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1.
Ecotoxicol Environ Saf ; 190: 110142, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911389

RESUMO

Cadmium (Cd) has been reported to induce reproductive toxicity. Recent study indicated that aberrant epigenetic regulation of Multidrug resistance 1b (Mdr1b) causes xenobiotic efflux failure at the blood-testis barrier (BTB). However, whether Mdr1b dysregulation is involved in Cd-mediated dyszoospermia and the underlying mechanism remain unknown. In this study, mice were intragastrically administered 0 or 2.5 mg/kg CdCl2 every other day for 2 months to investigate changes in spermatogenesis and epigenetic regulation of Mdr1b. Mouse Leydig cells TM3 were cultured to detect Mdr1b expression localization. We found that the Cd group revealed BTB disruption concomitant with obvious sperm abnormity and dynamic impairment. Hypermethylation and decreased nuclear factor Ya (Nfya) recruitment to the Mdr1b promoter were correlated with low sperm motility in response to Cd. In conclusion, these findings provide in vivo evidence that epigenetic dysregulation of Mdr1b in the BTB is a potential cause of dyszoospermia upon Cd exposure.


Assuntos
Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Espermatogênese/efeitos dos fármacos , Animais , Barreira Hematotesticular/metabolismo , Fator de Ligação a CCAAT , Núcleo Celular/metabolismo , Resistência a Múltiplos Medicamentos , Epigênese Genética , Infertilidade Masculina , Masculino , Camundongos , Regiões Promotoras Genéticas , Motilidade Espermática , Espermatozoides/metabolismo , Testículo/metabolismo
2.
Chem Biol Interact ; 315: 108869, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31682803

RESUMO

Spermatogenic dysfunction is one of the major secondary complications of male diabetes. Salidroside (SAL) is the important active ingredients isolated from Herba Cistanche, which exhibits numerous pharmacological activities such as antioxidant, anti-diabetic, and anti-inflammatory effects. The present study was designed to determine whether SAL contributes to the recovery from spermatogenic dysfunction in streptozotocin (STZ) induced type-1 diabetic mice. SAL (25, 50, or 100 mg/kg) and Clomiphene citrate (CC, 5 mg/kg) were orally administered to male type-1 diabetic mice for 10 weeks. Testis tissues were collected for histopathological and biochemical analysis. Moreover, reproductive organ weight, sperm parameters, and testicular cell DNA damage were estimated. The results revealed that SAL significantly improved the weight of the reproductive organs, sperm parameters and testicular morphology to different degrees in type-1 diabetic mice. Furthermore, reactive oxygen species (ROS) and malondialdehyde (MDA) levels were significantly reduced, and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), markedly increased in the testicular tissue after SAL treatment. In addition, our data also showed a marked downregulation the fluorescence expressions of p38 MAPK phosphorylation and upregulation the protein expressions of ZO-1, Occludin, Claudin-11 and N-cadherin after SAL administration (100 mg/kg) compared with the type-1 diabetic group. In conclusion, these results demonstrated that SAL exerts protective effects on type-1 diabetes-induced male spermatogenic dysfunction, which is likely mediated by inhibiting oxidative stress-mediated blood testis barrier damage.


Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Glucosídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fenóis/farmacologia , Substâncias Protetoras/farmacologia , Espermatogênese/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Barreira Hematotesticular/metabolismo , Catalase/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/metabolismo , Glutationa/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Contagem de Espermatozoides/métodos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Estreptozocina/farmacologia , Superóxido Dismutase/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo
3.
Ecotoxicol Environ Saf ; 189: 110053, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31862514

