RESUMO
Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.
Assuntos
Infertilidade Masculina , Mutação de Sentido Incorreto , Humanos , Animais , Camundongos , Masculino , Estudos Retrospectivos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Infertilidade Masculina/genética , Mutação , Ducto Deferente/anormalidades , Espermatogênese/genéticaRESUMO
Objective: To analyze the mechanism of LINC00461 regulating the recurrence of diffuse large B cell lymphoma (DLBCL) through microRNA (miR)-411-5p/BCL2 interacting protein 3 (BNIP3) pathway. Methods: DLBCL samples in TCGA and GSE12453 were used for differential analysis to find long noncoding RNA (lncRNA) related to DLBCL recurrence. The 4 DLBCL data with the highest and lowest expression levels of LINC00461 in the TCGA database were selected for GSEA enrichment analysis. The targeting relationships of miR-411-5p with LINC00461 and BNIP3 were verified by the dual luciferase report. Blood samples from DLBCL patients were used to analyze the correlation between miR-411-5p and LINC00461 or BNIP3. LINC00461, miR-411-5p, or BNIP3 was overexpressed or silenced by transfection, and a tumor-bearing nude mice model was constructed to detect their effects on proliferation and apoptosis. Results: The level of LINC00461 in DLBCL was significantly higher than that in normal cases, and the level in recurrence DLBCL was significantly higher than that in nonrecurrence. The enrichment analysis results showed that the function of LINC00461 was closely related to apoptosis. The results shown that miR-411-5p bound to LINC00461 and BNIP3 and was negatively correlated with LINC00461 and BNIP3 mRNA in blood of DLBCL patients. Suppressing the level of LINC00461 inhibited cell proliferation and induced apoptosis. The inhibition of LINC00461 or overexpression of miR-411-5p reduced the expression of BNIP3 protein, thereby inducing apoptosis at the in vivo and in vitro levels. Conclusion: LINC00461 may induce miR-411-5p to "sponge," thereby increasing the expression of BNIP3 protein, and exerting the function of inhibiting apoptosis and promoting DLBCL recurrence.
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Placental hypoxia serves a role in the early stages of normal pregnancy and is involved in the pathophysiology of preeclampsia. Previously, it was suggested that p57kinase inhibitory protein (KIP)2 regulates the cell cycle during embryogenesis and apoptosis. Recent evidence has indicated that p57KIP2 is increased in preeclamptic placentas and absence of p57KIP2 induces preeclampsiatype symptoms in rats. However, effects of p57KIP2 on apoptosis under hypoxic conditions remain to be elucidated. In the present study, HTR8/SVneo trophoblasts were cultured under hypoxic conditions (2% O2). Knockdown using small interfering (si)RNA and overexpression of p57KIP2 were utilized to explore the biological function of p57KIP2 in apoptosis and cell function in vitro. Furthermore, expression of p57KIP2 and apoptosis were evaluated by western blotting, flow cytometry and TUNEL assays, and the response of trophoblasts to hypoxia and the role of p57KIP2 in trophoblast migration and invasion was assessed. The role of p57KIP2 in the JNK signaling pathway in HTR8/SVneo trophoblasts was further studies. In vitro, protein expression of p57KIP2 was increased in HTR8/SVneo cells exposed to 2% O2. Exogenous p57KIP2 overexpression significantly decreased the expression of proapoptosis proteins, including p53, Bax and cleaved caspase3, under hypoxic conditions for 24 h. In addition, knockdown of p57KIP2 increased the response to apoptosis following hypoxia for 24 h. The present study revealed that overexpression of p57KIP2 decreased the levels of phosphorylatedJNK. JNK inhibitor treatment combined with the overexpression of p57KIP2 significantly decreased the levels of apoptosis and increased cell invasion and migration. Taken together, p57KIP2 knockdown significantly increased apoptosis in HTR8/SVneo cells exposed to 2% O2, whereas overexpression of p57KIP2 had opposite effects, mediated by the JNK/stress activated protein kinase (SAPK) signaling pathway. The results indicated that hypoxiainduced expression of p57KIP2 promoted trophoblast migration and invasion by mediating the JNK/SAPK signaling pathway, which is crucial during placentation. These results may provide a novel molecular mechanism to understand the involvement of p57KIP2 in the pathogenesis of preeclampsia.
