Human Reproduction vol.6 no 7 pp.987-991, 1991
Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa
T.Kobayashi, T.Miyazaki1, M.Natori and S.Nozawa Department of Obstetrics and Gynaecology, School of Medicine, Keio University, Tokyo, Japan 'To whom correspondence should be addressed at Department of Obstetrics and Gynaecology, Ogikubo Hospital, 3-1-24 Imagawa. Suginami, Tokyo 167, Japan
The levels of superoxide dismutase (SOD), a highly specific scavenging enzyme for superoxide anion radicals (O2~), and lipid peroxide produced by oxygen free radicals were measured in human seminal plasma and spermatozoa. Seminal plasma contained 366.8 ± 20.9 U/ml (mean ± SE) of SOD activity. SOD activity in human spermatozoa showed a significant correlation to the number of motile spermatozoa, while the activity in seminal plasma did not relate to the sperm concentration or motility. The lipid peroxide concentration in seminal plasma was 6.22 ± 0.46 nmol/ml and had no significant relationship to sperm concentration or motility. The malondialdehvde (MDA) concentration in spermatozoa was significantly related to the number of immotile spermatozoa. A decrease in the motility of spermatozoa incubated in medium without seminal plasma was observed after 120 min, while the MDA concentration of the spermatozoa increased. Addition of exogenous SOD (400 U/ml) to the sperm suspension significantly decreased this loss of motility and the increase of the MDA concentration. These data suggest a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may inhibit this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa for in-vitro fertilization (TVF) or artificial insemination. Key words: human spermatozoa/Iipid peroxide/oxygen free radical/sperm motility/superoxide dismutase
Introduction Toxic metabolites of oxygen, including the superoxide anion radical (O2~), hydrogen peroxide and the hydroxyl radical, are important mediators of tissue damage (McCord, 1974; Bulkley, 1983; Cross et ai, 1987). The peroxidation of polyunsaturated fatty acids in the phospholipids of the cell membrane is brought about by free radicals and this lipid peroxidation causes the loss of cell viability resulting in tissue necrosis (Fridovich, 1983). Superoxide dismutase (SOD) acts as a major intracellular © Oxford University Press Downloaded from https://academic.oup.com/humrep/article-abstract/6/7/987/584176 by Queen's University Belfast user on 17 January 2018
protective enzyme against oxygen toxicity by catalysing the ablation of O 2 ~. Over 40 years ago, it was reported that oxygen was toxic to the motility of human spermatozoa (McLeod, 1943) and it has been shown that lipid peroxidation due to oxygen free radicals causes the loss of sperm motility (Jones and Mann, 1977; Mann et ai, 1980; Alvarez et ai, 1987). Furthermore, oxygen free radicals seem to be generated by human spermatozoa (Alvarez et ai, 1987; Aitken and Clarkson, 1987, 1988). Although the existence of SOD activity in human seminal plasma and ejaculated spermatozoa has been reported (Menella and Jones, 1980; Nissen and Kreysel, 1983), the essential role of SOD in human sperm motility is not well understood. The objective of this study was to determine whether or not SOD activity in human spermatozoa or seminal plasma had any relationship to sperm concentration and motility. Moreover, to investigate the physiological role of SOD in human sperm motility, we examined the effect of exogenous SOD on sperm motility and spermatozoa] lipid peroxidation. Materials and methods Human semen samples were obtained from patients consulting the Keio University Hospital and from healthy donors by masturbation. A total of 130 samples were used. After liquefaction, the semen volume was measured and the sperm concentration and percentage motility were determined using a Makler counting chamber (Makler, 1980). These data are shown in Table I. Furthermore, the semen samples were screened for the presence of leukocytes. Only those samples with a leukocyte concentration of < 5 % relative to spermatozoa were utilized for the experiment. Superoxide dismutase activity Spermatozoa were separated from seminal plasma by centrifugation at 1800 g for 10 min and the supernatant was used for the measurement of SOD activity in seminal plasma. The sperm pellet was resuspended in a 0.25 M sucrose solution with 10 mM potassium phosphate buffer, the osmotic pressure of which was equivalent to that of seminal plasma, and centrifuged at 600 g for 10 min. This washing procedure was repeated three times.The washed spermatozoa then underwent hypo-osmotic treatment (Keyhani and Storey, 1973) by centrifugation at 1800 g for 10 min with 10 mM potassium phosphate buffer. The supernatant obtained was used for the measurement of SOD activity in spermatozoa. Sonication was also performed for the disruption of spermatozoa, but since it gave the same or less SOD activity, we chose the hypo-osmotic treatment method instead. 987
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Table I. Sperm concentration and percentage motility of semen samples in each experimental group Experiment
Sperm concentration (x lO4/™!)