RESUMO

Particulate matter with an aerodynamic diameter of less than 2.5 µm (PM2.5) derived from automobile exhaust can lead to serious male spermatogenesis dysfunction, but its specific molecular mechanism is unclear. In this experiment, we focused on the blood-testis barriers (BTB) and explored the intracellular mechanisms underlying the fertility toxicity of PM2.5 originating from automobile exhaust in the primary cultured Sertoli cells(SCs) of rats. After PM2.5 exposure, excessive reactive oxygen species (ROS) and increased apoptosis of SCs were detected. The expression of the BTB related proteins including ZO-1, Occludin, N-cadherin and ß-catenin were significantly decreased and the spatial arrangement of F-actin was completely disordered through Immunofluorescence and Western blots tests. The phosphorylation of Jun N-terminal kinase (JNK), extracellular signal regulatory kinase (ERK), p38 mitogen-activated protein kinase (MAPK) were upregulated and nuclear factor (erythroid-derived 2) -like 2-related factor (Nrf2) was downregulated respectively. However, combined utilization of vitamin C and E were observed to prevent the increase of ROS generation, reduce celluar apoptosis, increase the expression of BTB related proteins, reconstructed the spatial arrangement of F-actin as well as improved the Nrf2 expression and attenuated the phosphorylation of the MAPK kinases and cleaved caspase-3 levels. Furthermore, ERK inhibitor (SCH772984), JNK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580) obviously up-regulated BTB-related proteins expression as well as activated Nrf2 expression at varying degrees, indicating that ROS-MAPKs-Nrf2 is involved in the signaling pathway that leads to PM2.5-induced spermatogenesis dysfunction. These findings indicate that PM2.5 derived from automobile exhaust causes oxidative stress, which in turn causes cellular apoptosis of SCs and damage of the blood-testis barrier, resulting male spermatogenesis dysfunction, in which ROS-MAPK-Nrf-2 pathways may play a key role.


Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Material Particulado/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Células de Sertoli/efeitos dos fármacos , Emissões de Veículos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Barreira Hematotesticular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Ratos , Células de Sertoli/metabolismo , Células de Sertoli/patologia
4.
Exp Oncol ; 41(3): 224-234, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31569932

RESUMO

In this review, literature data on the study of precancerous changes in testicular tissue and molecular changes, as well as the influence of environmental factors that can initiate carcinogenesis, were analyzed and summarized for the future determination of early diagnosis of germ cell tumors of the testis and the development of preventive measures. The review also discusses the significant new changes presented in the Fourth Edition of the World Health Organization Classification of Urogenital Tumors, published in 2016, and modern concepts of the etiology and pathogenesis of these diseases. Among the environmental factors that can initiate carcinogenesis, the most noteworthy are the biological effects of low doses of ionizing radiation, such as the effect of radiation-induced genome instability, which increases the risk of carcinogenesis, the "bystander effect", and chronic oxidative stress. Disruption of ubiquitin-proteasomal proteolysis, impaired molecular-level components of the blood-testis barrier, and impaired regulatory action of TGF-ß on the cell cycle can play a crucial role in the pathogenesis of male infertility and the initiation of carcinogenesis in the testis. The effect of low doses of ionizing radiation as an additional etiological factor leads to changes in the structural, as well as molecular, components of the testis, including epigenetic changes, which can be characterized as environmental pathomorphosis, which leads to impaired spermatogenesis and increased risk of malignancy. Summarizing the literature review data, we can state that patients with blocked spermatogenesis, in which atypical germ cell neoplasia in situ cells are detected in testicular tissue, constitute a group at increased risk of testicular carcinogenesis. The presence of additional etiological factors, such as chronic low doses of ionizing radiation, can initiate the progression of carcinogenesis in the testicle.


Assuntos
Transformação Celular Neoplásica , Neoplasias Testiculares/etiologia , Neoplasias Testiculares/metabolismo , Testículo/metabolismo , Testículo/patologia , Biomarcadores , Barreira Hematotesticular/metabolismo , Humanos , Masculino , Modelos Biológicos , Radiação Ionizante , Espermatogênese , Neoplasias Testiculares/patologia , Testículo/efeitos da radiação , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo
5.
Chem Commun (Camb) ; 55(86): 12932-12935, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31599282

RESUMO

Here, we report the development of novel PET radiotracer ([11C]CW22) of BET proteins. In vivo imaging results in rodents and nonhuman primates (NHP) demonstrate that [11C]CW22 has excellent brain uptake, good specificity, good selectivity, suitable metabolism, appropriate kinetics and distribution in the brain. Our studies demonstrated that [11C]CW22 exhibits ideal properties as a PET imaging probe of BET proteins for further validation.


