Heavy Metals and Spermatozoa. 1. Inhibition of the Motility and Metabolism of Spermatozoa by Metals Related to Copper*

FERTILITY AND STERILITY Copyright' 1980 The American Fertility Society

Vol. 34, No.5, November 1980 Printed in V.SA.

HEAVY METALS AND SPERMATOZOA.!' INHIBITION OF THE MOTILITY AND METABOLISM OF SPERMATOZOA BY METALS RELATED TO COPPER*

MICHAEL K. HOLLAND, PH.D.t IAN G, WHITE, PH.D" D,Sc,*

Department of Veterinary Physiology, University of Sydney, Sydney, Australia

The toxicity to human spermatozoa of seven metals (nickel, palladium, platinum, silver, gold, zinc, and cadmium) and one alloy (brass: 80% copper, 20% zinc) related to copper was assessed in vitro, Only brass and cadmium significantly reduced the percentage of motile unwashed spermatozoa; however, washing the spermatozoa increased the spermicidal effectiveness of both brass and cadmium and also resulted in a significant reduction in motility caused by zinc and silver, Oxygen consumption by once-washed spermatozoa was apparently increased by zinc and brass, but the high rate of oxidation of these metals confounds interpretation of their effect, Silver caused a decline in the oxygen uptake of spermatozoa, Silver, zinc, brass, and, to a lesser extent, cadmium decreased the quantity of glucose utilized by spermatozoa and also decreased the glucose oxidized, Accumulation of lactate by washed spermatozoa was impaired severely by zinc and less severely by brass and cadmium, Fertil Steril34:483, 1980

in a highly successful trial with women. 4 Subsequent tests 5 • 6 on laboratory animals showed that cadmium, cobalt, lead, nickel, and zinc also proved effective. Despite a plethora of suggestions,7. 8 no single explanation for the mechanism of action of these heavy metals has emerged. This may be due to species differences in the relative importance of the extensive range of effects attributed to these metals, which includes the possibility of a spermicidal action. The only previous study 9 of the spermicidal properties of heavy metals, as opposed to heavy metal ions, established the supremacy of copper as the most spermicidal metal, followed by zinc and silver. This study extends these observations to a range of metals chemically and structurally related to copper in an attempt to ascertain the reason for the effectiveness of copper while simultaneously investigating the metabolic basis for this spermicidal effect.

De Quatrefages 1 in 1850 reported that heavy metal ions have spermicidal properties. Over a century later, White,2 as part of a study of heavy metal contamination of water used for preparing artificial insemination diluents, analyzed the detrimental effect ofCu2+, Zn2 +, Fe 3 +, Pb 2 +, Cd2 +, Co2 +, and Mn 2 + ions on the motility of human, ram, bull, and dog spermatozoa. Interest in the contraceptive potential of heavy metals was very much stimulated when Zipper et aI., 3 showed that copper and zinc placed within the lumen of the uterus rendered rabbits infertile. They utilized the most promising metal-copper-

Received April 15, 1980; revised July 14, 1980; accepted July 21,1980. *Supported in part by the World Health Organization and Australian Research Grants Committee funds provided to 1. G. W. . tRecipient of an Australian Government Postgraduate Research Studentship. Present address: Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104. *Reprint requests: Ian G. White, Ph.D., D.Sc., Department of Veterinary Physiology, University of Sydney, New South Wales 2006, Australia.

MATERIALS AND METHODS

Collection and Evaluation of Semen. Semen was collected by masturbation from healthy men (age 483

