RESUMEN
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.
Asunto(s)
Azoospermia/etiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/patología , Espermatogénesis/fisiología , Adenilil Ciclasas/metabolismo , Adulto , Animales , Azoospermia/metabolismo , Azoospermia/patología , Bicarbonatos/metabolismo , Western Blotting , AMP Cíclico/metabolismo , Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Fosforilación , Ratas , Ratas Sprague-Dawley , Células de Sertoli/metabolismo , Células de Sertoli/patología , Adulto JovenRESUMEN
BACKGROUND: This phase I study was designed to determine the maximum tolerated dose (MTD) and preliminary efficacy of docetaxel with concurrent radiotherapy (RT), in high-risk squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: Eligible patients had resected squamous cell carcinoma of the head and neck, histologically involved lymph nodes, and/or extranodal disease, and/or involved surgical margins and performance status 0 to 1. Treatment included weekly docetaxel with concurrent RT in a dose-finding study; a subsequent small cohort of patients was treated using the MTD of docetaxel. RESULTS: Twenty patients were enrolled. Planned accrual was 25, but the study was closed prematurely because of slow accrual. The MTD was 15 mg/m2. Dose-limiting toxicity was oral stomatitis. Therapy was well tolerated. Five patients experienced locoregional relapse at a median follow-up of 32 months. CONCLUSION: Docetaxel with concurrent RT has acceptable toxicity. This approach warrants further investigation in a phase II trial.
Asunto(s)
Carcinoma de Células Escamosas/secundario , Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeza y Cuello/terapia , Traumatismos por Radiación/prevención & control , Radioterapia de Intensidad Modulada/métodos , Taxoides/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/mortalidad , Terapia Combinada , Intervalos de Confianza , Docetaxel , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Femenino , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Invasividad Neoplásica/patología , Estadificación de Neoplasias , Probabilidad , Pronóstico , Estudios Prospectivos , Dosificación Radioterapéutica , Radioterapia Adyuvante , Medición de Riesgo , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
During spermatogenesis, leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) to gain entry into the adluminal compartment for further development. At the same time, these as well as other germ cell types in the epithelium must retain their close association with Sertoli cells via specialized cell junctions. In this study, we demonstrate that RAB13-a guanosine triphosphatase (GTPase) known to participate in tight junction function in other epithelia-also participates in the dynamics of the ectoplasmic specialization, a testis-specific type of anchoring junction. By immunohistochemistry microscopy, RAB13 localized to the ectoplasmic specialization. Moreover, RAB13 was found to associate with vinculin (VCL) and espin (ESPN), two putative ectoplasmic specialization actin (ACT)-binding proteins, by coimmunoprecipitation and immunofluorescence microscopy experiments. To address the role of RAB13 in ectoplasmic specialization dynamics, an in vivo model was used in which administration of Adjudin induced the disassembly of Sertoli-germ cell anchoring junctions. Following administration of this drug, the RAB13 level decreased steadily when the loss in testicular weight was taken into account. Similarly, the association of RAB13 with VCL decreased but was not completely lost during Adjudin-mediated ectoplasmic specialization restructuring. Taken collectively, these results suggest that RAB13 functions in ectoplasmic specialization dynamics in the testis.
