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1.
Ecotoxicol Environ Saf ; 209: 111798, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33360214

RESUMO

Di-(2-ethylhexyl) phthalate (DEHP), one of the most commonly used endocrine-disrupting chemicals, has been shown to cause reproductive dysfunction in humans and animal models. However, very few studies have investigated the impact of DEHP at the post-transcriptional level in mouse testes, and the underlying mechanisms remain unclear. In the present research, TM3 Leydig cells were treated with 200 µM phthalic acid mono-2-ethylhexyl ester (MEHP, bio-metabolite of DEHP), and then the mRNA and lncRNA sequencing of TM3 Leydig cells was performed. Mice were exposed prepubertally to 0 or 500 mg DEHP/kg/day. RNA sequencing of mouse testes was performed to verify the RNA-seq results in vitro. The expression patterns of relevant genes and proteins were verified using real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. DEHP and MEHP exposure led to testicular damage and accelerated cell aging via ROS accumulation. RNA sequencing analyses indicated that FOXO signaling and longevity regulation pathways were activated in resistance to ROS accumulation. FOXO signaling and longevity regulation pathway-related genes and proteins were also activated. By constructing a competing endogenous RNA (ceRNA) network, we observed that the ceRNA network might play a role in regulating FOXO signaling and longevity regulation pathways in response to excessive ROS accumulation and cell aging. In summary, our data here suggests that the ceRNA network may play a role in regulating FOXO signaling and longevity pathways in response to DEHP exposure in mouse testes.


Assuntos
Dietilexilftalato/toxicidade , Disruptores Endócrinos/toxicidade , RNA Longo não Codificante/metabolismo , Envelhecimento , Animais , Dietilexilftalato/metabolismo , Disruptores Endócrinos/metabolismo , Regulação da Expressão Gênica , Humanos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Longevidade , Masculino , Camundongos , Ácidos Ftálicos , Testículo/efeitos dos fármacos , Transcriptoma
2.
Ecotoxicol Environ Saf ; 209: 111671, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33360290

RESUMO

Lead (Pb) is a toxic heavy metal pollutants and can damage male reproductive function. Selenium (Se) possesses an ability of antagonizing Pb toxicity. However, biological events in the process of Pb toxicity and mitigative effect of Se are not well understood. The aim of present research was to investigate potential mechanism of Se against Pb toxicity from the perspective of oxidative stress, heat shock response and autophagy in the spermatogonia and Leydig cell of chicken. The cells from one-day-old male Hyline chickens were treated with Se (0.5 µmol/L) and/or Pb (20 µmol/L) for 24 h, respectively. Cell viability, cell ultrastucture, Pb and Se concentrations, testosterone level, oxidative stress indicators and relative expression of heat shock proteins (HSPs) and autophagy-related genes were measured. The results showed that spermatogonia was more tolerant to Pb than Leydig cell; cell injury was confirmed via histological assessment, cell viability and testosterone level; oxidative stress was further indicated by the decrease of catalase, glutathione peroxidase, glutathione-s-transferase and superoxide dismutase activities and the increase of malondialdehyde and reactive oxygen species contents. Pb increased expression of HSPs (27, 40, 60, 70 and 90). Meanwhile Pb induced autophagy through up-regulation of autophagy-related proteins 5, Beclin 1, Dynein, light chain 3 (LC3)-I and LC3-II and down-regulation of mammalian target of rapamycin in two type cells of chicken. However, Se intervention mitigated the aforementioned alterations caused by Pb. In conclusion, Pb led to oxidative stress, which triggered heat shock response and autophagy; Se administration mitigated reproductive toxicity of Pb through strengthening antioxidant defense in the spermatogonia and Leydig cell of chicken.