Assuntos
Apoptose , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Sistema de Sinalização das MAP Quinases , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismo , Caspase 3/metabolismo , Hipóxia Celular , Linhagem Celular , Feminino , Humanos , Pré-Eclâmpsia/patologia , Gravidez , Trofoblastos/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The essence of primary ovarian insufficiency (POI) is the premature exhaustion of primordial follicles in the follicle pool, which is caused by the excessive premature activation of primordial follicles after birth. Bisphenol A (BPA) exposure promotes the transition of primordial follicles to primary follicles, thus the number of primordial follicles in the primordial follicle pool decreases significantly. However, the molecular mechanisms underlying abnormal follicle activation are poorly understood. Phosphatase and tensin homologue (PTEN) signal system is a negative regulator of follicle activation, which is called the brake of follicle activation. Besides, BPA induces Michigan Cancer Foundation-7 breast cancer cells proliferation by dysregulating PTEN/serine/threonine kinase/p53 axis. Whether BPA initiates the excessive premature activation of primordial follicles in the mouse ovaries via PTEN signaling pathway is unclear. In this study, we treated 6-week-old female CD-1 mice with different concentrations of BPA to study the effect of BPA on follicular activation and development in vivo, as well as the role of PTEN signaling in this process. We observed that BPA in concentrations from 1 µg/kg to 10 mg/kg groups downregulated PTEN expression and initiated excessive premature activation of primordial follicles in the mouse ovaries, and this effect was partly reversible by PTEN overexpression. Our results improve the understanding of both the effect of BPA in occurrence of POI and molecular mechanisms underlying initiation of primordial follicle pool activation, thus providing insight for POI treatment and theoretical basis for reducing the risk of POI.
Assuntos
Compostos Benzidrílicos/farmacologia , Estrogênios não Esteroides/farmacologia , Folículo Ovariano/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Fenóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Feminino , Camundongos , Folículo Ovariano/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismoRESUMO
BACKGROUND: Growth arrest and DNA-damage inducible protein 45 beta (Gadd45b) is serving as a neuronal activity sensor. Brain ischemia induces the expression of Gadd45b, which stimulates recovery after stroke and may play a protective role in cerebral ischemia. However, little is known of the molecular mechanisms of how Gadd45b expression regulated and the down-stream targets in brain ischemia. Here, using an oxygen-glucose deprivation and reperfusion (OGD/R) model, we identified Huwe1/Mule/ARF-BP1, a HECT domain containing ubiquitin ligase, involved in the control of Gadd45b protein level. In this study, we also investigated the role of Huwe1-Gadd45b mediated pathway in BDNF methylation. RESULTS: We found that the depletion of Huwe1 by lentivirus shRNA mediated interference significantly increased the expression of Gadd45b and BDNF at 24 h after OGD. Moreover, treatment with Cycloheximide (CHX) inhibited endogenous expression of Gadd45b, and promoted expression of Gadd45b after co-treated with lentivirus shRNA-Huwe1. Inhibition of Gadd45b by lentivirus shRNA decreased the expression levels of brain derived neurotrophic factor (BDNF) and phosphorylated cAMP response element-binding protein (p-CREB) pathway, while inhibition of Huwe1 increased the expression levels of BDNF and p-CREB. Moreover, shRNA-Huwe1 treatment decreased the methylation level of the fifth CpG islands (123 bp apart from BDNF IXa), while shRNA-Gadd45b treatment increased the methylation level of the forth CpG islands (105 bp apart from BDNF IXa). CONCLUSIONS: These findings suggested that Huwe1 involved in the regulation of Gadd45b expression under OGD/R, providing a novel route for neurons following cerebral ischemia-reperfusion injury. It also indicated that the methylation of BDNF IXa was affected by Gadd45b as well as Huwe1 in the OGD/R model.