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19 to 35). Precautions were taken to prevent cold shock, and all ejaculates were delivered to the laboratory within 60 minutes of collection. On arrival the semen was maintained at 37° C for 15 to 30 minutes to ensure complete liquefaction before an aliquot (5 J.Ll) was removed and motility examined in 5 to 10 fields, using x 200 magnification, with a Wild Heerbrugg M20 microscope. Ejaculates were used if more than 60% of the spermatozoa were progressively motile and if other properties of the semen (e.g., color, viscosity, presence of extraneous cells) and spermatozoa (e.g., count, morphology, degree of agglutination) conformed to accepted international standards. 10 Preparation of Spermatozoa. Ejaculates were pooled and mixed by gentle agitation. They were then either used directly or, in experiments in which spermatozoa were washed free of seminal plasma, the pools were diluted with 5 volumes of calcium-free Krebs-Ringer phosphate (KRP) buffer (0.123 M NaCI, 0.005 M KCI, 0.0124 M MgS0 4, 0.016 M sodium phosphate, pH 7.4, at 37° C), centrifuged at 740 x g (rav = 17 cm) for 10 minutes at 20° C, the supernatant was discarded, and the spermatozoal pellet was resuspended in a small volume of KRP buffer. Aliquots were removed to formol-saline for counting spermatozoa in a hemocytometer so that the concentration of the spermatozoal suspension could be adjusted as desired. Estimation of Motility. Flasks containing 1.0 ± 0.1 x 108 spermatozoa, 200 sq mm of the metal to be tested, and 3 mM glucose in KRP buffer were incubated for 3 hours with constant shaking at 37° C in a water bath. Aliquots (5 J.Ll) were removed immediatley after the spermatozoa were added to the flasks and at 30, 60, 120, and 180 minutes thereafter. The percentage of motile spermatozoa was estimated to the nearest 5%, using the method previously described, by an observer unaware of the identity of the sample. Evaluation of Metabolism. A special microWarburg apparatus utilizing small flasks and in which the manometers were immersed in a thermostatically controlled bath enabled the low oxygen consumption of human spermatozoa to be measured accurately. Approximately 1.0 ± 0.1 x 108 spermatozoa were added to each flask, which contained 3 mM glucose (including 0.5 J.LCi of [U_ 14C] glucose) and 200 sq mm of the metal under test, in a total volume of 1 ml. The flasks were incubated for 3 hours at 37° C with constant shaking. Oxygen uptake, glucose utilization and oxidation, and lac-

tate accumulation were measured. Control flasks with metal omitted (to determine the metabolism of the spermatozoa) and others containing the metal but no spermatozoa (to determine the oxidation of the metal) were run in parallel. The reaction was terminated by tipping HCI from the side arm into the flasks, which were then removed and placed on ice, before ice-cold perchloric acid was added to a final concentration of 3% (w/v). The flasks were then left for 30 minutes on ice with occasional mixing. Precipitated protein was pelleted at 27,000 x g (rav = 11 cm) for 20 minutes at 2° C and the supernatant was decanted and neutralized to pH 6 with K 2C03. The potassium perchlorate was removed by centrifugation (750 x g, ray = 17 cm) for 10 minutes at 2° C, and the supernatant was stored at - 20° C for glucose and lactate assays. Determination of Glucose Utilization and Oxidation and Lactate Accumulation. Glucose and lactate were determined by enzymatic spectrophotometric analysis according to the methods of Bergmeyer et al. 11 and Gutmann and Wahlefeld, 12 respectively. The quantity of glucose oxidized was estimated from the 14C02 trapped in 50 f.LI of 2 N KOH (in Co2-free water) in the center well of the Warburg flasks and the specific activity of the original [U- 14 C]glucose. The scintillant used consisted of 0.25% (w/v) 2,5-diphenyloxazole (PPO) and 0.009% (w/v) 1,4-bis-(4-methyl-5-phenyloxazole-2-yl)benzene (dimethyl-POPOP) in toluenelBrydet-X10 (2:1, v/v). Statistical Analysis. The data were subjected to analysis of variance including partitioning into individual contrasts by use of sets of orthogonal polynomials. 13 Details of the number of replicates and any special treatment of data are provided with the individual experiments. Materials. Fine chemicals and enzymes were purchased from Boehringer Mannheim (Australia) Pty. Ltd., Sydney, Australia; all other chemicals were of analytic reagent quality and were obtained from the Ajax Chemical Co., Sydney. [U14C]Glucose was supplied by the Radiochemical Centre, Amersham, United Kingdom. The metals were the generous gift of Wright and Co., Sydney, Australia, metallurgists, and all were certified electrochemically as greater than 99.5% pure. RESULTS

Effect of Metals on the Percentage of Motile Unwashed Spermatozoa. Changes in the percentages of motile, unwashed spermatozoa incubated for 3

HEAVY METALS AND SPERMATOZOA. 1

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TABLE 1. Changes in the Percentages of Motile, Unwashed Spermatozoa in the Presence of Heavy Metals % Motile spennatozoaa at incubation time