Assuntos
Encéfalo/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Animais , Barreira Hematotesticular/metabolismo , Encéfalo/metabolismo , Radioisótopos de Carbono/química , Cristalografia por Raios X , Cinética , Macaca , Camundongos , Conformação Molecular , Proteínas do Tecido Nervoso/química , Neurônios , Domínios Proteicos , Compostos Radiofarmacêuticos/metabolismo , Receptores de Superfície Celular/química
6.
Theriogenology ; 138: 84-93, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31302435

RESUMO

Sertoli cells (SCs) are polarized epithelial cells and provide a microenvironment for the development of germ cells (GCs). The Wilms' tumor suppressor gene WT1 which support spermatogenesis is expressed explicitly in SCs. This study investigated the effect of WT1 on the polarity and blood-testis barrier (BTB) formation of bovine SCs in order to provide theoretical and practical bases for the spermatogenic process in mammals. In this study, newborn calf SCs were used as research material, and the RNAi technique was used to knockdown the endogenous WT1. The results show that WT1 knockdown did not affect the proliferation ability of SCs, but down-regulated the expression of polarity-associated proteins (Par3, Par6b, and E-cadherin), junction-associated protein (occludin) and inhibits transcription of downstream genes (WNT4, JNK, αPKC, and CDC42) in non-canonical WNT signaling pathway. WT1 also altered ZO-1 and occludin protein distribution. Overexpression of WNT1 did not affect the expression of Par6b, E-cadherin, and occludin, whereas the non-canonical WNT signaling pathway inhibitors wnt-c59, CCG-1423, and GO-6983 down-regulated the expression of Par6b, E-cadherin, and occludin respectively. This study indicates that WT1 mediates the regulation of several proteins involved in bovine SCs polarity maintenance and intercellular tight junctions (TJ) by non-canonical WNT signaling pathway.


Assuntos
Polaridade Celular/genética , Células de Sertoli/fisiologia , Junções Íntimas/genética , Proteínas WT1/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Animais Recém-Nascidos , Barreira Hematotesticular/metabolismo , Bovinos , Células Cultivadas , Masculino , Espermatogênese/genética , Junções Íntimas/metabolismo , Proteínas WT1/genética , Via de Sinalização Wnt/genética
7.
Endocrinology ; 160(8): 1832-1853, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31157869

RESUMO

During spermatogenesis, the blood-testis barrier (BTB) undergoes cyclic remodeling that is crucial to support the transport of preleptotene spermatocytes across the immunological barrier at stage VIII to IX of the epithelial cycle. Studies have shown that this timely remodeling of the BTB is supported by several endogenously produced barrier modifiers across the seminiferous epithelium, which include the F5-peptide and the ribosomal protein S6 [rpS6; a downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1)] signaling protein. Herein, F5-peptide and a quadruple phosphomimetic (and constitutively active) mutant of rpS6 [i.e., phosphorylated (p-)rpS6-MT] that are capable of inducing reversible immunological barrier remodeling, by making the barrier "leaky" transiently, were used for their overexpression in the testis to induce BTB opening. We sought to examine whether this facilitated the crossing of the nonhormonal male contraceptive adjudin at the BTB when administered by oral gavage, thereby effectively improving its BTB transport to induce germ cell adhesion and aspermatogenesis. Indeed, it was shown that combined overexpression of F5-peptide and p-rpS6-MT and a low dose of adjudin, which by itself had no noticeable effects on spermatogenesis, was capable of perturbing the organization of actin- and microtubule (MT)-based cytoskeletons through changes in the spatial expression of actin- and MT-binding/regulatory proteins to the corresponding cytoskeleton. These findings thus illustrate the possibility of delivering drugs to any target organ behind a blood-tissue barrier by modifying the tight junction permeability barrier using endogenously produced barrier modifiers based on findings from this adjudin animal model.


Assuntos
Barreira Hematotesticular/metabolismo , Laminina/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Proteína S6 Ribossômica/fisiologia , Actinas , Animais , Transporte Biológico/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Hidrazinas/farmacologia , Indazóis/farmacologia , Masculino , Fragmentos de Peptídeos/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Proteínas de Junções Íntimas/análise
8.
Am J Physiol Endocrinol Metab ; 317(1): E121-E138, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31112404

RESUMO

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.


