Oxidative stress, osmotic stress and apoptosis: Impacts on sperm function and preservation in the horse

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1 Animal Reproduction Science 107 (2008) Oxidative stress, osmotic stress and apoptosis: Impacts on sperm function and preservation in the horse Barry A. Ball Department of Population Health and Reproduction, University of California, Davis, CA 95616, USA Available online 29 April 2008 Abstract Oxidative stress is an important component of the cytopathology of equine spermatozoa undergoing storage as liquid or frozen semen. Damage to chromatin, membranes and proteins of sperm are important components of oxidative damage to sperm. Similarly, sperm are exposed to a variety of osmotic stresses during storage that result from exposure to hypertonic media or result as a consequence of osmotic changes induced during freezing. A number of changes induced during processing and storage of equine sperm also appear to induce apoptotic-like changes which may adversely affect sperm survival and function. These processes appear in many cases to be interrelated, and this review will examine current understanding of these processes on the equine sperm function Elsevier B.V. All rights reserved. Keywords: Equine; Sperm; Oxidative stress; Osmotic stress; Apoptosis 1. Introduction Although considerable progress has been made over the past few years in liquid or frozen storage of equine sperm, there remains a large inter-individual difference in the success of semen preservation for the stallion. During low temperature storage equine sperm are subjected to oxidative damage to membrane phospholipids, proteins and chromatin. Osmotic stress leads to damage This paper is part of the Special issue entitled Proceedings of the 5th International Symposium on Stallion Reproduction, Guest Edited by Terttu Katila. Tel.: ; fax: address: baball@ucdavis.edu /$ see front matter 2008 Elsevier B.V. All rights reserved. doi: /j.anireprosci

2 258 B.A. Ball / Animal Reproduction Science 107 (2008) to the plasma membrane and alteration in sperm metabolism. Furthermore, evidence from a number of species also suggests that ejaculated sperm undergo apoptotic-like changes as a consequence of cryopreservation. It appears likely that these three processes are interlinked and may impact various compartments in the sperm cell via similar pathways. Therefore, an understanding of these processes and their common metabolic pathways may be important in attempts to obviate adverse affects on equine sperm during storage. 2. Oxidative stress 2.1. General impacts Oxidative stress is a well-defined component of many disease processes. The generation of reactive oxygen species (ROS) may occur as a normal consequence of oxidative metabolism or may result from specific mechanisms within particular cell types, such as the oxidative burst of leukocytes. Oxidative stress was suggested as an important factor in disruption of sperm function over 60 years ago (MacLeod, 1943); however, it is only recently that the importance of oxidative stress in normal and abnormal sperm function has become more apparent. It is now clear that ROS have an important role in normal sperm function and that an imbalance in either the production or degradation of ROS may have serious adverse effects on sperm. The effects of oxidative stress are particularly important during sperm storage by either cooling or cryopreservation, and this damage is further increased in situations where much of seminal plasma is removed from a semen sample because much of the antioxidant capacity in semen resides with seminal plasma ROS scavengers in equine semen The primary ROS scavengers described in semen are catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx). There appears to be a wide species variation in the relative abundance and importance of these scavengers in seminal plasma, and we determined the relative activities of these enzyme systems for equine semen. The activities of catalase (98.7 ± 29.2 U/mg protein), SOD (29.15 ± 6.64 U/mg protein) and GPx (0.87 ± 0.06 M NADPH oxidized/min/mg protein) were determined in equine seminal plasma (Ball et al., 2000; Baumber and Ball, 2005). Specific activity of catalase in tissue homogenates was significantly greater in the prostate gland than in the ampulla, bulbourethral gland, vesicular gland, cauda epididymal fluid, or testis. These data indicate that equine seminal plasma has a relatively greater activity of both catalase and superoxide dismutase with a significant variation between stallions in the activities of these scavengers. Sperm appear to have very limited amounts of ROS scavengers, and seminal plasma is a potent source of ROS scavengers which functions to protect ejaculated equine sperm from the adverse effects of ROS. The removal of seminal plasma during semen