Metal

No. of replications

o Hr None Nickel Zinc Palladium Silver Cadmium Platinum Gold Brass

5 4 5 3 4 5 3 3 3

72.0 70.0 70.0 76.7 65.0 73.0 75.0 76.7 70.0

± ± ± ± ± ± ± ± ±

0.5 Hr

2.5 3.5 3.5 4.4 3.5 2.5 2.9 4.4 2.9

70.0 71.3 67.0 75.0 62.5 71.0 71.7 71.7 68.3

± ± ± ± ± ± ± ± ±

1.0 Hr

3.2 4.3 3.0 2.9 3.2 1.0 1.7 1.7 1.7

64.0 67.5 60.0 71.7 60.0 62.0 66.7 68.3 63.3

± ± ± ± ± ± ± ± ±

3.0 Hr

2.0 Hr

2.9 3.2 3.5 1.7 3.5 1.2 1.7 1.7 1.7

58.0 61.3 58.0 65.0 56.3 52.0 61.7 61.7 50.0

± ± ± ± ± ± ± ± ±

2.5 2.4 3.7 2.9 3.1 1.2 3.3 1.7 2.9

57.0 55.0 48.0 60.0 50.0 43.0 58.3 58.3 31.7

± ± ± ± ± ± ± ± ±

2.5 4.6 3.4 2.9 4.1 2.0 4.4 3.3 4.4

aValues are the means ± standard error of the given number of replications. Approximately 10 8 spermatozoa were present in each flask.

hours with nickel, zinc, palladium, silver, cadmium, platinum, gold and brass are shown in Table 1. There was some decline in the percentages of motile spermatozoa in control flasks over this period. This could not be attributed to fluctuations in pH, which changed only from 7.5 to 7.2 during the period. In the presence of zinc, silver, cadmium, and brass, there was a more pronounced decline in motility (Table 1) which could only be attributed to an effect of the metal per se. As it was not until the 3rd hour that the effect of the metals became obvious, the percentage of motile spermatozoa at the 3rd hour in the relevant replicate of each metal was subtracted from the corresponding replicate ofthe control, and the differences were subjected to analysis of variance. The "between replications" and "metals x replication" interaction mean squares were combined and used as error term. This established that only brass (P < 0.001) and cadmium (P < 0.05) significantly reduced the percentage of motile spermatozoa. The apparent reduction in spermatozoal motility with zinc and silver was not statistically significant but may well have become significant if the duration ofthe experiment had been extended. This difference in time of onset of spermicidal effect is possibly due to variations in the time taken by each metal to achieve an effective concentration of the spermicidally-active intermediate in solution. This may in part reflect differences in oxidation rate of the metals. Effect of Metals on the Percentage of Motile, Washed Spermatozoa. Changes in the percentages of motile, washed spermatozoa incubated with nickel, zinc, palladium, silver, cadmium, platinum, gold and brass for 3 hours are shown in Table 2. Throughout the course of the incubations, the pH did not deviate from 7.4, thus none of the changes in motility could be attributed to the effects of pH. Analyses of variance were again

estimated, using the technique of differences between controls and metal-treated samples previously described. Most metals decreased the motility of washed spermatozoa more severely than unwashed spermatozoa, and the onset of the decline in motility occurred earlier. Cadmium (P < 0.001) and brass (P < 0.001) proved highly spermicidal. In the presence of cadmium, spermatozoal motility declined to approximately one-third of control values within 1 hour and was completely abolished within 3 hours. Brass was almost as effective; motility was reduced to two-thirds of control values within 1 hour, and only 5% of the spermatozoa were motile at 3 hours. Zinc (P < 0.001) also significantly reduced the percentage of motile spermatozoa, but the effect was delayed, and at 3 hours nearly half of the spermatozoa were still motile. Silver (P < 0.05) was the only other metal to cause a significant decline in spermatozoal motility. Silver, like zinc, was slower to act and over-all was less spermicidal, as motility was still two-thirds of control values after 3 hours. There was an indication (Table 2) that, if the incubation had been prolonged beyond 3 hours, some of the other metals (e.g., palladium) may have had a small spermicidal effect. Effect of Metals on the Metabolism of Washed Spermatozoa. An attempt was made to elucidate the metabolic basis for these spermicidal effects of the metals, using the micro-W arburg technique. Spermatozoa were washed because this made them more susceptible to the metals and removed extraneous substrates normally present in seminal plasma. Oxygen uptake (Table 3) by metals in the absence of spermatozoa was generally negligible except in the case of zinc, brass, and cadmium. This reflects the relative ease with which these metals oxidize. The addition of spermatozoa resulted in a

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TABLE 2. Changes in the Percentages of Motile, Washed Spermatozoa in the Presence of Heavy Metals % Motile spennatozoaa at incubation time