Assuntos
Barreira Hematotesticular/metabolismo , Anticoncepcionais Masculinos/farmacologia , Hidrazinas/farmacologia , Indazóis/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína S6 Ribossômica/metabolismo , Animais , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mutagênese Sítio-Dirigida , Ratos , Ratos Sprague-Dawley , Proteína S6 Ribossômica/genética , Epitélio Seminífero/metabolismo , Células de Sertoli/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espermatócitos/metabolismo , Espermatogênese/efeitos dos fármacos
9.
Front Biosci (Landmark Ed) ; 24: 1316-1329, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31136981

RESUMO

Blood-testis barrier (BTB) that is constructed by testicular Sertoli cells (SCs) is essential for spermatogenesis. Krüppel-like factor 6 (Klf6), a nuclear transcription regulator, is reported to be associated with tight junction molecules of BTB between SCs during spermatogenesis; however, the specific regulatory role and mechanism of Klf6 in BTB regulation are still unknown. Here, we primarily confirmed the temporal and spatial expression patterns of Klf6 in mouse testes. Then, Klf6 was silenced in mouse cultured SCs using either Klf6-siRNA or Klf6-shRNA lentivirus. We mainly found that: (i) Klf6 was indispensable for the proliferative activity of mouse SCs; (ii) Klf6 regulated the integrity and permeability of BTB; (iii) Klf6 knockdown led to the significant upregulation of Zo-1, Claudin-11 and Vimentin, and downregulation of Claudin-3. Furthermore, Zo-1 and Claudin-3, participated in the tight junction remolding, were determined as targets of transcription factor Klf6 by luciferase assay. In summary, our findings suggest that Klf6 regulates the BTB assembly and disassembly via mainly targeting Zo-1 and Claudin-3 in mouse SCs.


Assuntos
Barreira Hematotesticular/metabolismo , Fator 6 Semelhante a Kruppel/metabolismo , Células de Sertoli/metabolismo , Testículo/metabolismo , Junções Íntimas/metabolismo , Animais , Células Cultivadas , Claudina-3/genética , Claudina-3/metabolismo , Claudinas/genética , Claudinas/metabolismo , Regulação da Expressão Gênica , Fator 6 Semelhante a Kruppel/genética , Masculino , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Interferência de RNA , Espermatogênese/genética , Testículo/ultraestrutura , Vimentina/genética , Vimentina/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
10.
Endocrinology ; 160(6): 1448-1467, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30994903

RESUMO

During spermatogenesis, microtubule (MT) cytoskeleton in Sertoli cells confers blood-testis barrier (BTB) function, but the regulators and mechanisms that modulate MT dynamics remain unexplored. In this study, we examined the role of calmodulin-regulated spectrin-associated protein (CAMSAP)2 (a member of the CAMSAP/Patronin protein family), and a minus-end targeting protein (-TIP) that binds to the minus-end (i.e., slow-growing end) of polarized MTs involved in determining MT length, in Sertoli cell function. CAMSAP2 was found to localize at discrete sites across the Sertoli cell cytosol, different from end-binding protein 1 (a microtubule plus-end tracking protein that binds to the plus-end of MTs), and colocalized with MTs. CAMSAP2 displayed a stage-specific expression pattern, appearing as tracklike structures across the seminiferous epithelium in adult rat testes that lay perpendicular to the basement membrane. CAMSAP2 knockdown by RNA interference was found to promote Sertoli cell tight junction (TJ) barrier function, illustrating its role in inducing TJ remodeling under physiological conditions. To further examine the regulatory role of CAMSAP2 in BTB dynamics, we used a perfluorooctanesulfonate (PFOS)-induced Sertoli cell injury model for investigations. CAMSAP2 knockdown blocked PFOS-induced Sertoli cell injury by promoting proper distribution of BTB-associated proteins at the cell-cell interface. This effect was mediated by the ability of CAMSAP2 knockdown to block PFOS-induced disruptive organization of MTs, but also F-actin, across cell cytosol through changes in cellular distribution/localization of MT- and actin-regulatory proteins. In summary, CAMSAP2 is a regulator of MT and actin dynamics in Sertoli cells to support BTB dynamics and spermatogenesis.


