RESUMO
Abstract Chronic stress (CS) can contribute to dysfunction in several organs including liver and kidney. This study was performed to investigate the changes in serum biochemistry, histological structure, as well as in localization of tyrosine phosphorylated proteins (TyrPho) and Heat shock protein 70 (Hsp-70) in liver and kidney tissues of CS rats induced by two stressors (restrained and force swimming) for 60 consecutive days. Samples of blood, liver, and kidney were collected from adult male SpragueDawley rats in each group. Our results showed that serum biochemical parameters including corticosterone, blood sugar, urea nitrogen, creatinine, cholesterol, triglyceride, HDL-C, LDL-C, ALT, AST, alkaline phosphatase in CS group were significantly different from that in normal group in both liver and kidney tissues. Although histological structure was not changed. TyrPho expression was significantly increased in liver lysate but significantly decreased in kidney. Hsp-70 expression in liver increased whereas in kidney decreased. In conclusion, CS can induce changes in liver and kidney functions.
Resumo O estresse crônico (SC) pode contribuir para a disfunção em vários órgãos, incluindo fígado e rim. Este estudo foi realizado para investigar as alterações na bioquímica sérica, estrutura histológica, bem como na localização de proteínas tirosina fosforiladas (TyrPho) e proteína de choque térmico 70 (Hsp-70) em tecidos hepáticos e renais de ratos CS induzidas por dois estressores (restrito e natação forçada) por 60 dias consecutivos. Amostras de sangue, fígado e rim foram coletadas de ratos Sprague-Dawley machos adultos em cada grupo. Nossos resultados mostraram que os parâmetros bioquímicos séricos, incluindo corticosterona, glicemia, nitrogênio ureico, creatinina, colesterol, triglicerídeos, HDL-C, LDL-C, ALT, AST, fosfatase alcalina no grupo CS foram significativamente diferentes do grupo normal em ambos os fígados e tecidos renais. Embora a estrutura histológica não tenha sido alterada, a expressão de TyrPho aumentou significativamente no lisado hepático, mas diminuiu significativamente no rim. A expressão de Hsp-70 no fígado aumentou, enquanto que no rim diminuiu. Em conclusão, a CS pode induzir alterações nas funções hepáticas e renais.
RESUMO
Chronic stress (CS) can contribute to dysfunction in several organs including liver and kidney. This study was performed to investigate the changes in serum biochemistry, histological structure, as well as in localization of tyrosine phosphorylated proteins (TyrPho) and Heat shock protein 70 (Hsp-70) in liver and kidney tissues of CS rats induced by two stressors (restrained and force swimming) for 60 consecutive days. Samples of blood, liver, and kidney were collected from adult male Sprague-Dawley rats in each group. Our results showed that serum biochemical parameters including corticosterone, blood sugar, urea nitrogen, creatinine, cholesterol, triglyceride, HDL-C, LDL-C, ALT, AST, alkaline phosphatase in CS group were significantly different from that in normal group in both liver and kidney tissues. Although histological structure was not changed. TyrPho expression was significantly increased in liver lysate but significantly decreased in kidney. Hsp-70 expression in liver increased whereas in kidney decreased. In conclusion, CS can induce changes in liver and kidney functions.
O estresse crônico (SC) pode contribuir para a disfunção em vários órgãos, incluindo fígado e rim. Este estudo foi realizado para investigar as alterações na bioquímica sérica, estrutura histológica, bem como na localização de proteínas tirosina fosforiladas (TyrPho) e proteína de choque térmico 70 (Hsp-70) em tecidos hepáticos e renais de ratos CS induzidas por dois estressores (restrito e natação forçada) por 60 dias consecutivos. Amostras de sangue, fígado e rim foram coletadas de ratos Sprague-Dawley machos adultos em cada grupo. Nossos resultados mostraram que os parâmetros bioquímicos séricos, incluindo corticosterona, glicemia, nitrogênio ureico, creatinina, colesterol, triglicerídeos, HDL-C, LDL-C, ALT, AST, fosfatase alcalina no grupo CS foram significativamente diferentes do grupo normal em ambos os fígados e tecidos renais. Embora a estrutura histológica não tenha sido alterada, a expressão de TyrPho aumentou significativamente no lisado hepático, mas diminuiu significativamente no rim. A expressão de Hsp-70 no fígado aumentou, enquanto que no rim diminuiu. Em conclusão, a CS pode induzir alterações nas funções hepáticas e renais.
