Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility.

Under moderate conditions, reactive oxygen species (ROS) have been shown to inhibit sperm motility after several hours of incubation. The rapid decrease in flagellar beat frequency observed within the first hour of contact between ROS and spermatozoa was associated with a rapid loss of intracellular adenosine triphosphate (ATP). Motility of intact spermatozoa ceased when their ATP concentration was reduced by 85 +/- 5%. Axonemal damage was confirmed when ROS-treated spermatozoa could not reactivate motility after demembranation in a medium containing magnesium adenosine triphosphate (Mg.ATP). However, in conditions allowing rephosphorylation of the axonemes (addition of cyclic adenosine monophosphate, or cAMP, and protein kinase or sperm extracts to the demembranation medium), the motility could reactivate. Three lines of evidence suggested that ATP depletion induced by ROS treatment was responsible for the effects observed in spermatozoa. First, the rapid decrease in intracellular ATP observed after ROS treatment was closely followed by a decrease in beat frequency, loss of intact sperm motility, and axonemal damage due to insufficient phosphorylation. Second, incubation of spermatozoa with the combination pyruvate-lactate allowed maintenance of sperm ATP at a normal level and prevented the effects of ROS; furthermore, spermatozoa immobilized after ROS treatment, then supplemented with pyruvate-lactate, were able to reinitiate motility in parallel with an increase in their ATP level. Third, treatment of spermatozoa with rotenone, an ATP depleting agent, produced effects similar to ROS treatment and could also be reversed by the addition of pyruvate-lactate. These data are consistent with the conclusion that ROS treatment produced axonemal damage mostly as a result of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)     

A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa

Capacitation of human spermatozoa is essential for fertilization, and is characterized visually by hyperactivated motility. We have investigated whether reactive oxygen species could induce these two events in human spermatozoa. The addition of xanthine + xanthine oxidase + catalase (X+XO+CAT: generation of superoxide anion and removal of hydrogen peroxide) and foetal cord serum (FCS), a known biological inducer of capacitation and hyperactivation, to spermatozoa, induced levels of hyperactivation (15.4 ± 1.6% and 8.0 ± 1.0%, respectively) which were significantly higher than that of controls (5.4 ± 0.6%). The hyperactivation measured was part of the capacitation process. Furthermore, the addition of superoxide dismutase prevented the capacitation and hyperactivation induced by X+XO+CAT or by FCS. These results suggest that the superoxide anion may be involved in capacitation and hyperactivation of human spermatozoa.     

Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa

The objective of this study was to examine the effect of reactive oxygen species (ROS) and cryopreservation on DNA fragmentation of equine spermatozoa. In experiment 1, equine spermatozoa were incubated (1 hour, 38 degrees C) according to the following treatments: 1) sperm alone; 2) sperm + xanthine (X, 0.3 mM)-xanthine oxidase (XO, 0.025 U/mL); 3) sperm + X (0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, 38 degrees C) with X (1 mM)-XO (0.1 U/mL) and either catalase (200 U/mL), superoxide dismutase (SOD, 200 U/mL), or reduced glutathione (GSH, 10 mM). Following incubation, DNA fragmentation was determined by the single cell gel electrophoresis (comet) assay. In experiment 3, equine spermatozoa were cryopreserved, and DNA fragmentation was determined in fresh, processed, and postthaw sperm samples. In experiment 1, incubation of equine spermatozoa in the presence of ROS, generated by the X-XO system, increased DNA fragmentation (P <.005). In Experiment 2, the increase in DNA fragmentation associated with X-XO treatment was counteracted by the addition of catalase and GSH but not by SOD, suggesting that hydrogen peroxide and not superoxide appears to be the ROS responsible for such damage. In experiment 3, cryopreservation of equine spermatozoa was associated with an increase (P <.01) in DNA fragmentation when compared with fresh or processed samples. This study indicates that ROS and cryopreservation promote DNA fragmentation in equine spermatozoa; the involvement of ROS in cryopreservation-induced DNA damage remains to be determined.     

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.     