Assuntos
Barreira Hematotesticular/metabolismo , Citoesqueleto/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células de Sertoli/metabolismo , Actinas/metabolismo , Animais , Células Cultivadas , Humanos , Masculino , Permeabilidade , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/metabolismo , Células de Sertoli/citologia , Espermatogênese/fisiologia
11.
Ecotoxicol Environ Saf ; 171: 475-483, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30639874

RESUMO

Bisphenol A (BPA), an environmental contaminant, has been shown to disturb the dynamics of Sertoli cell blood-testis barrier (BTB) in mammal testis. However, the effects of BPA on Sertoli cell barrier (SC barrier) were little known in fish to date. To evaluate the potential mechanism of reproductive toxicity of BPA, we studied the damage of SC barrier using in vivo models. In this study, male adult rare minnow Gobiocypris rarus were exposed to 15 µg/L BPA for 7-35 days. Gonadal histology and the integrity of SC barrier were analyzed. Meanwhile, the expressions of SC barrier -associated proteins, tumor necrosis factor (TNFα) content, and the mRNA expressions of genes in the mitogen activated protein kinase (MAPK) pathway were detected. Histological analysis demonstrated 15 µg/L BPA promoted the infiltration of inflammatory cells in fish testes after 7-days exposure. The biotin tracer assay showed that 7-days BPA exposure increased permeability for spermatid cysts. In addition, the BPA treatment caused increased TNFα in testis, which was reportedly related to SC barrier impairment. The expressions of Occludin and ß-Catenin protein were significantly decreased in the testes after 7- and 21-days exposure. BPA also altered the mRNA expressions of occludin, ß-catenin, p38 MAPK and JNK. Therefore, the detrimental effects of BPA on reproduction of male fish may attribute to the disturbed expressions of SC junction proteins.


Assuntos
Compostos Benzidrílicos/toxicidade , Barreira Hematotesticular/efeitos dos fármacos , Cyprinidae , Fenóis/toxicidade , Células de Sertoli/efeitos dos fármacos , Animais , Barreira Hematotesticular/metabolismo , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Masculino , Ocludina/genética , Ocludina/metabolismo , Células de Sertoli/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
12.
Ecotoxicol Environ Saf ; 167: 161-168, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30326357

RESUMO

Long-term exposure to particulate matter 2.5 (PM2.5) from automobile exhaust impairs spermatogenesis through oxidative stress injury, but the underlying mechanism is unknown. To investigate the toxic mechanism of PM2.5-induced spermatogenesis impairment, we focused on the MAPK signaling pathway. We also examined the effects of treatment with vitamins C and E on spermatogenic function. Male SD rats were divided randomly into three groups: control (0.9% sterilized saline), PM2.5 exposure (20 mg/kg.b.w.), and PM2.5 exposure (20 mg/kg.b.w.) with vitamin intervention (vitamin C, 100 mg/kg.b.w.; vitamin E, 50 mg/kg.b.w.). Male rats showed a marked decline in fertility and decreased sperm quality after PM2.5 exposure. The expression of SOD and Nrf2 was significantly decreased, and that of MDA was increased markedly. The expression of blood-testis barrier-associated proteins, such as ZO-1, occludin, connexin 43, and ß-catenin, was significantly decreased, the Bcl-2/Bax ratio was downregulated, and the cleaved caspase-3 level was increased. Phosphorylation of MAPKs, including ERKs, JNKs, and p38, was upregulated. Treatment with vitamins C and E reversed the damage induced by PM2.5 exposure. These results suggest that PM2.5 from automobile exhaust disrupted spermatogenesis via ROS-mediated MAPK pathways, and that a combined vitamin C and E intervention effectively mitigated toxicity in the male reproductive system.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo , Material Particulado/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Espermatogênese/efeitos dos fármacos , Emissões de Veículos/toxicidade , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Barreira Hematotesticular/metabolismo , Caspase 3/metabolismo , Conexina 43/metabolismo , Fertilidade/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ocludina/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Análise do Sêmen , Transdução de Sinais , Espermatozoides/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Vitamina E/farmacologia , Proteína da Zônula de Oclusão-1/metabolismo , Proteína X Associada a bcl-2/metabolismo , beta Catenina/metabolismo
13.
J Vis Exp ; (142)2018 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-30582592

RESUMO

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.


