Other ROS Detection Systems

The foregoing discussion highlights the major strength and weaknesses of protocols for assessing the generation of ROS by human spermatozoa based around the chemi-luminescent analysis of redox activity and detection of O2- using flow cytometry. There are other fluorescent probes in use for measuring ROS generation by cells particularly dichlorofluorescin diacetate (DCFH-DA) [68] . This probe becomes fluorescent on oxidation and is purported to measure cellular H2 O2 production. However, in reality this oxidant has no effect on DCFH-DA fluorescence unless it is accompanied by peroxidase activity which in a highly compartmentalized, cytoplasm-deficient cell such as the spermatozoon may not always be the case. It is also possible that the DCFH/H2O2/peroxidase detection system may actually generate H2O2 in a scheme whereby H2O2 reacts with peroxidase to form compound 1, which then oxidizes DCFH to the DCF*- semiquinone-free radical. The latter then decays to fluorescent DCF with the release of an electron to oxygen, generating O2- which will rapidly dismutate to H2O2 and continue the cycle. Other ROS such as the per-oxynitrite, hypochlorous acid, and the hydroxyl radical can also oxidize this probe and might make significant contributions to the positive signals observed in defective human spermatozoa [68, 76]. Finally, the dye, nitroblue tetrazolium (NBT), has been used to detect ROS generation by human spermatozoa. NBT is held to be reduced by the donation of electrons from O2 -• to generate a detectable chemical signal; in this case, blue-black deposits of formazan [77]. The problem with this probe is that it is not specific for spermatozoa, and unless the sperm suspensions are carefully purified, will generate signals dominated by the presence of contaminating leukocytes [78]. Furthermore, this probe is not specific for O2- since it can be activated by cytochrome P450-reductase oxidoreductases which can effect a one-electron reduction of the probe [42] to produce unstable tetrazoinyl radical intermediates, which then give up their electrons to reduce O2 to O2- in a reversible process. SOD, by removing O2- displaces this oxidation to the right and thus prevents production of the formazan. For this reason, many aerobic tetrazolium reductions are inhibitable by SOD even though O2- was not actually being generated in the system in the absence of NBT [79],

The above discussion covers most of the methods that are currently being used to assess ROS production by human spermatozoa. Clearly, this is still an unresolved issue. We are still not certain which particular ROS are damaging to spermatozoa, how they originate, and which of the above methods is the most appropriate for their routine diagnostic assessment. Clearly, the field needs to undertake detailed comparative studies where the readout of selected assays is correlated with defective sperm function, in the form of lost motility or oxidative DNA damage, in order to determine which is the most sensitive procedure to incorporate into routine diagnostic analyses of semen quality.

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