Cryopreservation and Oxidative Stress

Spermatozoa and seminal plasma have several mechanisms that generate ROS and this is discussed below. However, sperm and seminal plasma possess a number of enzymes and low-molecular-weight antioxidants that scavenge ROS in order to prevent cellular damage. During spermiogenesis, when sperm shape is modified and streamlined by losing most of the cytoplasm and, thus, intrinsic cytoplasmic enzyme scavenger systems, sperm attain vulnerability to oxidative attack [36]. Spermatozoa subsequently contain a limited volume of cytoplasm and are predominantly dependent upon the antioxidant support of seminal plasma. Further, sperm preparation for cryopreservation often involves the removal of seminal plasma by centrifugation, thereby increasing the susceptibility of spermatozoa to oxidative stress. In addition, research suggests that the antioxidant activity of the spermatozoa themselves may be decreased by cryopreservation [37, 38]. Freeze thawing of equine, human, and bovine spermatozoa was associated with an increase in ROS generation [39-43]. Recent studies have demonstrated that rhesus macaque sperm also suffer from excessive ROS generation when frozen and subsequently thawed [44, 45] in addition to severe DNA and chromosomal damage [5].

Oxidative stress denotes a condition associated with an increased rate of cellular damage induced by ROS [46]. The cause of the pro-oxidant-antioxidant shift may be due to an increase in ROS production, a decrease in antioxidant capacity, or possibly a combination of the two. Oxidative stress induced by the generation of ROS in vitro results in a reduction in sperm motility, viability, ionophore-induced acrosome reaction and sperm-oocyte fusion. Hydrogen peroxide appears to be the primary ROS responsible for these changes, and membrane lipid peroxidation is believed to be an important mechanism of action [36, 47-50].

The lipid peroxidation cascade is initiated when ROS attack polyunsaturated fatty acids (PUFAs) in the sperm cell membrane [50-52]. Spermatozoa are particularly susceptible to oxidative attack because they contain high concentrations of unsaturated fatty acids and, as terminally differentiated cells, have limited repair mechanisms. As a consequence of lipid peroxidation, the plasma membrane loses the fluidity and integrity it requires for participation in the membrane fusion events associated with fertilization. In addition to membrane effects, lipid peroxidation can also damage DNA. Peroxidation of DNA can lead to chromatin cross-linking, base changes, and DNA strand breaks. Several researchers have reported DNA damage in spermatozoa associated with membrane lipid peroxidation and oxidative stress [36, 53-55] .

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