Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.     

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer‐assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.     

NOX5 in human spermatozoa: expression, function, and regulation

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+) chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the H(V)1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.     

Radicaux libres et spermatozoïdes humains: physiologie et physiopathologie

The role of reactive oxygen species in the physiopathology of human sperm function has been emphasized in recent years. Their production in semen has been associated with loss of motility, decreased capacity for spermoocyte fusion and loss of fertility. In semen preparations, there are two major sources of reactive oxygen species: leucocytes and spermatozoa themselve. It has been proposed that reactive oxygen species production by human spermatozoa was dependent upon a membrane-bound NADPH oxidase or a mitochondrial diaphorase. Hydrogen peroxide produced by the dismutation of superoxide anion has been recognized as the most toxic oxidizing species for human spermatozoa. Owing to their high content of polyunsaturated fatty acids, it has been proposed that lipid peroxidation of the sperm plasma membrane is largely responsible for defective sperm function. Reactive oxygen species also affect the sperm axoneme as a result of ATP depletion, inhibit mitochondrial functions, and synthesis of DNA, RNA and proteins, produce cytoskeletal modifications and inhibit sperm-oocyte fusion. Human spermatozoa possess enzymatic defence systems such as superoxide dismutase, glutathion peroxidas/reductase and catalase to counteract the toxic effects induced by reactive oxygen species. Correlations have been reported between their effectiveness and the duration of sperm motility. If the excessive production of reactive oxygen species is detrimental for human spermatozoa, they could also participate in the physiological function of the spermatozoa when present at low concentrations. Indeed, reactive oxygen species have been shown to be involved in the activation of several enzymes. Furthermore, sperm capacitation, acrosome reaction and sperm-zona interaction would be enhanced by reactive oxygen species.     

Sea urchin spermatozoa generate at least two reactive oxygen species; the type of reactive oxygen species changes under different conditions

Reactive oxygen species (ROS) cause oxidative stress and act as signal transduction molecules in many cells. Spermatozoa from several mammals generate ROS, which are involved in male infertility and signaling during capacitation. In the present study, we investigated ROS generation by sea urchin spermatozoa at the initiation of motility, during dilution with seawater, and following egg jelly treatment. In seawater containing an ROS indicator, 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), fluorescence increased after the addition of spermatozoa. The ROS generation rate was dependent upon the dilution ratio and respiratory rate of the spermatozoa. Spermatozoa in sodium-free seawater did not increase fluorescence, but fluorescence did increase with the addition of NaCl. Sodium chloride also led to the initiation of sperm motility and respiration. Using the indicator MitoSOX Red, ROS generation was detected from spermatozoa exposed to egg jelly dissolved in seawater, but not in normal seawater. Moreover, the respiratory inhibitor antimycin A prevented CM-H(2)DCFDA-detectable ROS and increased MitoSox-detectable ROS at a higher concentration. These findings revealed that the ROS generated were of different species, possibly hydrogen peroxide (H(2)O(2)) and superoxide anion (O(-)(2)), and their detected levels were altered by egg jelly. We concluded that sea urchin spermatozoa generate at least two species of ROS depending on the physiological conditions to which they are exposed. It is possible that the major ROS from sea urchin spermatozoa changes during the course of fertilization.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Stimulation of mitochondrial reactive oxygen species production by unesterified,unsaturated fatty acids in defective human spermatozoa

Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The etiology of this condition is thought to involve the excessive generation of reactive oxygen species by human spermatozoa; however, the cause of this aberrant activity is unknown. In this study we demonstrate that defective human sperm populations are characterized by high cellular contents of both esterified and unesterified fatty acids and a decrease in the proportion of the total fatty acid pool made up by docosahexaenoic acid. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation (P < 0.001). This relationship was causal and mediated by the range of unesterified, unsaturated fatty acids that are present in human spermatozoa. Thus direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We conclude that defective human spermatozoa are characterized by an abnormally high content of fatty acids that, in their unesterified, unsaturated form, promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence.     

Prostasomes, dérivés actifs de l’oxygène et sperme humain

Prostasomes are particular lipid vesicles secreted by the human prostate and found in the semen. No specific action has yet been attributed to prostasomes, but they appear to act at various levels. For example, prostasomes enhance sperm motility in vitro and participate in the immunomodulation properties of seminal plasma. Excessive production of reactive oxygen species (ROS) in human semen has a negative influence on the functional capacities of spermatozoa. The presence of leukocytes in semen is associated with increased production of ROS that can be harmful to sperm cells, under certain conditions. Previous results tend to suggest a possible role of prostasomes on ROS production in human semen. After reviewing the literature concerning the structural and functional characteristics of prostasomes and the role of ROS in human semen, we report our results concerning the influence of prostasomes on ROS production and the consequences on semen. We have demonstrated that prostasomes exert an antioxidant function in human semen. This function is effective both on polymorphonuclear neutrophils and on sperm cells. The mechanism of action of prostasomes is unusual, as they act on ROS production mainly on the plasma membranes of neutrophils. They induce a decrease of NADPH-oxidase activity associated with rigidification of the plasma membrane. Prostasomes protect the functional capacities of spermatozoa during an oxidative stress created by the presence of NADPH in the incubation medium.     

Trehalose improves rabbit sperm quality during cryopreservation

High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, acrosome integriy, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry.     

Electrophilic Aldehydes Generated by Sperm Metabolism Activate Mitochondrial Reactive Oxygen Species Generation and Apoptosis by Targeting Succinate Dehydrogenase

Oxidative stress is a major cause of defective sperm function in cases of male infertility. Such stress is known to be associated with high levels of superoxide production by the sperm mitochondria; however, the causes of this aberrant activity are unknown. Here we show that electrophilic aldehydes such as 4-hydroxynonenal (4HNE) and acrolein, generated as a result of lipid peroxidation, target the mitochondria of human spermatozoa and stimulate mitochondrial superoxide generation in a dose- and time-dependent manner. The activation of mitochondrial electron leakage by 4HNE is shown to involve the disruption of succinate dehydrogenase activity and subsequent activation of an intrinsic apoptotic cascade beginning with a loss of mitochondrial membrane potential and terminating in oxidative DNA adduct formation, DNA strand breakage, and cell death. A tight correlation between spontaneous mitochondrial superoxide generation and 4HNE content (R² = 0.89) in untreated populations of human spermatozoa emphasized the pathophysiological significance of these findings. The latter also provide a biochemical explanation for the self-perpetuating nature of oxidative stress in the male germ line, with the products of lipid peroxidation stimulating free radical generation by the sperm mitochondria in a positive feedback loop.     

Possible Role of Nitric Oxide on Fertile and Asthenozoospermic Infertile Human Sperm Functions

The capacity of human sperm fertilization is principally dependent on sperm motility and membrane integrity. Oxygen-derived free radicals, such as superoxide anion, are known to impair sperm motility and membrane integrity by inducing membrane lipid peroxidation (LPO). Nitric oxide (NO), a biologically active free radical, has recently been shown to inactivate superoxide and increase intracellular guanosine-3', 5'-cyclic monophosphate (cGMP). The aim of this study is to investigate the effects of NO on human sperm motility, viability, lipid peroxidation and cGMP in fertile and asthenozoospermic infertile individuals in vitro. Semen samples were obtained from 10 fertile volunteers and 10 asthenozoospermic infertile patients. Washed spermatozoa were incubated at 37°C in Ham's F-10 medium with 0, 25, 50, 100, 200, or 400nM sodium nitroprusside (SNP, Na₂ [Fe(CN) ₅NO] · 2H₂O), a nitric oxide releaser. Samples were analyzed for viability, determined by eosin-Y dye exclusion method at 0, 1, 2, 3, 5 and 6 h of incubation; motility, determined by the trans-membrane migration method within 2 h of incubation; LPO determined by malondi-aldehyde (MDA) -thiobarbituric acid method at 3 h of incubation; and the intracellular cGMP, determined by ¹²⁵I-cGMP radioimmunoassay at 3 h of incubation. The results showed: in both fertile and infertile samples, viability, trans-membrane migration ratio and the levels of intracellular cGMP in 25-100nM SNP-treated spermatozoa were significantly higher than those in control groups, while MDA contents in treated groups were significantly lower than those in controls. However, when concentrations of SNP increased to 200-400nM, the opposite effects were exhibited. The effects of SNP on these processes were biphasic within 25-400nM. The most effective concentration was 100nM. These data suggested that NO is beneficial to sperm viability and motility in both fertile and infertile individuals, and that reduction of lipid peroxidative damage to sperm membranes and increase of intracellular cGMP may be involved in these benefits.     

Reactive oxygen species generation and human spermatozoa: The balance of benefit and risk

Although the generation of reactive oxygen species is an activity normally associated with phagocytic leucocytes, mammalian spermatozoa were, in fact, the first cell type in which this activity was described. In recent years it has become apparent that spermatozoa are not the only nonphagocytic cells to exhibit a capacity for reactive oxygen species production, because this activity has been detected in a wide variety of different cells including fibroblasts, mesangial cells, oocytes, Leyding cells endothelial cells, thryroid cells, adipocytes, tumour cell and platelets. Since the capacity to generate reactive oxygen species is apparently so widespread, the risk-benefit equation for these potentially pernicious molecules becomes a matter of intese interest. In the case of human spermatozoa, the risk of manufacturing reactive oxygen metabolites is considerable because these cells are particularly vulnerable to lipid peroxidation. Indeed, there is now good evidence to indicate that oxygen radicals are involved in the initiation of peroxidative damage to the sperm plasma membrane, seen in many cases of male infertility. This risk is off-set by recent data suggesting that superoxide anions and hydrogen peroxide also participate in the induction of key biological events such as hyperactiavated motility and the acrosome reaction. Thus, human spermatozoa appear to use reactive oxygen species for a physiological purpose and have the difficult task of ensuring the balanced generation of these potentially harmful, but biologically important, modulators of cellular function.     

Peroxiredoxins: hidden players in the antioxidant defence of human spermatozoa

Spermatozoon is a cell with a precious message to deliver: the paternal DNA. Its motility machinery must be working perfectly and it should be able to acquire fertilizing ability in order to accomplish this mission. Infertility touches 1 in 6 couples worldwide and in half of the cases the causes can be traced to men. A variety of conditions such as infections of the male genital tract, varicocele, drugs, environmental factors, diseases, smoking, etc., are associated with male infertility and a common feature among them is the oxidative stress in semen that occurs when reactive oxygen species (ROS) are produced at high levels and/or when the antioxidant systems are decreased in the seminal plasma and/or spermatozoa. ROS-dependent damage targets proteins, lipids, and DNA, thus compromising sperm function and survival. Elevated ROS in spermatozoa are associated with DNA damage and decreased motility. Paradoxically, ROS, at very low levels, regulate sperm activation for fertilization. Therefore, the regulation of redox signaling in the male reproductive tract is essential for fertility. Peroxiredoxins (PRDXs) play a central role in redox signaling being both antioxidant enzymes and modulators of ROS action and are essential for pathological and physiological events. Recent studies from our lab emphasize the importance of PRDXs in the protection of spermatozoa as infertile men have significant low levels of PRDXs in semen and with little enzymatic activity available for ROS scavenging. The relationships between sperm DNA damage, motility and lipid peroxidation and high levels of thiol-oxidized PRDXs suggest the enhanced susceptibility of spermatozoa to oxidative stress and further support the importance of PRDXs in human sperm physiology. This review aims to characterize PRDXs, hidden players of the sperm antioxidant system and highlight the central role of PRDXs isoforms in the protection against oxidative stress to assure a proper function and DNA integrity of human spermatozoa.     

Improved Human Sperm Recovery Using Superoxide Dismutase and Catalase Supplementation in Semen Cryopreservation Procedure

The aim of this work was to evaluate the effects of ROS scavenger supplementation in human semen samples undergoing cryopreservation procedures.After screening out andrological pathologies, we selected 25 male partners of infertile couples with the following semen profile: volume >/= 2.0 ml, normal viscosity, sperm count >/=20 x 10(6)/ml, straight progressive motility (classes 1 and 2) >/= 40% (Mazzilli, Rossi, Delfino and Nofroni (1999) Andrologia 31: 187-194), atypical forms 相似文献   

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.     

Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation

Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.     

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production,while the Inhibition of Glycolysis Has Less Impact on Sperm Motility

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37°C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.     

Heparin- and superoxide anion-dependent capacitation of cryopreserved bovine spermatozoa: requirement of dehydrogenases and protein kinases

Capacitation is part of an oxidative process necessary for bovine spermatozoa to acquire fertilizing capacity. This process includes the generation of reactive oxygen species (ROS) and the participation of protein kinases such as A (PKA), C (PKC) and tyrosine kinase (PTK). A redox status is required to support both sperm motility and capacitation. Our aim was to determine the requirement of lactate dehydrogenase C4 (LDH-C4) and isocitrate dehydrogenase (NADP-ICDH) and of protein kinases in cryopreserved bovine sperm capacitation. The presence of inhibitors of both LDH-C4 and NADP-ICDH prevented the heparin-induced capacitation. H89, GF109203X or genistein blocked capacitation triggered by heparin or the superoxide (O(-*)(2))generator system xanthine-xanthine oxidase-catalase (XXOC) suggesting the requirement of PKA, PKC and PTK in this process. Taken together these results suggest that LDH-C4 and NADP-ICDH contribute with the redox status to support bovine sperm capacitation and that PKA, PKC and PTK are involved in different mechanisms induced by different inducers that lead bovine spermatozoa to be capacitated.     

Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species which lead to lipid peroxidation of sperm membranes. The objective was to determine an alpha-tocopherol concentration capable of improving the quality of cryopreserved porcine semen. Boar spermatozoa frozen with 200, 500 or 1000 microg/mL alpha-tocopherol were thawed and incubated at 37 degrees C for 4 h. Routine parameters of semen quality, susceptibility to lipid peroxidation 2-thiobarbituric acid (TBARS) and oxygen uptake were evaluated. Motility was higher (P<0.05) in samples treated with different concentrations of alpha-tocopherol up to 2 h of incubation. Viability and acrosome integrity significantly decreased during incubation (no significant differences between treatments). Two hundred micrograms per milliliter alpha-tocopherol protected spermatozoa against lipid peroxidation during incubation, but 1000 microg/mL failed to protect after 2 h of incubation. There was a negative association between TBARS and motility, suggesting that lipid peroxidation affected sperm motility. Both control and 200 microg/mL alpha-tocopherol samples preserved the capacity to generate oxidative energy up to 1 h of incubation. The addition of 200 microg/mL alpha-tocopherol in the semen extender could be useful to preserve boar spermatozoa against the oxidative stress generated by cryopreservation.