Asunto(s)
Uniones Intercelulares/metabolismo , Células de Sertoli/citología , Espermatocitos/citología , Testículo/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Células Cultivadas , Citoesqueleto/efectos de los fármacos , ADN Complementario/metabolismo , GTP Fosfohidrolasas/metabolismo , Hidrazinas/farmacología , Indazoles/farmacología , Uniones Intercelulares/efectos de los fármacos , Masculino , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Epitelio Seminífero/citología , Epitelio Seminífero/efectos de los fármacos , Epitelio Seminífero/metabolismo , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Espermatocitos/efectos de los fármacos , Espermatocitos/metabolismo , Testículo/citología , Testículo/efectos de los fármacos , Vinculina/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
A phase I/II trial was undertaken to determine the maximum tolerated dose of polyethylene glycol interferon-alpha-2b (PEG-IFN) with interleukin-2 (IL-2), and to evaluate the efficacy and toxicity in patients with metastatic renal cell carcinoma. Patients initially received subcutaneous PEG-IFN, 3.0 mcg/kg/wk, combined with IL-2, but owing to unexpected toxicity a revised phase I schedule ensued. Patients received 1.0, 1.5, 2.0, or 3.0 mcg/kg/wk of PEG-IFN on days 1, 8, 15, and 22; subcutaneous IL-2 was given at a dose of 5 x 10 IU/m2 every 8 hours x 3 on day 1, followed daily at 5 x 10 IU/m2 days 2, 3, 4, and 5 of week 1, then 5 times per week for 3 weeks, followed by 2 weeks off. The maximum tolerated dose of PEG-IFN was 2.0 mcg/kg/wk. Fifty-four patients were enrolled. Frequent grade III/IV cardiac and neurologic toxicities led to an expanded phase I trial. Eleven serious events in 33 patients in the phase II portion led to early termination. No patient died from treatment. The overall response rate in 53 evaluable patients was 30.2% (95% confidence interval 20.5-39.9), with 2 complete responses and 14 partial responses and at least 1 response at each dose level. The median duration of response was 11 months (range, 2 to 65+ mo); median survival was 20 months (range, 2 to 71+ mo); median time to progression was 4 months. Despite clinical efficacy, the study was closed prematurely owing to excess toxicity. Although all serious adverse events resolved, this degree of toxicity is unacceptable for an outpatient treatment regimen.
Asunto(s)
Carcinoma de Células Renales/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Interleucina-2/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Pacientes Ambulatorios , Lesión Renal Aguda/inducido químicamente , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/patología , Cardiomiopatías/inducido químicamente , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/efectos adversos , Interleucina-2/administración & dosificación , Interleucina-2/efectos adversos , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Polietilenglicoles , Proteínas Recombinantes , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
A plethora of evidence has recently accumulated to suggest that Rab guanosine triphosphates (GTPases) may have functions other than those originally proposed in vesicle formation, movement, docking, and fusion. Studies have shown, for example, that Rab proteins interact with actin filaments and microtubules, illustrating cross-talk between intracellular transport and cytoskeletal dynamics. In this report, we show that Rab4A associates with adherens junction signaling proteins in the testis. By immunoprecipitation, Rab4A was found to interact with alpha- and beta-catenin as well as with actin, vimentin, alpha- and beta-tubulin, and protein kinase C (PKC)-alpha and -epsilon. Additionally, administration of Adjudin to adult rats up-regulated the Rab4A level, which coincided with the loss of spermatocytes, round and elongating/elongated spermatids from the seminiferous epithelium. More importantly, the ability of Rab4A to associate with alpha- and beta-catenin increased during Adjudin-induced junction restructuring in the testis, illustrating that Rab4A-catenin interactions are likely to be involved in the disassembly of Sertoli-germ cell contacts. Taken collectively, these results suggest that Rab4A participates in adherens junction dynamics.
Asunto(s)
Uniones Adherentes/metabolismo , Testículo/metabolismo , alfa Catenina/metabolismo , beta Catenina/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Uniones Adherentes/efectos de los fármacos , Animales , Citoesqueleto/metabolismo , Hidrazinas/farmacología , Inmunoprecipitación , Indazoles/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Testículo/efectos de los fármacosRESUMEN
The cyclic change in hormonal profiles between the two main phases of the menstrual cycle mediate shifts in mate preference. Males who advertise social dominance are preferred over other men by females in the follicular phase of the cycle. The present study explored assignment of high or low status resources to dominant looking men by females in either phase of the menstrual cycle. Thirteen females who reported that they were free from any kind of hormonal intervention and experienced a 28 day cycle, were invited to participate in a mock job negotiation scenario. Participants were asked to assign either a minimum, low, high or maximum social status job package to a series of male 'employees' that were previously rated to look either dominant or non-dominant. The results showed that during the follicular phase of the cycle participants assigned dominant looking men more high status job resources than the non-dominant looking men. However, during the luteal phase the participants assigned low status resources to the non-dominant looking men. Females are not merely passive observers of male status cues but actively manipulate the environment to assign status.