Assuntos
Ratos , Estresse Fisiológico , Ratos Sprague-Dawley , Rim/anatomia & histologia , Fígado/anatomia & histologiaRESUMO
Endothelial nitric oxide synthase (eNOS) and receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) are one of the majors signaling pathways related to endothelial health in diabetes. Several reports have shown that the inhibition of VE-PTP can lead the nitric oxide production, although repeated studies showed that VE-PTP regulated the eNOS exclusive at Ser1177 in indirect-manner. A recent, exciting paper (Siragusa et al. in Cardiovasc Res, 2020. https://doi.org/10.1093/cvr/cvaa213 ), showing that VE-PTP regulates eNOS in a direct-manner, dephosphorylating eNOS at Tyr81 and indirect at Ser1177 and the effects of a VE-PTP inhibitor, AKB-9778, in the blood pressure from diabetic patients.
RESUMO
Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.
Assuntos
Citosol/enzimologia , Retina/enzimologia , Quinases da Família src/isolamento & purificação , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia/métodos , Eletroforese em Gel de Poliacrilamida , Peptídeos/metabolismo , Fosforilação , Análise de Sequência de Proteína/métodos , Especificidade por Substrato , Quinases da Família src/químicaRESUMO
Letrozole (Letro) is a drug commonly used for breast cancer treatment since it can decrease estrogen level. In experimental animal, the Letro has been used to induce the polycystic ovarian syndrome (PCOS) model. Tyrosine phosphorylation (TyrPho) is an essential process in various biological functions both normal and abnormal conditions especially reproduction. Although some side effects of Letro are reported, the alterations of TyrPho responsible for liver and kidney functions have never been demonstrated. In this study, the blood serum, liver, and kidney of control and PCOS rats induced with Letro (orally, 1 mg/ KgBW) for consecutive 21 days were used to determine the serum biochemical components and to investigate the TyrPho expression using western blot analysis. Histopathology of such tissues was observed by Masson's trichrome staining. The results showed that Letro did not affect histological structures but significantly increased the serum levels of urea nitrogen, cholesterol, triglyceride, HDL, LDL, ALT, AST, and alkaline phosphatase. Additionally, the TyrPho protein expressions of 32 and 27 kDas in liver and of 55 and 43 kDas in kidney were increased while of a kidney 26 kDa was decreased as compared to those of control. In conclusion, this recent study indicated that the changes of TyrPho proteins in liver and kidney induced with Letro associated with their functions by alteration of serum biochemical levels.
El letrozol (Letro) es un medicamento utilizado comúnmente para el tratamiento del cáncer de mama, debido a que puede disminuir el nivel de estrógeno. En animales de experimentación, el Letro se ha utilizado para inducir el modelo de síndrome de ovario poliquístico (PCOS). La fosforilación de tirosina (TyrPho) es un proceso esencial en diversas funciones biológicas, tanto en condiciones normales como anormales, especialmente en la reproducción. A pesar de informes que indican algunos efectos secundarios de Letro, no se han demostrado las alteraciones de TyrPho responsables de las funciones hepáticas y renales. En este estudio, el suero sanguíneo, el hígado y el riñón control y las ratas PCOS inducidas con Letro (por vía oral, 1 mg / KgBW) durante 21 días consecutivos se usaron para determinar los componentes bioquímicos del suero y para investigar la expresión de TyrPho usando análisis de transferencia Western. La histopatología de los tejidos se observó mediante la tinción tricrómica de Masson. Los resultados mostraron que Letro no afectó las estructuras histológicas, pero aumentó significativamente los niveles séricos de urea, colesterol, triglicéridos, HDL, LDL, ALT, AST y fosfatasa alcalina. Además, las expresiones de la proteína TyrPho de 32 y 27 kDas en el hígado y de 55 y 43 kDas en el riñón aumentaron mientras que en un riñón disminuyeron 26 kDa en comparación con el control. En conclusión, este estudio indicó que los cambios de las proteínas TyrPho en el hígado y los riñones inducidos con Letro se asociaron con sus funciones mediante la alteración de los niveles bioquímicos en suero.