Assuntos
Antioxidantes/farmacologia , Chumbo/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Selênio/farmacologia , Espermatogônias/fisiologia , Animais , Antioxidantes/metabolismo , Autofagia/efeitos dos fármacos , Catalase/metabolismo , Galinhas/metabolismo , Poluentes Ambientais/metabolismo , Glutationa Peroxidase/metabolismo , Proteínas de Choque Térmico/metabolismo , Chumbo/metabolismo , Células Intersticiais do Testículo/metabolismo , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espermatogônias/metabolismo
3.
Chemosphere ; 262: 127792, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32805656

RESUMO

Tebuconazole is a triazole fungicide, used in agriculture to treat phytopathogenic fungi, and as a biocide, has been reported to be related to reproductive and developmental toxicity. The purpose of this study was to investigate the effect of tebuconazole exposure on rat fetal Leydig cells and fetal testis during pregnancy. Pregnant Sprague-Dawley rats were randomly divided into 4 groups, daily gavaged with corn oil (as a control), 25, 50, and 100 mg/kg body weight tebuconazole for 10 days (from the 12th day of pregnancy). Tebuconazole increased fetal serum testosterone and progesterone levels at a dose of 100 mg/kg. Exposure to 100 mg/kg tebuconazole significantly caused an increase in the number of fetal Leydig cells per testis without inducing cell aggregation. Tebuconazole up-regulated the expression of Star, Cyp11a1, Hsd17b3, and Fshr and their proteins. Further investigation found that tebuconazole caused increased phosphorylation of AKT1, ERK1/2, and mTOR, the level of BCL2, as well as the decrease of Beclin1, LC3B, and BAX, which may contribute to the fetal Leydig cell autophagy and proliferation. In conclusion, in utero exposure of tebuconazole causes the proliferation of fetal Leydig cells.


Assuntos
Fungicidas Industriais/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Reprodução/efeitos dos fármacos , Triazóis/toxicidade , Animais , Feminino , Células Intersticiais do Testículo/metabolismo , Masculino , Fosfoproteínas/genética , Fosforilação , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Testículo/efeitos dos fármacos , Testículo/embriologia , Testículo/patologia , Testosterona/sangue , Regulação para Cima
4.
Zhonghua Nan Ke Xue ; 26(3): 258-264, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-33346967

RESUMO

Objective: To investigate the effects of Xiongcan Yishen Prescription (XYP) on the expressions of cholesterol transport proteins, steroidogenic enzymes and steroidogenic factor-1 (SF-1) in the Leydig cells of the rats with late-onset hypogonadism (LOH). METHODS: Twenty-five 18-month-old male SD rats were randomly divided into five groups of equal number, LOH model control, testosterone propionate (TP) and low-, medium- and high-dose XYP, and another 5 two-month-old male SD rats included as normal controls. After modeling, the animals in the TP group were treated by intramuscular injection of TP at 5.21 mg/kg qd alt, those in the low-, medium- and high-dose XYP groups intragastrically with XYP at 10.4, 20.8 and 41.6 g/kg qd alt respectively, and those in the LOH model and normal control groups with saline, all for 28 successive days. Then, all the rats were sacrificed for determination of the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the Leydig cells by Western blot. RESULTS: The expressions of StAR, TSPO, CYP11A1, HSD3B7, HSD17B4 and SF-1 in the Leydig cells were significantly decreased in the LOH model controls compared with those in the normal controls (P< 0.05), but remarkably increased in the low-, medium- and high-dose XYP groups in comparison with those in the LOH model control group (P< 0.05). CONCLUSIONS: Xiongcan Yishen Prescription can up-regulate the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the rat Leydig cells, which might be one of the possible mechanisms of the prescription in the treatment of LOH.