Metal

No. of replications OHr

None Nickel Zinc Palladium Silver Cadmium Platinum Gold Brass

5 3 5 3 4 5 3 3 3

70.0 66.7 64.0 70.0 63.8 63.0 70.0 66.7 63.3

± ± ± ± ± ± ± ± ±

0.5 Hr

0.0 1.7 2.9 0.0 3.8 3.7 0.0 1.7 3.3

66.0 66.7 59.0 61.7 57.5 45.0 70.0 68.3 53.3

± ± ± ± ± ± ± ± ±

l.OHr

1.0 1.7 1.9 1.7 1.4 3.5 0.0 1.7 3.3

61.0 63.3 58.0 58.3 55.0 22.0 65.0 61.7 38.3

± ± ± ± ± ± ± ± ±

2.0 Hr

1.3 3.3 2.5 1.7 2.0 4.6 2.9 1.7 6.0

62.5 57.7 41.0 55.0 48.3 6.0 58.3 55.0 15.0

± ± ± ± ± ± ± ± ±

3.0 Hr

1.4 1.7 4.3 2.9 3.1 3.7 1.7 2.9 5.0

61.0 51.7 24.0 48.3 38.8

± ± ± ± ± 0 53.3 ± 50.0 ± 5.0 ±

1.3 1.3 3.7 4.4 4.7 1.7 0.0 2.9

aValues are the means ± standard error of the given number of replications. Approximately 108 spermatozoa were present in each flask.

large decrease in the oxygen consumed in the presence of zinc and brass. Thus spermatozoa apparently impair the oxidation ofthese metals possibly by acting as an alternative electron "sink" to oxygen. Nevertheless, the oxygen consumed by zinc plus spermatozoa or brass plus spermatozoa was greater than any other metal plus spermatozoa combination and was above that consumed by spermatozoa alone. Silver was the only metal which reduced the oxygen consumption of spermatozoa to below control values. Utilization of glucose via oxidative or glycolytic metabolism is an excellent over-all index of the metabolism of spermatozoa. Rather unexpectedly, some metals, particularly zinc, silver, and brass, gave values for glucose utilization, in the absence of spermatozoa, which could not be dismissed as trivial. This utilization was too great to be attributed to contamination by bacteria or errors inherent in the enzymic assay of glucose. However, it will be recalled that the basis of Fehling's test for aldehyde sugars involves conversion of the cupric ion to the cuprous, with concomitant oxidation of the aldehyde to the carboxylic acid. 14 A proportion of glucose molecules is in the aldehydic form. This could explain the glucose utilized in the presence of brass (which is 80% copper) and possibly zinc. Silver nitrate is used in Tollen's test for aldehydes,14 and a similar explanation may hold true for silver. When the values for glucose utilization obtained with each metal in the absence of spermatozoa were subtracted from the corresponding values obtained with spermatozoa plus silver, zinc, cadmium, or brass, all metals decreased the ability of glucose to serve as an energy-yielding substrate for spermatozoa. This inhibition of glucose utilization could reflect either impaired oxidative or glycolytic metabolism. The ability of spermatozoa to oxidize glucose to

carbon dioxide and water, with concomitant generation of ATP through the activity of the electron transport chain, was assessed by determining the formation of 14C02 from [U- 14 C]glucose. In the absence of spermatozoa, only trace quantities of glucose were apparently oxidized with most metals, and this probably reflects accidental minor contamination. However, in the case of zinc and brass, larger quantities of radioactivity were trapped than seems reasonable to account for by accidental contamination. These two metals also had the highest oxygen uptake, i.e., they oxidize very readily. During oxidation, metal ions and other reactive intermediates form and enter solution. These entities could include free radicals which may react nonspecifically with glucose, thus generating fragments of the radioactive glucose molecule which may decay or be broken down and so yield false values for glucose oxidation. When the values for metal alone are subtracted from those obtained in the presence of spermatozoa, it is clear that zinc and brass severely decreased the quantity of glucose oxidized by spermatozoa, while cadmium and silver caused smaller declines. This indicated that some of the impairment of spermatozoal glucose utilization by these metals was attributable to decreased oxidative metabolism of the spermatozoa. As lactate accumulates as a result of glycolysis, impairment of glycolysis would result in a decline in glucose utilization and a lower accumulation oflactate. In the absence of spermatozoa, traces of lactate accumulated only with gold and zinc. This almost certainly reflects accidental contamination. When spermatozoa were present, zinc and brass greatly decreased lactate accumulation, while cadmium had a similar, although less severe, effect. None of the other metals affected the accumulation of lactate by spermatozoa.

