Droga , Compostos de Epóxi/antagonistas & inibidores , Compostos de Epóxi/farmacologia , Ginsenosídeos/química , Masculino , Camundongos , MicroRNAs/genética , Conformação Molecular , Fenantrenos/farmacologia , Substâncias Protetoras/química , Relação Estrutura-Atividade
14.
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
15.
Anim Reprod Sci ; 207: 21-35, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31266599

RESUMO

Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca2+) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds.


Assuntos
Compostos Benzidrílicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Fenóis/farmacologia , Receptores Estrogênicos/fisiologia , Receptores Acoplados a Proteínas-G/fisiologia , Testículo/efeitos dos fármacos , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Interação Gene-Ambiente , Células Intersticiais do Testículo/metabolismo , Masculino , MicroRNAs/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Receptores Estrogênicos/genética , Receptores Acoplados a Proteínas-G/genética , Maturidade Sexual/efeitos dos fármacos , Maturidade Sexual/genética , Suínos , Testículo/metabolismo
16.
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
17.
Chemosphere ; 235: 271-279, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31260867

RESUMO

Sertoli and Leydig cells provide key supporting roles in spermatogenesis. Various toxins have been studied in the TM3 and TM4 mouse testis cell lines to identify their regulatory effects. Alpha-solanine (α-solanine), a toxic compound found in the potato, has cytotoxic effects on various cells, including cancer cells. However, the effect of α-solanine on testis function has not been identified. In this study, we verified for the first time the anti-proliferative effect of α-solanine in mouse testes. α-Solanine reduced cell viability in TM3 and TM4 cells and reduced the expression of the cell cycle checkpoint genes Ccnd1 and Ccne1. We also detected changes in the mitochondrial membrane potential (MMP) and in the cytosolic calcium and intracellular signal pathways in both cell lines. α-Solanine induced AKT, P70S6K, S6, ERK1/2, and JNK activation in mouse testis cells. In addition, the inhibition of AKT with a pharmacological inhibitor (LY294002) demonstrated more synergic anti-proliferative effects than in the TM3 and TM4 cell lines treated only with α-solanine. Inha and Inhba mRNA expression also decreased in both cell lines and α-solanine i.p. injected mouse testes. Collectively, the results from this study verify the toxic effects of α-solanine on testes and male reproductive function.


Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inibinas/antagonistas & inibidores , Mitocôndrias/patologia , Transdução de Sinais/efeitos dos fármacos , Solanina/toxicidade , Testículo/metabolismo , Animais , Células Cultivadas , Técnicas In Vitro , Inibinas/genética , Inibinas/metabolismo , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espermatogênese , Testículo/efeitos dos fármacos , Testículo/patologia
18.
Physiol Res ; 68(4): 689-693, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31342755

RESUMO

The increasing worldwide production of bisphenols has been associated to several human diseases, such as chronic respiratory and kidney diseases, diabetes, breast cancer, prostate cancer, behavioral troubles and reproductive disorders in both sexes. The aim of the present in vitro study was to evaluate the potential impact bisphenols A, B, S and F on the cell viability and testosterone release in TM3 Leydig cell line. Mice Leydig cells were cultured in the presence of different concentrations of bisphenols (0.04-50 µg.ml-1) during 24 h exposure. Quantification of the cell viability was assessed using the metabolic activity assay, while the level of testosterone in cell culture media was determined by enzyme-linked immunosorbent assay. Within the panel of substances under investigations, the higher experimental concentrations (10; 25 and 50 µg.ml-1) significantly (P<0.001) decreased Leydig cells viability, while the same doses of BPA and BPB also reduced testosterone production significantly (P<0.001). Taken together, the results of our study reported herein is a consistent whit the conclusion that higher experimental doses of bisphenols have a cytotoxic effect and could have a dose-dependent impact on testosterone production.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fenóis/toxicidade , Testosterona/antagonistas & inibidores , Animais , Compostos Benzidrílicos/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Disruptores Endócrinos/administração & dosagem , Estrogênios não Esteroides/administração & dosagem , Estrogênios não Esteroides/toxicidade , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Fenóis/administração & dosagem , Testosterona/metabolismo
19.
Life Sci ; 233: 116694, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31351970