Assuntos
Colesterol/metabolismo , Medicamentos de Ervas Chinesas/uso terapêutico , Hidroxiesteroide Desidrogenases/metabolismo , Hipogonadismo , Células Intersticiais do Testículo/efeitos dos fármacos , Animais , Transporte Biológico , Proteínas de Transporte , Hipogonadismo/tratamento farmacológico , Células Intersticiais do Testículo/metabolismo , Masculino , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Esteroides/metabolismo , Testosterona
5.
PLoS One ; 15(12): e0244553, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33378407

RESUMO

Leydig cells represent the steroidogenic lineage of mammalian testis, which produces testosterone. Genetic evidence indicates the requirement of Notch signaling in maintaining a balance between differentiated Leydig cells and their progenitors during fetal development. In primary Leydig cells, Notch1 expression decreases with testicular development, while the expression of its ligand, Jagged1, remains relatively unchanged, suggesting that the roles of Jagged1 extend beyond Notch signaling. In addition, Jagged1 is known to be processed into its intracellular domain, which then translocate to the nucleus. In this study, we investigated the effect of Jagged1 intracellular domain (JICD) on steroidogenesis in Leydig cells. The independent overexpression of JICD in MA-10 Leydig cells was found to inhibit the activity of cAMP-induced Nur77 promoter. In addition, JICD suppressed Nur77 transactivation of the promoter of steroidogenic genes such as P450scc, P450c17, StAR, and 3ß-HSD. Further, adenovirus-mediated overexpression of JICD in primary Leydig cells repressed the expression of steroidogenic genes, consequently lowering testosterone production. These results collectively suggest that steroidogenesis in testicular Leydig cells, which is regulated by LH/cAMP signaling, is fine-tuned by Jagged1 during testis development.


Assuntos
Proteína Jagged-1/química , Proteína Jagged-1/genética , Células Intersticiais do Testículo/citologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Regiões Promotoras Genéticas , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Redes Reguladoras de Genes , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Domínios Proteicos , Transporte Proteico , Receptor Notch1/metabolismo , Transdução de Sinais , Esteroides/metabolismo
6.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374605

RESUMO

The immune privilege of the testes is necessary to prevent immune attacks to gamete-specific antigens and paternal major histocompatibility complex (MHC) antigens, allowing for normal spermatogenesis. However, infection and inflammation of the male genital tract can break the immune tolerance and represent a significant cause of male infertility. Different T cell subsets have been identified in mammalian testes, which may be involved in the maintenance of immune tolerance and pathogenic immune responses in testicular infection and inflammation. We reviewed the evidence in the published literature on different T subtypes (regulatory T cells, helper T cells, cytotoxic T cells, γδ T cells, and natural killer T cells) in human and animal testes that support their regulatory roles in infertility and the orchitis pathology. While many in vitro studies have indicated the regulation potential of functional T cell subsets and their possible interaction with Sertoli cells, Leydig cells, and spermatogenesis, both under physiological and pathological processes, there have been no in situ studies to date. Nevertheless, the normal distribution and function of T cell subsets are essential for the immune privilege of the testes and intact spermatogenesis, and T cell-mediated immune response drives testicular inflammation. The distinct function of different T cell subsets in testicular homeostasis and the orchitis pathology suggests a considerable potential of targeting specific T cell subsets for therapies targeting chronic orchitis and immune infertility.


Assuntos
Imunidade , Linfócitos T/imunologia , Linfócitos T/metabolismo , Testículo/imunologia , Testículo/metabolismo , Animais , Autoimunidade , Biomarcadores , Gerenciamento Clínico , Suscetibilidade a Doenças , Homeostase , Humanos , Imunomodulação , Células Intersticiais do Testículo/imunologia , Células Intersticiais do Testículo/metabolismo , Masculino , Células de Sertoli/imunologia , Células de Sertoli/metabolismo , Espermatogênese , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
7.
Ecotoxicol Environ Saf ; 203: 111053, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888615