processing may increase the susceptibility of sperm to oxidative stress because of the removal of these enzyme scavengers. In addition to the enzyme scavengers discussed above, a number of other components of seminal plasma likely counteract oxidative stress in neat semen. In particular, factors such as albumin, urate, taurine, hypotaurine, pyruvate, lactate, ascorbic acid, tocopherol and ergothioniene are present in seminal plasma and may act as antioxidants (Mann et al., 1963; Alvarez and Storey, 1983; De Lamirande and Gagnon, 1992; Halliwell and Gutteridge, 1999). Relatively less research has been conducted on the composition of equine seminal plasma and the role of low molecular weight antioxidants in equine semen; although assessment of the total antioxidant capacity of

3 B.A. Ball / Animal Reproduction Science 107 (2008) seminal plasma suggests that these low-molecular weight components may constitute most of the antioxidant capacity of semen (Thiele et al., 1995) Generation of ROS by equine sperm Production of ROS by equine sperm appears to be derived from either a sperm-specific NADPH oxidase (NOX5) present in the plasma membrane of the sperm head or from sperm mitochondria (Sabeur and Ball, 2006, 2007). Although the superoxide anion (O 2 ) appears to be the primary ROS generated by equine sperm, this short-lived specie rapidly dismutates to form hydrogen peroxide (H 2 O 2 )(Ball et al., 2001b; Burnaugh et al., 2007), and it is likely H 2 O 2 that accounts for the major cytotoxic effect in sperm (Baumber et al., 2000). Generation of ROS is significantly increased in the presence of cryodamaged, nonviable or morphologically abnormal sperm, particularly sperm characterized by the presence of proximal cytoplasmic droplets or abnormalities of the midpiece (Ball et al., 2001b). Under these conditions, generation of greater amounts of ROS is principally driven by electron leakage from the mitochondrial electron transport chain with subsequent reduction of molecular oxygen to form the superoxide anion (Sabeur and Ball, 2006). Damage to sperm mitochondria appears to be an important aspect of cryodamage to equine sperm during cryopreservation. Frequently, morphological evaluation of sperm after freezing and thawing demonstrate moderate to marked swelling of the midpiece representing distension of mitochondria which suggests that sperm mitochondria are a significant site of cryodamage to these cells with the potential for uncoupling of normal oxidative metabolism, generation of ROS and induction of apoptotic change Effects of ROS on equine sperm capacitation Under physiological conditions, lesser generation of ROS is stimulated in the presence of calcium, and the membrane-associated NADPH oxidase, NOX5, appears to be the likely mechanism for this generation of ROS. This lesser generation of ROS by equine sperm is important in induction of capacitation with a concomitant increase in tyrosine phosphorylation (Baumber et al., 2003a). This observation is further confirmed by the finding that during capacitation of equine sperm in vitro there is an increase in production of the superoxide anion (Burnaugh et al., 2007). The ability of low very concentrations of ROS to induce capacitation in equine sperm also impacts sperm preservation. During cryopreservation, sperm have an increased intracellular calcium concentration, an increased generation of ROS, and a reduced antioxidant capacity because of removal of seminal plasma. These factors in turn could lead to premature capacitation of sperm (Neild et al., 2003) with a subsequent reduction in longevity of cryopreserved sperm. This cryocapacitation of sperm results in similar but not identical changes in sperm membrane as detected during capaciation in vitro (Thomas et al., 2006) Adverse effects of ROS on sperm function As previously noted in this paper, an increased generation of ROS by equine sperm may occur in the presence of large numbers of morphologically abnormal or damaged sperm in a sample which may adversely affect the remaining viable sperm due to an increased oxidative stress. Although superoxide anion appears to be the primary product generated by sperm, hydrogen peroxide is much more membrane permeable and appears to be the most important ROS for

