Dihydroethidium

The first probe to be assessed in detail in this context was dihydroethidium or hydro-ethidine (DHE) [69]. This probe is nonfluorescent in its own right, but on oxidation will produce DNA-sensitive fluorochromes that will stain the mitochondrial and nuclear DNA (Fig. 14.6). The application of this probe is not straightforward, however, and its interpretation has to be considered with care. One of the major issues with DHE is that it is generated by the chemical reduction of ethidium bromide (Et+), a general DNA-sensitive fluorochrome. As a result, every commercial preparation of DHE will be contaminated with Et+, which will, in turn, stain every nonvi-able cell in the sperm suspension with a bright red fluorescence. Consequently, this probe cannot be used in isolation, but has to be coupled with a cell vitality marker. If a cell vitality marker is not used, then it will be impossible to distinguish free radical generating cells from those that are simply dead. SYTOX green is an appropriate vitality stain in this context because its emission spectrum (500-530 nm) is completely different from Et+ (>560 nm). This combination of probes therefore allows flow cytometry to be used to rapidly screen human spermatozoa in order to identify those cells that are alive and generating ROS ([69]; Fig. 14.6).

A second problem with DHE as a probe is that it can be nonspecifically oxidized to generate Et+ by a variety of metabolites and xenobiotic chemical reagents including hydrogen peroxide, hypochlorous acid, peroxynitrite as well as a certain qui-none species including o-chloranil or o--naphthoquinone ([69, 70]; Fig. 14.6). In order to be certain that the probe is capable of measuring superoxide anion (O2-0, it is necessary to demonstrate that the cellular oxidation of DHE generates

Fig. 14.6 DHE as a probe for ROS generation by human spermatozoa. (a) Diagrammatic representation of the chemistry underlying the use of DHE as a probe for ROS generation. DHE may either undergo a nonspecific 2-electron oxidation to generate Et+ or react with O2-^ to generate the specific reaction product, 2OHET+. Both of these fluorochromes will interact with DNA in the sperm nucleus to generate a red fluorescence. In order to be certain that spermatozoa were producing O2-\ it was essential to isolate the putative 2OHET+ by HPLC and verify the chemical identity of the product using a variety of analytical tools including NMR spectroscopy, mass spectrometry, and spectrofluorimetry. In the course of these studies, o-chloranil was used to drive the nonspecific oxidation of DHE to Et+, while the closely related compound, menadione, was used to stimulate the O2-Mnduced formation of 2OHET+. (b) Confocal image of human spermatozoa stained with DHE and SYTOX® Green; overlay image of red and green fluorescence. Nonviable cells are green or orange while viable cells that are generating ROS are red. Scale bar = 10 ^m. Reproduced from De Iuliis et al. [69], by kind permission from The Endocrine Society

2-hydroxyethidium (2OHET+), a unique reaction product that can only be created when DHE is oxidized by O2- [71]. Using HPLC, two reaction products were found to be formed when free radical generating spermatozoa were treated with DHE; mass spectrometry revealed these peaks to be Eth+ and 2OHET+ [69]. Appropriately, the 2OHET+ signal was found to be greatly augmented when O.- production by human spermatozoa, was artificially stimulated with menadione (vitamin K) in concert with a dramatic decline in sperm motility and a corresponding increase in the incidence of DNA damage [ 69 ] . Furthermore, using the DHE:Sytox Green assay to monitor O2 -• generation in viable cells, a clear negative correlation was observed between ROS generation and sperm motility. The fact that the mitochon-drial inhibitor CCCP (carbonyl cyanide m-chlorophenylhydrazone, a proton iono-phore which collapses the mitochondrial membrane potential] had no impact on the DHE signal generated by human spermatozoa, prompted us to propose that the O2- detected in these cells was not of mitochondrial origin. This, it turned out, was not true. Sperm mitochondria are very active generators of O2"; however, they are unusual in that the production of ROS, at least in the short term, does not depend on the mitochondria membrane potential [72],

The most commonly employed probe for measuring mitochondrial ROS generation is MitoSOX Red (MSR). This probe is based on DHE but carries an additional charge, which results in its selective accumulation within the mitochondria. Following reaction with O.-\ MSR produces DNA-sensitive fluorochromes that generate a red fluorescence when excited at 510 nm that can be detected by flow cytometry. As with DHE, it is important to ensure that only live cells are imaged when MSR fluorescence is being monitored. Failure to incorporate this step into the protocol may produce spurious results as a consequence of dead cells staining with the residual Eth+ contaminating commercial MSR preparations. In order to simultaneously monitor mitochondrial ROS and cell vitality, we again advocate the use of a Sytox green/MSR combination in a flow cytometry-based protocol [72].