RESUMO

Vinclozolin is a common dicarboximide fungicide used to protect crops from diseases. It is also an endocrine disruptor and is thought to be related to abnormalities of the reproductive tract. However, its mechanism of inducing abnormalities of the male reproductive tract is still unclear. The purpose of this study was to study the effect of gestational vinclozolin exposure on the development of rat fetal Leydig cells. Female pregnant Sprague-Dawley rats were exposed to vinclozolin (0, 25, 50, and 100 mg/kg body weight/day) by gavage from gestational day 14-21. Vinclozolin dose-dependently reduced serum testosterone levels at doses of 50 and 100 mg/kg and the anogenital distance at 100 mg/kg. RNA-seq, qPCR, and Western blotting showed that vinclozolin down-regulated the expression of Nr5a1, Sox9, Lhcgr, Cyp11a1, Hsd3b1, Hsd17b3, Amh, Pdgfa, and Dhh and their encoded proteins. Vinclozolin reduced the number of NR2F2-positive stem Leydig cells at a dose of 100 mg/kg and enhanced autophagy in the testes. In conclusion, vinclozolin disrupts reproductive tract development and testis development in male fetal rats via several pathways.


Assuntos
Disruptores Endócrinos/toxicidade , Fungicidas Industriais/toxicidade , Organogênese/efeitos dos fármacos , Oxazóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Testículo/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Testículo/embriologia , Testículo/patologia , Testosterona/sangue
8.
Toxicol Lett ; 332: 213-221, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32693021

RESUMO

Di-n-hexyl phthalate (DNHP) is commonly used as a plasticizer. However, whether DNHP influences Leydig cell development during puberty remains unexplored. In this study, DNHP (0, 10, 100, and 1000 mg/kg) was administered via gavage to 35-day-old male Sprague-Dawley rats for 21 days. Serum levels of testosterone, luteinizing hormone, follicle-stimulating hormone, Leydig cell number, the expression of Leydig and Sertoli cell genes and proteins were investigated. DNHP significantly increased serum testosterone levels at 10 mg/kg but lowered its level at 1000 mg/kg. DNHP significantly increased luteinizing hormone levels at 1000 mg/kg without affecting follicle-stimulating hormone levels. DNHP increased Leydig cell number at all doses but down-regulated the expression of Lhcgr, Hsd3b1, Hsd17b3, and Hsd11b1 in Leydig cell per se at 1000 mg/kg. DNHP elevated phosphorylation of ERK1/2 and GSK-3ß at 10 mg/kg but decreased SIRT1 and PGC-1α levels at 1000 mg/kg. In conclusion, DNHP exposure causes Leydig cell hyperplasia possibly via stimulating phosphorylation of ERK1/2 and GSK-3ß signaling pathways.


Assuntos
Células Intersticiais do Testículo/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Plastificantes/toxicidade , Animais , Tamanho Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/efeitos dos fármacos , Hiperplasia , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/ultraestrutura , Hormônio Luteinizante/sangue , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Maturidade Sexual , Transdução de Sinais/efeitos dos fármacos , Testosterona/sangue
9.
PLoS Genet ; 16(6): e1008810, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32497091

RESUMO

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.


Assuntos
Criptorquidismo/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Intersticiais do Testículo/patologia , Proteínas do Tecido Nervoso/metabolismo , Diferenciação Sexual/genética , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Criptorquidismo/patologia , Modelos Animais de Doenças , Proteínas Hedgehog/metabolismo , Humanos , Insulina/metabolismo , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas/metabolismo , Transdução de Sinais/genética , Testosterona/metabolismo , Proteína Gli3 com Dedos de Zinco/genética
10.
Life Sci ; 254: 117767, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32407848