Percentage motility (%)
Seminal plasma SOD
4893 ± 3501 (0-17000) 7058 ± 6 1 7 0 (50-26000)
53 5 ± 28 (0-9O) 42 8 ± 19 (5-90)
6224 ± 3 6 1 1 (2000-17000) 8182 ± 6050 (1000-27000)
61.2 ± 22 (10-90) 40 0 ± 24 (0-85)
Spermatozoa SOD lipid peroxide
Values presented as mean ± SD The range of values is given in parentheses.
SOD activity in seminal plasma and spermatozoa was measured by assessing the degree of inhibition of the reduction of nitroblue tetrazolium (NBT) by xanthine oxidase when specimens were added (Beauchamp and Fridovich, 1971). Briefly, 3 mM xanthine (Sigma Chemical Co., St Louis, MO) and xanthine oxidase (Boehringer Mannheim Gmbh, FRG), adjusted to a concentration that reduced NBT at a rate of 0.015-0.025 absorbance units/ml, were used as O2~ generators, while 0.75 mM NBT (Sigma) was used as the indicator of O2" generation. The reaction was performed at 25°C in 0.05 M sodium bicarbonate buffer (pH 10.2) containing 3 mM EDTA. Manganese-containing SOD (Mn-SOD) activity was measured in the presence of 1 mM potassium cyanide which inhibits copper, zinc-containing SOD (Cu-Zn SOD) activity. One unit of activity was defined as the amount of enzyme required to inhibit the change of light absorbance at 560 nm by 50%. Lipid peroxidation Spermatozoa were separated from seminal plasma by centrifugation at 1800 g for 10 min and the lipid peroxide level in seminal plasma was measured using a kit (Determiner LPO"; Kyowa Medics, Tokyo, Japan) (Ohishi et al., 1985). The sperm pellet was resuspended in 2 ml of phosphate-buffered saline (pH 7.2) and this solution was used for measurement of the lipid peroxides in spermatozoa. The level of lipid peroxidation of spermatozoa was assessed by malondialdehyde (MDA) production, while the spermatozoal MDA concentration was measured using thiobarbituric acid (Buege and Aust, 1978). To 1.0 ml of sperm suspension was added 2.0 ml of TBA reagent (15% w/v trichloroacetic acid, 0.375% w/v thiobarbituric acid, and 0.25 N hydrochloric acid). The mixture was heated in a boiling water bath for 15 min. After cooling, the suspension was centrifuged at 1000 g for 10 min, the supernatant was collected, and its absorbance was measured at 535 nm. The MDA concentration was determined by the specific absorbance coefficient (1.56 x 105/mol/cm). Effect of SOD on sperm motility and lipid peroxidation The progressively motile spermatozoa were collected by the continuous-step density gradient technique using isotonic 80% Percoll solution (Kaneko et al., 1987). Motile spermatozoa were 988 Downloaded from https://academic.oup.com/humrep/article-abstract/6/7/987/584176 by Queen's University Belfast user on 17 January 2018
resuspended in phosphate-buffered saline (pH 7.2) at a final cell concentration of 6.0 x 107/ml. Bovine erythrocyte SOD (400 units/ml; Sigma) was added to the experimental group of sperm samples, while the same amount of heat-inactivated SOD (Baker et al., 1985) was added to the control group of samples. The sperm suspension was left to stand in air at 25°C for 360 min. Aliquots were withdrawn at 0, 120, 240 and 360 min for the measurement of motility and spermatozoal lipid peroxidation. Changes of motility were expressed as the percentage motility, which was defined as the motility at each time point divided by the motility at 0 min. The change of lipid peroxide level was expressed as the percentage absorbance, which was defined as the difference of the absorbance of MDA at 535 nm between 0 min and each time point divided by the absorbance at 0 min. Statistical analysis Statistical analysis was performed using linear regression or Student's r-test. A probability value of P < 0.05 was considered to be significant. Results Superoxide dismutase activity SOD activity in seminal plasma ranged from 0 to 800 U/ml and the mean activity level was 366.8 ± 20.9 U/ml (mean ± SE). There was no significant relationship between the SOD activity in seminal plasma and the sperm concentration, sperm motility, or the number of motile or immotile spermatozoa. Mn-SOD activity was not detected in seminal plasma by the method we used and SOD activity was also observed in the seminal plasma of patients with azoospermia. Spermatozoal SOD activity showed a significant correlation (P < 0.001) with the number of motile spermatozoa (Figure 1), but the total number of spermatozoa bore no significant
motile sperm (x 10'/-/) Fig. 1. Correlation between SOD activity in spermatozoa and the number of motile spermatozoa. SOD activity in 1 ml of the supernatant after hypo-osmotic treatment correlated significantly with the number of motile spermatozoa in 1 ml of the medium used for hypo-osmotic treatment (r = 0.85. P < 0.001. n = 38).