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HEAVY METALS AND SPERMATOZOA. 1

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In vivo 15 and in vitro16 studies have shown that spermatozoa must be motile to penetrate the cumulus oophorus surrounding the ovum and thus achieve fertilization. Immotile spermatozoa, even if metabolically active, are incapable of fertilization. Motility can therefore be viewed as an index of the physiologic state ofthe spermatozoon, and indeed it is one of the major parameters upon which clinical assessment of ejaculates is based. 10 A large number oftechniques has been devised for estimating motility,17 but all have various deficiencies,18 including the following: (1) protracted time is required (e.g., photographic methods); (2) expensive equipment is needed (e.g., computerassisted techniques); or (3) generalizations must be made about large populations of spermatozoa derived from very small samples (e.g., hemocytometer technique). Direct microscopic observation, as used in these experiments, suffers the disadvantage of being subjective, although the results proved remarkably repeatable as judged by the small standard error terms. Bias was minimized by ensuring that the observer was unaware of the identify of samples, which were presented in random order for scoring. As these experiments involved only comparison of treatments with controls and with each other, an absolute measure of motility was not required. The deleterious effects of the metals on motility was increased by washing the spermatozoa. Washing enhanced the spermicidal activity of brass and cadmium and resulted in significantly decreased motility with zinc and silver. The toxicity of brass (80% copper, 20% zinc) was almost certainly due to its high content of copper, which is known to be spermicida1. 19, 20 It is not known whether the increase in the spermicidal effect of the metals on washing is due solely to removal of seminal plasma or is partially attributable to damage to the spermatozoa by the washing process. Interestingly, the action of all metals is not affected to the same extent by washing the spermatozoa. Cadmium, for example, is less effective than brass in immobilizing unwashed spermatozoa but is more toxic than brass to washed spermatozoa, which it also affects more rapidly. Thus the mechanism by which seminal plasma protects the spermatozoa may not be the same for all metals, or, alternatively, more than one mechanism may be involved. Considering the complex chemical nature of seminal plasma, the latter possibility is not unlikely.

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HOLLAND AND WHITE

The spermicidal potential of the various heavy metals is very much related to their metal ions; however, it is uncertain whether the spermicidal effects of the metals are due solely to their ions or are attributable to products formed during oxidation of the metal. Few data have been published on the mechanism of the spermicidal effect of heavy metals or their ions. In the absence of a glycolyzable substrate, human spermatozoa can generate sufficient ATP from oxidative metabolism to support motility2\ however, in vivo they usually rely on glycolysis to provide sufficient ATP for motility.22 Therefore, inhibition of glycolysis and, to a lesser extent, of oxidative metabolism would have severe consequences for the motility of spermatozoa. Brass clearly decreased spermatozoal glycolysis, as judged by reduced utilization of glucose and a decreased accumulation of lactate, but also depressed oxidative metabolism. It is uncertain whether the effect on oxidative metabolism is direct or a consequence of reduced availability of pyruvate as a result of inhibition of glycolysis. Zinc has effects essentially similar to those of brass but decreases oxidative metabolism more severely. Studies of the respiration of spermatozoa in the presence of brass and zinc are complicated by the finding that, as a result of oxidation, these metals themselves consume large quantities of oxygen when incubated with buffer in the absence of spermatozoa. When spermatozoa are present, oxygen consumption is reduced, presumably because spermatozoa act as an alternative electron acceptor to oxygen. It is. possible that intermediates formed during the oxidation of brass and zinc, rather than the metal ions themselves, are responsible for some or all of the spermicidal effect of these metals. Cadmium had the most detrimental effect on the motility of washed spermatozoa but only moderately depressed glycolysis and had even less effect on oxidative metabolism. It is possible, therefore, that cadmium may specifically inhibit the motility apparatus of spermatozoa over and above any effects on metabolism. . Silver reduced the motility of washed spermatozoa to two-thirds of control values after 3 hours. This was accompanied by reduced oxygen consumption and reduced glucose utilization and oxidation, but there were no changes in lactate accumulation. This finding suggests some impairment of the oxidative metabolism of the spermatozoa but only a small effect on glycolysis. Of the metals tested, only cadmium and brass