Assuntos
Animais , Feminino , Ratos , Síndrome do Ovário Policístico/induzido quimicamente , Letrozol/efeitos adversos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fosforilação/fisiologia , Tirosina/metabolismo , Western Blotting , Ratos Wistar , Modelos Animais de Doenças , Eletroforese em Gel de PoliacrilamidaRESUMO
Low molecular weight protein tyrosine phosphatases (LMW-PTP) are ubiquitous enzymes found across a spectrum of genera from prokaryotes to higher eukaryotes. LMW-PTP belong to the Cys-based PTP class II protein family. Here, we show that LMW-PTP can be categorized into two different groups, referred as class II subdivision I (class II.I) and subdivision II (class II.II). Using BPtpA from the opportunistic pathogen Burkholderia cenocepacia, as a representative member of the LMW-PTP class II.I, we demonstrated that four conserved residues (W47, H48, D80, and F81) are required for enzyme function. Guided by an in silico model of BPtpA, we show that the conserved residues at α3-helix (D80 and F81) contribute to protein stability, while the other conserved residues in the W-loop (W47 and H48) likely play a role in substrate recognition. Overall, our results provide new information on LMW-PTP protein family and establish B. cenocepacia as a suitable model to investigate how substrates are recognized and sorted by these proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia cenocepacia/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Infecções por Burkholderia/microbiologia , Burkholderia cenocepacia/química , Humanos , Modelos Moleculares , Fosforilação , Proteínas Tirosina Fosfatases/químicaRESUMO
Cells can communicate with other neighboring or distant cells through the secretion of extracellular vesicles (EV), composed of a lipid bilayer and bearing surface molecules that allow them to recognize target cells. In this way, EV induce signaling via different mechanisms, modulating the physiological state of the recipient cell. EV have been identified in both male and female reproductive fluids, however, the possible role of EV isolated from female reproductive fluids has become an emerging field only recently. It is known that ejaculated mammalian spermatozoa need to undergo physiological preparation in the female reproductive tract to fertilize the egg. EV secreted by different regions of the female tract constitute signals that may have a key role in regulating sperm functions. The aims of the present study were isolating EV from different regions of the bovine oviduct and analyzing their interaction and physiological effects on spermatozoa. Here, we report the characterization of bovine oviductal fluid EV from the isthmus and ampulla region and their effect on the induced acrosome reaction and signaling events associated with sperm capacitation. EV induced an increase in sperm protein tyrosine phosphorylation, while cell survival of cryopreserved bovine spermatozoa was maintained. We also show that EV uptake regulates the sperm calcium levels by inducing an immediate increase in the intracellular calcium concentration and sperm priming, after a pre-incubation period, of the progesterone-induced intracellular calcium rise. Our data contribute to understand the role of EV in the communication between the female reproductive tract and the sperm physiology, information that may be used to improve the efficiency of reproductive assisted technologies.
Assuntos
Reação Acrossômica , Oviductos/metabolismo , Espermatozoides/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Sobrevivência Celular , Criopreservação , Ejaculação , Tubas Uterinas/metabolismo , Feminino , Luz , Masculino , Fosforilação , Espalhamento de Radiação , Transdução de Sinais , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides , Tirosina/químicaRESUMO
N-ethylmaleimide-sensitive factor (NSF) disassembles fusion-incompetent cis soluble-NSF attachment protein receptor (SNARE) complexes making monomeric SNAREs available for subsequent trans pairing and fusion. In most cells the activity of NSF is constitutive, but in Jurkat cells and sperm it is repressed by tyrosine phosphorylation; the phosphomimetic mutant NSF-Y83E inhibits secretion in the former. The questions addressed here are if and how the NSF mutant influences the configuration of the SNARE complex. Our model is human sperm, where the initiation of exocytosis (acrosome reaction (AR)) de-represses the activity of NSF through protein tyrosine phosphatase 1B (PTP1B)-mediated dephosphorylation. We developed a fluorescence microscopy-based method to show that capacitation increased, and challenging with an AR inducer decreased, the number of cells with tyrosine-phosphorylated PTP1B substrates in the acrosomal domain. Results from bioinformatic and biochemical approaches using purified recombinant proteins revealed that NSF-Y83E bound PTP1B and thereupon inhibited its catalytic activity. Mutant NSF introduced into streptolysin O-permeabilized sperm impaired cis SNARE complex disassembly, blocking the AR; subsequent addition of PTP1B rescued exocytosis. We propose that NSF-Y83E prevents endogenous PTP1B from dephosphorylating sperm NSF, thus maintaining NSF's activity in a repressed mode and the SNARE complex unable to dissociate. The contribution of this paper to the sperm biology field is the detection of PTP1B substrates, one of them likely being NSF, whose tyrosine phosphorylation status varies during capacitation and the AR. The contribution of this paper to the membrane traffic field is to have generated direct evidence that explains the dominant-negative role of the phosphomimetic mutant NSF-Y83E.