Role of superoxide dismutase in human sperm raotility
immotile sperm (X10V-/)
Fig. 2. Correlation between the spermatozoal malondialdehyde (MDA) concentration and the number of immotile spermatozoa. The MDA concentration in 1 ml of sperm suspension in phosphatebuffered saline correlated significantly with the number of immotile spermatozoa in the sperm suspension (r = 0.80. P < 0.001, n = 51).
relationship to the SOD activity. Mn-SOD activity was not detected in spermatozoa. In the process of preparing spermatozoa for SOD activity measurement, no activity was detected in the medium from the third washing procedure. Lipid peroxidation Lipid peroxide levels in seminal plasma ranged from 0 to 13.5 nmol/mJ and the mean was 6.22 ± 0.46 nmol/ml (mean ± SE). The lipid peroxide concentration in seminal plasma showed no relationship with sperm concentration, sperm motility, or the number of immotile spermatozoa. The spermatozoal MDA concentration had a significant correlation (P < 0.001) with the number of immotile spermatozoa (Figure 2), but there was no relationship between MDA concentration and the other parameters.
2 4 Incubation time (mbi)
Fig. 3. Effect of SOD on sperm motility. Data points are the mean ± SE of 14 samples. The decrease in percentage motility (motility at each time point divided by the motility at 0 min) was significantly inhibited by SOD treatment compared to the control. *P < 0.025, **P < 0.005.
Effect of SOD on sperm motility and lipid peroxidation On the basis of the findings regarding SOD activity in seminal plasma, 400 U/ml of SOD was used in this experiment. The motility of seminal plasma-free spermatozoa declined 120 min after the start of incubation. However, the loss of motility was significantly decreased by SOD treatment in comparison to the control (Figure 3). The MDA concentration in spermatozoa increased with incubation. SOD affected the increase of the percentage absorbance, which indicated an increase of MDA production by spermatozoa, and the increase of MDA was significantly less (P < 0.01) at 360 min after the start of incubation as compared with the control (Figure 4).
Discussion In advanced organisms, SOD consists of two different metalcontaining enzymes, which are copper, zinc-containing SOD (CuZn SOD) in the cytosol, and manganese-containing SOD (MnSOD) in the mitochondria (Fridovich, 1978). Since Mn-SOD
tncubflttoo tfarw (min)
Fig. 4. Effect of SOD on MDA production by spermatozoa. Data points are the mean ± SE of 14 samples. The increase in the percentage absorbance (difference of absorbance of MDA at 535 nm between 0 min and each time point divided by the absorbance at 0 min) was affected by SOD treatment, and was significantly reduced after 360 min of incubation by SOD treatment compared to the control. *P < 0.01.
activity was not detected in this study, the SOD activity was thought to represent that due to Cu-Zn SOD. SOD activity in seminal plasma showed no relationship to sperm concentration and motility. Nissen and Kreysel (1983) reported that SOD activity in human seminal plasma had a significant relationship 989
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T.Kobayashi et al.
