Toxicity and uptake of heavy metals by human spermatozoa*

Transcription

1 FRTILITY AND STRlUTY Copyright 1982 The Amerian Fertility Soiety Printed in U.SA. r I Toxiity and uptake of heavy metals by human spermatozoa* Sharon Battersby, Ph.D. John A. Chandler, Ph.D. t Mihael S. Morton, Ph.D. Tenovus Institute for Caner Researh, Welsh National Shool of Mediine, Heath, Cardiff, Wales The effets of metalli opper on human spermatozoa are desribed. Inubation with the metal aused a fall in the perentage of motile sperm, whih was diretly related to the surfae area of opper employed and to the opper ontent of whole semen. These hanges were aompanied by a derease in semen zin levels and an uptake of opper by individual sperm ells, as determined by X-ray miroanalysis. Low onentrations of ioni opper aused a less marked fall in sperm motility, although the metal was generally more toxi than zin or admium ions. All three metals were aumulated by spermatozoa on inubation, with admium ausing a derease in sperm nulear zin onentrations. Fertil Steril37:230, 1982 The sperm atoxi effet of opper has long been known,l, 2 but renewed interest in this phenomenon has only arisen in reent years with the advent of opper intrauterine devies (IUDs).3, 4 Several workers have investigated the effets of the metal on sperm viability, with opper being shown to inhibit both the motility of5-7 and ervial muus penetration by8, 9 human sperm in vitro. Relatively few studies have, however, attempted to investigate the mode of ation of opper,7 although the spermatoxi behavior of the metal appears unlikely to be a major fator in the ontraeptive effiieny of IUDs. 10, 11 While major interest has been foused on the role of opper in both male and female reprodutive systems, zin may also be an important heavy metal in relation to fertility inthe human male. Spermatozoa and seminal fluid from many speies ontain very high zin onentrations,12 whih are at least in part derived from prostati Reeived May 18, 1981; revised and aepted September 2, *Supported by the World Health Organization (Contrat 74052). treprint requests: John A. Chandler, Ph.D., Tenovus Institute for Caner Researh, Welsh National Shool of Mediine, The Heath, Cardiff, CF4 4XX Wales. 230 Battersby et ai. Toxiity and uptake of heavy metals seretion. 13, 14 There is some evidene that zin is neessary for energy expenditure by sperm15, 16 and that zin-rih prostate fluid has a protetive role in sperm viabilityp' 17 Some investigators, however, have demonstrated a toxi effet of the metal on sperm ells,18 and exessive uptake of zin may be deleterious to human spermatozoa. 17 We thus onsidered determination of the effets of zin on human sperm viability and elemental ontent in vitro and investigation of the effetiveness of a range of onentrations of both metalli and ioni opper in inativating spermatozoa to be important. In addition to X-ray analyti studies of opper- and zin-treated sperm, a third metal, admium, was also studied, being both an element highly toxi to the male reprodutive system19 and one shown to interat with endogenous zin in biologi systems.20 MATRIALS AND MTHODS X-RAY MICROANALYSIS Mirosopi and analyti studies were performed with an eletron mirosope miroanalyzer (MMA 4, Kratos, U. K) fitted with two wavelength-dispersive rystal spetrometers and an energy dispersive solid state detetor (SSD) (Ke- Fertility and Sterility

