Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods.The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10⁶ dG were respectively 7.85 and 5.87 (p = 0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

Treatment with zinc, d-aspartate,and coenzyme Q10 protects bull sperm against damage and improves their ability to support embryo development

Reactive oxygen species (ROS) are physiologically generated during mitochondrial respiration and are involved in several signaling mechanisms. However, under pathological conditions, the concentration of ROS may exceed the antioxidant scavenging systems and subsequently lead to cell damage. High ROS levels have been proven to be detrimental to spermatozoa and furthermore compromise sperm function through lipid peroxidation, protein damage, and DNA strand breakage. Although the oral administration of antioxidants has been demonstrated to improve the semen quality in subfertile men, it is still a matter of debate if it can positively influence fertilization outcome and embryo developmental competence. Studies carried out in suitable animal models could resolve these fundamental questions. Hence, the main aims of the present study were to evaluate: (1) the effects of zinc, d-aspartate, and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on bull sperm motility and DNA fragmentation; and (2) whether treated spermatozoa have a superior competence in fertilization and in supporting the development of healthy embryos. Our data indicate that this treatment prevents the loss of sperm motility and the rise in sperm DNA fragmentation over time. Moreover, blastocyst rate was found to be significantly higher in oocytes fertilized by treated spermatozoa, and these blastocysts harbored a significantly lower percentage of apoptotic cells.     

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.     

The quantification of lipid and protein oxidation in stallion spermatozoa and seminal plasma: seasonal distinctions and correlations with DNA strand breaks, classical seminal parameters and stallion fertility

The goal of this work was to correlate oxidative stress caused by reactive oxygen species (ROS) and DNA damage with classic semen parameters in spermatozoa and seminal plasma of fertile and subfertile stallions. Oxidation was measured in both lipids and proteins, using the thiobarbituric acid reactive species (TBARS) assay and the DNPH carbonyl groups assay, respectively. Sperm DNA damage was monitored using the TUNEL assay. These parameters were monitored in samples obtained during the breeding and the non-breeding seasons. In general, fertile stallions showed better classical semen parameters, and those parameters improved from the non-breeding to the breeding season, although an increase in sperm production was accompanied by a decrease in the semen quality from subfertile stallions in the breeding season. In terms of oxidation levels we found that there were clear differences whether lipids or proteins were considered. In the breeding season there seemed to be a tendency towards normalizing lipid oxidation in spermatozoa and seminal plasma, and protein oxidation in the seminal plasma, of both fertile and subfertile animals. Thus, differences monitored in the non-breeding season were no longer visible. Interestingly, a higher level of protein oxidation was found in the sperm of fertile animals in the breeding season. Considering that there were positive correlations between sperm protein oxidation and sperm motility and vitality, these results suggests that the oxidation of semen proteins may be important for sperm function. On the other hand, lipid oxidation in the seminal plasma seemed to be a general indicator for sperm damage. In the non-breeding season positive correlations between lipid and protein oxidation levels in both sperm and seminal plasma and several defects in sperm function were found, but only for subfertile animals, thus suggesting that lipid and protein oxidation may aid in the identification of subfertile stallions during the non-breeding season. Levels of ROS production never seemed to result in compromised sperm DNA integrity, indicating that measurements were within physiological levels and/or that there is an efficient antioxidant activity in stallion sperm cells.     

Effect of trace elements on the seminal oxidative status and correlation to sperm motility in infertile Saudi males

The impact of trace elements, especially zinc, selenium, copper, and magnesium, on male fertility has gained great interest and significance. Increased oxidative stress and altered trace element levels are probable etiological factors underlying male reproductive dysfunction and infertility. The present study focused on the evaluation of seminal oxidative markers, such as reactive oxygen species (ROS), malondialdehyde (MDA), and total antioxidant capacity (TAC), and trace element levels in the normozoospermic fertile control group (n = 40) and asthenozoospermic infertile group (n = 30). Semen from infertile men exhibited significantly higher ROS and MDA levels accompanied with significant decline in TAC and trace element (zinc and magnesium) levels. Furthermore, a significant correlation was observed between trace elements and oxidative markers with sperm motility. The current study revealed increased lipid peroxidation and oxidant-reductant imbalance that leads to deterioration of semen quality and male infertility. Thus, oxidative stress and trace elements can be considered important biomarkers of male infertility. Measurement of seminal oxidative stress with conventional seminological parameters must be integrated in fertility assessment from early stages to ensure healthy semen characteristics and fertility in men.     