with the number of motile spermatozoa and that no SOD activity was detected in spermatozoa. One possible explanation for this discrepancy is the different method used for the measurement of SOD activity. SOD activity was detectable even in azoospermic patients suggesting that the SOD activity in seminal plasma may not be derived from spermatozoa. The evidence showing that spermatozoa] SOD activity had a significant correlation with the number of motile spermatozoa may suggest that spermatozoa containing SOD can avoid damage by various toxic agents, including oxygen free radicals. Spermatozoal SOD has been reported to play a major role in protecting the spermatozoa against lipid peroxidation (Alvarez and Storey, 1983; Alvarez et al., 1987) and the data presented here may support these observations. The result may be interpreted by the consideration that it is natural for dead cells not to have enzyme activity. Enzymes in the spermatozoa could leak out from dead cells into the seminal plasma, so that the SOD activity in seminal plasma might bear arelationshipto the number of immotile spermatozoa. However, the data presented here showed no significant relationship between SOD activity of seminal plasma and the number of immotile spermatozoa. It has been reported that poorly motile or immotile spermatozoa are more susceptible to lipid peroxidation than motile spermatozoa, although the reason was not discussed (Jones et al., 1979). This report may be explainable by our findings, i.e. since immotile spermatozoa do not have SOD activity, they seem to be subject to damage by O2~. It is known that phospholipid, which is a major component of the mitochondrial sheath of mammalian spermatozoa, is susceptible to lipid peroxidation (Jones and Mann, 1977). Furthermore, it has been reported that lipid peroxidation causes the loss of sperm motility (Alvarez and Storey, 1982; Jones et al., 1978, 1979). The result that the MDA concentration of spermatozoa was significantly correlated with the number of immotile spermatozoa indicates that the immotile spermatozoa underwent lipid peroxidation. Moreover, the fact that the MDA concentration in spermatozoa increased together with the loss of motility suggests that lipid peroxidation caused the loss of motility, and that oxygen free radicals are possibly one of the causes of impaired sperm motility. Although high centrifugal force such as 1800 or 2500 g did not affect sperm motility and vitality (Jeulin et al., 1982), the centrifugal pelleting of human sperm populations induces a massive release of oxygen free radicals (Aitken and Clarkson, 1988). This fact may have influenced the results of MDA concentration in spermatozoa in the present study. However, in this study, semen samples were centrifuged without dilution, while the experiments of Aitken and Clarkson (1988) involved diluted semen samples. Since seminal plasma has relatively high SOD activity, it is likely that O2~ released by the centrifugal pelleting will be scavenged. Consequently, it seems that the centrifugal pelleting in this study may not have much effect on the data with respect to MDA concentration in spermatozoa. There have been no previous reports with respect to lipid peroxide in human seminal plasma. Jones et al. (1979) reported that human spermatozoa became immobile within 5 min of the administration of 3 0 - 6 0 nmol/ml of lipid peroxide. The finding of the mean seminal plasma lipid peroxide concentration of 990 Downloaded from https://academic.oup.com/humrep/article-abstract/6/7/987/584176 by Queen's University Belfast user on 17 January 2018
6.22 ± 2.9 nmol/ml thus indicates that this concentration is unlikely to affect sperm motility. It appears that the lipid peroxide in seminal plasma does not arise from the spermatozoa, since it was detected in seminal plasma from patients with azoospermia. SOD protected sperm motility when the seminal plasma was removed by Percoll density gradient centrifugation. This suggests that one of the possible physiological roles of SOD in seminal plasma may be the maintenance of sperm motility. Furthermore, it may be that O2~ is one of the causes of the lipid peroxidation of spermatozoa, since SOD is a highly specific scavenger of
or. In conclusion, SOD in human seminal plasma may protect against a loss of sperm motility by lipid peroxidation. Further studies on the effects of SOD on the fertilization capacity of spermatozoa and on oocytes are required. It seems, however, that SOD may have a clinical application in the use of spermatozoa, from which seminal plasma is removed, for IVF or artificial insemination. References Aitken.R.J. and Clarkson.J.S. (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertii, 81, 459-469. Aitken,R.J. and Clarkson,J.S. (1988) Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J. Androl., 9, 367-376. AlvarezJ.G. and Storey,B.T. (1982) Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: Its effect on sperm motility. Biol. Reprod., 27, 1102-1108. Alvarez.J.G. and Storey,B.T. (1983) Role of superoxide dismutase in protecting rabbit spermatozoa from O2 toxicity due to lipid peroxidation. Biol. Reprod., 28, 1129-1136. Alvarez.J.G., Touchstone,J.C., Blasco,L. and Storey,B.T. (1987) Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: Superoxide dismutase as major enzyme protectant against oxygen toxicity. J. Androl., 8, 338 — 348. Baker.G.L., Corry.R.J. and Autor,A.P. (1985) Oxygen free radical induced damage in kidneys subjected to warm ischemia and reperfusion. Ann. Surg., 202, 628—641. Beauchamp.C. and Fridovich.I. (1971) Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem., 44, 276-287. Buege.J.A. and Aust,S.D. (1978) Microsomal lipid peroxidation.
