This study has shed light on the responsiveness of the current range of assays used to detect ROS generation by human spermatozoa and also increased our understanding of how specific reagents increase oxidative stress in the male germ line. In the following discussion, we shall begin by considering the assays themselves and then review the mechanisms by which different classes of reagent elicit a free radical response from human spermatozoa.
Comparison of probes for assessing ROS generation by human spermatozoa
MSR and DHE – These probes are both reduction products of ethidium bromide, but MSR has been chemically modified to give the molecule a positive charge that results in its concentration in the mitochondrial matrix. From a diagnostic perspective, it is important to emphasize that commercial preparations of these reagents are contaminated with small amounts of the parent ethidium compound, which can result in the spurious staining of non-viable cells. It is therefore imperative that these assays are run with vitality markers such as Sytox green, the emission characteristics of which permit simultaneous assessment of cell vitality and ROS-generating activity by flow cytometry (Koppers et al., 2008, 2011). In this study, all the reagents which acted primarily on the mitochondria to stimulate ROS production (2OHE2, AA, 4HNE), generated MSR and DHE signals within the live cell population that were highly correlated with each other. In general, the relationship was linear, but in the case of AA in particular, the MSR signal was the more responsive (Fig. 5C,D). As the physiological induction of ROS generation in defective human spermatozoa is thought to involve the ability of free, unesterified unsaturated fatty acids such as AA to trigger electron leakage from the mitochondrial electron transport chain, this result emphasizes the particular clinical utility of the MSR assay (Koppers et al., 2010). This conclusion was reinforced by the analysis of spontaneous redox activity in defective human spermatozoa recovered from the low-density region of Percoll gradients. Although H2DCFDA, DHE and MSR were all capable of detecting the enhanced redox activity associated with defective, low-density human spermatozoa, the MSR signals were significantly more intense than those observed with any other probe (Fig. 8D).
H2DCFDA – This probe becomes fluorescent on oxidation and is purported to measure cellular H2O2 production. In reality, this oxidant has no effect on H2DCFDA fluorescence unless it is accompanied by peroxidase activity, which in a highly compartmentalized, cytoplasm-deficient cell such as a spermatozoon, may be a rate limiting factor in the genesis of activity. Other ROS such as ONOO- and the hydroxyl radical can also directly oxidize this probe and might make significant contributions to the positive signals observed in defective human spermatozoa (Myhre et al., 2003; Mahfouz et al., 2010). In practice, the signals generated by H2DCFDA were most effective when the oxidants were being generated in the cytoplasm at or near the plasma membrane, as when menadione was used to enhance cellular redox activity (Fig. 3E). Leucocytes were also found to have a confounding effect on the H2DCFDA signal (Fig. 8C). When these cellular contaminants were removed, H2DCFDA could only detect an intense redox signal in around 3% of defective low-density human spermatozoa, whereas MSR detected excessive ROS generation in 30% of such cells (Fig. 8D). Furthermore, when mitochondrial ROS was triggered by incubation with rotenone or antimycin, no change was observed in the H2DCFDA signal (Fig. 8E) whereas MSR is known to detect the increased ROS generated under such circumstances (Koppers et al., 2008).
Luminol and lucigenin – Like H2DCFDA, these chemiluminescent probes appeared to be relatively insensitive to the mitochondrial ROS generated on exposure to 4HNE (Fig. 7G,H), AA (Fig. 5G,H) or 2OHE2 (Fig. 2G,H). In contrast, luminol-HRP was, by far, the most sensitive probe for detecting the ROS generated in response to menadione, where a significant proportion of the ROS are released to the outside of the cell. In the extracellular space, HRP is able to catalyse the oxidation of luminol to generate a luminol radical (Lֹ). The latter interacts with ground state oxygen to produce O2ֹֿ which then participates in the oxygenation of Lֹ to create an unstable endoperoxide, which breaks down with the release of light (Aitken et al., 2004, 2012a). Again, like H2DCFDA, this method is also highly vulnerable to the confounding influence of leucocytes, which are clearly capable of releasing ROS into the extracellular space (Aitken & West, 1990; Hipler et al., 1998; Aitken et al., 2004). When these cells were removed from the sperm suspensions with anti-CD45-coated Dynabeads, the ability of the luminol system to discriminate between spermatozoa from high- and low-density Percoll fractions was lost (Fig. 8B).
Lucigenin could detect the mitochondrial ROS generated in response to 2OHE2, but only at the highest dose of reagent used. It also detected the increased ROS activity generated in response to menadione and AA treatment, but the data were so variable that statistical significance was not achieved. Lucigenin appeared to be insensitive to the increased redox activity associated with 4HNE exposure. The problem with this probe is that the chemistry of its chemiluminescence is very complex. Activation of the probe requires a one-electron reduction, rather than the one-electron oxidation associated with luminol-dependent chemiluminescence (Aitken et al., 2012a). This one-electron reduction creates a radical (LucH+ֹ) from lucigenin (Luc2+) that rapidly gives up its electron to ground state oxygen to create O2ֹֿ and return the lucigenin to its parent state. The LucH+ֹ generated from the one-electron reduction of lucigenin then combines with O2ֹֿ to produce the dioxetane that, in turn, decomposes with the generation of light. The O2ֹֿ involved in the last reaction could come from an independent cellular source, such as the mitochondria, in which case the chemiluminescence recorded would reflect the generation of O2ֹֿ. However, an unknown proportion of the O2ֹֿ involved in this reaction is an artefact created by the reaction between LucH+ֹ and ground state oxygen. Chemiluminescence created by the cellular generation of O2ֹֿ or the redox cycling of lucigenin cannot be readily distinguished, as both sources of ROS are suppressible by SOD. Furthermore, we have clearly demonstrated that lucigenin chemiluminescence in the presence of NADH or NADPH does not represent O2ֹֿ production, but rather the respective abilities of cytochrome b5 reductase, and cytochrome P450 reductase to reduce Luc2+ to LucH+ֹ and artificially trigger a redox cycle that generates O2ֹֿ as a by-product. In light of these considerations, lucigenin is not regarded as a suitable probe for ROS generation by human spermatozoa (Baker et al., 2004, 2005).
