Since ROS affects living cells as a result of freezing, cryogenic storage, and subsequent thawing, we should consider some of the effects from low-temperature storage. The enzyme-driven chemical reactions described in Fig. 3.1 and the generation of ROS occur in nature at body temperature. Sperm function has been studied at body temperatures (37°C) as well as at ambient environmental temperatures since it has been shown that maintaining sperm at body temperature after ejaculation or collection is highly detrimental to maintenance of sperm motility and viability. Ejaculated sperm, depending on species, can function close to optimally at these temperatures. It has not been studied how low temperature influences spermatozoal enzyme systems, although it is likely that in mammalian systems they are optimized for mammalian body temperatures and any ROS that are generated due to osmotic and oxidative stress are scavenged suboptimally at subzero temperatures. As alluded to earlier, low temperatures can predispose cells to oxidative attack by affecting primary metabolic processes, membrane integrity of the plasma, and mitochondrial membranes, and compromise the activity of enzymes that may protect cells from ROS and RNS. Much of our knowledge of low-temperature cell biology derives from studies in plants, yeast, and cell-free frozen solutions . Plants are highly vulnerable to oxidative stress since O2 is a terminal electron acceptor in respiration and is produced by normal photosynthetic mechanisms. The technique of electron paramagnetic resonance (EPR) spectroscopy which monitors unpaired electrons has been used to determine that superoxide anion trapped in ice at -196°C remains unaltered for a minimum of 7 days  . This suggests that there are several main considerations for sperm stored at subzero temperatures: (1) production and stability of cooling-induced ROS, particularly free radicals, at low temperature; (2) functionality of SOD, catalase, and glutathione peroxidase at low temperatures; and (3) presence of unfrozen water in the intracellular and extracellular compartments. Regarding the first, any ROS formed prior to attainment of cellular freezing could remain stable during the cryopreserva-tion and subzero period. It is not known as yet for mammalian cells whether new ROS can be formed at subzero temperatures. Secondly, it is not likely that enzyme scavenging systems would be functional at temperatures associated with cooling to subzero temperatures nor warming during the thaw process until ambient temperature is reached to allow normal enzyme conformation and subsequent function. For scavenging to be at least partially optimal, temperatures would likely need to be in the 15-37°C range to prevent additional oxidative damage from surviving and newly generated ROS. A paucity of information exists for these subjects currently. In plants, experimental overexpression of SOD genes in transgenic alfalfa crops demonstrated enhanced winter survival indicating that SOD expression minimizes ROS damage when applied to this crop . Plants have a number of other adaptive responses to surviving winter extreme temperatures that mammalian cells do not have. However, animal cells have some similar capabilities and can be potentially modified or thera-peutically managed to better survive cryopreservation.
Very small amounts of water are known to be associated with proteins, carbohydrates, and membranes associated with cells and there is controversy surrounding the role of this water when cells are cryopreserved . It has been estimated that intact cells retain up to 5% of total intracellular water that may be excluded from freezing due to very tight association with protein and membrane structure. Some researchers consider this water to be nonfreezable and that it may play a significant role in tissue damage. An association of ROS with this unfreezable cellular water has not been made, but it is tempting to speculate that unfrozen water could potentially serve as a reservoir for ROS produced during cryopreservation and its associated dehydration process.
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A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.