Using MSR, we have found that the mitochondria of normal human spermatozoa can be induced to generate O2- with inhibitors of complex III in the mitochondrial electron transport chain (ETC). Thus, while normal human spermatozoa exhibit extremely low rates of spontaneous ROS generation, addition of antimycin A and myxothiazol, both of which act at complex III of the ETC, resulted in a dramatic increase in redox activity (Fig. 14.7). Myxothiazol binds close to the bL heme of this complex, allowing ubiquinol (QH2) to access the Rieske iron sulfur center in order to undergo a one-electron oxidation to create the semiquinone radical, Q~\ which is unstable and rapidly reverts to the parent quinone (Q) with the release of an electron which is avidly taken up by oxygen to create O2-\ Antimycin A treatment also leads to the generation of Q- by inhibiting the reoxidation of heme bL through its capacity to disrupt electron transfer from heme bH to Q- [72]. As a consequence of these interactions, both myxothiazol and antimycin A stimulate the generation of Q-\ which then reduces O2 to O2- in the intramembranous space. This O2- then dismutates to H2O2 under the influence of SOD and escapes to the outside of the cell, where it can be detected by the luminol-peroxidase monitoring system. Rotenone (an inhibitor of electron transfer from FeSN-2 cluster to ubiquinone) was found to have a minor stimulatory effect on H2O2 release at 10 |M, possibly because the O2- generated with this

Fig. 14.7 Schematic diagram showing the main pathways of electron flux through the mitochondrial electron transport chain. Sites of action for the mitochondrial inhibitors used in this study are also indicated. VDAC voltage-dependent anion channel; SDH succinate dehydrogenase; Q ubiquinone; QH2 ubiquinol; Q- ubisemiquinone; SOD superoxide dismutase; GPx glutathione peroxidase; FMN flavin mononucleotide; NADH nicotinamide adenine dinucleotide; FAD flavin adenine dinucleotide. With antimycin and myxothiazol, electron leakage leads to the generation of O2- in the intermembranous space, while rotenone leads to O2- formation in the mitochondrial matrix. It is primarily the generation of ROS in the matrix that appears to damage spermatozoa. Reproduced from Koppers et al. [72], with kind permission from The Endocrine Society

Fig. 14.7 Schematic diagram showing the main pathways of electron flux through the mitochondrial electron transport chain. Sites of action for the mitochondrial inhibitors used in this study are also indicated. VDAC voltage-dependent anion channel; SDH succinate dehydrogenase; Q ubiquinone; QH2 ubiquinol; Q- ubisemiquinone; SOD superoxide dismutase; GPx glutathione peroxidase; FMN flavin mononucleotide; NADH nicotinamide adenine dinucleotide; FAD flavin adenine dinucleotide. With antimycin and myxothiazol, electron leakage leads to the generation of O2- in the intermembranous space, while rotenone leads to O2- formation in the mitochondrial matrix. It is primarily the generation of ROS in the matrix that appears to damage spermatozoa. Reproduced from Koppers et al. [72], with kind permission from The Endocrine Society compound is formed in the mitochondrial matrix and may be neutralized by the antioxidant enzymes (SOD and glutathione peroxidase) that abound at this site (Fig. 14.7). Stigmatellin (10 |M), which inhibits the transfer of electrons from ubiquinol (QH2) to the Rieske iron sulfur cluster and prevents semiquinone (Q"0 formation (Fig. l4.7), failed to induce mitochondrial ROS generation. The generation of ROS with these mitochondrial electron transport inhibitors is not suppressed by collapsing the mito-chondrial membrane potential with CCCP; indeed, the addition of these reagents actually enhances ROS generation [72],

Biologically, the spontaneous generation of mitochondrial ROS is associated with a significant decline in sperm motility as a result of lipid peroxidation occurring in the midpiece of the cell [72]. On the basis of the damaging effects observed with rotenone, it has been suggested that mitochondrial ROS are particularly damaging to sperm function if they are generated in the mitochondrial matrix, rather than in the intermembranous space [72]. In the latter position, the O2- appears to escape rapidly to the cytosol where it dismutates to H2O2 and leaves the cell. However, when the ROS are generated in the mitochondrial matrix, they do not escape so readily and precipitate the peroxidative damage that leads to motility loss [72]. Since the MSR probe is targeted to the mitochondrial matrix, it is in the perfect position to detect ROS formation in this strategically important location.

The triggers for mitochondrial ROS generation are varied, including electromagnetic radiation, the local availability of lipid peroxides, and polyunsaturated fatty acids [73, 74], The fact that the spontaneous generation of mitochondrial ROS is highly correlated with the polyunsaturated fatty acid content of human spermatozoa strongly suggests that the etiology of oxidative stress in these cells involves a lipid imbalance generated by errors in metabolism or, possibly, diet [75].

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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