Assuntos
Barreira Hematotesticular/fisiologia , Animais , Barreira Hematotesticular/metabolismo , Barreira Hematotesticular/fisiopatologia , Masculino , Camundongos
14.
Mol Med Rep ; 18(6): 5647-5651, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30365105

RESUMO

Varicocele (VC) is an abnormal tortuosity and venous distension of the pampiniform plexus in the spermatic cord. VC is the most common surgically correctable cause of male infertility. The purpose of the present study was to investigate the effects of VC on the tight junctions and the blood­testis barrier (BTB) of Sertoli cells in the bilateral testes of rats. A model of VC was established by left renal vein narrowing in Sprague­Dawley rats; control rats underwent dissection of the vein without narrowing. The bilateral testes were harvested at 4, 6 and 8 weeks after the operation. The relative expression of claudin­11 and transforming growth factor (TGF)­ß in the testis was determined by reverse transcription­polymerase chain reaction analysis and immunohistochemistry (IHC). The expression level of claudin­11 was prominently downregulated in the VC model group compared with the control group, while the level of TGF­ß in the testes was higher in the VC group. IHC examination demonstrated that VC led to destruction of the integrity of the BTB, and the degree of destruction increased with time. Furthermore, it was also observed that unilateral VC affected contralateral testicular function. In conclusion, the present study partially explained the molecular mechanisms underlying the pathogenesis of VC and provided grounds for further research into the treatment of male infertility.


Assuntos
Barreira Hematotesticular/metabolismo , Claudinas/genética , Expressão Gênica , Varicocele/genética , Varicocele/metabolismo , Animais , Biomarcadores , Claudinas/metabolismo , Modelos Animais de Doenças , Masculino , Ratos , Varicocele/patologia
15.
Toxicol Appl Pharmacol ; 360: 257-272, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30291936

RESUMO

Methamidophos (MET) is an organophosphate (OP) pesticide widely used in agriculture in developing countries. MET causes adverse effects in male reproductive function in humans and experimental animals, but the underlying mechanisms remain largely unknown. We explored the effect of MET on mice testes (5 mg/kg/day/4 days), finding that this pesticide opens the blood-testis barrier and perturbs spermatogenesis, generating the appearance of immature germ cells in the epididymis. In the seminiferous tubules, MET treatment changed the level of expression or modified the stage-specific localization of tight junction (TJ) proteins ZO-1, ZO-2, occludin, and claudin-3. In contrast, claudin-11 was barely altered. MET also modified the shape of claudin-11, and ZO-2 at the cell border, from a zigzag to a more linear pattern. In addition, MET diminished the expression of ZO-2 in spermatids present in seminiferous tubules, induced the phosphorylation of ZO-2 and occludin in testes and reduced the interaction between these proteins assessed by co-immunoprecipitation. MET formed covalent bonds with ZO-2 in serine, tyrosine and lysine residues. The covalent modifications formed on ZO-2 at putative phosphorylation sites might interfere with ZO-2 interaction with regulatory molecules and other TJ proteins. MET bonds formed at ZO-2 ubiquitination sites likely interfere with ZO-2 degradation and TJ sealing, based on results obtained in cultured epithelial cells transfected with ZO-2 mutated at a MET target lysine residue. Our results shed light on MET male reproductive toxicity and are important to improve regulations regarding the use of OP pesticides and to protect the health of agricultural workers.


Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Inseticidas/farmacologia , Organofosfatos/farmacologia , Compostos Organotiofosforados/farmacologia , Proteína da Zônula de Oclusão-2/metabolismo , Animais , Barreira Hematotesticular/metabolismo , Claudinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ocludina/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/metabolismo , Espermatogênese/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
16.
J Reprod Dev ; 64(6): 511-522, 2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30175719

