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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 MitoPY1 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. [ABSTRACT FROM AUTHOR]

As a natural by-product of mitochondrial respiration, reactive oxygen species (ROS) in sperm play a role in promoting fertilization, by intervening in a series of events. Nevertheless, an abnormal and uncounteracted increase in ROS production leads to oxidative stress (OS) which can, ultimately, culminate in cell death. An established relationship between OS and male infertility highlights the importance of an accurate detection method for ROS content that can be easily implemented and reproduced in any andrology lab. More recently, reactive nitrogen species (RNS) production and subsequent nitrosative stress have also been described. Here we describe the use of fluorescent probes, including some that targeted to the mitochondria due to the coupling of a cation (TPP + ), in order to assess the levels of different ROS and RNS in human sperm using flow cytometry and/or fluorescent microscopy. This methodology is user friendly and accurate and can be safely applied in research- and/or clinical-based contexts., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A positive relationship between mitochondrial functionality and gamete quality, ultimately contributing to fertilization success and normal embryo development has been established for some years now. Both mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production are major indicators of mitochondrial function, and the need for accurate biomarkers mirroring gamete quality highlights the importance of a precise assessment of mitochondrial quality and function. In this chapter, we discuss the use of some mitochondrial fluorescent probes coupled to flow cytometry and/or fluorescence microscopy to specifically assess mitochondrial ROS production and MMP in both sperm and oocytes. Furthermore, as the distribution/aggregation of mitochondria in the oocyte is of interest to determine its quality, a detailed protocol is also given. These methodologies are easy, accurate and can be safely applied in research- and/or clinical-based contexts.