Mitochondrial ROS and Spermatozoa 2211 Mitochondrial ROS Production

The first reports of mitochondrial ROS generation in mammalian spermatozoa were established in the rabbit using cytochrome c peroxidase assays [26] and an in-depth study in the rat via chemiluminescence [9]. The first analysis of human spermatozoa using the novel MitoSOX Red superoxide fluorescent probe by Koppers et al. [27] indicated that the mitochondria was indeed a major source of ROS and oxidative stress observed in defective human spermatozoa. The level of mitochondrial ROS generation within the dysfunctional sperm population highly correlated with decreased sperm motility. Confirming these results, stimulation of mitochondrial ROS via inhibition of different ETC complexes resulted in decreased motility and lipid peroxidation, a hallmark of oxidative stress. Although only a recent advance regarding the understanding of the subcellular sources of ROS in mammalian spermatozoa, a number of studies provide further support for this hypothesis. Mitochondrial Membrane Potential, ROS, and Oxidative DNA Damage

The rate of ROS formation from the ETC is found to be increased when the electron flow slows down (resulting in a reduced state) and/or when the concentration of oxygen increases [30]. However, the generation of ROS from mitochondria is not straight forward and is becoming an increasingly complex process. The contribution of ROS between different sites of ROS formation, e.g., Complex I vs. III, can greatly vary between different tissues and can also change depending on the mitochondrial membrane potential (DYm) [43], For example, complex III is responsible for the majority of O- produced in both heart and lung mitochondria [44, 45]; in contrast, complex I is the major ROS site in the brain [43].

In human spermatozoa, a number of studies have now shown that the DYm can be used as an indicator of sperm function and therefore quality. Spermatozoa that exhibit high DYm generally have been shown to have high fertilizing capacity as represented by increased ability to acrosome react, higher motility, and normal morphology [46], In contrast, spermatozoa with low MMP showed correlations with decreased DNA stability [47] and motility [48-50]. Indicative of all these results is that low DYm spermatozoa cells are associated with reduced IVF rates [49].

Given the relationship between DYm and ROS generation, the most critical study to date was performed by Wang et al. [51], whom in accordance with other studies showed that patients exhibiting abnormal semen parameters had a significantly lower DYm than control subjects. More importantly, they also showed patients had higher rOs levels and the two parameters negatively correlated with the ROS produced (r = -0.45). The DYm also was positively correlated with sperm concentration (r=0.62). As such, the DYm has been linked as a strong indicator of sperm function, which may be due to its inverse relationship with ROS generation.

While these studies do not provide causative evidence for a link between DYm and ROS generation, it is further supported in a subsequent study by De Iuliis et al. [52], which found that the 8-hydroxy-2'-deoxyguanosine (8OHdG) formation in human spermatozoa is negatively associated with DYm. To further emphasize such a relationship, exposure of purified human spermatozoa to electromagnetic radiation (EMR) results in significant increases in mitochondrial ROS and 8OHdG formation, which are highly correlative with each other (R2 = 0.727) [53]. While no study to date has analyzed the correlation between all three factors, mitochondrial ROS, DYm, and oxidative DNA damage, these data combined does suggest all three have significant relationship. Lipid Peroxidation in Midpiece

Along with decreased motility, one of the best markers of oxidative stress in spermatozoa is increased levels of lipid peroxidation. Validated in a number of subsequent studies, it has also now been shown that lipid peroxidation directly affects sperm functions including motility and sperm-oocyte fusion [54, 55],

The direct stimulation of mitochondrial ROS by inhibition of the ETC results in the subsequent increase in lipid peroxidation in human spermatozoa [27], Interestingly, the lipid peroxidation observed in this study is distinctly localized to only the midpiece. A similar result was also observed following stimulation with fatty acids (FAs) (discussed below). This is indicative that lipid peroxidation within the midpiece is also a marker of mitochondrial ROS generation due to the unique localization of mitochondria to the midpiece. In light of these results, it is important to note that a number of external stimuli have been used to generate oxidative stress in human spermatozoa such as iron (II) [56],

While still a novel concept, there is now an increasing evidence that the mitochondria is a major source of ROS in mammalian spermatozoa, and as such, is likely to be a key focus in future research of oxidative stress-related male infertility.

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