Reactive oxygen species(ROS)production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state,a requirement for functional spermatozoa.However,an increase in oxidative ...Reactive oxygen species(ROS)production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state,a requirement for functional spermatozoa.However,an increase in oxidative stress,due to an abnormal production of ROS,has been shown to be related to loss of sperm function,highlighting the importance of an accurate detection of sperm ROS,given the specific nature of this cell.In this work,we tested a variety of commercially available fluorescent probes to detect ROS and reactive nitrogen species(RNS)in human sperm,to define their specificity.Using both flow cytometry(FC)and fluorescence microscopy(FM),we confirmed that MitoSOX™Red and dihydroethidium(DHE)detect superoxide anion(as determined using antimycin A as a positive control),while DAF-2A detects reactive nitrogen species(namely,nitric oxide).For the first time,we also report that RedoxSensor™Red CC-1,CellROX®Orange Reagent,and MitoPYl seem to be mostly sensitive to hydrogen peroxide,but not superoxide.Furthermore,mean fluorescence intensity(and not percentage of labeled cells)is the main parameter that can be reproducibly monitored using this type of methodology.展开更多
基金the Portuguese funding agency for science and technology(PD/BD/128237/2016-PhD Programme in Experimental Biology and Biomedicine)CNC is funded by FEDER,through Programa Operacional Factores de Competitividade-COMPETE 2020 and National funds via FCT under the project POCI-01-0145-FEDER-007440+1 种基金the European Regional Development Fund(ERDF),through the Centro 2020 Regional Operational Programme:project CENTRO-01-0145-FEDER-000012-HealthyAging2020,the COMPETE 2020-Operational Programme for Competitiveness and Internationalisationthe Portuguese national funds via FCT-Foundation for Science and Technology LP:project POCI-01-0145-FEDER 007440 and UID/NEU/04539/2019.
文摘Reactive oxygen species(ROS)production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state,a requirement for functional spermatozoa.However,an increase in oxidative stress,due to an abnormal production of ROS,has been shown to be related to loss of sperm function,highlighting the importance of an accurate detection of sperm ROS,given the specific nature of this cell.In this work,we tested a variety of commercially available fluorescent probes to detect ROS and reactive nitrogen species(RNS)in human sperm,to define their specificity.Using both flow cytometry(FC)and fluorescence microscopy(FM),we confirmed that MitoSOX™Red and dihydroethidium(DHE)detect superoxide anion(as determined using antimycin A as a positive control),while DAF-2A detects reactive nitrogen species(namely,nitric oxide).For the first time,we also report that RedoxSensor™Red CC-1,CellROX®Orange Reagent,and MitoPYl seem to be mostly sensitive to hydrogen peroxide,but not superoxide.Furthermore,mean fluorescence intensity(and not percentage of labeled cells)is the main parameter that can be reproducibly monitored using this type of methodology.