RESUMO

OBJECTIVE: Heat stress shock affects the generation of free radicals and can have a harmful effect on spermatogenesis. Photobiomodulation (PBM) is very effective in andrology for treating male infertility. This research aimed at the evaluation of the impacts of PBM on spermatogenesis on the transient scrotal hyperthermia-induced oligospermia mouse model. MATERIALS AND METHODS: This experimental research divided 24 mice into the following four groups: (1) Control, (2) Scrotal hyperthermia, (3) Scrotal hyperthermia receiving laser 0.03 J/cm2 for 30 s for each testis, 35 days after induction of scrotal hyperthermia every other day for 35 days, and (4) Scrotal hyperthermia receiving laser 0.03 J/cm2 for 30 s for each testis, immediately after induction of scrotal hyperthermia every other day for 35 days. Scrotal hyperthermia was induced by water bath with 43 °C for 30 min. Then, the mice were euthanized, and their sperm samples were collected for sperm parameters analysis. Then, we took the testis samples for histopathological experimentations, serum testosterone level, reactive oxygen species (ROS), RNA extraction for the examination of IL1-α, IL6 and TNF-α genes expression as well as production and glutathione disulfide (GSH) activity. KEY FINDINGS: Our outputs indicated that PBM could largely improve the sperms parameters and stereological parameters, like spermatogonia, primary spermatocyte, round spermatid and Leydig cells together with an increasing level of the serum testosterone and GSH activity compared to the scrotal hyperthermia induced mice. In addition, it was found that the diameter of seminiferous tubules, ROS production, as well as the expression of IL1-α, IL6, and TNF-α genes significantly decreased in the treatment groups by PBM compared to the scrotal hyperthermia induced mice, but there was not a significant difference in terms of testis weight and Sertoli cells between the studied groups. SIGNIFICANCE: It could be concluded that PBM may be regarded as an alternative treatment for improving the spermatogenesis process in the scrotal hyperthermia induced mice.


Assuntos
Terapia com Luz de Baixa Intensidade/métodos , Escroto/metabolismo , Espermatogênese/efeitos dos fármacos , Animais , Febre/metabolismo , Resposta ao Choque Térmico/fisiologia , Temperatura Alta , Hipertermia Induzida/métodos , Infertilidade Masculina/patologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Escroto/patologia , Células de Sertoli/metabolismo , Espermatozoides/patologia , Testículo/efeitos dos fármacos
11.
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
12.
Chemosphere ; 251: 126336, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32145574

RESUMO

1-Nitropyrene (1-NP) is a representative nitro-polycyclic aromatic hydrocarbon from diesel exhaust. Recently, we found that maternal 1-NP exposure caused fetal growth retardation and disturbed cognitive development in adolescent female offspring. To investigate long-term 1-NP exposure on spermatogenesis and steroidogenesis, male mice were exposed to 1-NP (1.0 mg/kg/day) by gavage for 70 days. There was no significant difference on relative testicular weight, number of testicular apoptotic cells and epididymal sperm count between 1-NP-exposed mice and controls. Although long-term 1-NP exposure did not influence number of Leydig cells, steroidogenic genes and enzymes, including STAR, P450scc, P45017α and 17ß-HD, were downregulated in 1-NP-expoed mouse testes. Correspondingly, serum and testicular testosterone (T) levels were reduced in 1-NP-exposed mice. Additional experiment showed that testicular GRP78 mRNA and protein were upregulated by 1-NP. Testicular phospho-IRE1α and sliced xbp-1 mRNA, a downstream molecule of IRE1α, were elevated in 1-NP-exposed mice. Testicular phospho-PERK and phospho-eIF2α, a downstream molecule of PERK pathway, were increased in 1-NP-exposed mice. Testicular NOX4, a subunit of NAPDH oxidase, and HO-1, MDA, two oxidative stress markers, were increased in 1-NP-exposed mice. Testicular GSH and GSH/GSSG were decreased in 1-NP-exposed mice. These results suggest that long-term 1-NP exposure induces reactive oxygen species-evoked ER stress and disrupts steroidogenesis in mouse testes.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Poluentes Ambientais/toxicidade , Pirenos/toxicidade , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Endorribonucleases , Epididimo , Feminino , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Tamanho do Órgão , Proteínas Serina-Treonina Quinases , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Testosterona/sangue
13.
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
14.
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
15.
Gene ; 738: 144488, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32087275