RESUMO

Stem cell homing is a complex phenomenon that involves multiple steps; thus far, attempts to increase homing efficiency have met with limited success. Spermatogonial stem cells (SSCs) migrate to the niche after microinjection into seminiferous tubules, but the homing efficiency is very low. Here we report that reversible disruption of the blood-testis barrier (BTB) between Sertoli cells enhances the homing efficiency of SSCs. We found that SSCs on a C57BL/6 background are triggered to proliferate in vitro when MHY1485, which stimulates MTORC, were added to culture medium. However, the cultured cells did not produce offspring by direct injection into the seminiferous tubules. When acyline, a gonadotropin-releasing hormone (GnRH) analogue, was administered into infertile recipients, SSC colonization increased by ~5-fold and the recipients sired offspring. In contrast, both untreated individuals and recipients that received leuprolide, another GnRH analogue, remained infertile. Acyline not only decreased CLDN5 expression but also impaired the BTB, suggesting that increased colonization was caused by efficient SSC migration through the BTB. Enhancement of stem cell homing by tight junction protein manipulation constitutes a new approach to improve homing efficiency, and similar strategy may be applicable to other self-renewing tissues.


Assuntos
Barreira Hematotesticular/metabolismo , Células de Sertoli/metabolismo , Espermatogônias/metabolismo , Testículo/metabolismo , Animais , Barreira Hematotesticular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Claudina-5/metabolismo , Masculino , Camundongos , Morfolinas/farmacologia , Oligopeptídeos/farmacologia , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Testículo/efeitos dos fármacos , Triazinas/farmacologia
17.
Toxicol Lett ; 295: 277-287, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29981920

RESUMO

There are reports of fluorochloridone (FLC)-induced male reproductive toxicity, but the underlying toxicological mechanisms remain unknown. In this study, we looked at how FLC exposure affected the integrity of the blood-testis barrier (BTB) and the Sertoli cell barrier and studied the molecular mechanisms. Male rats received gavage administration of FLC (30 mg/kg/d) for 14 consecutive days with sample collection at the 7th and 14th day; and primary cultured Sertoli cells were treated with 0-10 µM FLC in vitro for 24 h. Our in vivo findings revealed that FLC exposure caused time-dependent testicular injuries, sperm quality decrease as well as adverse changes in BTB integrity, F-actin organization, and expressions of claudin-11 and Arp3. In Sertoli cells isolated from FLC-treated rat testis, Sertoli cell barrier tightness was increased. In Sertoli cells in vitro exposed to FLC, abnormal changes in the barrier permeability were also observed, and the protein expressions of occludin, claudin-11, ZO-1, connexin-43, and Arp3 were significantly decreased in a dose- and time-dependent manner. Furthermore, the FLC-induced adverse changes in Sertoli cell barrier and F-actin were partly alleviated by the induction of Arp3 overexpression. In conclusion, our findings revealed that FLC perturbed BTB/Sertoli cell barrier function through Arp3-mediated F-actin disorganization.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Proteína 3 Relacionada a Actina/metabolismo , Actinas/metabolismo , Poluentes Ocupacionais do Ar/toxicidade , Barreira Hematotesticular/efeitos dos fármacos , Pirrolidinonas/toxicidade , Reprodução/efeitos dos fármacos , Células de Sertoli/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Proteína 3 Relacionada a Actina/genética , Animais , Barreira Hematotesticular/metabolismo , Barreira Hematotesticular/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Masculino , Permeabilidade , Ratos Sprague-Dawley , Medição de Risco , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Transdução de Sinais/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Fatores de Tempo
18.
Development ; 145(13)2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29899137

RESUMO

Spermatogenesis in mammals is a very complex, highly organized process, regulated in part by testosterone and retinoic acid (RA). Much is known about how RA and testosterone signaling pathways independently regulate this process, but there is almost no information regarding whether these two signaling pathways directly interact and whether RA is crucial for steroidogenic cell function. This study uses a transgenic mouse line that expresses a dominant-negative form of RA receptor α (RAR-DN) and the steroidogenic cell-specific Cre mouse line, Cyp17iCre, to generate male mice with steroidogenic cells unable to perform RA signaling. Testes of mutant mice displayed increased apoptosis of pachytene spermatocytes, an increased number of macrophages in the interstitium and a loss of advanced germ cells. Additionally, blocking RA signaling in Leydig cells resulted in increased permeability of the blood-testis barrier, decreased levels of the steroidogenic enzyme cytochrome P450 17a1 and decreased testosterone levels. Surprisingly, the epididymides of the mutant mice also displayed an abnormal phenotype. This study demonstrates that RA signaling is required in steroidogenic cells for their normal function and, thus, for male fertility.


