RESUMO
Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.
Assuntos
Armadilhas Extracelulares , Neutrófilos , Espermatozoides , Animais , Cães , Armadilhas Extracelulares/metabolismo , Masculino , Espermatozoides/metabolismo , Neutrófilos/metabolismo , Motilidade dos Espermatozoides , Feminino , Elastase de Leucócito/metabolismo , Técnicas de Cocultura , Acrossomo/metabolismoRESUMO
Excessive levels of reactive nitrogen species (RNS), such as peroxynitrite, promote nitrosative stress, which is an important cause of impaired sperm function. The metalloporphyrin FeTPPS is highly effective in catalyzing the decomposition of peroxynitrite, reducing its toxic effects in vivo and in vitro. FeTPPS has significant therapeutic potential in peroxynitrite-related diseases; however, its effects on human spermatozoa under nitrosative stress have not been described. This work aimed to evaluate the in vitro effect of FeTPPS against peroxynitrite-mediated nitrosative stress in human spermatozoa. For this purpose, spermatozoa from normozoospermic donors were exposed to 3-morpholinosydnonimine, a molecule that generates peroxynitrite. First, the FeTPPS-mediated peroxynitrite decomposition catalysis was analyzed. Then, its individual effect on sperm quality parameters was evaluated. Finally, the effect of FeTPPS on ATP levels, motility, mitochondrial membrane potential, thiol oxidation, viability, and DNA fragmentation was analyzed in spermatozoa under nitrosative stress conditions. The results showed that FeTPPS effectively catalyzes the decomposition of peroxynitrite without affecting sperm viability at concentrations up to 50 µmol/L. Furthermore, FeTPPS mitigates the deleterious effects of nitrosative stress on all sperm parameters analyzed. These results highlight the therapeutic potential of FeTPPS in reducing the negative impact of nitrosative stress in semen samples with high RNS levels.
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Infectious vaginitis is a microbiological syndrome of great importance in public health that affects millions of women worldwide. However, no studies have explored the phenomenon of the production of the neutrophil extracellular traps (NETs) that are released into the female reproductive tract in these pathologies. This study aimed to determine the presence of NETosis in vaginal discharges of women with bacterial vaginosis, candidiasis, and trichomoniasis by characterizing NETs. Extracellular DNA with neutrophil elastase and citrullinated histones was identified to confirm the NET components (n = 10). The concentration, phenotypes of NETs, and number of NETotic cells were determined. The results showed an increase in NETotic cells in women with Candida albicans (CA) and Trichomonas vaginalis (TV) and an increase in NETs in TV-induced vaginitis. Samples of CA- and TV-infected women showed different NET phenotypes (diffNETs, sprNETs, and aggNETs); diffNETs were found in high concentrations in samples with CA and were increased in three types of NETs in TV infections. Samples with intermediate microbiota and bacterial vaginosis showed increased NETotic cells while the intermediate microbiota presented a higher concentration of NETs. Therefore, alterations in the microbiota and the presence of fungal and parasitic infections are important stimuli for the activation and induction of NETosis, and their cytotoxic effects could enhance tissue damage.
Assuntos
Candidíase Vulvovaginal , Armadilhas Extracelulares , Vaginite por Trichomonas , Trichomonas vaginalis , Descarga Vaginal , Vaginose Bacteriana , Feminino , Humanos , Vaginose Bacteriana/microbiologia , Elastase de Leucócito , Candidíase Vulvovaginal/microbiologia , Histonas , Vaginite por Trichomonas/microbiologia , Candida albicansRESUMO
Neutrophil extracellular traps (NETs) play a key role in fertilisation by eliminating microorganisms and entrapping spermatozoa in the female reproductive tract (FRT). The deleterious effects of NETs on spermatozoa have been previously described; however, individual exposure to NET-derived components in bull spermatozoa has not been explored. The aim of this study was to evaluate the effects of the main NET-derived proteins, histone 2A (H2A), neutrophil elastase (ELA), myeloperoxidase (MPO), pentraxin 3 (PTX), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), at concentrations of 1, 10, and 30 µg/mL, on sperm parameters. Sperm were selected and incubated with different NET-derived proteins for 4 h. Membrane and acrosome integrity, lipoperoxidation, and membrane phospholipid disorders were also evaluated. Bovine polymorphonuclear neutrophil (PMN)/sperm co-cultures were evaluated by scanning electron microscopy and immunofluorescence. All NET-derived proteins/enzymes resulted in a reduction in membrane integrity, acrosome integrity, and lipoperoxidation at a concentration of 30 µg/mL. Bovine PMN/sperm co-cultures showed marked NET formation in the second hour. In conclusion, all NET-derived proteins/enzymes exerted cytotoxic effects on bull sperm, and this effect should be considered in future investigations on the uterine microenvironment and the advancement of spermatozoa in the FRT.
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STUDY QUESTION: Does oxidative stress (OS) activate autophagy in human sperm? SUMMARY ANSWER: Human spermatozoa subjected to OS activate an autophagic response. WHAT IS KNOWN ALREADY: Autophagy is a regulated pathway of lysosomal degradation which helps eukaryotic cells to maintain or restore homeostasis, being a cellular stress response mechanism. OS is a main cause of impaired sperm function and is linked to male infertility; however, whether OS activates autophagy in human spermatozoa is unknown. STUDY DESIGN, SIZE, DURATION: Human spermatozoa were exposed separately to ionomycin and hydrogen peroxide in order to induce OS. An untreated control group was included. Sperm cells under OS were then exposed to chloroquine in order to block autophagy. An untreated control and a control incubated only with the OS inducer were included in each experimental setting. PARTICIPANTS/MATERIALS, SETTING, METHODS: For this study, semen samples from normozoospermic donors were used and motile sperm cells were selected by the swim up technique. First, the generation of OS under our experimental conditions was demonstrated by analyzing sperm parameters including viability, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) motility and thiol oxidation. Then, proteins involved in autophagy, including the microtubule-associated protein light chain 3 (LC3), particularly LC3-I and LC3-II, autophagy-related 5 (ATG5) and autophagy-related 16 (ATG16) proteins as well as the phosphorylated form of AMP-activated protein kinase (pAMPK) were evaluated in spermatozoa exposed to OS and compared to the untreated control. Finally, the impact of autophagy blocking by chloroquine treatment on sperm quality, metabolic parameters, including glycolysis and oxidative phosphorylation, as well as the cell death markers phosphatidylserine externalization and caspase activation was analyzed. Sperm quality parameters, cell death markers and autophagy-related proteins were analyzed by flow cytometry. Motility was evaluated by the computer-assisted sperm analysis system and metabolic parameters were analyzed using an extracellular flux analyzer. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to ionomycin and hydrogen peroxide promotes OS resulting in increased ROS production and decreased viability, ΔΨm and motility, while increasing thiol oxidation. These alterations were accompanied by a decrease in LC3-I, indicating that autophagy was activated upon OS exposure. Ionomycin also caused an increase in LC3-II, ATG5, ATG16 and pAMPK content. Autophagy blocking of sperm exposed to OS caused deterioration in sperm quality and metabolic parameters as well as an increase in cell death markers. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from normozoospermic donors; tests on sperm from infertile patients were not carried out. The autophagy blocking plus OS might generate a non-specific response to a highly stressful situation leading to the induction of cell death. WIDER IMPLICATIONS OF THE FINDINGS: Human spermatozoa subjected to OS activate an autophagic response and its blockage results in increased oxidative damage and commits spermatozoa to cell death. These results suggest a crucial role of autophagy as a stress response by male gametes, which contributes to maintaining the functionality and lifespan of ejaculated sperm cells. Detection of autophagy activation in sperm cells ex vivo could be included in semen analysis as a marker of OS, especially in men displaying high levels of seminal ROS. Novel strategies that aim to activate this cellular stress response could improve sperm quality/functionality under natural ejaculate conditions in which increased ROS levels are expected. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Fondo Nacional de Investigación Científica y Tecnológica, Chile (ANID/FONDECYT, Grant number 11170758 to P.U.); the Comisión Nacional de Investigación Científica y Tecnológica, Chile (ANID/CONICYT, Grant number PAI79160030 to P.U.) and the Dirección de Investigación, Universidad de La Frontera. The authors disclose no potential conflicts of interest.
Assuntos
Estresse Oxidativo , Espermatozoides , Autofagia , Morte Celular , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismoRESUMO
SUMMARY: The association of alcohol consumption with type 2 diabetes has been explained by increased insulin sensitivity, anti-inflammatory effects, or effects of adiponectin. The aim was to launch a consistent relation between alcohol intake and insulin sensitivity. Several databases (MEDLINE, EMBASE, Scopus and Web of Science) were searched from 1990 to April 2020 for studies in English, using MeSH terms and text words involving to alcohol consumption and insulin sensitivity. Protocol registered on PROSPERO CRD42020205107. A total of seven original articles were analyzed, where four collected data through cross-sectional study, two papers with randomized crossover design, and one used a non-randomized study. The protective effect of moderate alcohol consumption on type 2 diabetes has been described, where an improvement on insulin levels has been shown in adults between 26.5-57 years old. Our research shows that alcohol effects on blood insulin levels could vary depending of the type of alcoholic drink ingested; and that alcohol intake increased leptin and adiponectin levels, suggesting that alcohol consumption may increase glucose catabolism promoting insulin sensitivity via leptin and adiponectin. However, original studies should consider time of exposure, age, dosage, ethnicity, and alcohol type in order to conclude right affirmations.
RESUMEN: La asociación del consumo de alcohol con la diabetes tipo 2 se ha explicado por una mayor sensibilidad a la insulina, efectos antiinflamatorios o efectos de la adiponectina. El objetivo fue establecer una relación coherente entre la ingesta de alcohol y la sensibilidad a la insulina. Se realizaron búsquedas en varias bases de datos (MEDLINE, EMBASE, Scopus y Web of Science) desde 1990 hasta abril de 2020 en busca de estudios en inglés, utilizando términos MeSH y palabras de textos relacionadas con el consumo de alcohol y la sensibilidad a la insulina. Protocolo registrado en PROSPERO CRD42020205107. Se analizaron un total de siete artículos originales, donde cuatro recopilaron datos a través de un estudio transversal, dos artículos con diseño cruzado aleatorizado y uno utilizó un estudio no aleatorizado. Se ha descrito el efecto protector del consumo moderado de alcohol sobre la diabetes tipo 2, donde se ha demostrado una mejora de los niveles de insulina en adultos entre 26,5 y 57 años. Nuestra investigación muestra que los efectos del alcohol sobre los niveles de insulina en sangre pueden variar según el tipo de bebida alcohólica ingerida; y que la ingesta de alcohol aumenta los niveles de leptina y adiponectina, lo que sugiere que el consumo de alcohol puede aumentar el catabolismo de la glucosa promoviendo la sensibilidad a la insulina a través de la leptina y la adiponectina. Sin embargo, los estudios originales deben considerar el tiempo de exposición, la edad, la dosis, el origen étnico y el tipo de alcohol para concluir afirmaciones correctas.
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Humanos , Consumo de Bebidas Alcoólicas/epidemiologia , Diabetes Mellitus Tipo 2/epidemiologia , Insulina/fisiologiaRESUMO
Human spermatozoa activate neutrophil extracellular traps (NETs) in vitro. NETosis is an efficient mechanism through which polymorphonuclear neutrophils (PMN) capture sperm in vitro. The objective of this study was to establish the role of store-operated Ca+2 entry (SOCE) in human sperm-triggered NETs and its impact on sperm integrity and oocyte binding capacity. PMN isolated from donors were exposed to spermatozoa isolated from normozoospermic donors using the swim-up technique and were divided into the following groups: (1) sperm, (2) PMN, (3) PMN + sperm, (4) PMN (pretreated with 2-APB, SOCE inhibitor) + sperm, (5) (PMN + DNase) + sperm, and (6) (PMN + PMA) + sperm (positive control). NETs were quantified using PicoGreen® and visualised by scanning electron microscopy and immunofluorescence of extracellular DNA and neutrophil elastase. Plasma membrane, acrosome, and DNA integrity were analysed by flow cytometry, and oocyte binding was evaluated using the hemizona pellucida assay. Sperm-triggered NETosis negatively affected the sperm membrane and acrosome integrity and decreased the oocyte binding capacity. These effects were negated by an SOCE inhibitor, thus improving sperm function and achieving high oocyte binding capacity. The SOCE inhibitor significantly reduced NET formation compared with that in control PMN/sperm (P < 0.05). Collectively, these results advance the knowledge about the role of PMN in reproduction and will allow the development of strategies to block NET formation in situations of reduced fertilisation success.
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Cálcio/metabolismo , Armadilhas Extracelulares/metabolismo , Neutrófilos/fisiologia , Espermatozoides , Adulto , Compostos de Boro , Proteína C-Reativa/metabolismo , Feminino , Voluntários Saudáveis , Histonas/metabolismo , Humanos , Masculino , Microscopia Eletrônica de Varredura , Componente Amiloide P Sérico/metabolismo , Adulto JovemRESUMO
PURPOSE: To study the effector mechanism against pathogens of polymorphonuclear neutrophils (PMN) and macrophages, called ETosis, involving the release of extracellular traps (ETs) in patients with acute epididymitis. To assess the different ET phenotypes present in semen samples and to identify correlations between ETosis and clinical parameters. MATERIALS AND METHODS: Samples from patients diagnosed with acute epididymitis were examined and compared with samples from uninfected controls. Biochemical analyses of seminal fluid included determination of peroxidase, α-glucosidase, fructose, and elastase levels. ETosis in semen was determined through presence of citrullinated histones, global histones, and extracellular DNA. Different ETosis phenotypes such as spread ETs, aggregated ETs, and diffuse ETs were identified by co-localisation of extruded DNA with myeloperoxidase and global histones. Anti-CD15+ and anti-CD68+ antibodies were used to identify different cell lines. RESULTS: Revealed a high number of ETs compared with the control group. The mean number of CD15+PMN and CD68+ macrophages was higher in the acute epididymitis group. ETosis increase in ejaculates correlated with clinical parameters such as enhancement of elastase concentrations and diminution of fructose in the semen. CONCLUSIONS: This work shows for the first time the presence of ETs and their components in semen from patients with acute epididymitis. The presence of infections is an important factor for induction of ETs in semen. Furthermore, the presence of ETosis in ejaculates is suggestive of developing infectious processes and might possibly have a diagnostic value.