Assuntos
Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Fosforilação/fisiologia , Proteínas SNARE/metabolismo , Reação Acrossômica/fisiologia , Western Blotting , Catálise , Biologia Computacional , Exocitose/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Plasmídeos , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Espermatozoides/metabolismo , Tirosina/metabolismoRESUMO
Increased protein tyrosine phosphorylation and the appearance of a phosphorylated protein of 32 kD (p32) are reported among the capacitation-like changes in cryopreserved boar sperm. Egg yolk freezing extenders are composed by two fractions: insoluble granules and soluble plasma, which contains the low density lipoproteins (LDL) proposed as responsible for the egg yolk cryoprotective action. The aim of this work was to analyze the effects of complete egg yolk and its insoluble, soluble and LDL fractions on boar sperm quality and protein tyrosine phosphorylation after the first stage of a standard cryopreservation protocol. Semen samples in Androstar® Plus diluent were centrifuged and resuspended in the different egg yolk extenders. Temperature was decreased from 17⯰C to 5⯰C and sperm quality, protein tyrosine phosphorylation and protein pattern were analyzed. Results showed that complete egg yolk as well as soluble and LDL egg yolk fractions maintained sperm quality after temperature decrease. Cooling without any lipid component or in the presence of the insoluble fraction, significantly reduced sperm motility. About sperm protein tyrosine phosphorylation analysis, the p32 band appeared before treatments or after cooling in Androstar® Plus diluent. Complete egg yolk and its insoluble fraction interfered with sperm tyrosine phosphorylation even after cells were extensively washed. Analysis of extenders revealed a high amount of tyrosine phosphorylated proteins in the insoluble fraction, which may have co-precipitate with sperm in experiments. Samples submitted to temperature decrease from 17⯰C to 5⯰C in the presence of soluble and LDL egg yolk fractions in Androstar® Plus diluent did not show any change in the p32 band associated with sperm capacitation. However, a tyrosine-phosphorylated protein of 33 kD present in clarified egg yolk was also observed in sperm treated with this extender. Protein transference from plasma and LDL egg yolk extenders was also observed in sperm protein profile. Results suggested that soluble and LDL fractions might have a protective action preventing sperm protein tyrosine phosphorylation during cooling from 17⯰C to 5⯰C. Further studies are needed to expand the knowledge of the LDL protection mechanism as well as to determine the possible benefits of clarified egg yolk in freezing protocols.
Assuntos
Crioprotetores/farmacologia , Lipoproteínas/farmacologia , Espermatozoides/metabolismo , Suínos , Tirosina/metabolismo , Animais , Crioprotetores/química , Lipoproteínas/química , Masculino , Proteínas de Membrana , Fosfoproteínas , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , TemperaturaRESUMO
Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca2+ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca 2+ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca 2+ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca 2+ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca 2+ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca 2+ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH 4 Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca 2+ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Capacitação Espermática , Espermatozoides/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Quinase 2 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Potenciais da Membrana , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , TirosinaRESUMO
SUMMARY: Valproic acid (VPA), an antiepileptic drug, has been demonstrated to damage histology and to change tyrosine phosphorylation patterns with increased oxidative stress in perirenal tissues. This study aimed to investigate the effect of VPA on microstructure, tyrosine phosphorylation, and lipid peroxidation of rat kidney. Adult male rats were divided into control and VPA-treated groups intraperitoneally injected with normal saline and VPA 500 mg/kgBW for 10 consecutive days, respectively (n = 7 each). The blood serum was examined for biochemical levels. The kidney tissues were routinely processed for histological observation. Total proteins from kidney were extracted to assay the malondialdehyde (MDA) levels and phosphorylation expression. The results showed that VPA significantly decreased blood glucose levels while tend to increase urea nitrogen and creatinine. MDA levels in VPA group were significantly higher that of control. Renal cortex of VPA-treated animals revealed vasodilatations. Although the ratio of a renal phosphorylated 72 kDa protein/ beta actin expression seemed to be not different in both groups, VPA significantly decreased the intensity of beta actin. In conclusion, VPA dilates renal microvasculature with increasing of MDA but suppresses the actin expression.