Asunto(s)
Conducta de Elección/fisiología , Toma de Decisiones/fisiología , Ciclo Menstrual/psicología , Predominio Social , Percepción Social , Adulto , Análisis de Varianza , Femenino , Humanos , Ciclo Menstrual/fisiología , Estimulación LuminosaRESUMEN
For the past several years, studies from other laboratories, as well as ours, have begun to unravel the mechanism of germ cell movement in the testis by using several in vitro and in vivo models of tight and adherens junction assembly and disassembly, two cellular phenomena that confer cell movement. However, for cell movement to be fully appreciated, the importance of "intracellular" cell movements, such as those involving actin and microtubule filaments, must be better understood. Recent research on Rab GTPases has shown that members of this superfamily function in the trafficking of vesicles containing cargo to distinct subcellular sites such as the plasma membrane while utilizing actin and microtubule filaments as tracks. In this mini-review, we provide an overview of Rab GTPase structure, function, and regulation, while placing added emphasis on the role of Rabs in cell junction dynamics in the testis.
Asunto(s)
Uniones Intercelulares/fisiología , Proteínas de Unión al GTP rab/fisiología , Animales , Transporte Biológico , Citoesqueleto/ultraestructura , Endocitosis , Exocitosis , Homeostasis , Humanos , Masculino , Fosforilación , Prenilación de Proteína , Relación Estructura-Actividad , Testículo/ultraestructura , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/clasificaciónRESUMEN
Using multiple high-performance liquid chromatography steps, we have identified and purified a polypeptide to apparent homogeneity from primary Sertoli cell conditioned culture medium that consisted of 2 molecular variants of 31 and 29 kDa when electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel run under reducing conditions. Partial N-terminal amino acid sequence analysis of these 2 proteins revealed a sequence of NH(2)-IKMAKMLKGFDAVGNATG, which is homologous to tissue inhibitor of metalloproteases-1 (TIMP-1). Studies by semiquantitative reverse transcription-polymerase chain reaction using a primer pair specific to rat TIMP-1 demonstrated that both Sertoli and germ cells express TIMP-1. During maturation, the steady-state TIMP-1 mRNA level in the testis increased significantly from 40 to 60 days of age, which suggests its role in the restructuring of the epithelium during spermiation. This increase in testicular TIMP-1 expression was apparently not due to the increase in germ cell number, because TIMP-1 expression decreased approximately fivefold in germ cells isolated from testes of aging rats. Using Sertoli cells cultured at low (0.05 x 10(6) cells/cm(2)) and high (0.5 x 10(6) cells/cm(2)) densities, it was found that TIMP-1 expression increased transiently but significantly during junction assembly. A similar induction of TIMP-1 mRNA was also detected in Sertoli-germ cell cocultures during germ cell adhesion onto Sertoli cells. More important, the inclusion of either alpha(2)-macroglobulin (a protease inhibitor produced by Sertoli cells) or aprotinin (a serine protease inhibitor) into an in vitro germ cell adhesion assay facilitated the attachment of fluorescently labeled germ cells onto the Sertoli cell epithelium when compared to control, which suggests that the assembly of adherens junctions may involve protease inhibitors.
Asunto(s)
Uniones Adherentes/metabolismo , Células de Sertoli/citología , Espermatocitos/citología , Uniones Estrechas/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular/fisiología , Células Cultivadas , Expresión Génica , Masculino , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Células de Sertoli/enzimología , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/aislamiento & purificaciónRESUMEN
Throughout spermatogenesis, germ cells migrate from the basal to the adluminal compartment while remaining attached to Sertoli cells via actin-based adherens and intermediate filament-based anchoring junctions. However, the events that trigger deadhesion and adhesion remain largely unknown. As part of our continued effort in elucidating the mechanism of germ cell movement, we have examined the role of Rab8B, a GTPase probably participating in intracellular trafficking events at the site of the adherens junction. By RT-PCR Rab8B mRNA was found in the brain, testis, heart, kidney, and spleen. Immunohistochemical studies revealed that Rab8B was concentrated predominantly in the basal compartment, localizing to a similar site at which immunoreactive E-cadherin was found. Additional experiments demonstrated that Rab8B associated with the actin, intermediate filament, and microtubule cytoskeletal networks. When Sertoli cells were cultured at high density or germ cells were cocultured with Sertoli cells, Rab8B increased significantly during junction assembly. Moreover, inclusion of germ cell-conditioned medium in Sertoli cell cultures resulted in stimulation of Rab8B expression. Conversely, treatment of adult rats with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide reduced Rab8B mRNA and protein levels, coinciding with the time of germ cell loss from the epithelium. Taken collectively, these studies suggest that Rab8B participates in adherens junction dynamics in the testis.