Assuntos
Antígenos de Diferenciação/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Ilhas de CpG , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Metilação de DNA , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glucose/farmacologia , Hipóxia-Isquemia Encefálica , Técnicas In Vitro , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Oxigênio/farmacologia , Complexo de Endopeptidases do Proteassoma , Mapeamento de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Transdução de Sinais , Ubiquitina/fisiologia , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
MicroRNAs (miRNAs) are noncoding RNAs with a short length of about 22 nucleotides. As major modulators participating in RNA interference, they affect cellular behaviors by regulating the expression of diverse genes at post-transcriptional levels. miR-15b is a member of the miR-15/16 family, which is broadly expressed in major tissues and specially enriched in the endovascular system of human beings. miR-15/16 affects cellular proliferation, apoptosis, invasion and angiogenesis. In this review, we summarize the role and the underlying mechanism of miR-15b as well as other miR-15/16 family members in different cells, especially in endothelial cells. We focus on the diverse roles of miR-15b in the occurrence, progression and prognosis of vascular diseases, with particular emphasis on preeclampsia, a hypertensive disorder related to endovascular dysfunction in the placenta.
Assuntos
Células Endoteliais/fisiologia , MicroRNAs/fisiologia , Doenças Vasculares/etiologia , Animais , Ciclina D1/genética , Ciclina E/genética , Humanos , Neuropilina-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: To study the expression of insulin signaling-related genes and oxidative stress markers in the visceral adipose tissue obtained from polycystic ovary syndrome (PCOS) patients and healthy control subjects and to investigate the relationships among abdominal obesity, insulin resistance, and oxidative stress at the tissue level. DESIGN: Case-control study. SETTING: University teaching hospital. PATIENT(S): In total, 30 PCOS patients and 30 healthy control subjects, who underwent laparoscopic surgery, were included in the study. INTERVENTION(S): Abdominal obesity was defined based on waist circumference (WC). The homeostasis model index was used to assess insulin resistance (HOMA-IR). MAIN OUTCOME MEASURE(S): Gene expression of glucose transporter 4 (GLUT4) and insulin receptor substrate 1 (IRS1) in visceral adipose tissue (VAT) and the parameters of oxidative stress, such as superoxide dismutase, enzyme glutathione reductase, and dimethylarginine, were measured, and the expression of protein oxidative damage product 3-nitro-tyrosine residues (nitrotyrosine) in VAT was identified with the use of immunohistochemistry. RESULT(S): PCOS was associated with lower expression of GLUT4 and IRS1 and a higher level of oxidative stress in VAT, which was strongly correlated with WC and HOMA-IR. Presence of abdominal obesity further intensified the correlations observed in our measurements. The nitrotyrosine expression in VAT was stronger in PCOS patients. CONCLUSION(S): The strong correlation of insulin resistance with oxidative stress at the VAT level suggests that local oxidative stress and abnormalities of insulin signaling in adipose tissue play critical roles in the pathogenesis of PCOS.