A final comment about chemiluminescence is that the photomultipliers that transduce the light emitted in such reactions into a digital signal are not standardized, so the response characteristics of every instrument will be different. Furthermore, the chemistry of chemiluminescence is complex and the read out will be influenced by minor differences in such factors such as pH, temperature, presence of transition metals, oxygen tension, tube agitation, air bubbles in the reaction chamber etc. (Aitken et al., 2004). As a result, the chemiluminescence signals will vary significantly between runs creating so much variance that statistical differences between group means are difficult to attain, even when clear dose-dependent trends are evident. These sources of variation also mean that the calculation of numerical threshold values for chemiluminescence in a diagnostic context is an unachievable objective (Desai et al., 2009). The technology is really best suited to longitudinal studies where time-dependent changes in chemiluminescence are monitored in response to the presence of a particular reagent (e.g. Fig. 8B).
NBT – The problems with NBT reduction as a probe for ROS are similar to those described above for lucigenin. Thus, any enzyme capable of effecting NBT reduction using NADH or NADPH as an electron donor, will generate a response that masquerades as a ROS signal (Baker et al., 2004, 2005). So, while O2ֹֿ is theoretically capable of reducing NBT, the same response can be generated by a number of reductases using alternative electron donors. The result is that NBT exhibits high background levels of reduction and although the technique was capable of detecting the mitochondrial ROS generated by 2OHE2 as well as the release of extracellular ROS in response to menadione, it failed to detect the mitochondrial redox activity elicited with either AA or 4HNE.
DAF-DA – It was evident from the experiments with DAF-DA that human spermatozoa are constantly generating NO as long as they are in a viable state (Fig. 8C,D) and that this probe is not capable of detecting the differences in redox activity between low- and high-density Percoll fractions (Fig. 8A,C,D).
Mechanisms for the activation of ROS generation
The results obtained in this study have also shed some light on the complex cellular mechanisms by which the various activators of ROS generation exert their biological effects on spermatozoa. Thus, 2OHE2 elicited ROS generation from the mitochondria, generating powerful responses that could be readily detected when MSR and DHE was used as the probe. For 2OHE2 to redox cycle, it has be oxidized to the corresponding semiquinone by oxidoreductases including cytochrome P450 isoforms that are located on the inner mitochondrial membrane, with their active sites facing the mitochondrial matrix (Zhang et al., 2007; Sangar et al., 2010). In the case of AA or 4HNE, the site of action is thought to be electron transport within the inner mitochondrial membrane, which results in a significant discharge of O2ֹֿ into the inter-membranous space which, again, can be readily detected by MSR or DHE (Fig. 5C,D; Fig. 7C,D).
Menadione was different from all of the other compounds tested in that it redox cycled at or close to the surface of the spermatozoa generating significant quantities of ROS in the extracellular space, even when covalently bound to a protein scaffold that would prevent its passage across the plasma membrane (Fig. 4D). Such activity suggests that plasma membrane redox systems must be present in the sperm plasma membrane, which are capable of transmitting electrons across the plasma membrane to acceptor molecules on the sperm surface. It is presumably in this manner that spermatozoa are able to maintain the surface expression of surface thiols as they engage the capacitation process (de Lamirande & Gagnon, 1998; Gualtieri et al., 2009).
Finally, AA was different from the other inducers of ROS generation in that while it clearly stimulated mitochondrial ROS generation, it did not induce a change in sperm motility. This was not because this fatty acid did not create a state of oxidative stress. On the contrary, AA stimulated higher levels of 8OHdG formation than any of the other ROS-inducers within the first 4 h (Fig. 6A,B). The most likely explanation for this difference is while oxidative stress can precipitate motility loss, the lipid peroxidation process that underpins this process takes a matter of hours to be maximally effective (Aitken et al., 2006). On the other hand, electrophiles can adduct proteins in the sperm axoneme and induce a complete loss of motility within minutes (Hughes et al., 2009). All the reagents inducing rapid motility loss in this study were either electrophiles (menadione, 4HNE) or have the potential to rapidly transform into electrophiles (2OHE2) capable of paralysing sperm movement by adducting to axonemal proteins. In contrast, AA could only influence sperm movement by generating ROS and this mechanism alone was incapable of significantly influencing sperm motility within the 15 min observation period, although it could precipitate long-term DNA damage (Fig. 6).
Free radical generation by human spermatozoa is a complex process involving several different mechanistic pathways which may result in the generation of ROS in the mitochondria, cytoplasm or cell surface (Koppers et al., 2010; Musset et al., 2012). The current consensus is that mitochondria constitute the major source of ROS in defective human spermatozoa and that MSR is the method of choice from a clinical diagnostic perspective. Luminol–peroxidase-dependent chemiluminescence is an excellent probe for detecting ROS in the extracellular space and represents the most sensitive method for detecting seminal leucocytes, particularly when combined with a leucocyte-specific agonist such as opsonized zymosan. Conversely, lucigenin, H2DCFDA and NBT appear to lack the sensitivity, specificity and, in lucigenin's case, the repeatability needed to effectively monitor ROS generation by human spermatozoa, while flow cytometry employing DAF-DA cannot discriminate between good and poor quality spermatozoa because NO is constitutively generated by viable cells. This study should help achieve a measure of rationalization and standardization in the methods used to detect the ROS generation that underpins a significant proportion of male infertility.