Reactive oxygen species and human spermatozoa: physiology and pathology

The role of reactive oxygen species (ROS) in the pathophysiology of human sperm function has been emphasized in recent years. ROS production in semen has been associated with loss of sperm motility, decreased capacity for sperm–oocyte fusion and loss of fertility. There is a current presumption that the most prolific source of ROS in sperm suspensions is an NADPH oxidase located in leukocytes or in spermatozoa which produces superoxide which is further converted to peroxide by the action of superoxide dismutase. Hydrogen peroxide has been recognized as the most toxic oxidizing species for human spermatozoa, which are very sensitive to lipid peroxidation owing to the high content of polyunsaturated fatty acids in their plasma membrane, though this is not the sole mechanism by which sperm function might be impaired by ROS. Although the excessive production of ROS is detrimental to human spermatozoa, there is a growing body of evidence which suggests that ROS are also involved in the physiological control of some sperm functions. This review focuses on the nature and source of the ROS generated by human spermataozoa as well as their operational mechanisms and their effects, which may be detrimental or beneficial.     

Antioxidant effects of penicillamine against in vitro-induced oxidative stress in human spermatozoa

Oxidative stress contributes importantly to the aetiology of male infertility, impairing sperm function. The protective effect of antioxidants on seminal parameters has been established, and the antioxidant penicillamine has shown beneficial effects; however, its protective effect on human spermatozoa exposed to oxidative stress has not been reported. The objective of this work was to evaluate the effect of penicillamine on human spermatozoa exposed in vitro to oxidative stress. First, the effect of penicillamine on spermatozoa from normozoospermic donors was evaluated. Then, the effect of penicillamine on spermatozoa exposed to oxidative stress induced separately by ionomycin and hydrogen peroxide (H₂O₂) was analysed. An untreated control and a control treated only with the oxidative stress inducer were included. Reactive oxygen species (ROS) levels, viability, mitochondrial membrane potential (MMP) and motility were analysed. The results showed that penicillamine, added to the incubation medium, decreased the ROS levels induced by ionomycin and H₂O₂, and this effect was associated with better preservation of MMP, motility, and ATP levels. These results highlight the potential advantages of penicillamine supplementation of sperm culture medium, especially for semen samples with high ROS levels and also in circumstances where laboratory handling can cause an increase in ROS production.     

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility,free thiol content and seasonality

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.     

Effects of reactive oxygen species from activated leucocytes on human sperm motility, viability and morphology

The accumulated data suggest that inflammation can increase the level of reactive oxygen species (ROS), which contribute to impaired sperm function and male infertility. Therefore, we propose that inflammation-mediated production of ROS in male and female reproductive tracts hinder sperm fertilisation. To test this hypothesis, phorbol myristate acetate (PMA) with polymorphonuclear leucocytes (PMNs) was applied to generate endogenous ROS. We evaluated the time-dependent effects of ROS on human sperm motility, viability and mitochondrial membrane potential (MMP). The results showed that after treatment with PMA and PMNs, the motility of human spermatozoa significantly decreased to 50% on Day 1 and 15% on Day 4 compared with that of the, respectively, negative controls (P = 0.012). The viability of human spermatozoa decreased on Day 4 of PMA + PMNs treatment (P = 0.028). The MMP of human spermatozoa significantly decreased from Day 2 to Day 4 in the PMA + PMN group compared with that of the controls (P = 0.019). Taken together, the 4-day cultivation approach provided an accurate evaluation of sperm quality, especially sperm motility and MMP. Our findings indicated that endogenous inflammation increased ROS levels, which might induce sperm oxidative damage. Additionally, sperm motility might be one of the earliest and most sensitive indicators of this damage.     

Suppression of lipid peroxidation in human spermatozoa by prostatic inhibin

Loss of sperm motility owing to the production of hydrogen peroxide by lipid peroxidation is regulated by yet unidentified prostatic factor(s). Inhibinlike peptide (HSPI) of prostatic origin isolated from human seminal plasma and having a molecular weight of about 10,400 daltons was studied for its effect on ascorbate-induced lipid peroxidation in human spermatozoa. Dose-related suppression of lipid peroxidation occurred at a dose level of 0.25, 0.5, and 1.0 micrograms. HSPI may be one of the factors involved in the regulation of lipid peroxidation and therefore sperm motility.     