4 260 B.A. Ball / Animal Reproduction Science 107 (2008) damage to equine sperm (Baumber et al., 2000). When equine sperm were exposed to exogenous ROS (generated by incubation with xanthine/xanthine oxidase) there was an increase in H 2 O 2 production and a decrease in sperm motility (Baumber et al., 2000). The addition of the enzyme scavengers for ROS was used to assess the relative importance of superoxide anion (O 2 ) and H 2 O 2. Catalase (which catabolizes H 2 O 2 ), but not superoxide dismutase (SOD), which catabolizes the superoxide anion, maintained normal motility secondary to an induced oxidative stress. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential. Therefore, sperm motility appears to be a sensitive indicator of oxidative stress and may be one of the first parameters affected during oxidative stress Effects of ROS and sperm storage on DNA integrity of equine sperm Reactive oxygen species are well characterized as a cause of DNA damage in a variety of cell types, including sperm. In equine sperm, there was a dose-dependent increase in DNA damage when these cells were exposed to increasing concentrations of ROS (Baumber et al., 2003b). DNA damage was blocked in the presence of catalase or reduced glutathione (GSH) but not superoxide dismutase, which indicates that H 2 O 2 was the major ROS responsible for DNA damage in equine sperm (Baumber et al., 2003b). Damage to sperm DNA appears to be initiated when oxidative stress is comparable to that previously shown to affect sperm motility. During sperm storage, there is also a measurable increase in DNA damage as detected by the comet assay with both cooled storage (Linfor and Meyers, 2002) and frozen storage (Baumber et al., 2003b) of equine sperm. However, the addition of antioxidants ( -tocopherol, GSH, ascorbic acid) or enzyme scavengers (catalase, superoxide dismutase) to cryopreservation extenders did not reduce the extent of DNA fragmentation detected subsequent to freezing and thawing of equine sperm (Baumber et al., 2005). The addition of SOD to cryopreservation extender significantly increased DNA fragmentation, suggesting again that H 2 O 2 is the primary ROS resulting in DNA damage in equine sperm. In human sperm, DNA damage secondary to oxidative stress increased before there were any measurable changes in sperm oocyte fusion or alterations in sperm motility (Aitken et al., 1989) and as noted earlier, less oxidative stress may enhance capacitation of sperm with subsequent ability to initiate fertilization. Therefore, less oxidative stress may allow sperm with DNA damage to fertilize the oocyte. The sperm cell has limited to no ability to repair DNA damage, and studies from other species indicates that although fertilization may occur, the rate of subsequent embryonic development is reduced and the rate of early embryonic death is increased in situations in which fertilization is initiated by DNA-damaged sperm (Ahmadi and Ng, 1999; Morris et al., 2002). Similar data are not available for the horse; however, it appears likely that DNA damage subsequent to oxidative damage might also lead to increased rates of early embryonic loss Effect of ROS on membrane lipid peroxidation Sperm membranes are characterized by relatively greater concentrations of polyunsaturated fatty acids (Parks and Lynch, 1992) that are susceptible to peroxidative damage (Aitken, 1995). Because H 2 O 2 and O 2 are not energetic enough to initiate lipid peroxidation, a transition metal catalyst is typically required to cause lipid peroxidation (Ball and Vo, 2002). The presence of a transition metal catalyst (such as Fe 2+ ) can initiate lipid peroxidation which then proceeds in a chain reaction with the formation of lipid peroxides and ultimately the formation of cytotoxic

5 B.A. Ball / Animal Reproduction Science 107 (2008) aldehydes including malondialdehyde and the more potent 4-hydroxynonenol (Aitken, 1995). Membrane lipid peroxidation alters membrane fluidity and in sperm, this alteration can affect the ability of sperm membranes to fuse, an important function during acrosomal exocytosis. Although lipid peroxidation is well characterized for mammalian sperm, equine spermatozoa appear relatively more resistant to membrane peroxidation than sperm of other domestic animals (Baumber et al., 2000; Neild et al., 2005). Cryopreservation, however, increased susceptibility of equine sperm to lipid peroxidation, which was more pronounced over the region of the sperm midpiece (Neild et al., 2005). Likewise, storage of liquid semen at 5 C resulted in a detectable increase in lipid peroxidation in equine sperm (Ball and Vo, 2002). In vitro, the addition of tocopherol significantly reduced lipid peroxidation in equine sperm (Ball and Vo, 2002). The vitamin E analog, tocopherol succinate, is more water soluble and appears to load more readily into mitochondria than native vitamin E. Experimentally, tocopherol succinate was superior to tocopherol in preventing lipid peroxidation of equine sperm; however, the succinate ester of tocopherol suppressed motility of equine sperm to a greater extent than did -tocopherol (Almeida and Ball, 2005) Effects of antioxidants on cooled storage of equine sperm We also evaluated the effect of addition of the ROS scavenger, catalase, as well as lipid and water-soluble antioxidants on the maintenance of equine sperm motility during storage at 5 C (Ball et al., 2001a). Semen was collected, and catalase was added in nonfat, dried skim-milk extender (NFDSM). Motility was determined by computerized semen analysis (CASA). The addition of catalase did not improve maintenance of motility during 72-h storage at 5 C. In a second experiment, lipid-soluble antioxidants were evaluated. Semen was diluted to a final concentration of sperm/ml in NFDSM containing butylated hydroxytoluene (BHT), -tocopherol, or the synthetic antioxidant, Tempo. The addition of BHT significantly reduced (P < 0.05) progressive motility during storage, and there were no positive treatment effects of either -tocopherol or Tempo on maintenance of motility. In a third experiment, water-soluble antioxidants were evaluated. Semen was diluted in NFDSM containing the vitamin E analog, Trolox, Vitamin C, bovine serum albumin, or combinations of these antioxidants although no positive effects of treatment were detected. A positive effect of addition of ascorbic acid on preservation of membrane integrity of cooled equine sperm has been observed; however, there was no effect of addition of catalase under similar conditions (Aurich et al., 1997). Likewise, the addition of N-acetyl cysteine had no effect on maintenance of equine sperm motility or membrane integrity during cooled storage (Pagl et al., 2006). In conclusion, the addition of catalase or a variety of lipid- or water-soluble antioxidants did not improve the maintenance of motility during short-term cooled storage of equine sperm in the presence of skim-milk-based extenders. Similarly, most studies that have examined the addition of antioxidants to cryopreserved equine sperm have not shown a positive effect on post-thaw parameters or fertility. The addition of the enzyme scavengers, catalase or SOD, or low molecular weight antioxidants such as reduced glutathione, ascorbic acid or -tocopherol, did not decrease DNA fragmentation, or increase mitochondrial membrane potential, viability or motility of frozen equine sperm after thawing (Baumber et al., 2005). In contrast, Aguero et al. (1995) reported a positive effect of addition of -tocopherol to cryopreserved equine sperm. In a large study evaluating multiple antioxidants and combinations of antioxidants to both liquid and frozen bovine sperm, antioxidants were generally not beneficial (Foote et al., 2002). However, there were interactions between extender type (skim-milk-based vs. egg yolk-based extenders) and the addition of antioxidants. These

6 262 B.A. Ball / Animal Reproduction Science 107 (2008) authors suggest that the addition of casein and other milk proteins in extenders may provide abundant ROS scavenging capability to many extender formulations and may, therefore, obviate the need for addition of lipid or water-soluble antioxidants to semen extenders containing milk products (Foote et al., 2002). As noted above, this notion may also apply to equine semen as evidenced by the lack of positive effect of addition of antioxidants in many studies utilizing skim-milk-based extenders. Although the literature concerning the addition of vitamin E ( -tocopherol) to semen extenders appears to have at best variable response on maintenance of sperm function and fertility, a number of studies suggest that dietary supplementation of vitamin E may positively impact semen quality and maintenance of sperm during in vitro storage. Dietary supplementation with vitamin E appears to be effective in limiting lipid peroxidation of sperm membranes in both turkeys and in chickens (reviewed by Breque et al. (2003)). The addition of organic selenium to the diet also increases activity of selenium-dependent glutathione peroxidase in seminal plasma (Breque et al., 2003). Interestingly, supplementation of the female with dietary vitamin E and organic selenium appeared to improve fertility, perhaps through effects on sperm storage in the oviduct (Breque et al., 2003). In boars, dietary amounts of vitamin E and selenium affected the percentage of motile, morphologically normal sperm present in the ejaculate as well as the fertilization rate in gilts mated (Marin-Guzman et al., 1997). At present, similar reports are not available for the stallion; however, the results from both avian and other mammalian species suggests that dietary amounts of vitamin E and selenium may affect sperm quality and possibly sperm storage in this species. 3. Osmotic stress 3.1. General impacts on spermatozoa There has been considerable interest in the effects of osmotic stress on sperm as one of the important factors associated with damage during cryopreservation (reviewed by Watson (1995)). In particular, osmotic stress induced by changes in cell volume resulting from the movement of water and solutes across the sperm plasma membrane appears to be an important feature of damage to sperm (Gao et al., 1997). Equine sperm appear to behave as linear osmometers, and cell death occurs if sperm swell or shrink beyond fairly narrow osmotic tolerances (Ball and Vo, 2001; Pommer et al., 2002). Equine sperm are exposed to changes in osmotic pressure during several steps of the cryopreservation process. The addition of glycerol or other permeable cryoprotectants (CPA) as a part of most cryopreservation media results in a transient increase in osmotic pressure prior to equilibration of the CPA across the sperm membrane (Ball and Vo, 2001). As the extracellular media freezes during cooling, it is thought that water freezes leaving sperm suspended in small pockets of media that are increasingly hyperosmotic due to the presence of increasing solute concentrations in the unfrozen water. Differences in the permeability of glycerol and water and the temperature dependence of these permeabilities appear to account for a considerable portion of osmotic stress (Ball and Vo, 2001; Pommer et al., 2002). When glycerol-loaded cells are abruptly transferred to an isotonic, glycerol-free medium (such as skim-milk extenders or female reproductive tract secretions) the intracellular environment is hyperosmotic relative to the extracellular environment. Because the permeability of glycerol is less than that of water, there is a net influx of water with resulting increase in cell volume due to a relative hypoosmotic condition. If changes in cell volume exceed its osmotic tolerance, cell lysis may result. Recent experimental evidence further corroborates the observation that osmotic stress rather than formation of intracellular ice

7 B.A. Ball / Animal Reproduction Science 107 (2008) appears to be responsible for most of the damage to equine sperm during freezing and thawing in the presence of glycerol (Morris et al., 2007). Exposure of equine sperm to effectively variable concentrations of glycerol occurs during freezing and is dependent upon the cooling rate. The addition of increasing concentrations of the permeant cryoprotectant, glycerol decreased the motility of equine sperm (Ball and Vo, 2001). When sperm that were equilibrated with glycerol were abruptly returned to near isosmotic conditions by dilution, there was a significant reduction in motility for all concentrations of glycerol tested. Exposure of sperm to increasing tonicity by increasing glycerol concentrations increased the uptake of propidium iodide at greater than 1200 mosm; however, return of equine sperm to near isosmotic conditions significantly increased the uptake of PI compared to sperm maintained at anisosmotic conditions at all osmolalities >300 mosm. Mitochondrial membrane potential was not significantly altered by increased osmolality associated with increased glycerol. When sperm were abruptly returned to near isosmotic conditions, however, there was a significant decline in mitochondrial membrane potential. The percentage of live, acrosome-intact sperm did not decrease during exposure to increased concentrations of glycerol; however, the abrupt removal of glycerol by returning sperm to near isosmotic conditions significantly reduced the percentage of live, acrosome-intact sperm. Changes in the percentage of live, acrosome-intact sperm were due entirely to changes in viability because the percentage of acrosome-intact sperm was not significantly affected by changes in osmolality or the subsequent removal of glycerol by dilution. These data indicate that the rapid addition and removal of glycerol from equine sperm adversely affects various aspects of sperm function including motility, viability and mitochondrial membrane potential. In addition to near molar concentrations of cryoprotectants added and changes in osmotic pressure during freezing and thawing, water-soluble lubricants used for artificial vagina preparation represent another possible source of osmotic stress to sperm during collection and handling of semen (Devireddy et al., 2002a). Many of these lubricants are based upon sodium methylcellulose and are extremely hyperosmotic with osmolarities of approximately 2000 mosm. Significant contamination of semen with these water-soluble lubricants during semen collection can result in osmotic stress to sperm cells with subsequent changes in sperm motility (Devireddy et al., 2002a). This is particularly likely in situations where low volume ejaculates are collected in an artificial vagina that has been excessively lubricated with water-soluble lubricants. If sperm are subjected to osmotic stress (as might be induced by contamination with a watersoluble lubricant) and subjected to thermal stress (cold shock) at temperatures >0 C, it appears that these factors may lead to alterations in sperm membrane function that are detrimental to sperm during freezing and thawing. In particular, the combination of osmotic and thermal stress to sperm resulted in a decrease in water transport