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Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma*
FERTILITY AND STERILITY Copyright
Vol. 59, No. 6, June 1993
1993 The American Fertility Society
Printed on acid-free paper in U.S.A.
Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma*
Eve de Lamirande, Ph.D. t Claude Gagnon, Ph.D. Urology Research Laboratory, Royal Victoria Hospital, and Faculty of Medicine, McGill University, Montreal, Quebec, Canada
Objectives: To characterize the very vigorous type of motility observed in the semen of some infertile men and to compare the superoxide anion scavenging capacity of the seminal plasma from these men and that from normal men. Subjects: Patients consulting for infertility related to sperm motility problems and men presenting as sperm donors. Methods: Motility patterns and measurements of sperm motility parameters were evaluated by computer-assisted digital image analysis system. The superoxide anion scavenging capacity of seminal plasma was measured by inhibition of nitroblue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase. Results: Spermatozoa from 9 of 68 semen samples with normal sperm concentration, morphology, and percentage of motility showed the typical motility patterns observed during hyperactivation (HA) and a significant level of HA (16% ± 3%) as compared with those in semen (2.3% ± 0.3%) from fertile volunteers. The superoxide anion scavenging capacity of the seminal plasma from men with sperm HA was 37% lower than that of control seminal plasma. Conclusions: Sperm HA can occur in whole semen and is associated with a low superoxide anion scavenging capacity in seminal plasma. It could be one of the causes for idiopathic infertility. Fertil Steril 1993;59:1291-5 Key Words: Male infertility, idiopathic infertility, superoxide anion, superoxide dismutase, reactive oxygen species, spermatozoa, sperm motility
Oligozoospermia ( <20 X 106 spermatozoajmL) and asthenozoospermia ( <40% motile spermatozoa) are major causes of male infertility (1). However, in some cases, the number and motility of spermatozoa appear normal according to accepted standards (for example, the criteria defined by the World Health Organization [WHO]) (1), and yet no conception occurs despite female partners being normal. We
Received November 9, 1992; revised and accepted February 17, 1993. * Supported by a grant from the Medical Research Council of Canada (Ottawa, Ontario, Canada) and by a chercheur boursier from the Fonds de Ia Recherche en Sante du Quebec (Quebec, Canada) to C.G. t Reprint requests: Eve de Lamirande, Ph.D., Urology Research Laboratory, H6.47, Royal Victoria Hospital, 687 ave des Pins Ouest, Montreal, Quebec, Canada H3A 1Al. Vol. 59, No.6, June 1993
have recently observed in a group of infertile patients that sperm motility in whole semen was very vigorous and characterized by very high velocity and lateral displacement of the head and low linearity. This type of movement presented similarities with that observed during hyperactivation (HA) when sperm motility dramatically changes from the linear, relatively slow, and low amplitude type of movement observed in seminal plasma to a high velocity, low progression, whiplash-like type of movement (2-5). Very low percentages of sperm HA are found in semen of fertile men (2% to 3%) (3). The first aim of our study was to further characterize this type of vigorous motility found in the semen of these men. Human spermatozoa can produce reactive oxygen species such as the superoxide anion ("0 2 -) and hydrogen peroxide (H 20 2 ) (6, 7). Although hydrogen peroxide is known to be toxic for human spermatozoa
de Lamirande and Gagnon
Sperm hyperactivation in whole semen
1291
(6, 8), we have recently shown that the superoxide anion, generated by the combination xanthine plus xanthine oxidase (in the presence of catalase), induced HA and capacitation in human spermatozoa (9, 10). Furthermore, superoxide dismutase (SOD, the enzyme responsible for the degradation of the superoxide anion to hydrogen peroxide) not only blocked the HA and capacitation induced by the superoxide anion but also by fetal cord serum, a biological inducer of HA and capacitation (9). Seminal plasma has superoxide anion scavenging capacity because of the presence of SOD itself (11) and also the presence of proteins (such as albumin) that act as sacrificial scavengers (12) and of small molecules, such as the glutathione (12), vitamin E and C (13, 14), taurine and hypotaurine (15). The second aim of our study was to investigate whether the occurrence of HA in semen was associated with a low SOD-like activity in seminal plasma. MATERIALS AND METHODS Semen Samples
Semen samples were collected after 3 days of sexual abstinence from patients consulting for infertility (n = 115) related mostly to sperm motility problems (second referral) and men presenting as sperm donors (n = 34) without any prior selection. Sperm concentrations ranged from 1 to 840 X 106 spermatozoa/mL (mean± SEM, 87 ± 10 X 106 spermatozoa/mL; median, 48 X 106 spermatozoa/mL) and sperm motility ranged from 0% to 92% (mean ± SEM, 39% ± 2%; median, 35%). Spermograms indicated that 81 of these men suffered from oligozoospermia and/or asthenozoospermia ( <20 ± 106 spermatozoa/mL and/or <40% sperm motility). The present study concerns the 68 men (44 patients and 24 potential donors among whom were 3 fertile men) whose semen characteristics were considered normal according to the WHO. In this group of 68 men, the average sperm concentration was 150 ± 20 X 106 spermatozoa/mL (median, 88 X 106 spermatozoa/mL), motility was 64% ± 2% (median, 63%), and at least 40% of spermatozoa were morphologically normal. Motility Analysis
Thirty minutes after collection, semen samples were placed in a Makler counting chamber (SefiMedical Instruments, Haifa, Israel) kept at 37°C on a heated plate (Microwarm plate 30; Kita Zato, Japan). Motility was analyzed using the CellSoft com1292
de Lamirande and Gagnon
puter-assisted digital analysis system (Cryo Resources, Montgomery, NY) as previously described (16, 17). Criteria for HA were the following: curvilinear velocity (VCL, total distance traveled divided by the total time the cell was tracked) >80 J.Lm/sec, linearity (LIN, ratio of the straight line distance to the actual track distance times 10) <6.5, and amplitude of the lateral head displacement (ALH, deviation of the sperm head from the mean trajectory) >6.5 11m (17). Motility measurements were determined for at least 200 motile spermatozoa. The percentage of HA was then obtained using a program written to select from individual cell data files, only data from those cells that showed the specific characteristics of HA (17). Samples showing sperm HA .:::; 4% of motile spermatozoa were considered normal and included in the control group because fertile volunteers never had >4% sperm HA. Samples showing >4% sperm HA were included in the test group. Superoxide Dismutase-Like Activity
Superoxide dismutase-like activity (total superoxide anion scavenging capacity) was measured by the inhibition of nitroblue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase (18, 19). The reaction mixture contained HEPES-balanced saline (130 Mm NaCl, 4 mM KCl, 0.5 mM MgC1 2 , 14 mM fructose, and 25 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid [HEPES] adjusted to pH 8), xanthine (0.25 mM), nitroblue tetrazolium (2 mM), bovine serum albumin (10 mg/mL), and various volumes of seminal plasma. The reaction was initiated by the addition of xanthine oxidase (0.05 U /mL) and monitored at 22°C by the increase of absorbance at 570 nm. Under these conditions, the reduction of nitroblue tetrazolium is linear for the first 40 minutes. Measurements of the SOD-like activity were taken at 20 minutes. One unit of SODlike activity was defined as the amount of seminal plasma capable of decreasing by 50% the reduction of nitroblue tetrazolium. Seminal plasma samples used for the determination of SOD-like activity were obtained by centrifugation of semen at 600 X g for 15 minutes and recentrifugation of the supernatant at 10,000 X g for 20 minutes. Clear seminal plasma was frozen ( -20°C) until used. Statistics
Student's t-test (2 tails, unpaired values) was used to determine the difference between groups.
Sperm hyperactiuation in whole semen
Fertility and Sterility
Motility Measurements
Control
~~~
/
~/~~ lOU'm
Hyperactivated
Figure 1 Representative sperm motility patterns observed in the semen of control men and in the semen of men with sperm HA. Fertile volunteers had sperm motility patterns similar to those of control spermatozoa (upper panel) and very few of their spermatozoa had motility patterns characteristic of HA (lower panel). High numbers of spermatozoa with motility patterns characteristic of HA were found in the semen of 9 of 68 men with normal sperm concentration, morphology, and percentage of motility. Tracks were obtained using the CellSoft Research Module. Cells were tracked for ~ second at a rate of 30 frames/sec, and the bar corresponds to 10 11m. T, trashing; S, starspin; C, circlinghigh curvature; H, helical; B, biphasic.
RESULTS Motility Patterns
Spermograms performed on the 149 semen samples studied revealed that 68 of the samples were normal according to the WHO criteria. We observed in 9 (6 infertile men and 3 men presenting as sperm donors) of these 68 cases that sperm motility in whole semen was very vigorous and differed greatly from that observed in semen samples from fertile donors. The motility patterns of spermatozoa from fertile donors and from these 9 men were therefore compared. Spermatozoa in semen from fertile donors had relatively straight trajectories and low speed, and the ALH was narrow (Fig. 1). However, in the subgroup of 9 men with very vigorous sperm motility, there was a significant proportion of spermatozoa displaying the typical motility patterns of hyperactivated spermatozoa (3, 17) (Fig. 1). Trashing, helical, starspin, circling-high curvature, and biphasic motility patterns were always observed in these samples. Vol. 59, No.6, June 1993
Motility parameters were determined for semen samples from the 9 men with sperm HA and from 12 control men (3 fertile men and 9 sperm donors with normal spermograms according to the WHO criteria) (Table 1). Curvilinear velocity and ALH were significantly higher, and linearity was lower in semen showing motility patterns typical of HA as compared with control semen values. These differences in the motility parameters are consistent with the presence of HA (3, 17). The percentages of sperm HA were 2.6% ± 0.3% and 16% ± 3% in control and test (sperm HA) groups, respectively (P < 0.0001) (Table 1; Fig. 2). The sperm HA present in semen did not appear to be a transient phenomenon. Three of the 9 men with sperm HA in semen came back for a second test and had the same type of sperm motility and similar percentages of sperm HA (data not shown). Superoxide Dismutase-Like Activity
Superoxide dismutase was shown to block the HA induced by the superoxide anion and by fetal cord serum (9). The levels of SOD-like activity (the superoxide anion scavenging capacity) of seminal plasma from 7 of the 9 men with sperm HA and from 7 of the 12 sperm donors investigated for motility patterns were therefore determined (Fig. 2). There was a 37% lower SOD-like activity in the seminal plasma from semen with sperm HA as compared with that found in control seminal plasma (P < 0.002). Two ofthe 7 men with sperm HA in semen came back for a second test, and SOD-like activities were again found to be lower than that of control. DISCUSSION
Our data indicate that sperm HA can occur in whole semen and that this HA may be related to a Table 1 Motility Parameters in Semen Samples From Control Men and Men With Sperm HA * Group Control HA
Motility
VCL
%
p,m/sec
73 ± 4 59 ± 4
LIN
ALH
HA %
58 ± 3 6.6 ± 0.2 2.3 ± 0.1 2.6 ± 0.3 72 ± 3t 5.:3 ± 0.2:j: :3.6 ± 0.2:j: 16 ± :>:j:
* Motility parameters and percentages of HA were determined as described in Materials and Methods. Semen samples from 12 control men (:3 fertile men and 9 sperm donors with normal spermograms according to the WHO criteria and 9 men with sperm HA in the semen were analyzed). t Statistically different, P < 0.001, as compared with control group. :j: Statistically different, P < 0.0001, as compared with control group.
de Lamirande and Gagnon
Sperm hyperactivation in whole semen
1293
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Figure 2 Sperm HA and seminal plasma SOD-like activity in control semen and in semen with sperm HA. Sperm HA (top panel) and seminal plasma SOD-like activity (bottom panel) were determined as described in Materials and Methods. Semen samples tested for HA are described in the footnote of Table 1. For SOD-like activity, seminal plasma from 7 control men (3 fertile men and 4 men with sperm motility characteristics similar to those of the fertile men) and from 7 men with sperm HA in semen were analyzed. Values for sperm HA and SOD-like activity differ between the two groups, (P < 0.0001 andP < 0.002, respectively).
low level of SOD-like activity in seminal plasma. In 9 of 68 (13%) semen samples, a significant number of spermatozoa (>4% of the motile spermatozoa) had motility patterns and parameters similar to those found during HA (Fig. 1, 2; Table 1). The percentage of HA found in these samples ranged from 7% to 33%. Sperm HA occurs at low levels in semen from fertile men (Table 1; Fig. 2) (Burkman [3]) and the absence of sperm capacitation in semen was previously related to the presence, in seminal plasma, of antifertility and/or decapacitation factors (20). The present data indicate that the presence of sperm HA in semen is associated with levels of SODlike activity in the seminal plasma (Fig_ 2) that were 37% lower than those found in control seminal plasma. These results are in agreement with the involvement of the superoxide anion in human sperm HA (9, 10). The superoxide anion generated by the combination xanthine plus xanthine oxidase (in the presence of catalase) induced higher levels of HA and capacitation than fetal cord serum, a biological inducer of these processes. Furthermore, SOD blocked the HA and capacitation induced by the soperoxide anion and by fetal cord serum. The fact 1294
de Lamirande and Gagnon
that SOD reversed the HA once induced suggested that a constant generation of superoxide anion by spermatozoa is needed to maintain HA and that the superoxide anion generated by spermatozoa is, at least in part, released outside the cells (9, 10). The low levels of seminal plasma SOD-like activity associated with the presence of HA in the semen do not exclude that deficiencies in other factors, such as the antifertility and/or decapacitation factors, (20) or an overstimulation of the superoxide generating enzyme present in spermatozoa, could participate in the induction of sperm HA in the semen of these nine men. As it is often observed in biological systems, several control mechanisms participate in the regulation of a given phenomenon. Although sperm HA is a necessary step for spermatozoa to acquire a full fertilizing capacity (2, 3), the presence of HA in semen is nevertheless abnormal and could be a cause of infertility. Hyperactivation confers a mechanical advantage on spermatozoa in the oviduct where they may encounter viscous oviductal fluid and a viscoelastic cumulus matrix (21), but it remains to be investigated whether this high amplitude type of motility would favor the passage of spermatozoa through cervical mucus. A positive correlation was found between the degree of lateral head displacement and the success of sperm-cervical mucus interaction (22), but donor spermatozoa was used for this study and the sperm lateral head displacement was therefore lower than what is observed during HA. It was recently shown that mouse spermatozoa pretreated with calcium (therefore hyperactivated and capacitated) fertilized significantly fewer eggs after artificial insemination than did spermatozoa preincubated in calcium deficient medium (progressive motility, no HA) (23). The authors concluded that sperm transport to the oviduct was impaired by the calcium pretreatment probably because of loss of progressive motility (24). Similarly, lower numbers of hamster spermatozoa were found in oviducts of mice inseminated with capacitated spermatozoa as compared with that found with uncapacitated spermatozoa (25), and the number of spermatozoa found in the oviducts appeared to be inversely proportional to the percentage of hyperactivated spermatozoa in the sample used for insemination (25). It is possible that the same type of phenomenon happens in humans, and that, when HA occurs in semen, there is an impaired transport of spermatozoa through the female genital tract. It remains also possible that the HA observed in semen is leading to premature capacitation and ac-
Sperm hyperactivation in whole semen
Fertility and Sterility
rosome reaction. Some previous studies have shown a close correlation between these events (3, 10). These prematurely hyperactivated spermatozoa possibly undergo the acrosome reaction too early and/or die before they reach the fertilization site. The present data emphasize the need for a more careful examination of sperm motility characteristics in semen samples classified normal. Sperm samples showing hyperactivated motility can easily be missed by an untrained eye and may be considered as samples with very good motility, especially when a visual evaluation is performed at room temperature (HA has to be evaluated at 37°C). The presence of sperm HA in semen (found in 13% of the semen samples classified normal according to WHO criteria) could be a cause for male idiopathic infertility. Further studies are needed to understand the role of the SOD-like activity of seminal plasma in this phenomenon.
8.
9.
10.
11. 12.
13. 14. 15.
16.
Acknowledgments. The authors thank Mrs. Lina Ordonselli and Mrs. Andree F. Wilkinson for their secretarial assistance and the volunteers who participated in this study.
REFERENCES
17.
18.
1. World Health Organization. WHO Laboratory manual for
2.
3.
4.
5.
6.
7.
the examination of human semen and semen-cervical mucus interaction. 2nd edition. Cambridge: The Press Syndicate of the University of Cambridge, 1987:27. Katz D F, Y anagimachi R. Movement characteristics of hamster spermatozoa within the oviduct. Bioi Reprod 1980;22: 749-64. Burkman LJ. Hyperactivated motility of human spermatozoa during in vitro capacitation and implications for fertility. In: Gagnon C, editor. Controls of sperm motility. Boca Raton: CRC Press, 1990:303-29. Burkman LJ. Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human spermatozoa using computerized analysis. Fertil Steril 1991;55: 363-71. Mortimer ST, Mortimer D. Kinematics of human spermatozoa incubated under capacitating conditions. J Androl 1990;11:195-203. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 1987;8:338-48. Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive ox-
Vol. 59, No.6, June 1993
19.
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21.
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25.
ygen species by human spermatozoa. J Reprod Fertil 1987;81: 459-69. de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J Androl1992;13:368-78. de Lamirande E, Gagnon C. A positive role for the superoxide anion in the triggering of human sperm hyperactivation and capacitation. Int J Androl1993;16:21-5. de Lamirande E, Gagnon C. Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Radic Bioi Med 1993;14:157-66. Nissen HP, Kreysel HW. Superoxide dismutase in human semen. Klin Wochenscr 1983;61:63-5. Halliwell .B, Gutteridge JMC. Free radicals in biology and medicine. 2nd edition. Oxford: Clarendon Press, 1989:1527; 253-9. Chow CK. Vitamin E and oxidative stress. Free Radic Bioi Med 1991;11:215-32. Niki E. Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am J Clin Nutr 1991;54:119S-24S. Alvarez JG, Storey BT. Taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility. Bioi Reprod 1983;29: 548-55. de Lamirande E, Gagnon C. Quantitative assessment of the serum-induced stimulation of human sperm motility. Int J Androl1991;14:11-22. Murad C, de Lamirande E, Gagnon C. Hyperactivated motility is coupled with interdependent modifications at axonemal and cytosolic levels in human spermatozoa. J Androl1992;13: 323-31. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276-87. Spitz DR, Oberley LW. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal Biochem 1989;179:8-18. Han H-L, Mack SR, DeJonge C, Zaneveld LJD. Inhibition of the human sperm acrosome reaction by a high molecular weight factor from human seminal plasma. Fertil Steril 1990;54:1177-9. Suarez SS, Katz DF, Owen DH, Andrew JB, Powell RL. Evidence for the function of hyperactivated motility in sperm. Bioi Reprod 1991;44:375-81. Aitken RJ, Sutton M, Warner P, Richardson DW. Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes. J Reprod Fertil 1985;73:441-9. Olds-Clarke P, Wivell W. Impaired transport and fertilization in vivo of calcium-treated spermatozoa from+/+ or congenic tw32 /+ mice. Bioi Reprod 1992;47:621-8. Olds-Clarke P, Sego R. Calcium alters capacitation and progressive motility of uterine spermatozoa from+/+ and congenic tw32/+ mice. Bioi Reprod 1992;47:629-35. Shalgi R, Smith T, Yanagimachi R. A quantitative comparison of the passage of capacitated and uncapacitated hamster spermatozoa through the uterotubal junction. Bioi Reprod 1992;46:419-24.
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Sperm hyperactivation in whole semen
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Vol. 59, No. 6, June 1993
1993 The American Fertility Society
Printed on acid-free paper in U.S.A.
Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma*
Eve de Lamirande, Ph.D. t Claude Gagnon, Ph.D. Urology Research Laboratory, Royal Victoria Hospital, and Faculty of Medicine, McGill University, Montreal, Quebec, Canada
Objectives: To characterize the very vigorous type of motility observed in the semen of some infertile men and to compare the superoxide anion scavenging capacity of the seminal plasma from these men and that from normal men. Subjects: Patients consulting for infertility related to sperm motility problems and men presenting as sperm donors. Methods: Motility patterns and measurements of sperm motility parameters were evaluated by computer-assisted digital image analysis system. The superoxide anion scavenging capacity of seminal plasma was measured by inhibition of nitroblue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase. Results: Spermatozoa from 9 of 68 semen samples with normal sperm concentration, morphology, and percentage of motility showed the typical motility patterns observed during hyperactivation (HA) and a significant level of HA (16% ± 3%) as compared with those in semen (2.3% ± 0.3%) from fertile volunteers. The superoxide anion scavenging capacity of the seminal plasma from men with sperm HA was 37% lower than that of control seminal plasma. Conclusions: Sperm HA can occur in whole semen and is associated with a low superoxide anion scavenging capacity in seminal plasma. It could be one of the causes for idiopathic infertility. Fertil Steril 1993;59:1291-5 Key Words: Male infertility, idiopathic infertility, superoxide anion, superoxide dismutase, reactive oxygen species, spermatozoa, sperm motility
Oligozoospermia ( <20 X 106 spermatozoajmL) and asthenozoospermia ( <40% motile spermatozoa) are major causes of male infertility (1). However, in some cases, the number and motility of spermatozoa appear normal according to accepted standards (for example, the criteria defined by the World Health Organization [WHO]) (1), and yet no conception occurs despite female partners being normal. We
Received November 9, 1992; revised and accepted February 17, 1993. * Supported by a grant from the Medical Research Council of Canada (Ottawa, Ontario, Canada) and by a chercheur boursier from the Fonds de Ia Recherche en Sante du Quebec (Quebec, Canada) to C.G. t Reprint requests: Eve de Lamirande, Ph.D., Urology Research Laboratory, H6.47, Royal Victoria Hospital, 687 ave des Pins Ouest, Montreal, Quebec, Canada H3A 1Al. Vol. 59, No.6, June 1993
have recently observed in a group of infertile patients that sperm motility in whole semen was very vigorous and characterized by very high velocity and lateral displacement of the head and low linearity. This type of movement presented similarities with that observed during hyperactivation (HA) when sperm motility dramatically changes from the linear, relatively slow, and low amplitude type of movement observed in seminal plasma to a high velocity, low progression, whiplash-like type of movement (2-5). Very low percentages of sperm HA are found in semen of fertile men (2% to 3%) (3). The first aim of our study was to further characterize this type of vigorous motility found in the semen of these men. Human spermatozoa can produce reactive oxygen species such as the superoxide anion ("0 2 -) and hydrogen peroxide (H 20 2 ) (6, 7). Although hydrogen peroxide is known to be toxic for human spermatozoa
de Lamirande and Gagnon
Sperm hyperactivation in whole semen
1291
(6, 8), we have recently shown that the superoxide anion, generated by the combination xanthine plus xanthine oxidase (in the presence of catalase), induced HA and capacitation in human spermatozoa (9, 10). Furthermore, superoxide dismutase (SOD, the enzyme responsible for the degradation of the superoxide anion to hydrogen peroxide) not only blocked the HA and capacitation induced by the superoxide anion but also by fetal cord serum, a biological inducer of HA and capacitation (9). Seminal plasma has superoxide anion scavenging capacity because of the presence of SOD itself (11) and also the presence of proteins (such as albumin) that act as sacrificial scavengers (12) and of small molecules, such as the glutathione (12), vitamin E and C (13, 14), taurine and hypotaurine (15). The second aim of our study was to investigate whether the occurrence of HA in semen was associated with a low SOD-like activity in seminal plasma. MATERIALS AND METHODS Semen Samples
Semen samples were collected after 3 days of sexual abstinence from patients consulting for infertility (n = 115) related mostly to sperm motility problems (second referral) and men presenting as sperm donors (n = 34) without any prior selection. Sperm concentrations ranged from 1 to 840 X 106 spermatozoa/mL (mean± SEM, 87 ± 10 X 106 spermatozoa/mL; median, 48 X 106 spermatozoa/mL) and sperm motility ranged from 0% to 92% (mean ± SEM, 39% ± 2%; median, 35%). Spermograms indicated that 81 of these men suffered from oligozoospermia and/or asthenozoospermia ( <20 ± 106 spermatozoa/mL and/or <40% sperm motility). The present study concerns the 68 men (44 patients and 24 potential donors among whom were 3 fertile men) whose semen characteristics were considered normal according to the WHO. In this group of 68 men, the average sperm concentration was 150 ± 20 X 106 spermatozoa/mL (median, 88 X 106 spermatozoa/mL), motility was 64% ± 2% (median, 63%), and at least 40% of spermatozoa were morphologically normal. Motility Analysis
Thirty minutes after collection, semen samples were placed in a Makler counting chamber (SefiMedical Instruments, Haifa, Israel) kept at 37°C on a heated plate (Microwarm plate 30; Kita Zato, Japan). Motility was analyzed using the CellSoft com1292
de Lamirande and Gagnon
puter-assisted digital analysis system (Cryo Resources, Montgomery, NY) as previously described (16, 17). Criteria for HA were the following: curvilinear velocity (VCL, total distance traveled divided by the total time the cell was tracked) >80 J.Lm/sec, linearity (LIN, ratio of the straight line distance to the actual track distance times 10) <6.5, and amplitude of the lateral head displacement (ALH, deviation of the sperm head from the mean trajectory) >6.5 11m (17). Motility measurements were determined for at least 200 motile spermatozoa. The percentage of HA was then obtained using a program written to select from individual cell data files, only data from those cells that showed the specific characteristics of HA (17). Samples showing sperm HA .:::; 4% of motile spermatozoa were considered normal and included in the control group because fertile volunteers never had >4% sperm HA. Samples showing >4% sperm HA were included in the test group. Superoxide Dismutase-Like Activity
Superoxide dismutase-like activity (total superoxide anion scavenging capacity) was measured by the inhibition of nitroblue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase (18, 19). The reaction mixture contained HEPES-balanced saline (130 Mm NaCl, 4 mM KCl, 0.5 mM MgC1 2 , 14 mM fructose, and 25 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid [HEPES] adjusted to pH 8), xanthine (0.25 mM), nitroblue tetrazolium (2 mM), bovine serum albumin (10 mg/mL), and various volumes of seminal plasma. The reaction was initiated by the addition of xanthine oxidase (0.05 U /mL) and monitored at 22°C by the increase of absorbance at 570 nm. Under these conditions, the reduction of nitroblue tetrazolium is linear for the first 40 minutes. Measurements of the SOD-like activity were taken at 20 minutes. One unit of SODlike activity was defined as the amount of seminal plasma capable of decreasing by 50% the reduction of nitroblue tetrazolium. Seminal plasma samples used for the determination of SOD-like activity were obtained by centrifugation of semen at 600 X g for 15 minutes and recentrifugation of the supernatant at 10,000 X g for 20 minutes. Clear seminal plasma was frozen ( -20°C) until used. Statistics
Student's t-test (2 tails, unpaired values) was used to determine the difference between groups.
Sperm hyperactiuation in whole semen
Fertility and Sterility
Motility Measurements
Control
~~~
/
~/~~ lOU'm
Hyperactivated
Figure 1 Representative sperm motility patterns observed in the semen of control men and in the semen of men with sperm HA. Fertile volunteers had sperm motility patterns similar to those of control spermatozoa (upper panel) and very few of their spermatozoa had motility patterns characteristic of HA (lower panel). High numbers of spermatozoa with motility patterns characteristic of HA were found in the semen of 9 of 68 men with normal sperm concentration, morphology, and percentage of motility. Tracks were obtained using the CellSoft Research Module. Cells were tracked for ~ second at a rate of 30 frames/sec, and the bar corresponds to 10 11m. T, trashing; S, starspin; C, circlinghigh curvature; H, helical; B, biphasic.
RESULTS Motility Patterns
Spermograms performed on the 149 semen samples studied revealed that 68 of the samples were normal according to the WHO criteria. We observed in 9 (6 infertile men and 3 men presenting as sperm donors) of these 68 cases that sperm motility in whole semen was very vigorous and differed greatly from that observed in semen samples from fertile donors. The motility patterns of spermatozoa from fertile donors and from these 9 men were therefore compared. Spermatozoa in semen from fertile donors had relatively straight trajectories and low speed, and the ALH was narrow (Fig. 1). However, in the subgroup of 9 men with very vigorous sperm motility, there was a significant proportion of spermatozoa displaying the typical motility patterns of hyperactivated spermatozoa (3, 17) (Fig. 1). Trashing, helical, starspin, circling-high curvature, and biphasic motility patterns were always observed in these samples. Vol. 59, No.6, June 1993
Motility parameters were determined for semen samples from the 9 men with sperm HA and from 12 control men (3 fertile men and 9 sperm donors with normal spermograms according to the WHO criteria) (Table 1). Curvilinear velocity and ALH were significantly higher, and linearity was lower in semen showing motility patterns typical of HA as compared with control semen values. These differences in the motility parameters are consistent with the presence of HA (3, 17). The percentages of sperm HA were 2.6% ± 0.3% and 16% ± 3% in control and test (sperm HA) groups, respectively (P < 0.0001) (Table 1; Fig. 2). The sperm HA present in semen did not appear to be a transient phenomenon. Three of the 9 men with sperm HA in semen came back for a second test and had the same type of sperm motility and similar percentages of sperm HA (data not shown). Superoxide Dismutase-Like Activity
Superoxide dismutase was shown to block the HA induced by the superoxide anion and by fetal cord serum (9). The levels of SOD-like activity (the superoxide anion scavenging capacity) of seminal plasma from 7 of the 9 men with sperm HA and from 7 of the 12 sperm donors investigated for motility patterns were therefore determined (Fig. 2). There was a 37% lower SOD-like activity in the seminal plasma from semen with sperm HA as compared with that found in control seminal plasma (P < 0.002). Two ofthe 7 men with sperm HA in semen came back for a second test, and SOD-like activities were again found to be lower than that of control. DISCUSSION
Our data indicate that sperm HA can occur in whole semen and that this HA may be related to a Table 1 Motility Parameters in Semen Samples From Control Men and Men With Sperm HA * Group Control HA
Motility
VCL
%
p,m/sec
73 ± 4 59 ± 4
LIN
ALH
HA %
58 ± 3 6.6 ± 0.2 2.3 ± 0.1 2.6 ± 0.3 72 ± 3t 5.:3 ± 0.2:j: :3.6 ± 0.2:j: 16 ± :>:j:
* Motility parameters and percentages of HA were determined as described in Materials and Methods. Semen samples from 12 control men (:3 fertile men and 9 sperm donors with normal spermograms according to the WHO criteria and 9 men with sperm HA in the semen were analyzed). t Statistically different, P < 0.001, as compared with control group. :j: Statistically different, P < 0.0001, as compared with control group.
de Lamirande and Gagnon
Sperm hyperactivation in whole semen
1293
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Figure 2 Sperm HA and seminal plasma SOD-like activity in control semen and in semen with sperm HA. Sperm HA (top panel) and seminal plasma SOD-like activity (bottom panel) were determined as described in Materials and Methods. Semen samples tested for HA are described in the footnote of Table 1. For SOD-like activity, seminal plasma from 7 control men (3 fertile men and 4 men with sperm motility characteristics similar to those of the fertile men) and from 7 men with sperm HA in semen were analyzed. Values for sperm HA and SOD-like activity differ between the two groups, (P < 0.0001 andP < 0.002, respectively).
low level of SOD-like activity in seminal plasma. In 9 of 68 (13%) semen samples, a significant number of spermatozoa (>4% of the motile spermatozoa) had motility patterns and parameters similar to those found during HA (Fig. 1, 2; Table 1). The percentage of HA found in these samples ranged from 7% to 33%. Sperm HA occurs at low levels in semen from fertile men (Table 1; Fig. 2) (Burkman [3]) and the absence of sperm capacitation in semen was previously related to the presence, in seminal plasma, of antifertility and/or decapacitation factors (20). The present data indicate that the presence of sperm HA in semen is associated with levels of SODlike activity in the seminal plasma (Fig_ 2) that were 37% lower than those found in control seminal plasma. These results are in agreement with the involvement of the superoxide anion in human sperm HA (9, 10). The superoxide anion generated by the combination xanthine plus xanthine oxidase (in the presence of catalase) induced higher levels of HA and capacitation than fetal cord serum, a biological inducer of these processes. Furthermore, SOD blocked the HA and capacitation induced by the soperoxide anion and by fetal cord serum. The fact 1294
de Lamirande and Gagnon
that SOD reversed the HA once induced suggested that a constant generation of superoxide anion by spermatozoa is needed to maintain HA and that the superoxide anion generated by spermatozoa is, at least in part, released outside the cells (9, 10). The low levels of seminal plasma SOD-like activity associated with the presence of HA in the semen do not exclude that deficiencies in other factors, such as the antifertility and/or decapacitation factors, (20) or an overstimulation of the superoxide generating enzyme present in spermatozoa, could participate in the induction of sperm HA in the semen of these nine men. As it is often observed in biological systems, several control mechanisms participate in the regulation of a given phenomenon. Although sperm HA is a necessary step for spermatozoa to acquire a full fertilizing capacity (2, 3), the presence of HA in semen is nevertheless abnormal and could be a cause of infertility. Hyperactivation confers a mechanical advantage on spermatozoa in the oviduct where they may encounter viscous oviductal fluid and a viscoelastic cumulus matrix (21), but it remains to be investigated whether this high amplitude type of motility would favor the passage of spermatozoa through cervical mucus. A positive correlation was found between the degree of lateral head displacement and the success of sperm-cervical mucus interaction (22), but donor spermatozoa was used for this study and the sperm lateral head displacement was therefore lower than what is observed during HA. It was recently shown that mouse spermatozoa pretreated with calcium (therefore hyperactivated and capacitated) fertilized significantly fewer eggs after artificial insemination than did spermatozoa preincubated in calcium deficient medium (progressive motility, no HA) (23). The authors concluded that sperm transport to the oviduct was impaired by the calcium pretreatment probably because of loss of progressive motility (24). Similarly, lower numbers of hamster spermatozoa were found in oviducts of mice inseminated with capacitated spermatozoa as compared with that found with uncapacitated spermatozoa (25), and the number of spermatozoa found in the oviducts appeared to be inversely proportional to the percentage of hyperactivated spermatozoa in the sample used for insemination (25). It is possible that the same type of phenomenon happens in humans, and that, when HA occurs in semen, there is an impaired transport of spermatozoa through the female genital tract. It remains also possible that the HA observed in semen is leading to premature capacitation and ac-
Sperm hyperactivation in whole semen
Fertility and Sterility
rosome reaction. Some previous studies have shown a close correlation between these events (3, 10). These prematurely hyperactivated spermatozoa possibly undergo the acrosome reaction too early and/or die before they reach the fertilization site. The present data emphasize the need for a more careful examination of sperm motility characteristics in semen samples classified normal. Sperm samples showing hyperactivated motility can easily be missed by an untrained eye and may be considered as samples with very good motility, especially when a visual evaluation is performed at room temperature (HA has to be evaluated at 37°C). The presence of sperm HA in semen (found in 13% of the semen samples classified normal according to WHO criteria) could be a cause for male idiopathic infertility. Further studies are needed to understand the role of the SOD-like activity of seminal plasma in this phenomenon.
8.
9.
10.
11. 12.
13. 14. 15.
16.
Acknowledgments. The authors thank Mrs. Lina Ordonselli and Mrs. Andree F. Wilkinson for their secretarial assistance and the volunteers who participated in this study.
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