PDFlib PLOP: PDF Linearization, Optimization, Protection Page inserted by evaluation version www.pdflib.com – [email protected]
Reactive Oxygen Species and Human Spermatozoa" CLAUDE GAGNON,bsCAKIRA IWASAKI,d EVE DE LAMIRANDE,b AND NEIL KOVALSKIb Urology Research Laboratory Royal Victoria Hospital Faculty of Medicine, McGill University Montrkal, Qukbec, Canada H3A 1Al dDepartment of Urology Yokohama City University Yokohama, 232 Japan
INTRODUCTION Reactive oxygen species (ROS) such as hydrogen peroxide (HzOz), the superoxide anion ( . 0 2 ) and the hydroxyl radical ('OH), can be generated by living cells incubated under aerobic conditions.' To counteract the effect of ROS, cells possess systems that scavenge ROS to prevent internal cellular damage. Whereas there is little known on the sperm enzymatic systems involved in the generation of ROS, the presence of several enzymes involved in their neutralization, such as superoxide dismutase,2-6 catalase,' and the glutathione peroxidaseheductase system, has been documented.8-10 Thus, imbalance between the generation of ROS and their destruction can result in cellular damage. It has been reported more than four decades ago that ROS can be toxic to spermatozoa and that catalase can confer protection. 11-13 Human spermatozoa are especially sensitive to lipid peroxidation induced by oxygen reactive species because of their high content of polyunsaturated fatty a c i d ~ . ~ JThe ~ J 5lipoperoxidative lethal point, which corresponds to the level of peroxidation that will kill spermatozoa, has been calculated at 100 nmole of malonaldehyde formed per 108 cells.9 The formation of malonaldehyde, a by-product of membrane lipoperoxidation, has been inversely correlated with the percentage of motile cells and positively correlated with mid-piece morphological defects in human spermatozoa. l6 The group of Aitken has reported that damaged or deficient spermatozoa form higher levels of ROS than normal motile spermatozoa.17J8It was suggested that ROS formation could be a cause for idiopathic infertility. 17.18 In this chapter, we will first report on the incidence of ROS formation in the semen
The authors acknowledge the financial support of the Medical Research Council of Canada. Address all correspondence to Dr. Claude Gagnon, Director, Urology Research Laboratory, Royal Victoria Hospital, Room H6.47, 687 Pine Ave. W., Montreal, Quebec, Canada, H3A 1Al. a
C
436
GAGNON et 01.: ROS AND HUMAN SPERMATOZOA
431
of a population of infertile patients. We will then address the possibility that leukocytes present in the male reproductive tract can cause damage to spermatozoa, and finally, we will discuss the mechanism by which ROS can cause sperm damage in conditions where viability is not affected.
INCIDENCE OF ROS FORMATION IN INFERTILE PATIENTS To investigate whether ROS formation could be a significant factor in male infertility, the incidence of ROS formation was determined in the semen of an unselected population of patients consulting for infertility. Measurements of ROS formation were performed on whole semen rather then washed spermatozoa since several factors, both of cellular and secretory origins, could affect ROS formation and sperm function. For these experiments, ROS formation was determined 2-3 hours after semen collection by luminescenceusing luminol and a LKB computer-drivenluminometer, at 27.7"C. Results indicated that 25% of the semen from 172 patients who were not azoospermic generated easily detectable amounts of ROS (>lo m V/s/lO9 spermatozoa). On the other hand, none of the six semen specimens from azoospermic patients and none of the 12 from fertile donors currently used in our laboratory produced ROS.19.20 In the group of patients, there was no difference in the incidence of ROS formation in semen samples in which the volume was smaller than 1.5 ml, higher than 2.5 ml or between these two values. However, there was a significant negative correlation (r = 0.35, p < 0.05) between the level of ROS formation and semen volume in these specimens.20Since the seminal vesicles contribute the majority of the semen volume, these results may suggest that secretions from these glands contain scavenging systems that decrease the detection of ROS measured by chemiluminescence. The level of ROS formation was also inversely correlated with the percentage of motile cells (r = 0.46,p < 0.05), and the incidence of ROS formation was the lowest in sperm populations with more than 60%motile cells. Similarly to motility, linearity was inversely correlated with ROS formation (r = 0.33, p < 0.05).19.20 These results suggest that damaged spermatozoa were mainly responsible for the generation of ROS. Separation of cellular elements from semen, on a discontinuous Percoll gradient made of 20, 40, 65 and 95% Percoll layers, indicated that seminal plasma did not produce ROS, and that cells accumulating at the 40-65% and 65-95% interface (the 95% layer was combined with the latter interface) were responsible for the majority of ROS formed. The 40-65%interface contained few motile spermatozoa, occasional round cells, and a majority of immotile and morphologically abnormal spermatozoa. On the other hand, the 65-95% interface contained spermatozoa that were morphologically normal with an adequate intrinsic density. Whereas in gradients on which semen from fertile men were centrifuged, the latter Percoll interface contained 70-85% of the initial sperm population with a motility above 70-80%,*l the same interface from gradients on which semen from patients were centrifuged may contain only 20-50% of the spermatozoa in semen, and be characterized by a motility ranging from 0 to 50%.Thus, spermatozoa that are morphologically normal under the light microscope, and have the right density, may be biochemically and functionally abnormal. We hypothesize that these spermatozoa are responsible for the ROS detected in the lower Percoll interface. The level of ROS generated in this Percoll layer was also inversely correlated with the percentage of motile spermatozoa and their linearity.*O
438
ANNALS NEW YORK ACADEMY OF SCIENCES
EFFECT OF SPERM WASHING PROCEDURES ON ROS FORMATION It has long been known that washing spermatozoa by repeated centrifugationresuspension cycles results in a decrease in the quality of sperm motility. To investigate whether centrifugation damage could be linked to ROS formation, semen samples from patients were either centrifuged on Percoll gradient and the 65-95% interface analyzed or subjected to repeated (3 X ) centrifugation-resuspension cycles with fresh physiological buffer and the pellet analyzed. Analysis of the data revealed that washed spermatozoa in the 65-95% interface produced 4-fold more ROS than the whole semen. On the other hand, spermatozoa washed by repeated centrifugation-resuspension cycles generated 21-fold higher levels of ROS than the original semen when normalized for the number of spermatozoa. Adding back seminal plasma to spermatozoa washed by multiple centrifugations reduced ROS detected from 406 mV/s/109 spermatozoa to 61 mV/s/lOy spermatozoa, a value similar to that obtained with spermatozoa subjected to 3 cycles of centrifugation-resuspension but without removal of seminal plasma (71 mV/s/109 spermatozoa). However, the latter two values were still 8-fold higher than the 8 mV/s/lOy spermatozoa detected in the original semen.10 Thus, it would appear that morphologically abnormal and biochemically/functionally abnormal (but not dead) spermatozoa present in semen cause ROS formation. Centrifugation and removal of seminal plasma increase ROS detection.
NEUTROPHILS, INFECTION AND POSSIBLE DAMAGE TO SPERM FUNCTION Concentrations of neutrophils above 1 million/ml are occasionally found in semen of patients consulting for infertility. The detection of these polymorphonuclear cells is currently based on the content of myeloperoxidase22or the presence of specific antigens. However, in the last case, antibodies that generally recognize all leukocytes are often used.2' Since the presence of active neutrophils is a better reflection of an active acute infection than the whole leukocyte population, a test to determine active neutrophil concentration was developed. This new test, in contrast to the previous tests, such as the myeloperoxidase assay by histochemical techniques, determines the concentration of functionally active neutrophils. The new assay is based on the capacity of active neutrophils to generate ROS upon stimulation by invading microorganisms or by synthetic stimulants such as phorbol myristate acetate (PMA). The superoxide anion generated reduces nitroblue tetrazolium (pale yellow color) to formazan (blue color).2J Although this test is less sensitive than the histochemical myeloperoxidase assay, the sensitivity obtained (0.5 X loh polymorphonuclear leukocytes per ml of semen) is 2-fold below the level considered by the World Health Organization25as evidence of possible genital tract infection. The advantages of this assay include its simplicity, its low cost, and an objective assessment of functionally active polymorphonuclear leukocytes in semen. To address the question whether active neutrophils could affect sperm function, PMA-stimulated neutrophils were incubated with spermatozoa. Although neutrophils only produced ROS in the first 30 min. the percentage of motile spermatozoa was affected 2-3 hours later for neutrophil concentrations above 2 X loh cells/ml. However. at a later time ( 5 h). neutrophil concentrations as low as 0.6 X 106 cells/ml
GAGNON et al.: ROS AND HUMAN SPERMATOZOA
439
caused a decrease in motility of at least 30%. Addition of superoxide dismutase significantly reduced the amount of ROS detected by chemiluminescence and NBT, but without conferring any protection on sperm motility. On the other hand, catalase was able to prevent most of the damage done to sperm motility.26 Whereas pools of human seminal plasma can prevent most of the damage caused by ROS produced by neutrophils, individual sperm samples seemed to confer a wide range of protection from full protection to none (unpublished data).
EFFECTS OF ROS ON MOTILITY AND VIABILITY OF HUMAN SPERMATOZOA To further investigate the effects of ROS on sperm viability and motility, we have used xanthine (X) and xanthine oxidase (XO), which produces the superoxide anion (and subsequently H202 after dismutation), as a source of ROS. When X + X O at concentrations above 1 mM and 0.05 U/ml, respectively, were incubated with Percollwashed motile spermatozoa from a fertile donor, sperm motility was arrested within 30-60 min. A drop of 30-50% in sperm viability was always associated with these experimental conditions. However, at lower ROS concentrations (0.3-0.6 mM X 0.05 U XO/ml), viability was only marginally affected. Under these conditions, although ROS were present in solution for only one hour, the percentage of motile cells was only affected at 1.5 h after the addition of ROS. The drop in the percentage of motile cells occurred abruptly, in contrast to the flagellar beat frequency which was affected shortly but progressively after ROS addition. The decrease in beat frequency reached a value of 27% of control within the first hour after the addition of X + X O (TABLEl).*’.*8 ROS have long been known to cause damage to cells by interfering with membrane function. To determine whether the effects on sperm motility could also be caused by modifications on the sperm axoneme. the motor responsible for sperm movement, demembranated sperm models were prepared as previously d e ~ c r i b e d . ~ ~ . ~ ~ These sperm models are prepared by demembranation of motile intact spermatozoa with Triton X-100; the motility of these immotile, demembranated spermatozoa is
+
TABLE1. Effect of ROS on the Motilitv Parameters and ATP Level of Intact Soermatozoa Intact Spermatozoa Time (h) 0 0.5 1 1.5 2.5 24
Motility ( % of control)
Beat Frequency (% of control)
ATP Level ( % of control)
100
100 55
100 25 12 5 5
100 100
0 0 25
21 0 0 25
25
Percoll-washed spermatozoa from a fertile donor were incubated with xanthine (0.6 mM) and xanthine oxidase (0.05 U/ml). The percentage of motile spermatozoa and their flagellar beat frequency were measured at various time periods after addition of ROS. Sperm ATP levels were determined by the luciferine-luciferase assay. These results from one experiment are representative of 5 other experiments.
440
ANNALS NEW YORK ACADEMY OF SCIENCES
TABLE2. Effect of ROS on the Motilitv Parameters of Demembranated SDermatozoa Time (h) 0
0.5 1
1.5 2.5 24
Motility (% of control)
Beat Frequency (% of control)
100 100 50 25 0 25
100 100 100 100 0 100
Percoll-washed spermatozoa were treated as described in TABLEI . At various time intervals after the initiation of ROS treatment, spermatozoa were demembranated with Triton X-100, and motility was reinitiated by the addition of 0.5 mM Mg ATP.30 These results from the same experiment reported in TABLEI are representative of 5 other experiments.
then reactivated by the addition of a source of energy, ATP. Sperm models from ROS-treated intact spermatozoa could reactivate motility with a decreased percentage and duration of reactivation when compared to control untreated spermatozoa (TABLE2 ) . On the other hand, their flagellar beat frequency was not affected. It is worth mentioning that when intact spermatozoa were immobilized by ROS treatment, the reactivation of the sperm models was still possible, but for only an additional 30 min.27.28 The most surprising aspect of these experiments was that the motility of intact spermatozoa could spontaneously recover after several hours of arrest (TABLE1). This was associated with the reappearance of motility in sperm models when challenged with ATP (TABLE2).
IMPORTANCE OF ATP IN ROS DAMAGE TO SPERMATOZOA Since ATP concentration determines the flagellar beat frequency of demembranated spermatozoa,31 and ROS treatment caused a rapid decrease in flagellar beat frequency of intact spermatozoa, the level of ATP in control and ROS-treated cells was investigated. The level of ATP decreased rapidly following ROS treatment. The fall in ATP concentration was even steeper than that of the flagellar beat frequency. This discrepancy in the kinetics of these two parameters is not unexpected since the concentration of ATP within human spermatozoa is 4.5 mM,32 a value much higher than the 0.3-0.4 mM ATP required for half maximal beat frequency in demembranated sperm models.33 At the time when intact sperm motility ceased after ROS treatment, the level of ATP was 10- to 20-fold lower than that found in control ~ p e r m a t o z o a . ~ ~ The importance of the drop in ATP concentration after ROS treatment was further emphasized by the following series of observations. The addition of lactate and pyruvate together with ROS to intact spermatozoa completely prevented the decrease in intracellular ATP concentration, the drop in flagellar beat frequency and the fall in sperm motility generally seen a few hours after ROS treatment. Furthermore, spermatozoa immobilized by ROS treatment, and supplemented with pyruvate + lactate reinitiated motility much earlier than their counterparts supplemented with buffer only (unpublished data).
ANNALS NEW YORK ACADEMY OF SCIENCES
442
EFFECTS OF ROS SCAVENGERS ON X TREATED SPERMATOZOA
+
XO-
+
T o determine which of ROS produced by X XO caused the damage to sperm motility, ROS-treated spermatozoa were incubated with various scavengers. Since X XO can produce the superoxide anion which can be spontaneously or enzymically dismutated into HzOz and 0 2 , and that '0; and H20. can react together through the Haber Weiss reaction to produce the very toxic hydroxyl radical, superoxide dismutase, catalase and DMSO were used to scavenge '01,HzOz and 'OH, respectively. Of these scavengers only catalase at concentrations as low as 0.008 mg/ml conferred full protection on spermatozoa incubated with X and XO (TABLE3). The addition of superoxide dismutase at a high concentration (1 mg/ml) conferred only partial protection, whereas 140 mM DMSO mainly helped the recovery of motility without affecting its fall. It would then appear from these results that HzOz is the primary toxic ROS responsible for most damage done to spermatozoa. However, '0; and 'OH may, to some extent, also contribute to the toxicity of X XO on human spermatozoa.
+
+
CONCLUSION Normally, there is a fine balance between the amounts of ROS produced and scavenged by a cell. Cellular damage arises when this equilibrium is disturbed, especially when the increase in ROS produced cannot be eliminated by the reserve of scavenging systems. Such a disruption occurs in at least 25 % of the semen of patients consulting for infertility. However, whether the disruption in ROS equilibrium is due to a decreased scavenging capacity or to an enhanced ROS production remains to be seen. The clinical significance of leukocytes in male and female reproductive tracts remains controversial. However, the data summarized above indicate that stimulated leukocytes can produce an outburst of ROS that will decrease sperm motility over long periods of time, even at concentrations below 1 X I 0 6 cellsiml. Whereas human seminal plasma has enormous scavenging capacity for the superoxide anion produced by activated leukocytes, the situation may be quite different in extracellular fluids surrounding spermatozoa in the male and female reproductive tracts. The observation that exogenously added ROS affect sperm motility by decreasing the intracellular ATP level before affecting viability, raises the possibility that the poor sperm velocity frequently seen in the semen of infertile patients, which is a reflection of flagellar beat frequency, may be due to abnormally low levels of ATP. Whether this deficiency can be corrected by incubations with metabolic precursors known to increase cellular ATP levels remains to be investigated.
ACKNOWLEDGMENT We are grateful to Mrs. Lina Ordonselli for her secretarial assistance.
GAGNON et al.: ROS AND HUMAN SPERMATOZOA
443
REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9.
10. 11.
12.
13. 14.
15. 16.
17. 18.
19. 20. 21. 22.
GRISHAM, M. B. & J. M. MCCORD.1986. Chemistry and cytotoxicity of reactive oxygen nietabolites. In Biology of Oxygen Radicals. A. E. Taylor ct a/. , Eds.: 1 - 18. American Physiological Society. Bethesda, MD. ABU-ERREIZH, G., L. MAGNES& T. K. LI. 1978. Isolation and properties of superoxide dismutase from ram spermatozoa and erythrocytes. Bid. Reprod. 18: 554-560. MANELLA, M. R. F. & R . JONES.1980. Properties of spermatozoa superoxide dismutase and lack of involvement of superoxides in metal-ion-catalysed lipid-peroxidation reactinn5 in semen. Biochem. J. 191: 289-296. NISSEN,H. P. & H. W. KREYSEL. 1983. Superoxide disniutase in human semen. Klin. Wnchenschr. 61: 63-65. ALVAREZ, J. G. & B. T. STOREY.1983. Role of superoxide dismutase in protecting rabbit spermatozoa from oxygen toxicity due to lipid peroxidation. Biol. Reprod. 28: 1129-1136. ALVAREZ, J. G. & B. T. STOREY.1984. Lipid peroxidation and the reactions of superoxide and hydrogen peroxide in mouse spermatozoa. Biol. Reprod. 30: 833-841. P. WEBER,D. LAVA~.-MARTIN & R. CALVAYRAC. 1989. Catalase activity JEULIN. C., J. C. SOUFIR, in human spermatozoa and seminal plasma. Gamete Res. 24: 185-196. LI, T. K. 1975. The glutathione and thiol content of mammalian spermatozoa and seminal plasma. Biol. Reprod. 12: 641-646. ALVAREZ, J. G., J. C. TOUCHSTONE, L. BLASCO& B. T. STOREY.1987. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human 5permatozoaSuperoxide dismutase as major enzyme protectant against oxygen toxicity. J. Androl. 8: 338-348. ALVAREZ, J. G. & B. T. STOREY. 1989. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res. 23: 77-88. MCLEOD,J. 1943. The role of oxygen in the metabolism and motility of human spermatozoa. Am. J, Physiol. 138: 512-518. Tosic, J. 1947. Mechanism of hydrogen peroxide formation by spermatozoa and the role of amino acids in sperm motility. Nature 159: 544-545. Tosrc, J. & A. WALTON.1950. Metabolism of spermatozoa. Formation of hydrogen peroxide by spermatozoa and its effects on motility and survival. Biochem. J. 47: 199-206. JONES,R., T. MANN& R. SHERINS. 1979. Peroxidative breakdown of phospholipids in human spermatozoa. spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil. Steril. 31: 531-537. AITKEN,R. J. & J. S. CLARKSON. 1987. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil. 81: 459-469. RAO,B., J. C. SOUFIR, M. MARTIN & G. DAVID.1989. Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res. 24: 127-134. 1988. Significance of reactive oxygen species and antioxidants AITKEN.J. R. & J. S. CLARKSON. in defining the efficacy of sperm preparation techniques. J. Androl. 9: 367-376. & S. FISHEL.1989. Generation of reactive oxygen species, lipid AITKEN,J. R.. J. S. CLARKSON peroxidation and human sperm function. Bid. Reprod. 40: 183-197. IWASAKI, A. & C. GAGNON.1989. Free radical formation in human semen: Incidence and correlation with motility. 35th Annual Meeting of the Canadian Fertility and Andrology Society. Vancouver. Abstract 25. IwAsAKi, A. & C. GAGNON.1991. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil. Steril. In press. CHAPEAU, C. & C. GAGNON.1987. Nitrocellulose and polyvinyl coatings prevent sperm adhesion to glass without affecting the motility of intact and demembranated human spermatozoa. J. Androl. 8: 34-40. R. C. S P E N C ~G. R ,R . KINGHORN. A. WHITE.P. E. HARRISON, BARRATT, C. L. R., A. ROBINSON. E. KESSOPOUL~N & I. D. COOKE.1990. Seminal peroxidase positive cells are not an adequate indicator of asymptomatic urethral genital infection. Int. J. Androl. 13: 361-370.
444
ANNALS NEW YORK ACADEMY OF SCIENCES
R. J. & K. M. WEST.1990. Analysis ofthe relationship between reactive oxygen species 23. AITKEN, production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. Int. J. Androl. 13: 433-451. N . , E. DE LAMIRANDE & C. GAGNON.1991. Determination of neutrophil concentra24. KOVALSKI, tion in semen by measurement of superoxide radical formation. Fertil. Steril. In press. 2 5 . WHO. 1987. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge University Press. Cambridge, UK. N., E. DE LAMIRANDE & C. GAGNON.1991. Dimethylsulfoxide and catalase protect 26. KOVALSKI, against neutrophil induce loss of sperm motility. Canadian Fertility and Andrology Society, Quebec. Abstract TP27. E., A. IWASAKI & C. GAGNON.1992. Reactive oxygen species affect sperm 27. DE LAMIRANDE, axoneme. In press. E., A. IWASAKI& C. GAGNON.1990. Free radicals also affect sperm axoneme. 28. DE LAMIRANDE, 36th Annual Meeting of the Canadian Fertility and Andrology Society, L'Esterel, Quebec. Abstract 14. 1980. Characteristics of motor apparatus in testicular epididymal 29. MOHRI,H. & R. YAMAGIMACHI. and ejaculated spermatozoa. Exp. Cell Res. 127 191-196. E. & C. GAGNON.1983. Aprotinin and a seminal plasma factor inhibit the mo30. DE LAMIRANDE, tility of demembranated reactivated rabbit spermatozoa. Biol. Reprod. 28: 788-796. B. H. & I. R. GIBBONS.1972. Flagellar movement and adenosine triphosphatase ac31. GIBBONS, tivity in sea urchin extracted with Triton X-100. J. Cell Biol. 54: 75-97. 1983. D-[1-i4C] mannitol and [U-14C]sucrose as extracellular 32. FORD,W. C. C. & A. HARRISON. space markers for human spermatozoa and the rupture of 2-deoxyglucose. J. Reprod. Fertil. 69: 479-487. 33. COSSON,M. P. & C. GAGNON.1988. Protease inhibitor and substrates block motility and microtubule sliding of sea urchin and carp spermatozoa. Cell Motil. Cytoskeleton 10: 518-527. E. & C. GAGNON.1991. ATP depletion plays an important role in the inhibition 34. DE LAMIRANDE, of sperm motility observed after treatment with reactive oxygen species. 11th North American Testis Workshop, Montreal. Abstract 116.
Reactive Oxygen Species and Human Spermatozoa" CLAUDE GAGNON,bsCAKIRA IWASAKI,d EVE DE LAMIRANDE,b AND NEIL KOVALSKIb Urology Research Laboratory Royal Victoria Hospital Faculty of Medicine, McGill University Montrkal, Qukbec, Canada H3A 1Al dDepartment of Urology Yokohama City University Yokohama, 232 Japan
INTRODUCTION Reactive oxygen species (ROS) such as hydrogen peroxide (HzOz), the superoxide anion ( . 0 2 ) and the hydroxyl radical ('OH), can be generated by living cells incubated under aerobic conditions.' To counteract the effect of ROS, cells possess systems that scavenge ROS to prevent internal cellular damage. Whereas there is little known on the sperm enzymatic systems involved in the generation of ROS, the presence of several enzymes involved in their neutralization, such as superoxide dismutase,2-6 catalase,' and the glutathione peroxidaseheductase system, has been documented.8-10 Thus, imbalance between the generation of ROS and their destruction can result in cellular damage. It has been reported more than four decades ago that ROS can be toxic to spermatozoa and that catalase can confer protection. 11-13 Human spermatozoa are especially sensitive to lipid peroxidation induced by oxygen reactive species because of their high content of polyunsaturated fatty a c i d ~ . ~ JThe ~ J 5lipoperoxidative lethal point, which corresponds to the level of peroxidation that will kill spermatozoa, has been calculated at 100 nmole of malonaldehyde formed per 108 cells.9 The formation of malonaldehyde, a by-product of membrane lipoperoxidation, has been inversely correlated with the percentage of motile cells and positively correlated with mid-piece morphological defects in human spermatozoa. l6 The group of Aitken has reported that damaged or deficient spermatozoa form higher levels of ROS than normal motile spermatozoa.17J8It was suggested that ROS formation could be a cause for idiopathic infertility. 17.18 In this chapter, we will first report on the incidence of ROS formation in the semen
The authors acknowledge the financial support of the Medical Research Council of Canada. Address all correspondence to Dr. Claude Gagnon, Director, Urology Research Laboratory, Royal Victoria Hospital, Room H6.47, 687 Pine Ave. W., Montreal, Quebec, Canada, H3A 1Al. a
C
436
GAGNON et 01.: ROS AND HUMAN SPERMATOZOA
431
of a population of infertile patients. We will then address the possibility that leukocytes present in the male reproductive tract can cause damage to spermatozoa, and finally, we will discuss the mechanism by which ROS can cause sperm damage in conditions where viability is not affected.
INCIDENCE OF ROS FORMATION IN INFERTILE PATIENTS To investigate whether ROS formation could be a significant factor in male infertility, the incidence of ROS formation was determined in the semen of an unselected population of patients consulting for infertility. Measurements of ROS formation were performed on whole semen rather then washed spermatozoa since several factors, both of cellular and secretory origins, could affect ROS formation and sperm function. For these experiments, ROS formation was determined 2-3 hours after semen collection by luminescenceusing luminol and a LKB computer-drivenluminometer, at 27.7"C. Results indicated that 25% of the semen from 172 patients who were not azoospermic generated easily detectable amounts of ROS (>lo m V/s/lO9 spermatozoa). On the other hand, none of the six semen specimens from azoospermic patients and none of the 12 from fertile donors currently used in our laboratory produced ROS.19.20 In the group of patients, there was no difference in the incidence of ROS formation in semen samples in which the volume was smaller than 1.5 ml, higher than 2.5 ml or between these two values. However, there was a significant negative correlation (r = 0.35, p < 0.05) between the level of ROS formation and semen volume in these specimens.20Since the seminal vesicles contribute the majority of the semen volume, these results may suggest that secretions from these glands contain scavenging systems that decrease the detection of ROS measured by chemiluminescence. The level of ROS formation was also inversely correlated with the percentage of motile cells (r = 0.46,p < 0.05), and the incidence of ROS formation was the lowest in sperm populations with more than 60%motile cells. Similarly to motility, linearity was inversely correlated with ROS formation (r = 0.33, p < 0.05).19.20 These results suggest that damaged spermatozoa were mainly responsible for the generation of ROS. Separation of cellular elements from semen, on a discontinuous Percoll gradient made of 20, 40, 65 and 95% Percoll layers, indicated that seminal plasma did not produce ROS, and that cells accumulating at the 40-65% and 65-95% interface (the 95% layer was combined with the latter interface) were responsible for the majority of ROS formed. The 40-65%interface contained few motile spermatozoa, occasional round cells, and a majority of immotile and morphologically abnormal spermatozoa. On the other hand, the 65-95% interface contained spermatozoa that were morphologically normal with an adequate intrinsic density. Whereas in gradients on which semen from fertile men were centrifuged, the latter Percoll interface contained 70-85% of the initial sperm population with a motility above 70-80%,*l the same interface from gradients on which semen from patients were centrifuged may contain only 20-50% of the spermatozoa in semen, and be characterized by a motility ranging from 0 to 50%.Thus, spermatozoa that are morphologically normal under the light microscope, and have the right density, may be biochemically and functionally abnormal. We hypothesize that these spermatozoa are responsible for the ROS detected in the lower Percoll interface. The level of ROS generated in this Percoll layer was also inversely correlated with the percentage of motile spermatozoa and their linearity.*O
438
ANNALS NEW YORK ACADEMY OF SCIENCES
EFFECT OF SPERM WASHING PROCEDURES ON ROS FORMATION It has long been known that washing spermatozoa by repeated centrifugationresuspension cycles results in a decrease in the quality of sperm motility. To investigate whether centrifugation damage could be linked to ROS formation, semen samples from patients were either centrifuged on Percoll gradient and the 65-95% interface analyzed or subjected to repeated (3 X ) centrifugation-resuspension cycles with fresh physiological buffer and the pellet analyzed. Analysis of the data revealed that washed spermatozoa in the 65-95% interface produced 4-fold more ROS than the whole semen. On the other hand, spermatozoa washed by repeated centrifugation-resuspension cycles generated 21-fold higher levels of ROS than the original semen when normalized for the number of spermatozoa. Adding back seminal plasma to spermatozoa washed by multiple centrifugations reduced ROS detected from 406 mV/s/109 spermatozoa to 61 mV/s/lOy spermatozoa, a value similar to that obtained with spermatozoa subjected to 3 cycles of centrifugation-resuspension but without removal of seminal plasma (71 mV/s/109 spermatozoa). However, the latter two values were still 8-fold higher than the 8 mV/s/lOy spermatozoa detected in the original semen.10 Thus, it would appear that morphologically abnormal and biochemically/functionally abnormal (but not dead) spermatozoa present in semen cause ROS formation. Centrifugation and removal of seminal plasma increase ROS detection.
NEUTROPHILS, INFECTION AND POSSIBLE DAMAGE TO SPERM FUNCTION Concentrations of neutrophils above 1 million/ml are occasionally found in semen of patients consulting for infertility. The detection of these polymorphonuclear cells is currently based on the content of myeloperoxidase22or the presence of specific antigens. However, in the last case, antibodies that generally recognize all leukocytes are often used.2' Since the presence of active neutrophils is a better reflection of an active acute infection than the whole leukocyte population, a test to determine active neutrophil concentration was developed. This new test, in contrast to the previous tests, such as the myeloperoxidase assay by histochemical techniques, determines the concentration of functionally active neutrophils. The new assay is based on the capacity of active neutrophils to generate ROS upon stimulation by invading microorganisms or by synthetic stimulants such as phorbol myristate acetate (PMA). The superoxide anion generated reduces nitroblue tetrazolium (pale yellow color) to formazan (blue color).2J Although this test is less sensitive than the histochemical myeloperoxidase assay, the sensitivity obtained (0.5 X loh polymorphonuclear leukocytes per ml of semen) is 2-fold below the level considered by the World Health Organization25as evidence of possible genital tract infection. The advantages of this assay include its simplicity, its low cost, and an objective assessment of functionally active polymorphonuclear leukocytes in semen. To address the question whether active neutrophils could affect sperm function, PMA-stimulated neutrophils were incubated with spermatozoa. Although neutrophils only produced ROS in the first 30 min. the percentage of motile spermatozoa was affected 2-3 hours later for neutrophil concentrations above 2 X loh cells/ml. However. at a later time ( 5 h). neutrophil concentrations as low as 0.6 X 106 cells/ml
GAGNON et al.: ROS AND HUMAN SPERMATOZOA
439
caused a decrease in motility of at least 30%. Addition of superoxide dismutase significantly reduced the amount of ROS detected by chemiluminescence and NBT, but without conferring any protection on sperm motility. On the other hand, catalase was able to prevent most of the damage done to sperm motility.26 Whereas pools of human seminal plasma can prevent most of the damage caused by ROS produced by neutrophils, individual sperm samples seemed to confer a wide range of protection from full protection to none (unpublished data).
EFFECTS OF ROS ON MOTILITY AND VIABILITY OF HUMAN SPERMATOZOA To further investigate the effects of ROS on sperm viability and motility, we have used xanthine (X) and xanthine oxidase (XO), which produces the superoxide anion (and subsequently H202 after dismutation), as a source of ROS. When X + X O at concentrations above 1 mM and 0.05 U/ml, respectively, were incubated with Percollwashed motile spermatozoa from a fertile donor, sperm motility was arrested within 30-60 min. A drop of 30-50% in sperm viability was always associated with these experimental conditions. However, at lower ROS concentrations (0.3-0.6 mM X 0.05 U XO/ml), viability was only marginally affected. Under these conditions, although ROS were present in solution for only one hour, the percentage of motile cells was only affected at 1.5 h after the addition of ROS. The drop in the percentage of motile cells occurred abruptly, in contrast to the flagellar beat frequency which was affected shortly but progressively after ROS addition. The decrease in beat frequency reached a value of 27% of control within the first hour after the addition of X + X O (TABLEl).*’.*8 ROS have long been known to cause damage to cells by interfering with membrane function. To determine whether the effects on sperm motility could also be caused by modifications on the sperm axoneme. the motor responsible for sperm movement, demembranated sperm models were prepared as previously d e ~ c r i b e d . ~ ~ . ~ ~ These sperm models are prepared by demembranation of motile intact spermatozoa with Triton X-100; the motility of these immotile, demembranated spermatozoa is
+
TABLE1. Effect of ROS on the Motilitv Parameters and ATP Level of Intact Soermatozoa Intact Spermatozoa Time (h) 0 0.5 1 1.5 2.5 24
Motility ( % of control)
Beat Frequency (% of control)
ATP Level ( % of control)
100
100 55
100 25 12 5 5
100 100
0 0 25
21 0 0 25
25
Percoll-washed spermatozoa from a fertile donor were incubated with xanthine (0.6 mM) and xanthine oxidase (0.05 U/ml). The percentage of motile spermatozoa and their flagellar beat frequency were measured at various time periods after addition of ROS. Sperm ATP levels were determined by the luciferine-luciferase assay. These results from one experiment are representative of 5 other experiments.
440
ANNALS NEW YORK ACADEMY OF SCIENCES
TABLE2. Effect of ROS on the Motilitv Parameters of Demembranated SDermatozoa Time (h) 0
0.5 1
1.5 2.5 24
Motility (% of control)
Beat Frequency (% of control)
100 100 50 25 0 25
100 100 100 100 0 100
Percoll-washed spermatozoa were treated as described in TABLEI . At various time intervals after the initiation of ROS treatment, spermatozoa were demembranated with Triton X-100, and motility was reinitiated by the addition of 0.5 mM Mg ATP.30 These results from the same experiment reported in TABLEI are representative of 5 other experiments.
then reactivated by the addition of a source of energy, ATP. Sperm models from ROS-treated intact spermatozoa could reactivate motility with a decreased percentage and duration of reactivation when compared to control untreated spermatozoa (TABLE2 ) . On the other hand, their flagellar beat frequency was not affected. It is worth mentioning that when intact spermatozoa were immobilized by ROS treatment, the reactivation of the sperm models was still possible, but for only an additional 30 min.27.28 The most surprising aspect of these experiments was that the motility of intact spermatozoa could spontaneously recover after several hours of arrest (TABLE1). This was associated with the reappearance of motility in sperm models when challenged with ATP (TABLE2).
IMPORTANCE OF ATP IN ROS DAMAGE TO SPERMATOZOA Since ATP concentration determines the flagellar beat frequency of demembranated spermatozoa,31 and ROS treatment caused a rapid decrease in flagellar beat frequency of intact spermatozoa, the level of ATP in control and ROS-treated cells was investigated. The level of ATP decreased rapidly following ROS treatment. The fall in ATP concentration was even steeper than that of the flagellar beat frequency. This discrepancy in the kinetics of these two parameters is not unexpected since the concentration of ATP within human spermatozoa is 4.5 mM,32 a value much higher than the 0.3-0.4 mM ATP required for half maximal beat frequency in demembranated sperm models.33 At the time when intact sperm motility ceased after ROS treatment, the level of ATP was 10- to 20-fold lower than that found in control ~ p e r m a t o z o a . ~ ~ The importance of the drop in ATP concentration after ROS treatment was further emphasized by the following series of observations. The addition of lactate and pyruvate together with ROS to intact spermatozoa completely prevented the decrease in intracellular ATP concentration, the drop in flagellar beat frequency and the fall in sperm motility generally seen a few hours after ROS treatment. Furthermore, spermatozoa immobilized by ROS treatment, and supplemented with pyruvate + lactate reinitiated motility much earlier than their counterparts supplemented with buffer only (unpublished data).
ANNALS NEW YORK ACADEMY OF SCIENCES
442
EFFECTS OF ROS SCAVENGERS ON X TREATED SPERMATOZOA
+
XO-
+
T o determine which of ROS produced by X XO caused the damage to sperm motility, ROS-treated spermatozoa were incubated with various scavengers. Since X XO can produce the superoxide anion which can be spontaneously or enzymically dismutated into HzOz and 0 2 , and that '0; and H20. can react together through the Haber Weiss reaction to produce the very toxic hydroxyl radical, superoxide dismutase, catalase and DMSO were used to scavenge '01,HzOz and 'OH, respectively. Of these scavengers only catalase at concentrations as low as 0.008 mg/ml conferred full protection on spermatozoa incubated with X and XO (TABLE3). The addition of superoxide dismutase at a high concentration (1 mg/ml) conferred only partial protection, whereas 140 mM DMSO mainly helped the recovery of motility without affecting its fall. It would then appear from these results that HzOz is the primary toxic ROS responsible for most damage done to spermatozoa. However, '0; and 'OH may, to some extent, also contribute to the toxicity of X XO on human spermatozoa.
+
+
CONCLUSION Normally, there is a fine balance between the amounts of ROS produced and scavenged by a cell. Cellular damage arises when this equilibrium is disturbed, especially when the increase in ROS produced cannot be eliminated by the reserve of scavenging systems. Such a disruption occurs in at least 25 % of the semen of patients consulting for infertility. However, whether the disruption in ROS equilibrium is due to a decreased scavenging capacity or to an enhanced ROS production remains to be seen. The clinical significance of leukocytes in male and female reproductive tracts remains controversial. However, the data summarized above indicate that stimulated leukocytes can produce an outburst of ROS that will decrease sperm motility over long periods of time, even at concentrations below 1 X I 0 6 cellsiml. Whereas human seminal plasma has enormous scavenging capacity for the superoxide anion produced by activated leukocytes, the situation may be quite different in extracellular fluids surrounding spermatozoa in the male and female reproductive tracts. The observation that exogenously added ROS affect sperm motility by decreasing the intracellular ATP level before affecting viability, raises the possibility that the poor sperm velocity frequently seen in the semen of infertile patients, which is a reflection of flagellar beat frequency, may be due to abnormally low levels of ATP. Whether this deficiency can be corrected by incubations with metabolic precursors known to increase cellular ATP levels remains to be investigated.
ACKNOWLEDGMENT We are grateful to Mrs. Lina Ordonselli for her secretarial assistance.
GAGNON et al.: ROS AND HUMAN SPERMATOZOA
443
REFERENCES I. 2. 3. 4.
5. 6. 7. 8. 9.
10. 11.
12.
13. 14.
15. 16.
17. 18.
19. 20. 21. 22.
GRISHAM, M. B. & J. M. MCCORD.1986. Chemistry and cytotoxicity of reactive oxygen nietabolites. In Biology of Oxygen Radicals. A. E. Taylor ct a/. , Eds.: 1 - 18. American Physiological Society. Bethesda, MD. ABU-ERREIZH, G., L. MAGNES& T. K. LI. 1978. Isolation and properties of superoxide dismutase from ram spermatozoa and erythrocytes. Bid. Reprod. 18: 554-560. MANELLA, M. R. F. & R . JONES.1980. Properties of spermatozoa superoxide dismutase and lack of involvement of superoxides in metal-ion-catalysed lipid-peroxidation reactinn5 in semen. Biochem. J. 191: 289-296. NISSEN,H. P. & H. W. KREYSEL. 1983. Superoxide disniutase in human semen. Klin. Wnchenschr. 61: 63-65. ALVAREZ, J. G. & B. T. STOREY.1983. Role of superoxide dismutase in protecting rabbit spermatozoa from oxygen toxicity due to lipid peroxidation. Biol. Reprod. 28: 1129-1136. ALVAREZ, J. G. & B. T. STOREY.1984. Lipid peroxidation and the reactions of superoxide and hydrogen peroxide in mouse spermatozoa. Biol. Reprod. 30: 833-841. P. WEBER,D. LAVA~.-MARTIN & R. CALVAYRAC. 1989. Catalase activity JEULIN. C., J. C. SOUFIR, in human spermatozoa and seminal plasma. Gamete Res. 24: 185-196. LI, T. K. 1975. The glutathione and thiol content of mammalian spermatozoa and seminal plasma. Biol. Reprod. 12: 641-646. ALVAREZ, J. G., J. C. TOUCHSTONE, L. BLASCO& B. T. STOREY.1987. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human 5permatozoaSuperoxide dismutase as major enzyme protectant against oxygen toxicity. J. Androl. 8: 338-348. ALVAREZ, J. G. & B. T. STOREY. 1989. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res. 23: 77-88. MCLEOD,J. 1943. The role of oxygen in the metabolism and motility of human spermatozoa. Am. J, Physiol. 138: 512-518. Tosic, J. 1947. Mechanism of hydrogen peroxide formation by spermatozoa and the role of amino acids in sperm motility. Nature 159: 544-545. Tosrc, J. & A. WALTON.1950. Metabolism of spermatozoa. Formation of hydrogen peroxide by spermatozoa and its effects on motility and survival. Biochem. J. 47: 199-206. JONES,R., T. MANN& R. SHERINS. 1979. Peroxidative breakdown of phospholipids in human spermatozoa. spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil. Steril. 31: 531-537. AITKEN,R. J. & J. S. CLARKSON. 1987. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil. 81: 459-469. RAO,B., J. C. SOUFIR, M. MARTIN & G. DAVID.1989. Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res. 24: 127-134. 1988. Significance of reactive oxygen species and antioxidants AITKEN.J. R. & J. S. CLARKSON. in defining the efficacy of sperm preparation techniques. J. Androl. 9: 367-376. & S. FISHEL.1989. Generation of reactive oxygen species, lipid AITKEN,J. R.. J. S. CLARKSON peroxidation and human sperm function. Bid. Reprod. 40: 183-197. IWASAKI, A. & C. GAGNON.1989. Free radical formation in human semen: Incidence and correlation with motility. 35th Annual Meeting of the Canadian Fertility and Andrology Society. Vancouver. Abstract 25. IwAsAKi, A. & C. GAGNON.1991. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil. Steril. In press. CHAPEAU, C. & C. GAGNON.1987. Nitrocellulose and polyvinyl coatings prevent sperm adhesion to glass without affecting the motility of intact and demembranated human spermatozoa. J. Androl. 8: 34-40. R. C. S P E N C ~G. R ,R . KINGHORN. A. WHITE.P. E. HARRISON, BARRATT, C. L. R., A. ROBINSON. E. KESSOPOUL~N & I. D. COOKE.1990. Seminal peroxidase positive cells are not an adequate indicator of asymptomatic urethral genital infection. Int. J. Androl. 13: 361-370.
444
ANNALS NEW YORK ACADEMY OF SCIENCES
R. J. & K. M. WEST.1990. Analysis ofthe relationship between reactive oxygen species 23. AITKEN, production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. Int. J. Androl. 13: 433-451. N . , E. DE LAMIRANDE & C. GAGNON.1991. Determination of neutrophil concentra24. KOVALSKI, tion in semen by measurement of superoxide radical formation. Fertil. Steril. In press. 2 5 . WHO. 1987. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge University Press. Cambridge, UK. N., E. DE LAMIRANDE & C. GAGNON.1991. Dimethylsulfoxide and catalase protect 26. KOVALSKI, against neutrophil induce loss of sperm motility. Canadian Fertility and Andrology Society, Quebec. Abstract TP27. E., A. IWASAKI & C. GAGNON.1992. Reactive oxygen species affect sperm 27. DE LAMIRANDE, axoneme. In press. E., A. IWASAKI& C. GAGNON.1990. Free radicals also affect sperm axoneme. 28. DE LAMIRANDE, 36th Annual Meeting of the Canadian Fertility and Andrology Society, L'Esterel, Quebec. Abstract 14. 1980. Characteristics of motor apparatus in testicular epididymal 29. MOHRI,H. & R. YAMAGIMACHI. and ejaculated spermatozoa. Exp. Cell Res. 127 191-196. E. & C. GAGNON.1983. Aprotinin and a seminal plasma factor inhibit the mo30. DE LAMIRANDE, tility of demembranated reactivated rabbit spermatozoa. Biol. Reprod. 28: 788-796. B. H. & I. R. GIBBONS.1972. Flagellar movement and adenosine triphosphatase ac31. GIBBONS, tivity in sea urchin extracted with Triton X-100. J. Cell Biol. 54: 75-97. 1983. D-[1-i4C] mannitol and [U-14C]sucrose as extracellular 32. FORD,W. C. C. & A. HARRISON. space markers for human spermatozoa and the rupture of 2-deoxyglucose. J. Reprod. Fertil. 69: 479-487. 33. COSSON,M. P. & C. GAGNON.1988. Protease inhibitor and substrates block motility and microtubule sliding of sea urchin and carp spermatozoa. Cell Motil. Cytoskeleton 10: 518-527. E. & C. GAGNON.1991. ATP depletion plays an important role in the inhibition 34. DE LAMIRANDE, of sperm motility observed after treatment with reactive oxygen species. 11th North American Testis Workshop, Montreal. Abstract 116.