Potent humanin analogue (HNG) protects human sperm from freeze-thaw-induced damage

This study was aimed to investigate the protective effect of potent humanin analogue (HNG) supplementation to freezing media on freezing-thawing induced human sperm damage. We collected semen samples with normal sperm parameters from 15 healthy men. After the swim-up processing, the motile spermatozoa from each of the men were allocated to four equal groups: In the control group, the spermatozoa were frozen in media without HNG supplementation. In the other three groups, the spermatozoa were frozen in media supplemented with different concentrations of HNG (2 μM, 10 μM and 20 μM, respectively). We analyzed the sperm motility, viability, sperm mitochondrial membrane potential, apoptosis, sperm DNA fragmentation index (DFI), reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and caspase-3 activity for the sperm in each group. As a result, supplementation of HNG with 2 μM, 10 μM and 20 μM to the freezing media all significantly improved sperm motility and viability (all p < 0.05) when compared with the control group. Similarly, we found that supplementation of HNG reduced the damage to the mitochondrial membrane and DNA integrity, and inhibited the reaction of oxidative stress and the activity of caspase-3 in sperm. Although these protective effects increased with the elevated concentration of HNG in the freezing media, a final HNG concentration of 20 μM failed to exert significant improvements when compared with the concentration of 10 μM (all p > 0.05). In conclusion, our results suggested that HNG supplementation to the freezing media could protect sperm cells from freezing-thawing induced sperm damage.     

Chromosomal aberrations, Yq microdeletion, and sperm DNA fragmentation in infertile men opting for assisted reproduction

Male infertility is a multi‐factorial disorder, and identification of its etiology in an individual is critical for treatment. Systematically elucidating the underlying genetic causes (chromosomal and Yq microdeletion) and factors, such as reactive oxygen species (ROS) levels and total antioxidant capacity (TAC), which contribute to sperm DNA damage, may help to reduce the number of men with idiopathic infertility and provide them with the most suitable therapeutics and counseling. This study was done to comprehensively investigate genetic and oxidative stress factors that might be the etiology of a large percentage of men with idiopathic infertility. One hundred twelve infertile men and 76 fertile controls were screened for chromosomal aberrations and Yq microdeletions. ROS, TAC, and sperm DNA damage were assessed in cytogenetically normal, non‐azoospermic men with intact Y chromosome (n = 93). ROS was assessed in neat and washed semen by chemiluminescence; seminal TAC with a commercially available kit; and sperm DNA damage by the comet assay. Two men had cytogenetic abnormalities and seven men harbored Yq microdeletions. ROS levels in neat and washed semen of infertile men were significantly higher (P < 0.01) than controls. Infertile men had significantly lower (P < 0.01) TAC levels (1.79 mM), whereas sperm DNA fragmentation in infertile men was significantly higher (P < 0.01) than controls. Genetic factors and oxidative stress cumulatively account for large number of idiopathic infertile cases. Unlike, genetic causes, which cannot be cured, timely identification and management of oxidative stress may help to reverse/reduce the effects on induced DNA damage, and improve the outcomes for infertile males. Mol. Reprod. Dev. 79: 637–650, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

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.     

Composition lipidique des spermatozoides humains et susceptibilité au stress oxydant avant et après migration dans le mucus cervical

Spermatozoa are particularly susceptible to damage induced by ROS, especially as their plasma membrane contains large amounts of polyunsaturated fatty acids. Mammalian sperm cells develop the capacity to fertilise ova during transport in the male and female reproductive tracts. The nature and quality of the micro-environment of the female reproductive tract are important factors for sperm selection, capacitation and subsequent acrosome reaction.In vitro experiments using capacitating media have shown remodeling of the lipid composition of the sperm membrane during these steps and the same approaches have also shown that a low level of ROS was necessary. The oxidative status of the female genital tract is therefore certainly of primary importance for the physiological maturation of the sperm cell. It has been previously reported that an inappropriate oxidative balance in the male genital tract (ie, an excessive ROS production overwhelming all antioxidant strategies) impairs the structure and several functions of sperm cells. This phenomenon may arise in the female genital tract, but has never been investigated. The present paper is a review of the literature on these subjects and also reports our results concerning the changes in semen lipid content during cervical mucus migration and the effect of cervical mucus polymorphonuclear (PMN) cells on sperm characteristics. We showed that the sperm levels of vitamin E, cholesterol, phospholipids, sphingomyelin and plasmalogen assessed by HPLC decreased after migration through cervical mucus. These modifications were observed in parallel with lipid enrichment of the cervical mucus, suggesting an efflux of cholesterol and lipids from sperm cells. The spermatozoa recovered postmigration in the cervical mucus were characterised by low levels of the various lipid classes. Spermatozoa that migrated in cervical mucus samples with a considerable quantity of polymorphonuclear leukocytes (PMN) also showed significantly increased levels of sphingomyelin, diacyl phospholipids and plasmalogens in comparison to spermatozoa that migrated in cervical mucus devoid of PMN. Finally, we also found that PMA-induced ROS production was significantly increased for spermatozoa treated with cervical mucus containing PMN.     