RESUMO

Kisspeptin, encoded by the Kiss1 gene, and its receptor GPR54 have a central regulatory role in the male reproduction. However, their functions in peripheral tissues, such as testes, remain unclear. This study investigated the local expressions and function of Kiss1/GPR54 in goats' testes. The mRNA expression of Kiss1/GPR54 in pubertal goat Leydig cells was detected through reverse transcriptase PCR (RT-PCR), and its protein expression in Leydig cells or the testis was examined by immunohistochemistry and Western blot analysis. Isolated and cultured Leydig cells were treated with different concentration of kisspeptin (0, 1, 10 and 100 µM) and kisspeptin antagonist for 4, 24 or 72 h. The direct effect of kisspeptin on testosterone secretion and Kiss1/GPR54 mRNA expression was evaluated by ELISA and RT-PCR. Kiss1/GPR54 mRNA and protein were expressed in Leydig cells and spermatids, and GPR54 were expressed in Sertoli cells. Kisspeptin treatment significantly stimulated testosterone secretion in Leydig cells, with the highest levels found under 24 h of treatment with 10 µM kisspeptin. Treatment with kisspeptin + kisspeptin antagonist significantly reduced the kisspeptin-stimulated testosterone secretion in Leydig cells. Kisspeptin treatment significantly enhanced the expression of Kiss1/GPR54 mRNA in Leydig cells. These data suggest the local expressions of Kiss1/GPR54 in goats' testes and its autocrine role in Leydig cells, which is helpful in understanding the regulation role of kisspeptin/GPR54 system in other peripheral tissues.


Assuntos
Cabras/genética , Kisspeptinas/genética , Testículo/metabolismo , Animais , China , Células Germinativas/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Células Intersticiais do Testículo/metabolismo , Masculino , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas-G/genética , Células de Sertoli/metabolismo
16.
Ecotoxicol Environ Saf ; 192: 110266, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32058163

RESUMO

Despite the well-known acknowledgement of both the toxicity of cadmium (Cd) and the ameliorative effect of selenium (Se), the mechanism of the protective effect of selenium on cadmium-induced Mouse Leydig (TM3) cell apoptosis remains unknown. In this study, we hypothesized that the reactive oxygen species (ROS)-mediated c-jun N-terminal kinase (JNK) signaling pathway is involved in anti-apoptosis of selenium against cadmium in TM3 cells. We found that exposure to cadmium caused evident cytotoxicity, in which cell viability was inhibited, followed by inducement of apoptosis. Moreover, the level of ROS generation was elevated, leading to the phosphorylation of JNK. In addition, following cadmium exposure, the nuclear transcription factor c-jun was significantly activated, which led to increased expression of downstream gene c-jun, resulting in downstream activation of the apoptosis-related protein Caspase3 and upregulation of Cleaved-PARP, as well as inhibition of the anti-apoptosis protein Bcl-2. However, pretreatment with selenium remarkably suppressed cadmium-induced TM3 cell apoptosis. Furthermore, the level of ROS declined, and the JNK signaling pathway was blocked. Following this, the gene expression of c-jun decreased while Bcl-2 increased, which was consistent with the effects on proteins, that Caspase3 activity and Cleaved-PARP were inhibited while Bcl-2 level was restored. In order to explain the relationship between molecules of the signaling pathway, N-acetyl-L-cysteine (NAC), the ROS inhibitor, and JNK1/2 siRNA were administered, which further indicated the mediatory role of the ROS/JNK/c-jun signaling pathway in regulating anti-apoptosis of selenium against cadmium-induced TM3 cell apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Cádmio/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Intersticiais do Testículo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Selênio/farmacologia , Acetilcisteína/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Fosforilação , Transdução de Sinais/efeitos dos fármacos
17.
Hum Cell ; 33(2): 318-329, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32034722

RESUMO

95% of the body's testosterone is produced by the Leydig Cells (LCs) in adult testis, and LC functional degradation can cause testosterone deficiency ultimately leading towards hypogonadism. The transplantation of LCs derived from stem cells is a very promising therapy to overcome the testosterone deficiency. The isolated umbilical cord mesenchymal stem cells (UMSCs) were identified by flow cytometry and adipogenic and osteogenic differentiation. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were used for the differentiated Leydig-like cell identification. The comparisons of the testosterone levels, gene expression levels, and cyclic adenosine monophosphate (cAMP) productions were performed through radioimmunoassay, quantitative polymerase chain reaction (qPCR), and cAMP assay kit, respectively. Here, it is stated that our isolated human UMSCs, which could positively express CD29, CD44, CD59, CD90, CD105, and CD166 but negatively express CD34 as well as could be differentiated into adipocytes and osteocytes, could be differentiated into Leydig-like cells (UMSC-LCs) using a novel differentiation method based on molecular compounds. The enrichment UMSC-LCs could secrete testosterone into the medium supernatant and produce considerable cAMP at the stimulation of luteinizing hormone (LH), and positively expressed LC lineage-typical markers LHCGR, SCARB1, SATR, CYP11A1, CYP17A1, HSD3B1, HSD17B3, and SF-1 as well as negatively expressed mesenchymal stem cell typical markers CD29, CD44, and CD105. The expression levels of NR3C4, PDGFRA, and NR3A1 in UMSC-LCs were higher than those of UMSCs and were comparable with LCs. These results illuminated that UMSCs could be differentiated into Leydig-like cells using the defined molecular compounds, which might further support MSC-derived Leydig cell transplantation therapy for testosterone insufficiency.


Assuntos
Diferenciação Celular , Células Intersticiais do Testículo , Células-Tronco Mesenquimais/fisiologia , Testosterona , Cordão Umbilical/citologia , Feminino , Humanos , Hipogonadismo/etiologia , Células Intersticiais do Testículo/metabolismo , Masculino , Transplante de Células-Tronco Mesenquimais , Testosterona/deficiência , Testosterona/metabolismo
18.
Sci Rep ; 10(1): 1576, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005928

RESUMO

The pharmaceutical 17α-ethynylestradiol (EE2) is considered as an endocrine-disrupting chemical that interferes with male reproduction and hormonal activation. In this study, we investigated the molecular mechanism underlying EE2-regulatory testosterone release in vitro and in vivo. The results show that EE2 treatment decreased testosterone release from rat Leydig cells. Treatment of rats with EE2 reduced plasma testosterone levels and decreased the sensitivity of human chorionic gonadotropin (hCG). EE2 reduced luteinizing hormone receptor (LHR) expression associated with decreased cAMP generation by downregulation of adenylyl cyclase activity and decreased intracellular calcium-mediated pathways. The expression levels of StAR and P450scc were decreased in Leydig cells by treatment of rats with EE2 for 7 days. The sperm motility in the vas deferens and epididymis was reduced, but the histopathological features of the testis and the total sperm number of the vas deferens were not affected. Moreover, the serum dihydrotestosterone (DHT) level was decreased by treatment with EE2. The prostate gland and seminal vesicle atrophied significantly, and their expression level of 5α-reductase type II was reduced after EE2 exposure. Taken together, these results demonstrate an underlying mechanism of EE2 to downregulate testosterone production in Leydig cells, explaining the damaging effects of EE2 on male reproduction.


Assuntos
Etinilestradiol/farmacologia , Receptores do LH/metabolismo , Testosterona/metabolismo , Adenilil Ciclases/metabolismo , Animais , Gonadotropina Coriônica/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores do LH/efeitos dos fármacos , Testosterona/sangue
19.
Life Sci ; 245: 117365, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32001267