RESUMEN: Se ha demostrado que el ácido valproico (AVP), un fármaco antiepiléptico, daña la histología y cambia los patrones de fosforilación de la tirosina con el aumento del estrés oxidativo en los tejidos perirrenales. Este estudio tuvo como objetivo investigar el efecto del AVP en la microestructura, la fosforilación de la tirosina y la peroxidación lipídica del riñón de rata. Se dividieron ratas macho adultas en grupos control y tratados con AVP. Durante 10 días consecutivos fueron inyectadas por vía intraperitoneal con solución salina normal y 500 mg / kg de PC respectivamente (n = 7 cada uno). Se analizó el suero sanguíneo para determinar los niveles bioquímicos. Los tejidos renales se procesaron de forma rutinaria para la observación histológica. Las proteínas totales del riñón se extrajeron para analizar los niveles de malondialdehído (MDA) y la expresión de la fosforilación. Los resultados mostraron que el AVP disminuyó significativamente los niveles de glucosa en la sangre, mientras que tienden a aumentar el nitrógeno ureico y la creatinina. Los niveles de MDA en el grupo de AVP fueron significativamente más altos que los del control. La corteza renal de los animales tratados con AVP reveló vasodilataciones. Aunque la proporción de una expresión de proteína / actina de 72 kDa fosforilada renal no parece ser diferente en ambos grupos, el AVP disminuyó significativamente la intensidad de la actina beta. En conclusión, el AVP dilata la microvasculatura renal al aumentar el MDA, pero suprime la expresión de actina.
Assuntos
Animais , Masculino , Ratos , Tirosina/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Ácido Valproico/farmacologia , Rim/efeitos dos fármacos , Anticonvulsivantes/farmacologia , Tamanho do Órgão , Fosforilação , Vasodilatação/efeitos dos fármacos , Western Blotting , Ratos Wistar , Eletroforese em Gel de Poliacrilamida , MalondialdeídoRESUMO
STUDY QUESTION: Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? SUMMARY ANSWER: Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. WHAT IS KNOWN ALREADY: Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. STUDY DESIGN SIZE, DURATION: Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. MAIN RESULTS AND THE ROLE OF CHANCE: Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported DGAPA (IN203116 to C. Treviño), Fronteras-CONACyT No. 71 and Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH (RO1 HD38082) to P.E. Visconti and by a Lalor Foundation fellowship to M.G. Gervasi. A. Matamoros is a student of the Maestría en Ciencias Bioquímicas-UNAM program supported by CONACyT (416400) and DGAPA-UNAM. A. Moreno obtained a scholarship from Red MacroUniversidades and L. Giojalas obtained a schloarhip from CONICET and Universidad Nacional de Cordoba. The authors declare there are not conflicts of interest.
Assuntos
Citometria de Fluxo/métodos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Tirosina/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Quinase 2 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/metabolismo , Immunoblotting , Masculino , Fosforilação/efeitos dos fármacos , Quinolonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Capacitação Espermática , Motilidade dos Espermatozoides/efeitos dos fármacos , Sulfonas/farmacologiaRESUMO
Intrauterine growth retardation (IUGR) is associated with the development of adult-onset diseases, including pulmonary hypertension. However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular dysfunction later in life is not fully understood. Here, we investigated the role of tyrosine phosphorylation of voltage-gated potassium channel 1.5 (Kv1.5) in this prenatal event that results in exaggerated adult vascular dysfunction. A rat model of chronic hypoxia (2 weeks of hypoxia at 12 weeks old) following IUGR was used to investigate the physiological and structural effect of intrauterine malnutrition on the pulmonary artery by evaluating pulmonary artery systolic pressure and vascular diameter in male rats. Kv1.5 expression and tyrosine phosphorylation in pulmonary artery smooth muscle cells (PASMCs) were determined. We found that IUGR increased mean pulmonary artery pressure and resulted in thicker pulmonary artery smooth muscle layer in 14-week-old rats after 2 weeks of hypoxia, while no difference was observed in normoxia groups. In the PASMCs of IUGR-hypoxia rats, Kv1.5 mRNA and protein expression decreased while that of tyrosine-phosphorylated Kv1.5 significantly increased. These results demonstrate that IUGR leads to exaggerated chronic hypoxia pulmonary arterial hypertension (CH-PAH) in association with decreased Kv1.5 expression in PASMCs. This phenomenon may be mediated by increased tyrosine phosphorylation of Kv1.5 in PASMCs and it provides new insight into the prevention and treatment of IUGR-related CH-PAH.
Assuntos
Animais , Masculino , Feminino , Gravidez , Organofosfatos/metabolismo , Polímeros/metabolismo , Canal de Potássio Kv1.5/análise , Hipóxia Fetal/complicações , Hipóxia Fetal/fisiopatologia , Retardo do Crescimento Fetal/metabolismo , Hipertensão Pulmonar/etiologia , Músculo Liso Vascular/química , Fosforilação , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Artéria Pulmonar/fisiopatologia , Artéria Pulmonar/patologia , Fatores de Tempo , RNA Mensageiro/análise , Imuno-Histoquímica , Immunoblotting , Distribuição Aleatória , Regulação para Cima , Imunofluorescência , Ratos Sprague-Dawley , Desnutrição/complicações , Modelos Animais de Doenças , Retardo do Crescimento Fetal/etiologia , Reação em Cadeia da Polimerase em Tempo Real , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/patologia , Músculo Liso Vascular/patologiaRESUMO
The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, ß-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and ß-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and ß-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and ß-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.
Assuntos
Núcleo Celular/metabolismo , Receptores ErbB/metabolismo , Transporte Ativo do Núcleo Celular , Betacelulina/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Neoplasias/metabolismo , Fosforilação , Fator de Crescimento Transformador alfa/metabolismoRESUMO
La Organización Mundial de la Salud en su edición 2010 ha publicado valores de referencia para los parámetros seminales. Sin embargo, los laboratorios deben verificarlos en su población para transferirlos a su práctica clínica. Para cumplimentar este requerimiento se propuso verificar los intervalos de referencia OMS 2010 en la población de la provincia y Ciudad Autónoma de Buenos Aires. También se determinó el rango de resultados para: número total de espermatozoides móviles progresivos y morfología normal en el eyaculado, parámetros cinéticos y pruebas funcionales. El proceso de verificación fue llevado a cabo de acuerdo a la guía C28-A2 CLSI (Clinical and Laboratory Standards Institute). Mediante difusión se convocó a hombres (n:20) argentinos residentes en Buenos Aires, con fertilidad probada en los últimos 12 meses. Se excluyeron individuos con patología andrológica. Las muestras se procesaron de acuerdo con los criterios OMS 2010. De los resultados se establece que los intervalos de referencia publicados en la última versión han sido verificados, con excepción del volumen seminal. Los rangos de valores obtenidos para los parámetros de movilidad progresiva y morfología en número totales, como también de los parámetros cinéticos y de las pruebas funcionales contribuirán a establecer futuros valores de referencia.
WHO 2010 provided reference values for semen analysis; however clinical laboratories should verify them in healthy fertile males. In order to be able to transfer this reference interval to our unit, it was proposed to examine the available data in the Buenos Aires population. The range of data obtained in this population was also provided for the following: total number of progressively motile and morphologically normal spermatozoa in the ejaculate,nematic parameter values and functional tests, in an attempt to establish standardization in these parameters. The process of reference value verification was carried out according to C28-A2 CLSI (Clinical and Laboratory Standards Institute) guidelines; to that aim, twenty men born in Argentina who reside in Buenos Aires province or city were recruited to participate. They were of proven fertility in the last 12 months. Individuals with pathologies by andrological consultation were excluded. Samples were processed according to 2010 WHO. In Buenos Aires City, WHO reference values were confirmed whereas semen volume was not confirmed. The range of values for total progressive and morphologically normal spermatozoa as well as kinetic parameter values, enrichment by swim up and functional test results were established for the male fertile population studied in an attempt to contribute to the construction of future reference values.
Organização Mundial da Saúde, em sua edição de 2010, tem publicado valores de referência para os parâmetros seminais. No entanto, os laboratórios devem verificá-los em sua população para serem transferidos a sua prática clínica. Para cumprir com esse requisito, decidiu-se verificar os intervalos de referência da OMS 2010, na população da província e da cidade de Buenos Aires. Também foram definidos intervalos de resultados para o número total de espermatozoides móveis progressivos e para a morfologia normal no ejaculado, os parâmetros cinéticos e os testes funcionáis. O processo de verificação foi realizado de acordo com o guia CLSI C28 A2 (Clinical and Laboratory Standards Institute). Por meio de uma convocação, (N 20) homens argentinos residentes em Buenos Aires, com comprovada fertilidade nos últimos 12 meses foram convidados. Indivíduos com patologia andrológica foram excluídos. As amostras foram processadas de acordo com os critérios da OMS de 2010. A partir dos resultados estabeleceu-se que os intervalos de referência publicados na versão mais recente foram verificados, com exceção do volume seminal. Os intervalos de valores obtidos para os parâmetros de motilidade progressiva e morfologia em número total, bem como os parâmetros cinéticos e os dos testes funcionais ajudarão a estabelecer valores de referência futura.
