Effects of ROS on Membranes

As mentioned before, the sperm plasma membrane is particularly susceptible to oxidative stress because of its extraordinary content of double-bonds bearing membrane lipids, which can easily be oxidized by excessive ROS levels produced by both, the sperm cells as well as leukocytes. The process by which these plasma membrane lipids are oxidatively damaged is called "lipid peroxidation" (LPO).

Eventually, this process results in decreased membrane fluidity. As a result, receptor-mediated signal transduction as well as ion gradients derail [60] leading to loss of sperm membrane function and therefore functional capacity to fertilize oocytes.

LPO can be subdivided into three phases, initiation, propagation and termination. During the initiation phase, ROS attack the PUFA at carbon atoms adjacent to the double bonds leading to hydrogen abstraction from neighbouring methylene groups, which are especially reactive. Eventually, this process creates a lipid radical and water. The free electron is transferred to the lipid creating a new radical, which is subsequently stabilized by delocalization of the free electron over the whole molecule resulting in an energetically more stable structure than the initiating free radical. Considering that the resulting lipid radical is also not a very stable molecule, it will spontaneously react with molecular oxygen to form lipid peroxide. The propagation of the reaction is characterized by a reaction of this lipid peroxide radical molecule with a neighbouring fatty acid creating another fatty acid radical in a process called "radical chain reaction". Ultimately, this process causes damage to numerous molecules and up to 60% of the unsaturated fatty acid contend present in the plasma membrane can be oxidized [15]. The propagation of LPO terminates when one radical reacts with another radical, thus producing a non-radical, stable product whereby the two free electrons from the two radicals form a covalent bond.

With regard to the impact of oxidative stress on membrane lipids Khosrowbeygi and Zarghami [55] showed that spermatozoa from patients with asthenozoospermia, asthenoteratozoospermia or oligoasthenoteratozoospermia had significantly higher PUFA levels in their plasma membranes than normozoospermic men and were therefore more susceptible to oxidative stress and LPO. Additionally, significantly higher malondialdehyde (MDA) concentrations, which correlated negatively with the sperm count, were detected in samples from infertile patients [ 61]. Another study revealed that the amount of MDA present in spermatozoa correlated negatively with fertilization rates in an IVF programme [62].

Essentially, sperm exposure to oxidative stress cause by leukocytes induces a decrease in membrane fluidity, which is directly related to the membrane function and its fusogenic capacity [46]. This loss of membrane fluidity and functionality might lead to a rapid loss of intracellular ATP causing decreases in motility with axonemal damages and viability [63]. Consequently, not only sperm motility but also capacitation [64], acrosomal function [65] and acrosin activity are impaired [66],

15.5.2 Effect of ROS on DNA

During the process of LPO, numerous stable carbonyl-containing by-products such as MDA and 4-hydroxy-2-alkenals such as 4-hydroxy-nonenal (HNE), resulting from o)6-fatty acids such as DHA are formed. These by-products themselves are either highly mutagenic (MDA) or genotoxic (HNE) [67] . Accordingly, these byproducts pose the danger for further DNA damage by adduct formation on spermatozoa [68]. In addition to the higher release ofMDA in semen of men with seminal oxidative stress, protein carbonyl and sialic acid are elevated [61] as an indication of oxidative damage to proteins and possible protective response of the body to such oxidative stress, respectively [69].

Among other factors, the different physico-chemical behaviour of different ROS (H2O2 vs. 'OH or ' O2-) is the reason for a differentiated action of membrane permeable and non-membrane permeable ROS on different sperm functions such as motility or DNA integrity. Also, the location of the production, extrinsic by leukocytes or intrinsic by the male germ cells themselves, appears to play a role as extrinsic ROS produced by leukocytes rather impairs sperm motility, while intrinsic ROS production seems to preferentially affect sperm nuclear DNA (nDNA) fragmentation [34]. Such nDNA damage has repeatedly and unequivocally been shown to be associated with fertilization and/or pregnancy failure after intrauterine insemination [70], in vitro fertilization [71-74] and ICSI [75-78] and is therefore predictive of the success of assisted reproduction.

Only until recently, investigations on sperm DNA damage merely focused on the nDNA. nDNA is surrounded and protected by nuclear proteins, namely, histones in somatic cells and protamines in spermatozoa. However, any eukaryotic cell contains a second type of DNA, mitochondrial DNA (mtDNA), which is, in contrast to nDNA, not protected. Furthermore, mtDNA replicates very fast without proper proof-reading and has only a very basic repair mechanism [79], thus making certain regions of the mitochondrial genome up to 100-times more susceptible to damage and mutations [80]. As a result, mtDNA is exceptionally susceptible to mutations and numerous diseases including male infertility such as asthenozoospermia or oli-goasthenozoospermia [81, 82].

mtDNA encodes for 13 polypeptides that are essential for the ETC on the inner mitochondrial membrane and 22 tRNAs and 2 rRNAs that are necessary for the translation of these polypeptides. These polypeptides are intimately involved in oxidative phosphorylation and ATP production in the mitochondria. During this process, mitochondria continuously oxidize different substrates while, at the same time, reducing oxygen to water [83]. However, as a by-product of energy production, the mitochondria also generate most of the endogenous ROS of the cell, and these damage the mitochondria, mtDNA and the cell. Consequently, mtDNA damages will result in decreased mitochondrial membrane potential (A^m) and defective mitochondrial function. Yet, the latter is essential for sperm motility [84] and is negatively correlated with seminal oxidative stress [85] . In turn, mitochondrial dysfunction has repeatedly been shown to cause an increased release of mitochon-drial ROS early events of apoptosis [86, 87] . Therefore, this parameter has been suggested as being a highly sensitive parameter [88] .

Ruiz-Pesini et al. [89] and Hoshi et al. [90] found a correlation between the quality of the semen and the functionality of the respiratory chain in sperm mitochondria. Moreover, it has been demonstrated that mtDNA point mutations, mtDNA single nucleotide polymorphisms and mtDNA haplogroups can significantly influence semen quality [91-94]. Thus, due to the high sensitivity of mtDNA to damage and the essential role mitochondria play in the cells' energy production, it is plausible that seminal oxidative stress may lead to mtDNA damage. This may result in dysfunction of the mitochondrial respiratory chain including further stimulation of mitochondrial ROS production and oxidative damage due to derailment of the ETC causing a vicious cycle [95].

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