across the sperm membrane (Devireddy et al., 2002a). Because water transport across the sperm membrane is an important feature of sperm survival during freezing and thawing, these alterations in water transport parameters that occur in sperm as a consequence of handling conditions prior to freezing should be considered as an important variable in the outcome of semen cryopreservation in the stallion (Devireddy et al., 2002b) Interaction between osmotic and oxidative stress Exposure of equine sperm to either hypo- or hyperosmotic conditions results in an increased generation of superoxide anion by viable sperm cells (Burnaugh et al., submitted) indicating that there is an active interplay between these two stressors. The mechanism of this interplay is not well characterized; however, studies from other cell types suggest that activation of membrane-associated phospholipase A2 (PLA2) with subsequent activation of an NADPH

8 264 B.A. Ball / Animal Reproduction Science 107 (2008) oxidase may represent a potential pathway for generation of superoxide anion in response to osmotic stress (Lambert, 2003; Lambert et al., 2006). In human sperm, products of PLA2, including arachadonic acid, appear to activate NADPH oxidase with a concomitant increase in superoxide anion generation (Aitken et al., 2006). The resulting increase in ROS may in turn be involved in activation of protein tyrosine kinases which regulate volume sensitive osmolyte channels important in maintaining homeostasis (Lambert et al., 2006). Altered protein tyrosine phosphorylation occurs after exposure of equine sperm to hyperosmotic conditions, and this increase can be blocked in the presence of inhibitors of NADPH oxidase as well as the protein tyrosine kinase inhibitor, staurosporine (Linfor et al., 2002; Burnaugh et al., submitted). Together, these studies indicate that there is cross-talk between osmotic and oxidative stress in equine sperm which may alter sperm function and possibly compound damage to cells exposed to these stressors during cryopreservation. 4. Apoptotic-like changes in equine sperm Apoptosis or programmed cell death is well characterized for a variety of somatic cells and plays an important role in regulation of spermatogenesis in most mammals, including the horse (Heninger et al., 2004, 2006). The potential role of apoptosis in mature sperm has been more controversial; however, we and others have reported that equine sperm demonstrate a number of changes characteristic of apoptosis (Brum et al., 2008; Desvousges et al., 2006). Separation of equine sperm via density-gradient centrifugation results in a lower-density subpopulation characterized by a lesser mitochondrial membrane potential, altered membrane permeability, increased DNA fragmentation, and activation of aspartic acid-directed cysteine proteases (caspases) which are features typically associated with apoptosis in somatic cells (Brum et al., 2008). Likewise, after cryopreservation, a subpopulation of equine sperm demonstrate evidence of apoptotic changes including decreased mitochondrial membrane potential, altered membrane structure, caspase activation (Brum et al., 2008) externalization of phosphatidylserine residues on the plasma membrane (Thomas et al., 2006) and increased DNA fragmentation (Baumber et al., 2003b). Similar results have been reported subsequent to cryopreservation of human (Paasch et al., 2004) and bovine sperm (Martin et al., 2004, 2007). Despite findings of apoptotic markers in subpopulations of sperm and after cryopreservation, controversy remains concerning the significance of this finding, and more research is needed to clarify the role of apoptotic changes subsequent to cryopreservation on the fertility of frozen-thawed equine sperm. 5. Summary and conclusions Although oxidative stress, osmotic stress and apoptosis have been reviewed separately as factors associated with sperm preservation, it appears very likely that there is considerable interplay among these stressors and their ultimate effects on sperm. In somatic cells, both oxidative and osmotic stress can induce apoptotic changes (Halliwell and Gutteridge, 1999; Lang et al., 2004; Halliwell, 2006; Burg et al., 2007). Osmotic stress is associated with increased superoxide generation in a variety of somatic cells as well as in equine sperm, and may serve as a signaling mechanism responsible for the cell s adaptive response. Oxidative stress may initiate apoptosis in human sperm (Agarwal and Said, 2005), and many features of oxidative damage to sperm appear similar to those initiated during apoptosis. Further research should evaluate the molecular pathways which may represent convergence of these stresses on the sperm cell with an aim to reducing their net detrimental effect on sperm during preservation.

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