Mitochondrial Antioxidants and Fertility

The deleterious effects resulting from the formation of ROS in the mitochondrion are, to a large extent, prevented by various antioxidant systems, and as such, mammalian mitochondria possess a relative abundance of enzymatic and nonenzymatic antioxidants designed to manage and counteract the ROS generated. An in-depth presentation of the literature is well beyond the scope of this chapter; however, a brief description is provided to represent the well-characterized ROS scavenging systems that act within the mitochondria and their known relationships with male fertility. Superoxide Dismutase

O- is one of the major ROS generated in the mitochondria and is critically maintained at the lowest possible concentration. O- can spontaneously dismutate or more commonly is enzymatically converted to H2O2 by a group of metalloenzymes called SOD [57]. O- is capable of propagating the generation of other ROS, either through the spontaneous reaction with NO creating peroxynitrite or via reducing transition metals within the mitochondrial ETC, which subsequently react with H2O2 producing hydroxyl radical. The mitochondrial matrix contains a specific form of SOD with manganese in the active site (MnSOD) [57], which eliminates the O2- formed in the matrix or on the inner side of the inner membrane. The inner membrane space contains a different SOD which contains copper and zinc instead of manganese (CuZnSOD) [58],

Not surprisingly, due to the important role of the enzyme, homozygous MnSOD knockout mice do not survive more than a few days following birth, in comparison to the heterozygous mutant mice which are viable and fertile [59, 60] . The life span and rate of aging are similar between heterozygous and wild-type animals, despite more accumulated DNA damage and increased prevalence of cancer during their life span [61]. In contrast, the overexpression of MnSOD to six to ten times above the normal level results in developmental abnormalities and decreased fertility of mice [62]. This decrease in fertility may be related to the importance of low-level ROS signals that have been shown to be critical during capacitative events, which will be discussed in Chap. 4 . Cytochrome c

Found in the intermembrane space of mitochondria, cytochrome c is an integral link in the ETC and has a dual role in O2- removal. In both functions, cytochrome c is reduced by either an electron from the ETC or by O2- and subsequently is regenerated (oxidized) by cytochrome c oxidase. A testis-specific isoform of cytochrome c has been identified; the knockout mouse model for the testis-specific isoform is fertile despite lower motility percentage in sperm from both vas deferens and epididymis. These knockout mice do exhibit loss of germ cells following aging (>4 months), due to increased levels of atrophy not apoptosis [63]. Glutathione Peroxidase

Glutathione peroxidases (GPxs) are a group of selenoenzymes that utilize reduced glutathione (GSH) for the reduction of H2O2 to H2O. GPxs participate in the recycling of ROS and some peroxidized compounds that are products from the attacks of organic molecules by ROS. The classical GPx (GPxl) is known to be ubiquitously expressed in all mammalian tissues [64] and can be detected in various cellular compartments including the cytosol, mitochondrial matrix, and intermembrane space. Homozygous knockout mice possessing no GPxl activity are healthy, fertile, develop normally, and do not show any signs of tissue damage and oxidative stress [65], Interestingly, exposure of GPxl knockout mice to toxins known to induce oxidative stress resulted in hypertension [66], suggesting that GPxl is more likely to be involved in protecting tissues and mitochondria against acute oxidative stress rather than the basal levels of mitochondrial ROS production. Its lack of importance in fertility is supported by Chabory et al. [67], which showed that Gpxl is expressed in very low levels compared to other GPx family members within the epididymis.

Phospholipid hydroperoxide GPx (GPx4) has broad selectivity, which allows it to reduce phospholipid hydroperoxides, H2O2, and cholesterol peroxides [68]. Three forms of GPx4 exist; nuclear, short, and the long form found exclusively in the mitochondria. The long form RNA transcript is predominately expressed in testis in mice [69]. Localized to the sperm midpiece, mitochondrial GPx4 has been hypothesized to play a role as a structural protein rather than an enzymatic antioxidant since it has been shown to have completely lost its solubility and its scavenging enzymatic properties [70]. Two independent knockout models have also confirmed that when mGPx4 is not expressed, it leads to male infertility due to structural malformations of the sperm midpiece and not due to a loss in antioxidant capability. Glutathione Reductase

GSH can either scavenge O- and hydroxyl radicals nonenzymatically or possibly more importantly by acting as an electron donor to several enzymes already discussed above. Once GSH is oxidized to GSSG (oxidized glutathione), it cannot be exported to cytosol and has to be reduced back to GSH in the mitochondrial matrix. The reduction is catalyzed by glutathione reductase (GRD), which is present in the matrix of mitochondria [71]. A strain of hypomorphic mice (Gr1a1Neu) that exhibited GRD activities that were just above basal levels (less than 10% in liver) compared to control mice were fertile and showed no phenotypic changes [72]. Peroxiredoxins

Peroxiredoxins are a recently discovered group of peroxidases that reduce H2O2 and lipid hydroperoxides, with at least six members currently identified in mammals [73]. Two isoforms of peroxiredoxins (PRDX3 and PRDX5) are found in mammalian mitochondria. The PRDX5 gene is also ubiquitously expressed in bovine tissues, with the highest expression found in testis [74]. In human spermatozoa, PRDX5 is localized to the acrosome, postacrosomal region, and midpiece. Therefore, PRDX5 may play a role in regulating ROS actions in the mitochondria as well as other functions. Although not all mitochondrial related, a number of peroxiredoxin knockout mice have been generated. PRDX1 knockout mice are fertile yet have a significant reduction in lifespan due to increased levels of oxidative protein and DNA damage and higher frequency of anemia and malignant cancer [75]. A similar anemic phenotype was also observed in the PRDX2 knockout [76]. PRDX3 knockout mice are fertile, but were shown to be more sensitive to lipopolysaccharide-induced oxidative stress [77]. PRDX6 knockout mice are also viable yet are shown to be more sensitive to hyperoxia and paraquat-induced oxidative stress [78, 79]. No knockout for PRDX5 is reported in the literature. Catalase

Catalase is another enzyme which catalyzes H2O2 into O2 and H2O. The role of catalase in H2O2 removal is thought to be insignificant compared to that of GPx [80]. Shown to be present at low levels in both human and rat spermatozoa [4, 81, 82], it is known to be absent from rabbit spermatozoa [26], mouse spermatozoa [7], and bull spermatozoa [83] . Unsurprisingly, due to its absence from mouse sperm, the knockout mouse models are fertile [84], although this does not preclude it from a role in spermatozoa in humans and the rat.

In general, knockout mouse models of mitochondrial antioxidants have not resulted in infertility, indicating that these enzymes are not critical for fertility. In contrast, they have indicated greater susceptibility to oxidative stress due to toxins or aging. The oxidative stress "challenged" mice in these experiments may better represent a more realistic model in humans considering the myriad environmental and external stimuli encountered in day-to-day activities.

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