Reactivation of motility of demembranated hamster spermatozoa: role of protein tyrosine kinase and protein phosphatases

Demembranated cauda epididymidal spermatozoa of hamster, following reactivation with 1 mm ATP, exhibited either a loop or planar type of motility. The spermatozoa with planar motility exhibited increased progressive velocity (VSL), straightness (STR), linearity (LIN) and beat cross frequency (BCF) compared to the spermatozoa with loop type motility. cAMP was observed to have differential effects on the motility parameters of the demembranated spermatozoa depending on the type of motility. For instance, in the loop type, average path velocity (VAP), curvilinear velocity (VCL) and VSL were increased in the presence of cAMP unlike in the planar type. Furthermore, in an attempt to understand the role of protein kinases and protein phosphatases in regulation of sperm motility, the effects of various inhibitors of these enzymes on the motility and phosphorylation of proteins of reactivated demembranated spermatozoa were studied. Inhibitors of PTKase (protein tyrosine kinase) and protein phosphatases inhibited the motility of reactivated demembranated hamster spermatozoa. The activity of the respective enzymes associated with demembranated spermatozoa was concurrently inhibited, thus providing evidence that the effect of the inhibitors on motility was mediated through their inhibitory effects on the activities of the enzymes. The results also demonstrated that two phosphotyrosinylated proteins of molecular weight 65 and 80 kDa showed reduced phosphorylation in the presence of PTKase inhibitors, thus indicating their possible role in reactivation of motility of demembranated hamster spermatozoa.     

Modulation of lipid peroxidation in human spermatozoa and human prostate by prostatic inhibin.

Loss of sperm motility as a result of the production of hydrogen peroxide by lipid peroxidation is regulated by as yet unidentified prostatic factor(s). Inhibinlike peptide of prostatic origin isolated from human seminal plasma, with a molecular size of about 10,400 daltons, was studied for its effect on ascorbate-induced lipid peroxidation in human spermatozoa. Dose-related suppression of lipid peroxidation was observed at dose levels of 0.25, 0.5, and 1.0 micrograms. The data suggest that inhibinlike peptide could be one of the factors involved in the regulation of lipid peroxidation and thereby of sperm motility. Inhibinlike peptide also exhibited local action in both normal and benign hyperplastic human prostate tissue by enhancing the rate of lipid peroxidation. These findings have implications in the pathophysiology of the prostate.     

In vitro effects of nicotine on human spermatozoa

Washed human spermatozoa from 12 normozoospermic donors were treated with different concentrations of nicotine 0.1, 1.0, 5.0 and 10.0 mm and were compared to spermatozoa suspended in nutrient medium only (control). Computer‐aided sperm analysis was used to assess sperm kinematic properties after 30, 60, 120 and 180 min of incubation. Viability was assessed by means of a dye exclusion staining technique (eosin/nigrosin), while acrosome‐reacted cells were identified under a fluorescent microscope using fluorescein isothiocyanate–Pisum sativum agglutinin as a probe. Nicotine significantly reduced total motility, progressive motility, curvilinear velocity, amplitude of lateral head displacement, beat cross‐frequency, viability and caused spontaneous acrosome reaction at concentrations of ≥5.0 mm after 2 and 3 h of exposure. Nicotine concentrations of 0.1 and 1.0 mm had no significant effect (P < 0.05) on spermatozoa except that 1.0 mm significantly decreased (P < 0.05) sperm progressive motility at 2 and 3 h of incubation as well as viability after 3 h of incubation. This study concludes that the occurrence of high levels of nicotine in the body and seminal fluid might adversely affect fertilisation capacity of human spermatozoa through a mechanism that involves decreased motility, viability and premature induction of the acrosome reaction.     

Acidification of intracellular pH in bovine spermatozoa suppresses motility and extends viable life

Intracellular pH (pHi) was determined in ejaculated bovine spermatozoa using a ratiometric absorbance technique under various incubation conditions that drastically altered sperm motility. The pHi was directly correlated with sperm motility. In a medium of Sodium, Potassium, and Magnesium [NKM] that supported active sperm motility, pHi was 6.9. In medium containing weak acids (NKM equilibrated with 100% CO2 or containing 80 mM 5,5-dimethyl-2,4-oxazolidinedione; DMO), pHi was depressed at least 0.5 pH unit and sperm motility was suppressed. After complete immobilization of sperm was established, removal of the weak acids indicated that suppression of motility was fully reversible for up to 48 hours in CO2 and up to 24 hours in DMO. This study shows that expression and conservation of sperm motility are inversely related, and that depression of pHi by weak acids can reversibly inhibit sperm motility. These findings may help to explain the mechanisms by which sperm are immobilized within the male reproductive tract, and could be applicable to the design of improved ambient temperature semen extenders.     