Assuntos
Resistência à Insulina , Gordura Intra-Abdominal/química , Obesidade Abdominal/metabolismo , Estresse Oxidativo , Síndrome do Ovário Policístico/metabolismo , Adulto , Biomarcadores/análise , Estudos de Casos e Controles , Feminino , Transportador de Glucose Tipo 4/genética , Hospitais Universitários , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Modelos Lineares , Análise Multivariada , Obesidade Abdominal/diagnóstico , Obesidade Abdominal/genética , Síndrome do Ovário Policístico/diagnóstico , Síndrome do Ovário Policístico/genética , RNA Mensageiro/análise , Transdução de Sinais , Circunferência da Cintura , Adulto JovemRESUMO
Since there is no consensus about the most reliable assays to detect invasive aspergillosis from samples obtained by minimally invasive or noninvasive methods, we compared the efficacy of an enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of invasive pulmonary aspergillosis. Neutropenic, male Sprague-Dawley rats (specific pathogen free; 8 weeks old; weight, 200 ± 20 g) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumigatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and examined with GM ELISA and qRT-PCR. The A. fumigatus DNA detection sequence was detected in the following number of samples from 12 immunosuppressed, infected rats examined on the scheduled days: day 1 (0/12), day 3 (0/12), day 5 (6/12), and day 7 (8/12) post-infection. The sensitivity and specificity of the qRT-PCR assay was 29.2% and 100%, respectively. Receiver operating characteristic curve (ROC) analysis indicated a Ct (cycle threshold) cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/ 12), day 5 (12/12), and day 7 (12/12) post-infection, and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal Ct cut-off value was 1.40 (AUC, 0.919). The GM assay was more sensitive than the qRT-PCR assay in diagnosing invasive pulmonary aspergillosis in rats.
Assuntos
Testes Diagnósticos de Rotina/métodos , Aspergilose Pulmonar Invasiva/diagnóstico , Mananas/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Modelos Animais de Doenças , Galactose/análogos & derivados , Técnicas Imunoenzimáticas/métodos , Mananas/imunologia , Curva ROC , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
In the present study, to identify the effective components of Chinese traditional herbs, Euphorbia hylonoma Hand.-Mazz. (Euphorbiaceae), a folk herb that has been used among the Qinling mountain area for hundreds of years, was investigated. 3,3'-Di-O-methyl ellagic acid-4'-O-ß-d-xylopyranoside (JNE2), an ellagic acid derivative, was isolated from the acetone extract of the herb and its antitumor activity against human hepatoma HepG2 cells was detected in vitro. The results showed that JNE2 inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner and blocked the cell cycle at the G1/S phase. A high dosage of JNE2 induced apoptosis of the tumor cells, but no significant differences were identified between the treatment groups. The invasiveness of HepG2 cells was also inhibited by JNE2. The mechanism of the antitumor effect of JNE2 at the molecular level was presumed to be due to the upregulation of the protein expression of Bax and caspase-3, and the downregulation of the protein expression of Bcl-2 and CCND1. The results suggested that JNE2 is a potential antitumor agent that merits further investigation.
RESUMO
A number of studies have demonstrated that inflammation plays a role in doxorubicin (DOX)-induced cardiotoxicity. However, the molecular mechanism by which DOX induces cardiac inflammation has yet to be fully elucidated. The present study aimed to investigate the role of the p38 mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) pathway in DOX-induced inflammation and cytotoxicity. The results of our study demonstrated that the exposure of H9c2 cardiac cells to DOX reduced cell viability and stimulated an inflammatory response, as demonstrated by an increase in the levels of interleukin-1ß (IL-1ß) and IL-6, as well as tumor necrosis factor-α (TNF-α) production. Notably, DOX exposure induced the overexpression of phosphorylated p38 MAPK and phosphorylation of the NF-κB p65 subunit, which was markedly inhibited by SB203580, a specific inhibitor of p38 MAPK. The inhibition of NF-κB by pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of NF-κB, significantly ameliorated DOX-induced inflammation, leading to a decrease in the levels of IL-1ß and IL-6, as well as TNF-α production in H9c2 cells. The pretreatment of H9c2 cells with either SB203580 or PDTC before exposure to DOX significantly attenuated DOX-induced cytotoxicity. In conclusion, our study provides novel data demonstrating that the p38 MAPK/NF-κB pathway is important in the induction of DOX-induced inflammation and cytotoxicity in H9c2 cardiac myocytes.