Assuntos
Humanos , Masculino , Sêmen , Organização Mundial da Saúde , Infertilidade , Valores de ReferênciaRESUMO
La Organización Mundial de la Salud en su edición 2010 ha publicado valores de referencia para los parámetros seminales. Sin embargo, los laboratorios deben verificarlos en su población para transferirlos a su práctica clínica. Para cumplimentar este requerimiento se propuso verificar los intervalos de referencia OMS 2010 en la población de la provincia y Ciudad Autónoma de Buenos Aires. También se determinó el rango de resultados para: número total de espermatozoides móviles progresivos y morfología normal en el eyaculado, parámetros cinéticos y pruebas funcionales. El proceso de verificación fue llevado a cabo de acuerdo a la guía C28-A2 CLSI (Clinical and Laboratory Standards Institute). Mediante difusión se convocó a hombres (n:20) argentinos residentes en Buenos Aires, con fertilidad probada en los últimos 12 meses. Se excluyeron individuos con patología andrológica. Las muestras se procesaron de acuerdo con los criterios OMS 2010. De los resultados se establece que los intervalos de referencia publicados en la última versión han sido verificados, con excepción del volumen seminal. Los rangos de valores obtenidos para los parámetros de movilidad progresiva y morfología en número totales, como también de los parámetros cinéticos y de las pruebas funcionales contribuirán a establecer futuros valores de referencia.(AU)
WHO 2010 provided reference values for semen analysis; however clinical laboratories should verify them in healthy fertile males. In order to be able to transfer this reference interval to our unit, it was proposed to examine the available data in the Buenos Aires population. The range of data obtained in this population was also provided for the following: total number of progressively motile and morphologically normal spermatozoa in the ejaculate, kinematic parameter values and functional tests, in an attempt to establish standardization in these parameters. The process of reference value verification was carried out according to C28-A2 CLSI (Clinical and Laboratory Standards Institute) guidelines; to that aim, twenty men born in Argentina who reside in Buenos Aires province or city were recruited to participate. They were of proven fertility in the last 12 months. Individuals with pathologies by andrological consultation were excluded. Samples were processed according to 2010 WHO. In Buenos Aires City, WHO reference values were confirmed whereas semen volume was not confirmed. The range of values for total progressive and morphologically normal spermatozoa as well as kinetic parameter values, enrichment by swim up and functional test results were established for the male fertile population studied in an attempt to contribute to the construction of future reference values.(AU)
A OrganizaþÒo Mundial da Saúde, em sua ediþÒo de 2010, tem publicado valores de referÛncia para os parÔmetros seminais. No entanto, os laboratórios devem verificá-los em sua populaþÒo para serem transferidos a sua prática clínica. Para cumprir com esse requisito, decidiu-se verificar os intervalos de referÛncia da OMS 2010, na populaþÒo da província e da cidade de Buenos Aires. Também foram definidos intervalos de resultados para o número total de espermatozoides móveis progressivos e para a morfologia normal no ejaculado, os parÔmetros cinéticos e os testes funcionáis. O processo de verificaþÒo foi realizado de acordo com o guia CLSI C28 A2 (Clinical and Laboratory Standards Institute). Por meio de uma convocaþÒo, (N 20) homens argentinos residentes em Buenos Aires, com comprovada fertilidade nos últimos 12 meses foram convidados. Indivíduos com patologia andrológica foram excluídos. As amostras foram processadas de acordo com os critérios da OMS de 2010. A partir dos resultados estabeleceu-se que os intervalos de referÛncia publicados na versÒo mais recente foram verificados, com exceþÒo do volume seminal. Os intervalos de valores obtidos para os parÔmetros de motilidade progressiva e morfologia em número total, bem como os parÔmetros cinéticos e os dos testes funcionais ajudarÒo a estabelecer valores de referÛncia futura.(AU)
RESUMO
Sperm capacitation involves an increase in intracellular Ca(2+) concentration as well as in protein kinase A (PKA)-dependent protein tyrosine (Tyr) phosphorylation. Interestingly, in humans, a decrease in extracellular Ca(2+) concentration ([Ca(2+)]e) during capacitation induces an increase in Tyr phosphorylation indicating the complexity of Ca(2+) signaling during this process. In view of this, in the present study we further investigated the Ca(2+)-mediated signaling pathways implicated in Tyr phosphorylation during human sperm capacitation. Results revealed that sperm incubation in a medium without added Ca(2+) (â Ca(2+)) increased Tyr phosphorylation but did not modify PKA-mediated phosphorylation. Moreover, inhibition of either PKA or Src family kinase signaling cascades in â Ca(2+) down-regulated both PKA substrate and Tyr phosphorylations, indicating