Effect of mitochondrial calcium uniporter blocking on human spermatozoa

Calcium (Ca²⁺) regulates a number of essential processes in spermatozoa. Ca²⁺ is taken up by mitochondria via the mitochondrial calcium uniporter (mCU). Oxygen‐bridged dinuclear ruthenium amine complex (Ru360) has been used to study mCU because it is a potent and specific inhibitor of this channel. In bovine spermatozoa, it has been demonstrated that mitochondrial calcium uptake inhibition adversely affects the capacitation process. It has been demonstrated in human spermatozoa that mCU blocking, through Ru360, prevents apoptosis; however, the contribution of the mCU to normal human sperm function has not been studied. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Spermatozoa obtained from apparently healthy donors were incubated with 5 and 10 μm Ru360 for 4 h at 37 °C. Viability was assessed using propidium iodide staining; motility was determined by computer‐aided sperm analysis, adenosine triphosphate (ATP) levels using a luminescence‐based method, mitochondrial membrane potential (ΔΨm) using JC‐1 staining and reactive oxygen species (ROS) production using dihydroethidium dye. Our results show that mCU blocking significantly reduced total sperm motility and ATP levels without affecting sperm viability, ΔΨm and ROS production. In conclusion, mCU contributes to the maintenance of sperm motility and ATP levels in human spermatozoa.     

An in vitro study on reproductive toxicology of Deltamethrin on rat spermatozoa

Recent findings indicate that synthetic pyrethroid insecticide may induce toxic manifestations by enhancing the production of reactive oxygen species and disrupting the balance between pro‐oxidants and antioxidants as a result of lipid peroxidation (LP) of cell membranes. The aim of the study was to examine the potency of Deltamethrin (Del) to induce oxidative stress response in rat spermatozoa in vitro. Spermatozoa were incubated with different concentrations (0, 10, 50, 100 and 200 μm ) of Del for 3 h at 37 °C. After that, sperm parameters (motility, viability and abnormal morphology), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined. We found that in vitro exposure to Del caused a significant decline of sperm motility and viability and increase of abnormal sperm morphology, MDA, SOD and CAT levels at different concentrations of Del. This study demonstrated that Del caused deterioration in sperm motility and viability, and induction in LP, abnormal morphology of spermatozoa and antioxidants enzyme activities.     

Hyperactivated motility is coupled with interdependent modifications at axonemal and cytosolic levels in human spermatozoa.

Whether the motility characteristics of hyperactivated spermatozoa were determined by stable changes at the axonemal level and whether the presence of cytosolic factors was required for the expression of these changes was investigated. Different degrees of sperm hyperactivation were produced in Percoll-washed spermatozoa after incubation for 1 hour to 3 hours at 37 degrees C in Ham's F-10 supplemented with human blood plasma or fetal cord serum. Decomplemented fetal cord serum induced the highest percentage of hyperactivation (19 +/- 3%), followed by human plasma (13 +/- 2%). Fetal cord serum that was not decomplemented did not induce a level of hyperactivation (1.7 +/- 0.2%) significantly different from control levels (0.9 +/- 0.2%). Dialyzed fetal cord serum induced intermediate levels of hyperactivation (6 +/- 1%). The motility characteristics of demembranated sperm models of hyperactivated spermatozoa induced by decomplemented fetal cord serum and nonhyperactivated spermatozoa were compared by videomicroscopy and computer-assisted digital image analysis. After demembranation with Triton X-100 and reactivation of motility by Mg. adenosine triphosphate (Mg.ATP), hyperactivated and nonhyperactivated spermatozoa showed similar motility characteristics. However, hyperactivated spermatozoa that were demembranated and reactivated in cytosolic extracts from hyperactivated spermatozoa had significantly higher (P less than 0.05) linear velocity (33 +/- 4 mu/sec) and lower linearity (0.23 +/- 0.04) than control spermatozoa that were demembranated and reactivated in control cytosolic extracts (velocity = 24 +/- 1 mu/sec; linearity = 0.32 +/- 0.02). The data suggest that the expression of hyperactivated motility requires interdependent changes at the axonemal and cytosolic levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

A human seminal plasma protein blocks the motility of human spermatozoa

An inhibitor of the motility of demembranated spermatozoa has been shown to be present in human seminal plasma. This seminal plasma motility inhibitor (SPMI) was purified to apparent homogeneity and tested on intact human spermatozoa. Motility parameters of spermatozoa incubated with the sperm motility inhibitor were evaluated with the video automated Cell Soft system. SPMI decreased the percentage of motile spermatozoa in a dose-dependent manner and motility was completely blocked in the presence of 1600 units/ml. Sperm velocity and beat/cross frequency showed a similar progressive decrease as the inhibitor was augmented. However, linearity was essentially not affected. The effects of SPMI on the percentage of motile spermatozoa increased with the time of contact between the inhibitor and spermatozoa. After 120 min., the IC50 was 35% lower than that observed at five min. The presence of seminal plasma did not prevent the inhibitory effects of the seminal plasma factor on sperm motility parameters. On the contrary, a potentiating effects was observed. The data suggest that the SPMI could play a significant role in cases of infertility caused by asthenospermia.     

Deoxynivalenol reduces quality parameters and increases DNA damage in mice spermatozoa

This study was performed to investigate in vitro effects of deoxynivalenol (DON) on mice sperm quality parameters including viability, motility and DNA damages at various concentrations and exposure times. Mice spermatozoa were exposed to DON at 0, 2.5, 5 and 10 µM for 1, 3 and 6 hr, motility parameters were evaluated by computer‐assisted analysis and viability was examined by colorimetric metabolic activity assay and HOS test. DNA damage was examined by acridine orange staining, and sperm damages via lipid peroxidation pathway were determined by malondialdehyde (MDA) content measurement. DON affected sperm parameters in a concentration‐ and time‐dependent manner. In all test groups, the average path velocity and progressive motile spermatozoa were remarkably reduced. In comparison with the controls, after 1, 3 and 6 hr exposure to DON, viability of spermatozoa was reduced 25, 30 and 49% respectively. DON exposure at 10 µM for 6 hr resulted in 15% DNA damage and 2.5‐fold more MDA generation, when compared with nonexposed spermatozoa. Our data suggest that DON causes sperm quality parameters decline in concentration‐ and time‐dependent fashion, which attribute to the reduction in sperm metabolic activity and membrane integrity and equally to increase in lipid peroxidation rate and DNA damage.     

Effects of methyl-beta-cyclodextrin on cryosurvival of boar spermatozoa

Methyl-beta-cyclodextrin (MBCD), which leads to the stimulation of cholesterol efflux from the cell membrane, was examined for its ability to increase the cryosurvival of spermatozoa from G?ttingen miniature pigs. The intactness of the acrosome and various motion parameters of spermatozoa after freezing and thawing were used to monitor cryosurvival. Spermatozoa were exposed to MBCD or a combination of MBCD and cholesterol-3-sulfate over a period of 3 hours while being cooled slowly from 25 degrees C to 5 degrees C, and were subsequently cryopreserved by the pellet method. After freezing and thawing, the acrosomal status was monitored by fluorescein isothiocyanate-labeled peanut agglutinin, and the motion parameters were assessed with a computer-assisted sperm motility analysis system. In post-thaw spermatozoa the values of the intact acrosome, motility, progressive motility, progressive velocity, straightness, and linearity of the cell path increased greatly with the concentration of MBCD (P < .05). In contrast, the lateral head displacement amplitude and the beat cross-frequency of post-thaw spermatozoa were not different among all treatments. On the contrary, the addition of cholesterol-3-sulfate to the diluent containing MBCD abolished the protective effect against freeze-thaw injury that MBCD provides to spermatozoa. These results indicate that cryosurvival of boar spermatozoa is enhanced by exposure to MBCD before freezing. This protective effect of MBCD may be due to active depletion of sperm membrane cholesterol.     

Influence of cigarette smoking on spermatozoa via seminal plasma

Numerous investigations have been conducted on the relationship between cigarette smoking and male infertility, however, the exact molecular mechanisms are not well understood in most of the cases. Few studies have indicated the direct effect of seminal plasma (SP) [in different dilutions with phosphate buffer solution (PBS)] from smokers (SM) on the sperm functional parameters from nonsmokers (non-SM). The aim of this study was to provide evidence that cigarette smoking affects male fertility via altering the sperm quality. Our results indicated that exposure of spermatozoa from the non-SM to the SP from the SM yielded a significant reduction in the sperm motility and acrosome reaction and an elevation in the amount of malondialdehyde (MDA), in a certain time course. Exposure of spermatozoa from the SM to the SP from the non-SM or with PBS resulted in the nonsignificant improvement in the altered sperm functional parameters indicating removal of SM's SP and then subsequent reconstitution with physiological media could be of clinical significance in the various assisted reproductive technologies applied for SM. However, the detrimental effect of SM's SP on non-SM's spermatozoa was prominent. In addition, as spermatozoa in SM's SP are susceptible to peroxidative damages, men with such cells who wish to have children should especially benefit from quitting smoking.