Assuntos
Doxorrubicina/toxicidade , Inflamação/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Inflamação/induzido quimicamente , Fosforilação/efeitos dos fármacos , Ratos , Fator de Transcrição RelA/metabolismoRESUMO
STUDY QUESTION: Does elevated temperature-induced cystic fibrosis transmembrane conductance regulator (CFTR) down-regulation in Sertoli cells in cryptorchid testis disrupt testicular tight junctions (TJs) through the nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway? SUMMARY ANSWER: Our results suggest that CFTR may be involved in regulating testicular TJs and the blood-testis barrier (BTB) through its negative regulation of the NF-κB/COX-2/PGE(2) pathway in Sertoli cells, a defect of which may result in the spermatogenesis defect in cryptorchidism. WHAT IS KNOWN ALREADY: Cryptorchidism, or undescended testes, is known to result in defective spermatogenesis. Although an elevated testicular temperature is regarded as an important factor affecting spermatogenesis in cryptorchidism, the exact mechanism remains elusive. It is known that the expression of functional CFTR is temperature sensitive. Our previous study has demonstrated that CFTR negatively regulates NF-κB/COX-2/PGE(2) in bronchial epithelial cells. Disruption of TJs by COX-2/PGE(2) has been found in tumour cells. STUDY DESIGN AND METHODS: Expression of CFTR, NF-κB, COX-2 and TJ proteins was examined in the testes of a surgical-induced cryptorchidism mouse model and a testicular hyperthermia mouse model, as well as in control or CFTR-inhibited/knocked down primary rat Sertoli cells. PGE(2) production was measured by ELISA. Sertoli cell barrier function was determined by transepethelial resistance (TER) measurements in rat Sertoli cell primary cultures. BTB integrity in the cryptorchidism model was monitored by examining tracker dye injected into seminiferous tubules. MAIN RESULTS: Down-regulation of CFTR accompanied by activation of NF-κB, up-regulation of COX-2 and down-regulation of TJ proteins, including ZO-1 and occludin, was observed in a cryptorchidism mouse model. BTB leakage revealed impaired BTB integrity in cryptorchid testes, confirming the destruction of TJs. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and CFTR knockout mouse model. Culturing primary Sertoli cells at 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression and PGE(2) production compared with the culture at the physiological 32°C. Inhibition or knockdown of CFTR led to increased COX-2 but decreased ZO-1 and occludin expression in Sertoli cells, which could be mimicked by PGE(2), but reversed by NF-κB or COX-2 inhibitor, suggesting that the regulation of TJs by CFTR is mediated by a NF-κB/COX-2/PGE(2) pathway. Inhibition of CFTR or administration of PGE(2) significantly decreased Sertoli cell TER. LIMITATIONS: This study has tested only the CFTR/NF-κB/COX-2/PGE(2) pathway in mouse testes in vivo and in rat Sertoli cells in vitro, and thus, it has some limitations. Further investigations in other species, especially humans, are needed. WIDER IMPLICATIONS OF THE FINDINGS: Our study may shed more light on one of the aspects of the complicated underlying mechanisms of defective spermatogenesis induced by cryptorchidism.