Assuntos
Barreira Hematotesticular/metabolismo , Fertilidade/fisiologia , Receptor alfa de Ácido Retinoico/metabolismo , Transdução de Sinais/fisiologia , Espermatócitos/metabolismo , Espermatogênese/fisiologia , Animais , Barreira Hematotesticular/citologia , Masculino , Camundongos , Camundongos Transgênicos , Receptor alfa de Ácido Retinoico/genética , Espermatócitos/citologia , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo
19.
Reprod Toxicol ; 81: 17-27, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29940330

RESUMO

As an environmental endocrine disruptor, Di-(2-ethylhexyl) phthalate (DEHP) affects blood-testis barrier (BTB)-associated proteins expression, which compromises BTB integrity and causes infertility. Notably, DEHP-induced testicular toxicity is related to oxidative stress, but the specific mechanism remains unclear. Therefore, we sought to investigate this mechanism and determine whether vitamin C and vitamin E administration would attenuate the BTB impairment induced by DEHP in vivo and by Mono-(2-Ethylhexyl) Phthalate (MEHP) in vitro, respectively. HE staining and EM found that DEHP exposure led to spermatogenesis dysfunction and BTB disruption, respectively. The Western blot and immunofluorescence results showed that DEHP exposure caused BTB impairment through oxidative stress-mediated p38 mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, Vitamin E and vitamin C could alleviate the oxidative stress, block DEHP-induced spermatogenesis dysfunction and BTB disruption by inhibiting p38 MAPK signaling pathway. In summary, vitamin E and vitamin C are good candidates for the treatment of DEHP-induced male infertility.


Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Barreira Hematotesticular/efeitos dos fármacos , Dietilexilftalato/toxicidade , Disruptores Endócrinos/toxicidade , Vitamina E/farmacologia , Vitaminas/farmacologia , Animais , Barreira Hematotesticular/metabolismo , Dietilexilftalato/análogos & derivados , Infertilidade Masculina/tratamento farmacológico , Masculino , Ratos Sprague-Dawley , Espermatogênese/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Reprod Biol Endocrinol ; 16(1): 55, 2018 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-29855380

RESUMO

BACKGROUND: Serum leptin levels are augmented in obese infertile men and in men with azoospermia. They also correlate inversely with sperm concentration, motility and normal forms. The mechanisms underlying the adverse effects of excess leptin on male reproductive function remain unclear. The present study aimed to evaluate the effects of exogenous leptin on sperm parameters in mice and to explore the underlying mechanisms. METHODS: We treated normal adult male mice with saline, 0.1, 0.5 or 3 mg/kg leptin daily for 2 weeks. After treatment, serum leptin levels, serum testosterone levels, sperm parameters and testicular cell apoptosis were evaluated. Blood testis barrier integrity and the expression of tight junction-associated proteins in testes were also assessed. We further verified the direct effects of leptin on tight junction-associated proteins in Sertoli cells and the possible leptin signaling pathways involved in this process. RESULTS: After treatment, there were no significant differences in body weights, reproductive organ weights, serum leptin levels and serum testosterone levels between leptin-treated mice and control mice. Administration of 3 mg/kg leptin reduced sperm concentration, motility and progressive motility while increasing the percentage of abnormal sperm and testicular cell apoptosis. Mice treated with 3 mg/kg leptin also had impaired blood testis barrier integrity, which was related to decreased tight junction-associated proteins in testes. Leptin directly reduced tight junction-associated proteins in Sertoli cells, JAK2/STAT, PI3K and ERK pathways were suggested to be involved in this process. CONCLUSIONS: Exogenous leptin negatively affects sperm parameters and impairs blood testis barrier integrity in mice. Leptin reduced tight junction-associated proteins in Sertoli cells, indicating that leptin has a direct role in impairing blood testis barrier integrity. Given the function of blood testis barrier in maintaining normal spermatogenesis, leptin-induced blood testis barrier impairment may be one of the mechanisms contributing to male subfertility and infertility.


Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Barreira Hematotesticular/metabolismo , Leptina/farmacologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Animais , Relação Dose-Resposta a Droga , Leptina/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo
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