November 1980

approached the reported effectiveness of copper in immobilizing spermatozoa. Brass, a combination of two metals with similar detrimental effects on metabolism, was arguably less effective as a spermicide than would be expected on the basis of its copper content. However, it is possible that a combination of two metals such as silver and cadmium with different spermicidal properties may be more effective. Cadmium, as well as exerting a possible specific effect on motility, essentially blocks glycolysis, while silver affects the oxidative metabolism of spermatozoa, and thus a combination of the two would be expected to block spermatozoal metabolism completely. Regrettably, cadmium has been reported to produce histologic changes in kidneys, liver, gastrointestinal tract, heart, testes, pancreas, bone, and blood vessels when ingested, inhaled, or injected. 23 Nevertheless, a more rational approach to contraceptive development based on sound biochemical principles may prove more effective than the indiscriminate testing of substances which has been in vogue. Acknowledgments. The competent and skillful technical assistance of Ms. K. Murdoch and Mr. P. Harlow is gratefully acknowledged. REFERENCES 1. De Quatrefages MA: Recherches experimentales sur les

2. 3.

4.

5.

6. 7. 8. 9.

10.

11.

spermatozoides des hermelles et des tarets. Ann Sci Nat 13:111, 1850 White IG: The toxicity of heavy metals to mammalian spermatozoa. Aust J Exp BioI 33:359, 1955 Zipper J, Medel M, Prager R: Suppression of fertility by intrauterine copper and zinc in rabbits. Am J Obstet Gynecol 105:529, 1969 ZipperJ, Tatum HJ, Pastene L, Medel M, Rivera M: Metallic copper as an intrauterine contraceptive adjunct to the "T" device. Am J Obstet GynecoI105:1274, 1969 Chang CC, Tatum HJ, Kincl FA: The effects ofintrauterine copper and other metals on implantation in rats and hamsters. Fertil Steril 21:274, 1970 Chang CC, Tatum HJ: A study of the antifertility effect of intrauterine copper. Contraception 1:265, 1970 Oster G, Salgo MP: The copper intrauterine device and its mode of action. New Engl J Med 293:432, 1975 Oster G, Salgo MP: Copper in mammalian reproduction. Adv Parmacol Chemother 14:327, 1977 Kesserii E, Le6n F: Effect of different solid metals and metallic pairs on human sperm motility. IntJ Fertil19:81, 1974 Freund M, Peterson RN: Semen evaluation and fertility. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St Louis, CV Mosby Co, 1976, p 344 Bergmeyer HU, Bernt E, Schmidt F, Stork H: D-glucose. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In Methods in Enzymic Analysis, Second Edition, Edited by HU Bergmeyer. Weinheim, Verlag Chemie, 1974, p 1196

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12. Gutmann I, Wahlefeld AW: L( + )lactate. Determination with lactate dehydrogenase and NAD. In Methods in Enzymic Analysis, Second Edition, Edited by HU Bergmeyer. Weinheim, Verlag Chemie, 1974, p 1464 13. Sokal RR, Rohlf FJ: Introduction to Biostatistics. San Francisco, WH Freeman Co, 1973, p 152 14. Vogel AI: A Textbook of Practical Organic Chemistry. London, Longmans Green and Co, 1956, p 330 15. Yanagimachi R: Time and process of sperm penetration into hamster ova in vivo and in vitro. J Reprod Fertil 11:359, 1966 16. Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation. J Reprod Fertil23: 193, 1970 17. Mitchell JA, Nelson L, Hafez ESE: Motility of spermatozoa. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St Louis, CV Mosby Co, 1976, p 83

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18. Atherton RW: Evaluation of sperm motility. In Techniques of Human Andrology, Vol 1, Edited by ESE Hafez. Amsterdam, Elsevier/North Holland Biomedical Press,1978, p 243 19. Ullman G, Hammerstein J: Inhibition of sperm motility in vitro by copper wire. Contraception 6:71, 1972 20. Rush F, Elstein M: The effect of incubating a copper releasing intrauterine device on sperm penetration and spinnbarkheit of cervical mucus. J Obstet Gynaecol Br Commonw 81:483, 1974 21. Suter DAI, Chow PYW, Martin ICA: Maintenance ofmotility in human spermatozoa by energy derived through oxidative phosphorylation and addition of albumin. BioI Reprod 20:505, 1979 22. Peterson RN, Freund M: ATP synthesis and oxidative metabolism in human spermatozoa. BioI Reprod 3:47, 1970 23. Fox MRS: Trace Elements and Human Disease, Vol 2, Edited by AS Prasad. New York, Academic Press, 1976, p 401