Methods Enzymol., 52, 302-310. Bulkley.G.B. (1983) The role of oxygen free radicals in human disease processes. Surgery, 94, 407—411. Cross.C.E., Halliwell.B.. Borish.E.T.. Pryor.W.A., Ames,B.N., Saul.R.L., McCordJ.M. and Harman.D. (1987) Oxygen radicals and human disease. Ann. Intern. Med., 107, 526-545. Fridovich.I. (1978) The biology of oxygen radicals. Science. 201, 875-880. Fridovich.I. (1983) Superoxide radical: An endogenous toxicant. Annu.
Rev. Pharmacol. Toxicoi, 23, 239-257. Jeulin,C, Serres,C. and Jouannet.P. (1982) The effects of centrifiagation. various synthetic media and temperature on the motility and vitality of human spermatozoa. Reprod. Nutr. Dev., 22, 8 1 - 9 1 . Jones,R. and Mann.T. (1977) Damage to ram spermatozoa by peroxidation of endogenous phospholipids. J. Reprod. Fertii , 50. 261-268. Jones,R., Mann.R. and Sherins.R. (1978) Adverse effects of peroxkiized lipid on human spermatozoa Proc. R. Soc. Lond., B201. 413-417
Role of superoxide dismutase in human sperm motility Jones,R., Mann.T. and Sherins,R. (1979) Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and action of seminal plasma. Fertil. Sieril., 31, 531-537. Kaneko,S., Sato,H., Kobanawa,K., Oshio.S., Kobayashi.T. and Iizuka.R. (1987) Continuous-step density gradient centrifugation for the selective concentration of progressively motile sperm for insemination with husband's semen. Arch. Androi, 19, 75-84. Keyhani,E. and Storey,B.T. (1973) Energy conservation capacity and morphological integrity of mitochondria in hypotonically treated rabbit epididymal spermatozoa. Biochem. Biophvs. Res. Commun., 305, 557-569. Makler.A. (1980) The improved ten-micrometer chamber for rapid sperm count and motility evaluation. Fertil. Steril., 33, 337 — 338. Mann,T., Jones,R. and Sherins.R. (1980) Oxygen damage, lipid peroxidation, and motility of spermatozoa. In Steinberger,A. and Steinberger,E. (eds), Tesricular Development, Structure and Function. Raven Press, New York, pp. 497-501. McCord.J.M. (1974) Free radicals and inflammation: Protection of synovial fluid by superoxide dismutase. Science, 185, 525 — 531. McLeod,J. (1943) The role of oxygen in the metabolism and motility of human spermatozoa. Am. J. Physiol., 138, 512—518. Mennella,M.R. and Jones,R. (1980) Properties of spermatozoa] superoxide dismutase and lack of involvement of superoxides in metalion-catalysed lipid-peroxidation reactions in semen. Biochem. J., 191, 289-297. Nissen.H.P. and Kreysel.H.W (1983) Superoxide dismutase in human semen. Klin. Wochenschr., 61, 63-65. Ohishi,N., Ohkawa,H., Miike,A., TatanoJ. and Yagi.K. (1985) A new assay method for lipid peroxides using a methylene blue derivative. Biochem. Int., 10, 205-211. Received on December 20, 1990; accepted on April 24, 1991
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