Should Seminal Oxidative Stress Measurement be Offered Routinely to Men Presenting for Infertility Evaluation?

ObjectiveTo determine if seminal oxidative stress measurement should be offered routinely to men presenting for infertility evaluation.MethodsWe performed an extensive review of the English-language literature by searching MEDLINE for studies published between 1980 and 2007.ResultsResearch conducted during the last decade has provided growing support for the concept that excessive production of reactive oxygen species (ROS) is related to abnormal semen parameters and sperm damage. Routine semen analysis remains the backbone of clinical evaluation in male infertility, but determining the levels and sources of excessive ROS generation in semen is currently not included in the routine evaluation of subfertile men. However, the diagnostic and prognostic capabilities of seminal oxidative stress measurement exceed the capabilities of conventional sperm quality tests. An oxidative stress test may accurately discriminate between fertile and infertile men and identify those with a clinical diagnosis of male factor infertility who are likely to initiate a pregnancy if they are followed over a period of time. In addition, such a test can help select subgroups of patients with infertility in which oxidative stress is an important factor and those who may benefit from antioxidant supplementation. Although consensus is still required about the type and dosage of antioxidants to be used, rationale and evidence exist supporting their use in infertile men with elevated oxidative stress.ConclusionConsensus is growing about the clinical utility of seminal oxidative stress testing in infertility clinics, but standardization of protocols to measure ROS is crucial before introducing these tests into routine clinical practice. (Endocr Pract. 2008;14:484-491)     

Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants

Antioxidants may be useful for supplementing sperm extenders. We have tested dehydroascorbic acid (DHA), TEMPOL, N-acetyl-cysteine (NAC) and rutin on epididymal spermatozoa from red deer, during incubation at 37 °C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mm or 0.1 mm of each antioxidant, including oxidative stress (Fe²⁺/ascorbate). Motility (CASA and clustering of subpopulations), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h. Lipoperoxidation, intracellular reactive oxygen species (ROS) and DNA damage (DNA) status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. Overall, antioxidants negatively affected motility and physiological parameters. Only DHA 1 mm protected motility, increasing the fast and progressive subpopulation. However, it had a detrimental effect on acrosomal and DNA status, in absence of oxidative stress. Tempol and rutin efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TEMPOL or rutin reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but its negative effects were lower than the other antioxidants when used at 1 mm, increasing the subpopulation of hyperactivated-like spermatozoa at 2 h. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable because of prooxidant effects, but TEMPOL, NAC and rutin may be considered for cryopreservation trials. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop.     

Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients

As the largest proportion of male infertility population, asthenozoospermia patients often resort to sperm cryopreservation to preserve fertility as well as to enrich motile sperm for assisted reproductive techniques (ART), although it may cause some cryodamage during the freezing–thawing process. The objective of this study was to investigate whether mitochondrial antioxidant Mito-Tempo was effective in preventing cryodamage of asthenozoospermic spermatozoa. Asthenozoospermic semen samples were collected and cryopreserved in media supplemented with different concentrations (0.0, 1.0, 10 and 100 μM) of Mito-Tempo. We measured sperm motility, viability, membrane integrity, DNA fragmentation, mitochondrial membrane potential, oxidation product, and antioxidant enzymes activities. Supplementation of the cryopreservation media with Mito-Tempo (10 and 100 μM) induced a significant improvement in sperm viability, motility, membrane integrity, mitochondrial membrane potential and chromatin integrity (P < 0.05). Significant enhancement of antioxidant enzymes activities accompanied by the decreased formation of oxidation products (ROS and MDA) was also observed in groups supplemented with Mito-Tempo (10 and 100 μM). It is concluded that mitochondria targeted antioxidant Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients after cryopreservation.     

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress

Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde–protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age.     

High Levels of Cadmium and Lead in Seminal Fluid and Blood of Smoking Men are Associated with High Oxidative Stress and Damage in Infertile Subjects