Assuntos
Epididimite/genética , Armadilhas Extracelulares/genética , Leucócitos/metabolismo , Sêmen/metabolismo , Adulto , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Linhagem Celular , Citrulinação/genética , Epididimite/diagnóstico , Epididimite/metabolismo , Epididimite/patologia , Armadilhas Extracelulares/metabolismo , Feminino , Frutose/metabolismo , Histonas/genética , Humanos , Leucócitos/patologia , Antígenos CD15/genética , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/metabolismo , Peroxidase/metabolismo , Projetos Piloto , alfa-Glucosidases/metabolismoRESUMO
BACKGROUND: The sperm vitrification developed by this group is based on the ultrarapid freezing of a vitrification solution composed of a non-permeable cryoprotectant (saccharides and protein), in which previously selected spermatozoa are resuspended, free of seminal plasma, and then plunged directly into liquid nitrogen. Compared to traditional sperm freezing, vitrification does not cause chemical or physical damage to the intracellular structures and reduces the damage to the plasma membrane because no ice crystals form, thus preserving motility and DNA integrity. OBJECTIVES: This manuscript is a review of the vitrification methodology developed by the authors' research group, including studies showing the application in human reproduction therapy. MATERIALS AND METHODS: The authors perform a review of the work initiated more than a decade ago by this research group, on the implementation of sperm vitrification, a more effective technique for cryopreservation of human spermatozoa, discussing the results obtained by other authors and the projection of this technique. RESULTS AND DISCUSSION: The vitrification technique has been developed in selected spermatozoa free of seminal plasma supplemented with saccharides such as sucrose, trehalose, and dextran, together with albumin, providing a high motility rate and protective structures of the cytoskeleton. In patients, it can be used to preserve their fertility for oncological reasons, genetics, inflammatory diseases, or reproductive medicine techniques. The possibility that vitrified spermatozoa can be preserved at temperatures of -80°C can simplify sample storage, optimizing the space and time as well as operator safety. CONCLUSION: Vitrification techniques have demonstrated the preservation of selected spermatozoa without seminal plasma and with non-permeable cryoprotectants and protein. Currently, it is one of the most effective ways to maintain sperm function and has been used in in vitro fertilization or intrauterine insemination in humans, achieving healthy live births.
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Criopreservação , Crioprotetores/uso terapêutico , Preservação da Fertilidade , Infertilidade/terapia , Preservação do Sêmen , Espermatozoides/efeitos dos fármacos , Crioprotetores/efeitos adversos , Difusão de Inovações , Feminino , Fertilidade , Preservação da Fertilidade/efeitos adversos , Humanos , Infertilidade/diagnóstico , Infertilidade/fisiopatologia , Masculino , Gravidez , Fatores de Risco , Preservação do Sêmen/efeitos adversos , Espermatozoides/patologia , Resultado do Tratamento , VitrificaçãoRESUMO
Male infertility is an increasing health problem, and oxidative/nitrosative stress plays an important role in the etiology of this condition. Nitrosative stress due to excessive levels of reactive nitrogen species (RNS) is associated with impaired male fertility. Flow cytometry may be a useful tool for semen evaluation, but the availability of multiparameter assays for analysis of sperm quality is limited. The present study standardized a multiparameter flow cytometry analysis for nitrosative stress status in human spermatozoa in a single assay. A suitable multicolor fluorochrome panel was designed and consisted of fluorescein-boronate to detect peroxynitrite, a highly RNS, propidium iodide to analyze viability, tetramethylrhodamine methyl ester perchlorate to detect mitochondrial membrane potential (MMP) and monobromobimane to analyze thiol oxidation. Proper positive and negative controls for each fluorochrome were used to establish the technique, and sperm cells of different qualities and spermatozoa subjected to cryopreservation were analyzed. The results showed that the controls clearly discriminated between the high and low fluorescence intensities for each fluorochrome. The analysis of sperm cells of different quality demonstrated that the assay properly detected differences in all parameters analyzed according to sperm quality. The results may be reported as the mean fluorescence intensity of each fluorochrome and the percentage of cells exhibiting different characteristics. In conclusion, a protocol was standardized to analyze nitrosative stress status, including peroxynitrite production, viability, MMP, and thiol oxidation, in a single analysis using flow cytometry. This protocol may be applied to research approaches and clinical andrology to improve the evaluation of sperm quality and provide a promising tool to increase the use of clinical flow cytometry. © 2020 International Society for Advancement of Cytometry.