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Nitric oxide synthase inhibitor NG-monomethyl-l -arginine preserves sperm motility after swim-up
FERTILITY AND STERILITY® Copyright ~, 1996 American Society for Reproductive Medicine
Vol. 66, No.5, November 1996
Printed on acid·free paper in U. S. A
Nitric oxide synthase inhibitor NG-monomethyl-L-arginine preserves sperm motility after swim-up
Doreen M. D. Perera, Ph.D.*t Maurice Katz, M.D.* Sujiwa R. Heenbanda, B.S.* Sally Marchant, B.S.* University College London Medical School, London, United Kingdom
Objective: To evaluate the effect of ~-monomethyl-L-arginine (L-NMMA) on sperm motility. Design: Controlled study using motile sperm suspensions obtained by the swim-up technique. Setting: Endocrine Laboratory, Department of Obstetrics and Gynaecology, University College London Medical School. Patients: Men attending the Reproductive Medicine Unit at University College London Hospitals, Obstetric Hospital, for routine fertility investigations. Interventions: Motile sperm suspensions were incubated with either culture medium (control) or L-NMMA (test) for 24 hours at 37°C. Main Outcome Measure: Motion characteristics were assessed manually and by computerassisted sperm motility analyzer. Results: In all 23 preparations either equivalent or significantly higher percentage total motility and percentage rapidly motile sperm were observed in the test compared with the control samples at 24 hours. There were no significant differences in motion characteristics between test and control specimens at 6 hours. Conclusions: ~-monomethyl-L-arginine inhibits the formation of nitric oxide; its ability to prevent sperm motility decline indicates that the arginine-nitric oxide pathway may playa role in modulating sperm motility and motility survival. Fertil Steril® 1996;66:830-3 Key Words: Semen, sperm, motility, swim-up, IVF, nitric oxide, inhibitors, L-NMMA
The maintenance of sperm motility for longer periods is desirable. Extended in vitro sperm survival is correlated with successful NF and pregnancy rates (1). Recently nitric oxide (NO) applied directly or via a NO donor compound was reported to reduce sperm motility whereas NO quencher hemoglobin prevented the deterioration (2). Nitric oxide is an inorganic radical gas formed by the action of NO synthase (NOS) on L-arginine. This reaction can be inhibited by a variety of compounds, including~ substituted L-arginine analogues. It is known that the L-arginine-NO pathway exists in many cell types such as endothelial, neuronal, and macrophages and Received January 2, 1996; revised and accepted June 5,1996. * Department of Obstetrics and Gynaecology. t Reprint requests: Doreen M. D. Perera, Ph.D., University College London Medical School, 86-96, Chenies Mews, London WC1E 6HX, United Kingdom (FAX: 44-0171-383-7429). :j: Assisted Conception Unit.
830
Perera et al. Communications-in-brief
that it has both regulatory and cytotoxic functions. Because NO appears to be detrimental to sperm motility, we hypothesized that the inhibition of NO synthesis may improve sperm motility. To test this hypothesis, we studied the effects of ~-mono methyl-L-arginine (L-NMMA), a nonselective competitive inhibitor of NOS, on sperm motion characteristics using motile sperm suspensions obtained by the swim-up technique. MATERIALS AND METHODS
Samples from patients attending the Reproductive Medicine Unit for routine fertility investigations were used. Semen was produced by masturbation after 3 days of abstinence. Assessments were made both manually according to WHO protocol (3) and by computer-assisted sperm motion analysis (CASA), using a Hamilton- Thorne Motility Analyzer with four-chambered microcells (Microm Ltd., Thame, Fertility and Sterility®
Oxford, United Kingdom). ~-monomethyl-L-argi nine and L-arginine (Tocris Cookson, Langford, Bristol, United Kingdom) stock solutions of 10-2 M were made with Ram's F-10 culture medium (Sigma-Aldrige Chemical Company, Poole, Dorset, United Kingdom). After liquefaction, the semen sample was divided into two parts; each half mixed with an equal volume of Ram's F-lO culture medium and was centrifuged for 10 minutes at 500 x g at room temperature. The supernatant was discarded and the pellet was overlayered with 200 J.lL of Ram's F-10 culture medium with 10% Albuminar (Armour Pharmaceuticals Ltd., East Bourne, East Sussex, United Kingdom) and motile sperm were allowed to swim-up for 45 minutes. Supernatant motile sperm suspensions with total motility >60% and rapidly motile sperm >25% were used for the study. Sperm counts varied from 5 X 106 to 80 X 106/mL. Samples with sperm counts <20 X 106 /mL were defined as oligospermia. Into a series of 1.5-mL Eppendorftubes (Starsted Ltd., Beaumont Leys, Leicestershire, United Kingdom) 30-J.lL aliquots of sperm suspensions were pipetted and mixed either with 60 J.lL of Ram's F-10 culture medium (control) or L-NMMA stock solution (test) and were incubated at 37°C in an atmosphere of 5% CO 2 • In a series of separate experiments (n = 6) L-NMMA concentrations (3.0, 6.0, and 9.0 x 10-7 M) in a total volume of 90 J.lL of sperm-L-NMMA mixture were incubated with sperm suspensions to assess the dose-response curve of the reaction. Motility assessments were made at 5 to 6 hours (n = 8), at 24 hours (n = 23), and hourly for 5 hours (n = 5) to determine the rate of decline in motility or the protection afforded by L-NMMA. To establish if the protective effect of L-NMMA could be reversed by the addition of L-arginine, another series of motile sperm suspensions (30-J.lL aliquots) was incubated with L-NMMA (60 J.lL of stock solution) and L-arginine in increasing concentrations ranging from 2.0 X 10- 7 to 12.0 X 10- 7 M (n = 6). To determine the influence of temperature on the reaction, we incubated a duplicate series of sperm suspensions, one at room temperature and the other at 37°C in the presence and absence of 6.0 X 10- 7 M L-NMMA. Motilities were assessed at 24 and 48 hours. A comparison between test and control measurements was performed using Mann-Whitney U test for nonparametric data.
plete immobilization by 24 hours. Four specimens treated with L-NMMA showed poor motility at 24 hours and three specimens failed to show any progression in both in the L-NMMA-added and the control samples. In three specimens with severe oligospermia and poor motility, it was difficult to obtain meaningful measurements using CASA and only manual means were used. The mean percentage total motility and percentage rapidly motile sperm in L-NMMA-added samples were 28% and 13%, respectively, compared with 5% and 0% in the controls (Fig. 1) after 24 hours of incubation. Specimens with sperm counts >20 X 106/mL showed no decline in motility characteristics up to 5 hours nor was there a difference between the control and the test samples. Two specimens with sperm counts <20 X 106/mL showed a decline from initial values within 5 hours only in the control samples. Specimens with oligospermia showed the steepest decline in motility at 24 hours even in the presence of L-NMMA. One of the three specimens that failed to show any motility and one of the four specimens with very low motility even in the presence of L-NMMA, however, did have initial post-swim-up sperm counts and motilities that were in the normal range. The concentration of L-NMMA conducive to preserving maximum sperm motility for up to 24 hours ranged from 6.0 X 10- 7 to 9.0 X 10- 7 M in a total volume of 90 J.lL of sperm-L-NMMA mixture. There was a noticeable decline in motility when 3.0 X 10- 7 M of L-NMMA was used. Protection of motility afforded by L-NMMA was prevented in a dose-dependent manner by the addition of L-arginine from 6.0 X 10- 7 to 12.0 X 10- 7 M. Complete inhibition of motility at 24 hours was seen in five of six samples treated with L-arginine at a concentration of 12.0 X 10- 7 M. There was, however, an initial increase in motility in three ofthe six samples when the L-arginine concentration was between 2.0 X 10- 7 and 5.0 X 10- 7 M. The motilities then declined rapidly as the L-arginine concentrations were increased in the samples. In the experiments investigating the influence of incubation temperature, specimens incubated at room temperature survived up to 48 hours and beyond, with or without added L-NMMA, but the percent total motilities were superior in the specimens with added L-NMMA. DISCUSSION
RESULTS
Specimens containing L-NMMA showed significantly higher total motilities than the corresponding controls, in 20 of23 samples at 24 hours (P < 0.001). Seventeen of the 23 control specimens showed comVol. 66, No.5, November 1996
The effect of NO on spermatozoa has been studied by several groups. Because macrophages possess the ability to produce NO under certain conditions, it was suspected that the poor sperm motility and survival observed in semen samples with excessive Perera et al. Communications-in-brief
831
35
30
25
~
20
g :::i!
"I-
15
10
Total motilHy
Total motilHy
Rapidly motile sperm
in the presence of L-NMMA
in the control
in the presence of L-NMMA
numbers of leukocytes may be the result of NO-induced damage to sperm. Using NO donor compounds, several investigators have reported a deleterious effect of NO on sperm but Hellstrom et aL (4) did not confirm this. The present study was undertaken to evaluate the potential protective effects of NOS inhibition on motile sperm. ~-monomethyl-L-arginine inhibits the formation of NO by inhibiting NOS action on L-arginine. Because L-NMMA preserved motility in the treated suspensions, it can be assumed reasonably that the deterioration of motility in the nontreated control suspensions was due to the presence or formation of NO. This indicates that NOS, the enzyme responsible for the synthesis of NO, may have been present in these samples. Because we were using isolated motile sperm suspensions obtained by the swim-up procedure, devoid of macrophages and other cellular elements, the indication was that the source of NOS could have been spermatozoa themselves. There is ample evidence that spermatozoa are able to produce a variety of radical oxygen species (5) although NO has not yet been measured directly in leukocyte-free semen. It is possible that spermatozoa may contain one or more isoforms of NOS, which have been identified in other cell types. We have therefore embarked on a study to identify and isolate NOS in human sperm. The lack of effect of L-NMMA on sperm motility at 6 hours (compared with 24 hours) suggests that NOS may be switched on with time. One possibility is that the NOS is not constitutive but was induced 832
Perera et al.
Communications-in-brief
Rapidly motile sperm
In the control
Figure 1 Comparison of motion characteristics of swim up sperm suspensions in the presence C.) and absence CD) of L-NMMA after incubating for 24 hours at 37°C. Values are expressed as means Cn = 23); error bars, ±SE. *Significantly different from control CP < 0.001).
by the experimental conditions. It is known that NOS expression in macrophages is induced by certain microbial products and cytokines. A similar mechanism may exist in seminal fluid. Nonsurvival or poor progression of sperm seen at 24 hours even with added L-NMMA may have been due to differences in the amounts of NOS and/or L-arginine present in individual sperm suspensions and/or variations in the rate of uptake and metabolism OfL-NMMA by NOS-containing sperm cells. It is possible that spermatozoa from these patients may respond to higher concentrations of the inhibitor. Although at the higher concentration range L-arginine was undoubtedly inhibitory to sperm motility, at lower concentrations it appeared to exert a stimulatory effect in some samples. This observation suggests that NO may have a biphasic effect on sperm motility. Similar effects have been reported in studies on the immune system and in bone resorption studies in which low NO concentrations stimulated cell growth, whereas higher concentrations were inhibitory. This biphasic effect might explain why Hellstrom et aL (4) seemingly paradoxically observed increased motility in their study. They were using lower concentrations of NO donors than any of the other investigators. The Ham's F-10 culture medium used in our study contained 1 X 10- 3 M L-arginine. It would be interesting to carry out further studies using an L-arginine-free culture medium to evaluate effects on sperm motility and survivaL The observation of nonsurvival at 24 hours in the control specimens is at variance with other studies Fertility and Sterility®
that have reported better motilities after extended incubation times. The reason for this deterioration may be the incubation temperature that was used. It appeared from our results that the deterioration in sperm motility was accelerated at 37°C, suggesting that the activity of the factor responsible for deterioration of sperm motility or the rate of production of such a factor was temperature dependent. When the initial sperm counts were low, however, motilities deteriorated much faster even at room temperature. Perhaps altered rates of NO production exist in patients with oligospermia, which affect in vitro sperm motility survival times. We have conducted preliminary experiments with other inhibitors such as ~-nitro-L-arginine and its methyl ester. The protective effects were not as evident as that seen with L-NMMA, and there appeared to be intersample variations. It is possible that LNMMA, in addition to its inhibitory action on NOS, may also interact with other enzymes in this system. Further studies are needed before conclusions can be drawn from these preliminary observations. The present study demonstrated for the first time that L-NMMA has potential value in delaying in vitro sperm motility decline in sperm suspensions isolated by the swim-up procedure. It suggests that
Vol. 66, No.5, November 1996
NO may have a crucial role in modulating sperm motility and motility survival. This finding has important implications for the clinical management of male factor infertility. One can only speculate at this stage about the possible impact of the use of NOS inhibitors on fertilization rates. We are continuing these studies with sperm suspensions prepared by Percoll density gradient centrifugation technique and with other NOS inhibitors. REFERENCES 1. Stovall DW, Guzick DS, Berga SL, Krasnow JS, Zeleznik AJ.
2.
3.
4.
5.
Sperm recovery and survival: two tests that predict in vitro fertilization outcome. Fertil Steril 1994;62:1244-9. Weinberg JB, Doty E, Bonaventura J, Haney AF. Nitric oxide inhibition of human sperm motility. Fertil Steril 1995;64: 408-13. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, 1987. Hellstrom WJG, Bell M, Wang R, Sikka SC. Effect of sodium nitroprusside on sperm motility, viability, and lipid peroxidation. Fertil Steril 1994;61:1117-22. Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos K, Richardson DW. Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligospermic patients and fertile donors. J Reprod Fert 1992;94:451-62.
Perera et al.
Communications-in-brief
833
Vol. 66, No.5, November 1996
Printed on acid·free paper in U. S. A
Nitric oxide synthase inhibitor NG-monomethyl-L-arginine preserves sperm motility after swim-up
Doreen M. D. Perera, Ph.D.*t Maurice Katz, M.D.* Sujiwa R. Heenbanda, B.S.* Sally Marchant, B.S.* University College London Medical School, London, United Kingdom
Objective: To evaluate the effect of ~-monomethyl-L-arginine (L-NMMA) on sperm motility. Design: Controlled study using motile sperm suspensions obtained by the swim-up technique. Setting: Endocrine Laboratory, Department of Obstetrics and Gynaecology, University College London Medical School. Patients: Men attending the Reproductive Medicine Unit at University College London Hospitals, Obstetric Hospital, for routine fertility investigations. Interventions: Motile sperm suspensions were incubated with either culture medium (control) or L-NMMA (test) for 24 hours at 37°C. Main Outcome Measure: Motion characteristics were assessed manually and by computerassisted sperm motility analyzer. Results: In all 23 preparations either equivalent or significantly higher percentage total motility and percentage rapidly motile sperm were observed in the test compared with the control samples at 24 hours. There were no significant differences in motion characteristics between test and control specimens at 6 hours. Conclusions: ~-monomethyl-L-arginine inhibits the formation of nitric oxide; its ability to prevent sperm motility decline indicates that the arginine-nitric oxide pathway may playa role in modulating sperm motility and motility survival. Fertil Steril® 1996;66:830-3 Key Words: Semen, sperm, motility, swim-up, IVF, nitric oxide, inhibitors, L-NMMA
The maintenance of sperm motility for longer periods is desirable. Extended in vitro sperm survival is correlated with successful NF and pregnancy rates (1). Recently nitric oxide (NO) applied directly or via a NO donor compound was reported to reduce sperm motility whereas NO quencher hemoglobin prevented the deterioration (2). Nitric oxide is an inorganic radical gas formed by the action of NO synthase (NOS) on L-arginine. This reaction can be inhibited by a variety of compounds, including~ substituted L-arginine analogues. It is known that the L-arginine-NO pathway exists in many cell types such as endothelial, neuronal, and macrophages and Received January 2, 1996; revised and accepted June 5,1996. * Department of Obstetrics and Gynaecology. t Reprint requests: Doreen M. D. Perera, Ph.D., University College London Medical School, 86-96, Chenies Mews, London WC1E 6HX, United Kingdom (FAX: 44-0171-383-7429). :j: Assisted Conception Unit.
830
Perera et al. Communications-in-brief
that it has both regulatory and cytotoxic functions. Because NO appears to be detrimental to sperm motility, we hypothesized that the inhibition of NO synthesis may improve sperm motility. To test this hypothesis, we studied the effects of ~-mono methyl-L-arginine (L-NMMA), a nonselective competitive inhibitor of NOS, on sperm motion characteristics using motile sperm suspensions obtained by the swim-up technique. MATERIALS AND METHODS
Samples from patients attending the Reproductive Medicine Unit for routine fertility investigations were used. Semen was produced by masturbation after 3 days of abstinence. Assessments were made both manually according to WHO protocol (3) and by computer-assisted sperm motion analysis (CASA), using a Hamilton- Thorne Motility Analyzer with four-chambered microcells (Microm Ltd., Thame, Fertility and Sterility®
Oxford, United Kingdom). ~-monomethyl-L-argi nine and L-arginine (Tocris Cookson, Langford, Bristol, United Kingdom) stock solutions of 10-2 M were made with Ram's F-10 culture medium (Sigma-Aldrige Chemical Company, Poole, Dorset, United Kingdom). After liquefaction, the semen sample was divided into two parts; each half mixed with an equal volume of Ram's F-lO culture medium and was centrifuged for 10 minutes at 500 x g at room temperature. The supernatant was discarded and the pellet was overlayered with 200 J.lL of Ram's F-10 culture medium with 10% Albuminar (Armour Pharmaceuticals Ltd., East Bourne, East Sussex, United Kingdom) and motile sperm were allowed to swim-up for 45 minutes. Supernatant motile sperm suspensions with total motility >60% and rapidly motile sperm >25% were used for the study. Sperm counts varied from 5 X 106 to 80 X 106/mL. Samples with sperm counts <20 X 106 /mL were defined as oligospermia. Into a series of 1.5-mL Eppendorftubes (Starsted Ltd., Beaumont Leys, Leicestershire, United Kingdom) 30-J.lL aliquots of sperm suspensions were pipetted and mixed either with 60 J.lL of Ram's F-10 culture medium (control) or L-NMMA stock solution (test) and were incubated at 37°C in an atmosphere of 5% CO 2 • In a series of separate experiments (n = 6) L-NMMA concentrations (3.0, 6.0, and 9.0 x 10-7 M) in a total volume of 90 J.lL of sperm-L-NMMA mixture were incubated with sperm suspensions to assess the dose-response curve of the reaction. Motility assessments were made at 5 to 6 hours (n = 8), at 24 hours (n = 23), and hourly for 5 hours (n = 5) to determine the rate of decline in motility or the protection afforded by L-NMMA. To establish if the protective effect of L-NMMA could be reversed by the addition of L-arginine, another series of motile sperm suspensions (30-J.lL aliquots) was incubated with L-NMMA (60 J.lL of stock solution) and L-arginine in increasing concentrations ranging from 2.0 X 10- 7 to 12.0 X 10- 7 M (n = 6). To determine the influence of temperature on the reaction, we incubated a duplicate series of sperm suspensions, one at room temperature and the other at 37°C in the presence and absence of 6.0 X 10- 7 M L-NMMA. Motilities were assessed at 24 and 48 hours. A comparison between test and control measurements was performed using Mann-Whitney U test for nonparametric data.
plete immobilization by 24 hours. Four specimens treated with L-NMMA showed poor motility at 24 hours and three specimens failed to show any progression in both in the L-NMMA-added and the control samples. In three specimens with severe oligospermia and poor motility, it was difficult to obtain meaningful measurements using CASA and only manual means were used. The mean percentage total motility and percentage rapidly motile sperm in L-NMMA-added samples were 28% and 13%, respectively, compared with 5% and 0% in the controls (Fig. 1) after 24 hours of incubation. Specimens with sperm counts >20 X 106/mL showed no decline in motility characteristics up to 5 hours nor was there a difference between the control and the test samples. Two specimens with sperm counts <20 X 106/mL showed a decline from initial values within 5 hours only in the control samples. Specimens with oligospermia showed the steepest decline in motility at 24 hours even in the presence of L-NMMA. One of the three specimens that failed to show any motility and one of the four specimens with very low motility even in the presence of L-NMMA, however, did have initial post-swim-up sperm counts and motilities that were in the normal range. The concentration of L-NMMA conducive to preserving maximum sperm motility for up to 24 hours ranged from 6.0 X 10- 7 to 9.0 X 10- 7 M in a total volume of 90 J.lL of sperm-L-NMMA mixture. There was a noticeable decline in motility when 3.0 X 10- 7 M of L-NMMA was used. Protection of motility afforded by L-NMMA was prevented in a dose-dependent manner by the addition of L-arginine from 6.0 X 10- 7 to 12.0 X 10- 7 M. Complete inhibition of motility at 24 hours was seen in five of six samples treated with L-arginine at a concentration of 12.0 X 10- 7 M. There was, however, an initial increase in motility in three ofthe six samples when the L-arginine concentration was between 2.0 X 10- 7 and 5.0 X 10- 7 M. The motilities then declined rapidly as the L-arginine concentrations were increased in the samples. In the experiments investigating the influence of incubation temperature, specimens incubated at room temperature survived up to 48 hours and beyond, with or without added L-NMMA, but the percent total motilities were superior in the specimens with added L-NMMA. DISCUSSION
RESULTS
Specimens containing L-NMMA showed significantly higher total motilities than the corresponding controls, in 20 of23 samples at 24 hours (P < 0.001). Seventeen of the 23 control specimens showed comVol. 66, No.5, November 1996
The effect of NO on spermatozoa has been studied by several groups. Because macrophages possess the ability to produce NO under certain conditions, it was suspected that the poor sperm motility and survival observed in semen samples with excessive Perera et al. Communications-in-brief
831
35
30
25
~
20
g :::i!
"I-
15
10
Total motilHy
Total motilHy
Rapidly motile sperm
in the presence of L-NMMA
in the control
in the presence of L-NMMA
numbers of leukocytes may be the result of NO-induced damage to sperm. Using NO donor compounds, several investigators have reported a deleterious effect of NO on sperm but Hellstrom et aL (4) did not confirm this. The present study was undertaken to evaluate the potential protective effects of NOS inhibition on motile sperm. ~-monomethyl-L-arginine inhibits the formation of NO by inhibiting NOS action on L-arginine. Because L-NMMA preserved motility in the treated suspensions, it can be assumed reasonably that the deterioration of motility in the nontreated control suspensions was due to the presence or formation of NO. This indicates that NOS, the enzyme responsible for the synthesis of NO, may have been present in these samples. Because we were using isolated motile sperm suspensions obtained by the swim-up procedure, devoid of macrophages and other cellular elements, the indication was that the source of NOS could have been spermatozoa themselves. There is ample evidence that spermatozoa are able to produce a variety of radical oxygen species (5) although NO has not yet been measured directly in leukocyte-free semen. It is possible that spermatozoa may contain one or more isoforms of NOS, which have been identified in other cell types. We have therefore embarked on a study to identify and isolate NOS in human sperm. The lack of effect of L-NMMA on sperm motility at 6 hours (compared with 24 hours) suggests that NOS may be switched on with time. One possibility is that the NOS is not constitutive but was induced 832
Perera et al.
Communications-in-brief
Rapidly motile sperm
In the control
Figure 1 Comparison of motion characteristics of swim up sperm suspensions in the presence C.) and absence CD) of L-NMMA after incubating for 24 hours at 37°C. Values are expressed as means Cn = 23); error bars, ±SE. *Significantly different from control CP < 0.001).
by the experimental conditions. It is known that NOS expression in macrophages is induced by certain microbial products and cytokines. A similar mechanism may exist in seminal fluid. Nonsurvival or poor progression of sperm seen at 24 hours even with added L-NMMA may have been due to differences in the amounts of NOS and/or L-arginine present in individual sperm suspensions and/or variations in the rate of uptake and metabolism OfL-NMMA by NOS-containing sperm cells. It is possible that spermatozoa from these patients may respond to higher concentrations of the inhibitor. Although at the higher concentration range L-arginine was undoubtedly inhibitory to sperm motility, at lower concentrations it appeared to exert a stimulatory effect in some samples. This observation suggests that NO may have a biphasic effect on sperm motility. Similar effects have been reported in studies on the immune system and in bone resorption studies in which low NO concentrations stimulated cell growth, whereas higher concentrations were inhibitory. This biphasic effect might explain why Hellstrom et aL (4) seemingly paradoxically observed increased motility in their study. They were using lower concentrations of NO donors than any of the other investigators. The Ham's F-10 culture medium used in our study contained 1 X 10- 3 M L-arginine. It would be interesting to carry out further studies using an L-arginine-free culture medium to evaluate effects on sperm motility and survivaL The observation of nonsurvival at 24 hours in the control specimens is at variance with other studies Fertility and Sterility®
that have reported better motilities after extended incubation times. The reason for this deterioration may be the incubation temperature that was used. It appeared from our results that the deterioration in sperm motility was accelerated at 37°C, suggesting that the activity of the factor responsible for deterioration of sperm motility or the rate of production of such a factor was temperature dependent. When the initial sperm counts were low, however, motilities deteriorated much faster even at room temperature. Perhaps altered rates of NO production exist in patients with oligospermia, which affect in vitro sperm motility survival times. We have conducted preliminary experiments with other inhibitors such as ~-nitro-L-arginine and its methyl ester. The protective effects were not as evident as that seen with L-NMMA, and there appeared to be intersample variations. It is possible that LNMMA, in addition to its inhibitory action on NOS, may also interact with other enzymes in this system. Further studies are needed before conclusions can be drawn from these preliminary observations. The present study demonstrated for the first time that L-NMMA has potential value in delaying in vitro sperm motility decline in sperm suspensions isolated by the swim-up procedure. It suggests that
Vol. 66, No.5, November 1996
NO may have a crucial role in modulating sperm motility and motility survival. This finding has important implications for the clinical management of male factor infertility. One can only speculate at this stage about the possible impact of the use of NOS inhibitors on fertilization rates. We are continuing these studies with sperm suspensions prepared by Percoll density gradient centrifugation technique and with other NOS inhibitors. REFERENCES 1. Stovall DW, Guzick DS, Berga SL, Krasnow JS, Zeleznik AJ.
2.
3.
4.
5.
Sperm recovery and survival: two tests that predict in vitro fertilization outcome. Fertil Steril 1994;62:1244-9. Weinberg JB, Doty E, Bonaventura J, Haney AF. Nitric oxide inhibition of human sperm motility. Fertil Steril 1995;64: 408-13. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, 1987. Hellstrom WJG, Bell M, Wang R, Sikka SC. Effect of sodium nitroprusside on sperm motility, viability, and lipid peroxidation. Fertil Steril 1994;61:1117-22. Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos K, Richardson DW. Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligospermic patients and fertile donors. J Reprod Fert 1992;94:451-62.
Perera et al.
Communications-in-brief
833