RESUMO

AIMS: The hypoxia-stimulated response of the endocrine system depends on the kind and duration of hypoxia. Hypoxia has been reported to stimulate testosterone (T) production in rats, but the mechanisms remain to be investigated. MATERIALS AND METHODS: Male rats were divided into two groups. The rats exposed to chronic intermittent hypoxia (CIH) at 8 h/day were housed in a hypoxic chamber (12% O2) for 14 days. Normoxic rats were used as control animals. T was measured after challenging the rat Leydig cells (LCs) with different stimulators, including hCG (0.01 IU/ml), forskolin (10-5 M), 8-bromo-cAMP (10-4 M), A23187 (10-5 M), cyclopiazonic acid (10-4 M), and androstenedione (10-8 M). Meanwhile, the LCs were incubated with trilostane (10-5 M) and/or 25-OH-hydroxycholesterol (10-5 M); thereafter the media were collected for pregnenolone assay. KEY FINDINGS: In the CIH group, plasma T levels were increased, but the serum luteinizing hormone (LH) was decreased. Furthermore, at several time intervals after hCG injection, plasma T levels were higher in the CIH group. The evoked-release of T and pregnenolone were significantly increased in the CIH group. Compared with the normoxic group, the CIH group had higher mRNA and protein expression levels of the LH receptor and CYP11A1 but not StAR. The plasma and testicular microvasculature VEGF levels were increased in the CIH group. The testicular vessel distribution was more obvious in CIH rats. SIGNIFICANCE: CIH-induced T secretion might be partially mediated by mechanisms involving the induction of LH receptor expression, testicular angiogenesis, CYP11A1 activity, 17ß-HSD activity, and calcium-related pathway.


Assuntos
Hipóxia Celular/fisiologia , Colforsina/farmacologia , Células Intersticiais do Testículo/metabolismo , Testosterona/metabolismo , Animais , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores do LH/genética , Receptores do LH/metabolismo , Vasodilatadores/farmacologia
20.
Molecules ; 24(11)2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163647

RESUMO

Lepidium meyenii is now widely consumed as a functional food and medicinal product, which is known as an enhancer of reproductive health. However, the specific chemical composition and mechanism of action for improving sexual function are unclear. The present study aims at screening and determining the potential compounds, which promote mouse leydig cells (TM3) proliferation. The partial least squares analysis (PLS) was employed to reveal the correlation between common peaks of high performance liquid chromatography (HPLC) fingerprint of L. meyenii and the proliferation activity of TM3. The results suggested that three compounds had good activities on the proliferation of TM3 and promoting testosterone secretion, there were N-benzyl-hexadecanamide, N-benzyl-(9z,12z)-octadecadienamide and N-benzyl-(9z,12z,15z)-octadecatrienamide which might be the potential bioactive markers related to the enhancing sexual ability functions of L. meyenii. The first step in testosterone synthesis is the transport of cholesterol into the mitochondria, and the homeostasis of mitochondrial function is related to cyclophilin D (CypD). In order to expound how bioactive ingredients lead to promoting testosterone secretion, a molecular docking simulation was used for further illustration in the active sites and binding degree of the ligands on CypD. The results indicated there was a positive correlation between the binding energy absolute value and testosterone secretion activity. In addition, in this study it also provided the reference for a simple, quick method to screen the promoting leydig cell proliferation active components in traditional Chinese medicine (TCM).


Assuntos
Lepidium/química , Células Intersticiais do Testículo/citologia , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Análise dos Mínimos Quadrados , Células Intersticiais do Testículo/efeitos dos fármacos , Ligantes , Masculino , Camundongos , Simulação de Acoplamento Molecular , Análise Multivariada , Compostos Fitoquímicos/química , Testosterona/metabolismo
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