Assuntos
Criptorquidismo/metabolismo , Ciclo-Oxigenase 2/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Dinoprostona/biossíntese , Regulação para Baixo , NF-kappa B/biossíntese , Junções Íntimas/patologia , Animais , Barreira Hematotesticular , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Ratos , Ratos Sprague-Dawley , Células de Sertoli/metabolismo , Temperatura , Regulação para CimaRESUMO
BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) and HCO(3)(-) conducting channel, mutations of which are known to be associated with male infertility. However, the underlying mechanisms remain elusive. METHODS: Literature databases were searched for papers on the topics related to CFTR and male fertility and infertility with relevant keywords. Unpublished data from authors' laboratory were also included for analysis. RESULTS: Clinical evidence shows increased mutation frequency or reduced CFTR expression in men with congenital bilateral absence of vas deferens (CBAVD) or sperm abnormalities, such as azoospermia teratospermia and oligoasthenospermia. Studies on primary rodent Sertoli cells and germ cells, as well as testes from CFTR knockout mice or a cryptorchidism model, yield findings indicating the involvement of CFTR in spermatogensis through the HCO(3)(-)/sAC/cAMP/CREB(CREM) pathway and the NF-κB/COX-2/PGE(2) pathway. Evidence also reveals a critical role of CFTR in sperm capacitation by directly or indirectly mediating HCO(3)(-) entry that is essential for capacitation. CFTR is emerging as a versatile player with roles in mediating different signaling pathways pertinent to various reproductive processes, in addition to its long-recognized role in electrolyte and fluid transport that regulates the luminal microenvironment of the male reproductive tract. CONCLUSIONS: CFTR is a key regulator of male fertility, a defect of which may result in different forms of male infertility other than CBAVD. It would be worthwhile to further investigate the potential of developing novel diagnostic and contraceptive methods targeting CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fertilidade , Infertilidade Masculina/genética , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fertilidade/genética , Humanos , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Mutação , Células de Sertoli/metabolismo , Capacitação Espermática , EspermatogêneseRESUMO
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent anion channel mostly expressed in epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased cyclooxygenase-2 (COX-2) expression and over-production of prostaglandin E(2) (PGE(2)) in human CF bronchial epithelia cell line (CFBE41o--) with elevated NF-κB activity compared to a wild-type airway epithelial cell line (16HBE14o--). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NF-κB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE(2) involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o-- cells were challenged with LPS as well as PGE(2), indicating possible involvement of CFTR in negative regulation of COX-2/PGE(2). In conclusion, CFTR is a negative regulator of PGE(2)-mediated inflammatory response, defect of which may result in excessive activation of NF-κB, leading to over production of PGE(2) as seen in inflammatory CF tissues.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/enzimologia , Dinoprostona/metabolismo , Pneumonia/enzimologia , Mucosa Respiratória/enzimologia , Infecções Respiratórias/enzimologia , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Retroalimentação Fisiológica , Humanos , Camundongos , Camundongos Endogâmicos CFTR , NF-kappa B/metabolismo , Pneumonia/genética , Pneumonia/microbiologia , Pseudomonas aeruginosa/patogenicidade , Mucosa Respiratória/microbiologia , Infecções Respiratórias/genética , Infecções Respiratórias/microbiologia , Fatores de Tempo , TransfecçãoRESUMO
Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO(3) (-) transport and the expression of the HCO(3) (-) sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO(3) (-) could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.
Assuntos
Azoospermia/etiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/patologia , Espermatogênese/fisiologia , Adenilil Ciclases/metabolismo , Adulto , Animais , Azoospermia/metabolismo , Azoospermia/patologia , Bicarbonatos/metabolismo , Western Blotting , AMP Cíclico/metabolismo , Fibrose Cística/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fosforilação , Ratos , Ratos Sprague-Dawley , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Adulto JovemRESUMO
Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.
Assuntos
Reação Acrossômica/fisiologia , Acrossomo/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Capacitação Espermática/fisiologia , Acrossomo/efeitos dos fármacos , Adulto , Animais , Cálcio/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Soros Imunes/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Fosfoinositídeo Fosfolipase C/imunologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
Our previous study demonstrated the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate that is necessary for sperm capacitation; however, whether its involvement is direct or indirect remains unclear. The present study investigated the possibility of a Cl-/HCO3- exchanger (solute carrier family 26, number 3 [SLC26A3]) operating with CFTR during guinea pig sperm capacitation. Incubating sperm in media with various concentrations of Cl- resulted in varied percentages of capacitated sperm in a concentration-dependent manner. Depletion of Cl-, even in the presence of HCO3-, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO3--dependent events, including increased intracellular pH, cAMP production, and protein tyrosine phosphorylation, also depend on Cl- concentrations. Similar Cl- dependence and inhibitor sensitivity were observed for sperm-hyperactivated motility and for sperm-egg fusion. The expression and localization of CFTR and SLC26A3 were demonstrated using immunostaining and Western blot analysis. Taken together, our results indicate that Cl- is required for the entry of HCO3- that is necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO3-, with CFTR providing the recycling pathway for Cl-.