We measured the levels of malondialdehyde (MDA), protein carbonyls, glutathione S-transferase (GST) and reducte glutathione (GSH) in seminal plasma and spermatozoa from 95 subjects including 50 infertile patients to evaluate the association between oxidative stress and damage and the components of the anti-oxidant defenses in seminal plasma and spermatozoa of infertile subjects and concentrations of cadmium (Cd) and lead (Pb) in the blood and seminal plasma because of tobacco smoke exposure. The reactive oxygen species (ROS) in spermatozoa were also evaluated by luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)-enhanced chemiluminescence assay. The sperm count, motility, and morphology in the smokers infertile group were found to be lower than those in the fertile male group and nonsmokers infertile group (p < 0.001). Concentrations of Cd, Pb, MDA, protein carbonyls, and ROS levels in the smokers infertile group were significantly higher than those in the fertile male and nonsmokers infertile male groups (p < 0.001). However, GSH levels and GST activities were decreased in the smokers infertile male group than those in the fertile male and nonsmokers infertile male groups (p < 0.001). The results indicate that smoking could affect semen quality and oxidative lipid and protein damage in human spermatozoa. From Pearson correlation analysis, positive correlations were demonstrated between the seminal plasma Cd and seminal plasma protein carbonyls and between seminal plasma Pb and spermatozoa ROS levels in smokers of the subfertile group, while there was a significant positive correlation between blood Cd and ROS levels in smokers of the fertile group. There was also a significant negative correlation of the Cd level of the blood and GSH levels of the sperm and seminal plasma. These findings suggest that cigarette smoking enhances the levels of Cd and Pb in seminal plasma and blood and the extent of oxidative damage associated with a decrease in components of the anti-oxidant defenses in the sperm of infertile males.     

The effects of radiation on sperm swimming behavior depend on plasma oxidative status in the barn swallow (Hirundo rustica)

Sperm are highly susceptible to reactive oxygen species (ROS) that can damage sperm DNA and structure, resulting in reduced fertilizing capacity. Exposure to radioactive contamination can also impair sperm swimming behavior and fertilizing ability, both through a reduction of sperm DNA integrity and via an increased generation of reactive oxygen species (ROS). However, the relationship between individual oxidative status and sperm swimming behavior has never been investigated in any wild population of animals exposed to radioactive contamination. We studied the motility of sperm collected from barn swallows, Hirundo rustica, breeding under different levels of radioactive contamination following the Chernobyl accident in 1986, in relation to individual oxidative status. We tested the hypothesis that the degree of impairment of sperm swimming behavior by radioactive contamination depended on plasma antioxidant capacity, the level of reactive oxygen metabolites (ROMs) and oxidative stress (sensu Costantini et al. 2006), a better oxidative status being associated with higher sperm motility. Sperm behavior parameters were subjected to principal component (PC) analysis, which extracted four PCs explaining 86% of the variance in sperm motility. PC2, representing sperm with high track velocity and ample lateral head displacement, was significantly predicted by the interaction between radiation level and either oxidative damage or oxidative stress. Contrary to our predictions, the highest values of PC2 were associated with relatively high radiation levels, particularly for high levels of either ROMs or oxidative stress. In addition, there was a tendency for values of PC3 (representing the percent of motile sperm) and PC4 (representing slow sperm with high beat cross frequency) to depend on the interaction between radiation level and total plasma antioxidant protection. Our results confirm the importance of oxidative status in determining the genetic and physiological outcome of exposure to radioactive contamination, complementing previous studies relating sperm abnormality to circulating levels of specific antioxidants. Our results also complement previous evidence that oxidative damage of sperm was negatively related to sperm motility, thus indicating a possible trade-off in quenching pro-oxidant compounds in the plasma and the seminal fluid.     

Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities

A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (β-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17β-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.     

Relationship between total superoxide dismutase activity with lipid peroxidation, dynamics and morphological parameters in canine semen

There is a lack of information regarding biochemical studies on canine semen. The knowledge of canine sperm metabolism is important because this species has differences in fertility among males, but biochemical studies are not well developed in this species. Reactive oxygen species (ROS) are active molecules produced during the oxygen reduction that have been implicated in several deleterious effects on the function and viability of spermatozoa when they are produced at high concentrations. Peroxidation of the plasma membrane phospholipids leads to a loss of motility, viability, and cytosolic elements, metabolic changes and structural alterations in spermatozoa. The most important antioxidant enzyme involved in ROS detoxification in mammalian spermatozoa is superoxide dismutase (SOD). The aims of the present study were to determine the presence of SOD-like activity in different fractions of canine semen, and to determine the relationship between SOD-like activity and different morphological, dynamical and biochemical parameters in canine spermatozoa. We demonstrated the presence of SOD-like activity in canine seminal plasma and spermatozoa; the negative correlation between SOD-like activity and lipid peroxidation concentrations in spermatozoa suggests a protective effect of this enzymatic activity against the potential oxidative stress in canine spermatozoa. A significant negative correlation between percentage of abnormal spermatozoa, and the percentage of progressive motility or vigor was found. Abnormal spermatozoa not only showed a deformed shape, moreover there was an energetic motility system failure. The presence of SOD-like activity in seminal plasma, spermatic, and postspermatic fractions of canine semen was demonstrated for the first time.     

Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome.