RESUMO
In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 106 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating, and capacitating plus 150 mM MnTBAP. There were incubations for 4 h at 37.5 °C in a humidified air atmosphere. Sample analysis was performed immediately after thawing (0 h), and at the end of the incubation period (4 h), unless otherwise indicated. The following variables were evaluated for spermatozoa: plasma membrane integrity and fluidity, acrosome integrity, intracellular calcium concentrations, intracellular pH, tyrosine phosphorylation, ATP concentrations, motility and heterologous zona-binding assay, using flow cytometry, fluorescent microscopy and/or chemiluminescence, depending on the most appropriate procedure for the variable being evaluated. Results indicated that capacitation-like changes were synergistically induced by the cAMP agonists, phosphodiesterase inhibitor and bicarbonate. The presence of bovine serum albumin was harmful to the plasma membrane. The MnTBAP supplementation had a positive effect on viability-related markers (plasma membrane integrity, membrane fluidity, associated with greater intracellular pH) when there were capacitating conditions. In conclusion, the activity of MnTBAP contributes to improving the in vitro incubation conditions of frozen-thawed stallion sperm.
Assuntos
Criopreservação/veterinária , Cavalos/fisiologia , Metaloporfirinas/farmacologia , Preservação do Sêmen/veterinária , Capacitação Espermática/efeitos dos fármacos , Animais , MasculinoRESUMO
Cryopreservation of stallion semen has not reached the level of efficiency and positive results described in other species. This is mainly due to the greater sensitivity of stallion sperm to the freezing process, showing higher rates of oxidative stress and plasma membrane damage, which trigger the activation of several cell damage pathways that ultimately culminate in DNA fragmentation and cell death. Therefore, finding molecules that improve the efficiency of this technique in stallion by preventing oxidative stress and cell damage is required. Thus, the aim of the present study was to evaluate the effect of adding three antioxidants (MnTBAP, NAC and FeTPPS) to the freezing medium on the quality and functional parameters of stallion sperm. Semen samples from three stallions frozen with the antioxidants were evaluated in two conditions: (a) adding the antioxidants before freezing, and (b) before and after freezing. Plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation, intracellular ROS levels, membrane lipid disorder, DNA damage, sperm motility and binding to the zona pellucida were assessed. The results showed that MnTBAP was the antioxidant treatment that best controlled the oxidative stress process and post-thaw cell damage, showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, number of spermatozoa bound to the zona pellucida of bovine oocytes and lower lipid disorder. Additionally, it was determined that a second post-thaw application of antioxidants is detrimental since induced higher cell damage and lower sperm motility, without showing any beneficial effect on the spermatozoa.
Assuntos
Antioxidantes/farmacologia , Criopreservação/veterinária , Crioprotetores/farmacologia , Preservação do Sêmen/veterinária , Acetilcisteína/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Criopreservação/métodos , Fragmentação do DNA , Cavalos , Masculino , Lipídeos de Membrana , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metaloporfirinas/farmacologia , Estresse Oxidativo , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/efeitos dos fármacos , Zona Pelúcida/fisiologiaRESUMO
Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.
Assuntos
Meios de Cultura/farmacologia , Fármacos para a Fertilidade Masculina/farmacologia , Cavalos , Análise do Sêmen/veterinária , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Aminopiridinas/farmacologia , Animais , Cafeína/farmacologia , Fertilização in vitro/veterinária , Fluoresceínas/farmacologia , Masculino , Potencial da Membrana Mitocondrial , Aglutinina de Amendoim/farmacologia , Fosforilação , Procaína/farmacologia , Sêmen/efeitos dos fármacos , Análise do Sêmen/métodos , Espermatozoides/efeitos dos fármacosRESUMO
Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.
Assuntos
Morte Celular , Mitocôndrias/patologia , Estresse Nitrosativo/fisiologia , Espermatozoides/patologia , Adulto , Caspases/metabolismo , Ativação Enzimática , Humanos , Masculino , Mitocôndrias/metabolismo , Necrose , Permeabilidade , Ácido Peroxinitroso/farmacologia , Fosfatidilserinas/metabolismo , Espermatozoides/metabolismo , Espermatozoides/ultraestruturaRESUMO
Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 °C) and (ii) incubated at 37 °C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (ΔΨM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and ΔΨM compared to the control, while the incubation at 37 °C promoted a decrease in these parameters. The ROS levels were increased at both incubation conditions. The progressive motility was decreased in all experimental groups and the decrease was more pronounced under incubation at RT. No increase in phosphatidylserine externalization was observed. In conclusion, prior to use in assisted reproduction procedures, devitrified spermatozoa at RT conserve a better viability and ΔΨM than at 37 °C.
Assuntos
Criopreservação/métodos , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/fisiologia , Temperatura , Vitrificação , Técnicas de Cultura de Células/métodos , Humanos , Masculino , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/fisiologiaRESUMO
OBJECTIVE: To determine the effect on human sperm of Escherichia coli strains separated on the basis of their ability to produce hemolysis. DESIGN: Experimental study. SETTING: University-based laboratory. PATIENT(S): Semen samples from healthy donors. INTERVENTION(S): Five million sperm, selected via the swim-up method, were incubated with 3 E. coli concentrations to obtain ratios of sperm to E. coli of 1:2, 1:16, and 1:128. The E. coli strains were: a hemolytic isolated strain (H), a nonhemolytic American Type Culture Collection strain (NH-ATCC), and a nonhemolytic isolated strain (NH-I). MAIN OUTCOME MEASURE(S): Aliquots of human sperm were used to measure progressive motility using computer-aided sperm analysis, mitochondrial membrane potential (ΔΨm) with a JC-1 (5,5',6,6' tetrachloro-1,1',3,3'-tetraethylbenzamidazolocarbocyanin iodide) and propidium iodide stain, and intracellular reactive oxygen species (iROS) with a dihydroethidium (DHE) stain. Sperm ΔΨm and iROS were measured by flow cytometry. Sperm vitality was considered the mean of propidium iodide-negative and DHE-negative cells. RESULT(S): Sperm incubated with the H strain in a 1:2 sperm to bacteria ratio demonstrated a significant decrease in motility and ΔΨm, and an increase of iROS. The NH-ATCC strain decreased sperm motility and ΔΨm, but in a ratio of sperm to bacteria of 1:128; it increased iROS at a ratio of 1:16. The NH-I strain did not affect the analyzed sperm functions, even at a 1:128 sperm to bacteria ratio. CONCLUSION(S): Results show a greater pathogenic effect on human sperm of E. coli strains with, versus without, hemolytic capacity.
Assuntos
Escherichia coli/patogenicidade , Hemólise , Espermatozoides/microbiologia , Sobrevivência Celular , Escherichia coli/classificação , Humanos , Masculino , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismoRESUMO
STUDY QUESTION: Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa? SUMMARY ANSWER: Studies conducted in vitro suggest that in human spermatozoa, MPT occurs in response to intracellular calcium increase and is associated with mitochondrial membrane potential (ΔΨm) dissipation, increased ROS production and DNA fragmentation. WHAT IS KNOWN ALREADY: Oxidative stress is a major cause of defective sperm function in male infertility. By opening calcium-dependent pores in the inner mitochondrial membrane (IMM), MPT causes, among other things, increased ROS production and ΔΨm dissipation in somatic cells. MPT as a mechanism for generating oxidative stress and DNA fragmentation in human spermatozoa has not been studied. STUDY DESIGN, SIZE, DURATION: Human sperm were exposed to ionomycin for 1.5 h (n = 8) followed by analysis of sperm IMM permeability, ΔΨm, ROS production and DNA fragmentation. PARTICIPANTS/MATERIALS, SETTING, METHODS: To evaluate the MPT in sperm cells, the calcein-AM and cobalt chloride method was used. The ΔΨm was evaluated by JC-1 staining, intracellular ROS production was evaluated with dihydroethidium and DNA fragmentation was evaluated by a modified TUNEL assay. Measurements were performed by fluorescence microscopy, confocal laser microscopy and flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Decreased calcein fluorescence after treatment with ionomycin (P < 0.05) suggests the opening of pores in the sperm IMM and this was accompanied by ΔΨm dissipation, increased ROS production and DNA fragmentation. ROS production occurred prior to the decrease in ΔΨm. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from healthy donors; tests on sperm from infertile patients were not carried out. WIDER IMPLICATIONS OF THE FINDINGS: We propose that the MPT, due to pores opening in sperm IMM, is an important mechanism of increased ROS and DNA fragmentation. Therefore, agents that modulate the opening of these pores might contribute to the prevention of damage by oxidative stress in human spermatozoa. STUDY FUNDING/COMPETING INTERESTS: This study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT Chile (F.T.). The authors disclose no potential conflicts of interest.