Assuntos
Antiporters/metabolismo , Bicarbonatos/farmacologia , Cloretos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/metabolismo , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/fisiologia , Animais , Antiporters/antagonistas & inibidores , Bicarbonatos/farmacocinética , Western Blotting , Cloretos/farmacocinética , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Relação Dose-Resposta a Droga , Feminino , Imunofluorescência , Cobaias , Concentração de Íons de Hidrogênio , Masculino , Capacitação Espermática/efeitos dos fármacos , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/efeitos dos fármacos , Transportadores de SulfatoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel regulated by cAMP-dependent phosphorylation, which is expressed in epithelial cells of a wide variety of tissues including the reproductive tracts. Mutations in the gene encoding CFTR cause cystic fibrosis, a common genetic disease in Caucasian populations with a multitude of clinical manifestations including infertility/subfertility in both sexes. However, the physiological role of CFTR in reproduction and its involvement in the pathogenesis of reproductive diseases remain largely unknown. This review discusses the role of CFTR in regulating fluid volume and bicarbonate secretion in the reproductive tracts and their importance in various reproductive events. We also discuss the contribution of CFTR dysfunction to a number of pathological conditions. The evidence presented is consistent with an important role of CFTR in reproductive health and disease, suggesting that CFTR might be a potential target for the diagnosis and treatment of reproductive diseases including infertility.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Doenças dos Genitais Femininos/metabolismo , Reprodução/fisiologia , Animais , Implantação do Embrião/fisiologia , Feminino , Humanos , Masculino , Capacitação Espermática/fisiologia , Transporte Espermático/fisiologiaRESUMO
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl(-) and HCO(3)(-) transport. Although >95% of all CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. Here we report that CFTR is detected in both human and mouse sperm. CFTR inhibitor or antibody significantly reduces the sperm capacitation, and the associated HCO(3)(-)-dependent events, including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower compared with that of the wild-type. These results suggest that CFTR in sperm may be involved in the transport of HCO(3)(-) important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fertilização/genética , Capacitação Espermática/genética , Espermatozoides/química , Análise de Variância , Animais , Bicarbonatos/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Mutantes , Microscopia de Fluorescência , Mutação/genética , Motilidade dos Espermatozoides/genéticaRESUMO
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl- channel expressed in a wide variety of epithelial cells, mutations of which are responsible for hallmark defective Cl- and HCO3- secretion seen in cystic fibrosis (CF). However, the physiological role of CFTR in reproductive tracts is far from understood although infertility has been observed in CF patients of both sexes. Previously we have demonstrated the expression of CFTR in the female reproductive tract and the involvement of CFTR in mediating anion secretion by the endometrium. Our recent results show that endometrial epithelial cells possess a cAMP-activated HCO3- transport mechanism, which could be impaired with channel blockers known to block CFTR or antisense against CFTR. Co-culture of sperm with CFTR antisense-treated endometrial cells or HCO3- secretion-defective CF epithelial cells resulted in reduced sperm capacitation and egg-fertilizing ability. Addition of HCO3- to the culture media and transfection of wild-type CFTR into CF cells rescued the fertilizing capacity of sperm. Immunostaining and Western blot revealed that CFTR is expressed in rodent sperm and intracellular measurement of pH during sperm capacitation indicated that the entry of HCO3- into sperm could be inhibited by CFTR inhibitor. These results are consistent with a critical role of CFTR in controlling uterine HCO3- secretion and sperm fertilizing capacity, suggesting that CFTR may be a potential target for post-meiotic regulation of fertility.