that the [Ca(2+)]e effects on Tyr phosphorylation depend on PKA targets. Inhibition of calmodulin or Ser/Thr protein phosphatase 2B also increased Tyr phosphorylation without affecting PKA-mediated phosphorylation, supporting the potential role of these Ca(2+) downstream effectors in the increase in Tyr phosphorylation observed in â Ca(2+). Experiments aimed to identify the kinase responsible for these observations revealed the presence of proline-rich tyrosine kinase 2 (PYK2), a focal adhesion kinase (FAK) family member, in human sperm, and the use of PF431396, an FAK inhibitor, supported the involvement of PYK2 in Tyr phosphorylation downstream of PKA activation. Results also showed that PYK2 was activated in â Ca(2+) as well as during capacitation and that PF431396 affected capacitated sperm motility, acrosome reaction and ability to penetrate both mouse cumulus matrix and zona-free hamster eggs. Together, our observations support PYK2 as an intermediary component of Ca(2+) signaling between PKA-mediated and Tyr phosphorylations that is required for achieving functional human sperm capacitation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 2 de Adesão Focal/fisiologia , Capacitação Espermática/fisiologia , Tirosina/metabolismo , Sinalização do Cálcio , Ativação Enzimática , Quinase 2 de Adesão Focal/metabolismo , Humanos , FosforilaçãoRESUMO
Crotalus durissus terrificus snake venom (CdtV) has long-lasting anti-inflammatory properties and inhibits the spreading andphagocytic activity of macrophages. Crotoxin (CTX), the main component of CdtV, is responsible for these effects.Considering the role of neutrophils in the inflammatory response and the lack of information about the effect of CdtV onneutrophils, the aim of this study was to investigate the effect of CdtV and CTX on two functions of neutrophils, namelyphagocytosis and production of reactive oxygen species, and on the intracellular signaling involved in phagocytosis,particularly on tyrosine phosphorylation and rearrangements of the actin cytoskeleton. Our results showed that theincubation of neutrophils with CdtV or CTX, at different concentrations, or the subcutaneous injection of CdtV or CTX inrats two hours or one, four or 14 days before or one hour after the induction of inflammation inhibited the phagocyticactivity of neutrophils. Furthermore, these in vitro and in vivo effects were associated with CdtV and CTX inhibition oftyrosine phosphorylation and consequently actin polymerization. Despite the inhibitory effect on phagocytosis, this study demonstrated that CdtV and CTX did not alter the production of the main reactive oxygen species. Therefore, this studycharacterized, for the first time, the actions of CdtV on neutrophils and demonstrated that CTX induces a long-lasting inhibition of tyrosine phosphorylation and consequently phagocytosis. We suggest that CTX represents a potential naturalproduct in controlling inflammatory diseases, since a single dose exerts a long-lasting effect on intracellular signaling involved in phagocytosis by neutrophils.
Assuntos
Animais , Antivenenos , Crotalus cascavella , Crotoxina/antagonistas & inibidores , Venenos de SerpentesRESUMO
Maspin is a tumor suppressor with many biological activities, multiple ligands and different subcellular localizations. Its underlying molecular mechanism remains elusive. We hypothesized that phosphorylation might regulate maspin localization and function. Using two-dimensional gel electrophoresis with different focusing power followed by Western blot we identified four different maspin forms with the same molecular weight (42 kDa), but different isoelectric points. Three of these forms were sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated. Sodium peroxidovanadate treatment, a protein-tyrosine phosphatase inhibitor, resulted in a rapid increase in maspin protein levels and cytoplasmic accumulation. These data show that there are three different maspin tyrosine phosphoforms. Inhibition of tyrosine phosphatases increased maspin protein levels and leads to its cytoplasmic accumulation.
RESUMO
Phosphorylation and dephosphorylation of protein tyrosine residues constitutes a major biochemical regulatory mechanism for the cell. We report a transient increase in the total tyrosine phosphorylation of the Aedes aegypti head during the first days after emergence from the pupal stage. This correlates with an initial reduction in total head protein tyrosine phosphatase (PTP) activity. Similarly, phosphotyrosine (pTyr)-containing bands are seen in extracts prepared from both male and female heads and are spread among a variety of structures including the antennae, proboscis and the maxillary palps combined with the proboscis. Also, mosquitoes treated with sodium orthovanadate, a classical PTP inhibitor, show reduced blood-feeding activity and higher head tyrosine phosphorylation levels. These results suggest that pTyr-mediated signalling pathways may play a role in the initial days following the emergence of the adult mosquito from the pupal stage.