RESUMO

AIMS: Hyperglycemia in combination with oxidative stress plays a significant pathophysiological role in diabetic testicular dysfunction, often leading to infertility. Activation of Toll-like receptor 4 (TLR4) has been reported to mediate oxidative stress during diabetes. However, engagement of the TLR4 signaling pathway in diabetic testicular dysfunction has not been previously explored. Herein, we investigated the role of TLR4 in reactive oxygen species (ROS) production and in the phosphorylation status of ERK1/2 in primary Leydig cells exposed to high glucose and in testis isolated from diabetic rats. MAIN METHODS: Testicular levels of TLR4 and phospho-ERK1/2 were determined by Western blotting. ROS production was detected with a fluorescent probe. Additionally, primary Leydig cells were exposed to normal (5.5 mmol/l) or elevated (33 mmol/l) glucose concentrations and treated with or without a TLR4 inhibitor, CLI095 (10-5 mol/l) for 24 h, followed by evaluation of TLR4 and phospho-ERK1/2 expression levels by Western blotting and immunofluorescence staining, respectively. KEY FINDINGS: We show that high glucose induces the expression of TLR4 in Leydig cells. Additionally, we demonstrate that blockade of this receptor in this cell population reduces oxidative stress and restores the levels of phospho-ERK1/2. SIGNIFICANCE: Our findings provide new insight into TLR4 interaction with ROS and MEK/ERK pathway in Leydig cells exposed to high glucose and present a rationale for the development of new therapeutics for diabetic testicular dysfunction.


Assuntos
Hiperglicemia/metabolismo , Células Intersticiais do Testículo/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estresse Oxidativo , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Western Blotting , Diabetes Mellitus Experimental/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptor 4 Toll-Like/metabolismo
20.
J Steroid Biochem Mol Biol ; 199: 105589, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31953167

RESUMO

Production of testosterone is under tight control by human chorion gonadotropin (hCG) during fetal life and luteinizing hormone (LH) in adulthood. Several animal and human studies have linked vitamin D status with sex steroid production although it is not clear whether there exist a direct or indirect involvement in androgen production. Few studies have investigated this crosslink in young healthy men and putative direct or synergistic effect of activated vitamin D (1,25(OH)2D3) and LH/hCG on sex steroid production in vitro. Here, we present cross-sectional data from 300 young men and 41 hCG-stimulated men with impaired Leydig cell function combined with data from an ex vivo culture of human testicular tissue exposed to 1,25(OH)2D3 alone or in combination with hCG. Serum 25-OHD was positively associated with SHBG (ß:0.002; p = 0.023) and testosterone/estradiol-ratio (ß:0.001; p = 0.039), and inversely associated with free testosterone (%) (free testosterone/total testosterone) (ß:-0.002; p = 0.016) in young men. Vitamin D deficient men had higher total and free estradiol concentrations than men with higher vitamin D status (19% and 18%, respectively; p < 0.01). Interestingly, men with impaired Leydig cell function and vitamin D deficiency had a significantly lower hCG-mediated increase in total and free testosterone compared with vitamin D sufficient men (p < 0.05). Accordingly, testicular tissue exposed to 100 nM 1,25(OH)2D3 had a 15% higher testosterone release into the media compared with vehicle treated specimens (p = 0.030). In conclusion, vitamin D deficiency is associated with lower testosterone/estradiol ratio in young men and lower Leydig cell sensitivity after hCG-stimulation in men with impaired gonadal function. The significant effect of 1,25(OH)2D3 on testosterone production in a human testis model supports that the stimulatory effect at least in part may be direct. Larger placebo-controlled studies are needed to determine whether vitamin D supplementation can influence testosterone production.


Assuntos
Hormônios Esteroides Gonadais/genética , Células Intersticiais do Testículo/metabolismo , Testosterona/biossíntese , Vitamina D/metabolismo , Adulto , Androgênios/biossíntese , Androgênios/genética , Animais , Gonadotropina Coriônica/genética , Estradiol/genética , Hormônios Esteroides Gonadais/biossíntese , Hormônios Esteroides Gonadais/metabolismo , Humanos , Células Intersticiais do Testículo/patologia , Hormônio Luteinizante/genética , Hormônio Luteinizante/metabolismo , Masculino , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Testosterona/genética , Vitamina D/genética , Deficiência de Vitamina D/genética , Deficiência de Vitamina D/metabolismo , Deficiência de Vitamina D/patologia , Adulto Jovem
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