2 60 I 40 Sf Q)! *' 20 O O o Surfae area of Cu(mm 2) Q...9- ID If) 400. :: u 8 % 200 "0 Figure 1 ffet of opper surfae area on the perentage of motile sperm and semen opper and zin onentrations following a 2-hour inubation of the metal with 0.2 ml semen. Results represent mean values for four sets of inubations. vex, Calif.). The detetor omprised a lithium drifted (SiLi) semi-ondutor with an X-ray energy resolution of 152 ev at 5.9 KeV. The transmission eletron mirosope was fitted with an extra ondenser lens, a "minilens," whih allowed the illumination to be foused to a probe 0.1 to 0.2 JJ.m in diameter. Analysis was performed at 80 kv, with a beam urrent of 0.02 JJ.A and a ounting time of 50 seonds. Speimens were plaed in a speimen holder with beryllium inserts, using arbon rings and aluminum irlips to redue extraneous opper signals. The analyti system was linked to an NS-880 multihannel analyzer (Traor, United Kingdom, Ltd.) and a PDP-U/05 omputer (DC, United Kingdom, Ltd). An average of 20 spermatozoa were analyzed per sample for nine elements in the nuleus, midpiee and arosome regions. The integrated peak minus bakground readings for eah element was measured and quantitation was performed using the method of Ha1l 21 and standards employed aording to Chandler. 22 HUMAN SMN Semen samples were obtained from donors at the Fertility Clini, UHW, Cardiff. The samples were olleted into sterile ontainers and allowed to liquefy for 30 minutes before use. The speimens used for inubation had > 70% motile I a sperm, > 50 x 10 6 sperm/ml, and < 30% abnormal forms. The perentage of motile sperm was estimated with the use of a field 0.1 sq mm in area in an improved Neubauer hemoytometer at a magnifiation of 400 x. SPCIMN PRPARATION Carbon-oated nylon grids (Agar Aids, United Kingdom) were oated with a layer of polyvinyl formal and a 5 JJ.I droplet of semen was plaed onto eah grid and allowed to flood over onto a filter paper, leaving a dispersion of sperm on the grid. When they were dry (within a few seonds), we oated the speimens with a layer of arbon 0:-10 nm) with an AI (United Kingdom) VAC 12 vauum oating unit to stabilize them during eletron-mirosopi examination. ATOMIC ABSORPTION SPCTROPHOTOMTRY A Varian-Tehtron AA 1200 spetrophotometer (Varian, West Germany) with a standard aetylene burner was employed. Piees of opper wire for analysis were washed with deionized water and dried between filter papers. They were dissolved in a minimum volume of 50% nitri aid (Aristar), and the solution was made up to 25 ml with deionized water. For whole semen analysis 20 drops of nitri aid were added to the solutions, and they were allowed to stand for 2 hours at 20 C. Volumes were then made up to.5 ml with the use of deionized water. RSULTS FFCT OF MTALLIC COPPR ON HUMAN SPRMATOZOA Motility and Copper in Semen Semen samples in 0.2-ml volumes were inubated with lengths of opper wire (> 99.99% pur- Table 1. Zin Levels in Copper Wire (Speifi Ativity = 500 sq mm) and Semen Samples (Atomi Absorption Spetrophotqmetry) Following Inubation for 2 Hours at 37' a Semen Copper Control (± 73.2) (± 1.1) Copper inubation 214.2b 12.0 (± 71.1) (± 2.2) appm - wet weight. Figures in parenthesis represent ± 1 standard deviation. bp < 0.05, Student's t-test. p < , Student's t-test. dfigures represent means for four opper semen inubations. Battersby et ai. Toxiity and uptake of heavy metals 231

3 .. r 0. K ::MJ o P " " :: a Figure 2 lemental distribution in head region of spermatozoa following 2-hour inubation with metalli opper. Vertial bars, standard deviation (SD). Asterisk, SD greater than the mean. Triangle, P < for Student's t-test. Open olumns, ontrol; hathed olumns, 50. sq mm opper; stippled olumns, sq mm opper; rosshathed olumns, sq mm opper; seond open olumns, sq mm opper. ity) of surfae area 50, 100,200, and 500 sq mm for 2 hours at 37 C. The perentage of motile sperm, as assessed by a hemoytometer, was related to the surfae area of opper employed (Fig. 1), motility falling to zero in 500 sq mm inubations. This fall in motility was aompanied by an inrease in the opper ontent of eah semen sample, as determined by atomi absorption spetrophotometry (Fig. 1) and a onomitant derease in semen zin levels. It was found that the derease in zin levels in semen samples following inubation was aompanied by a signifiant rise (P < ) in the zin ontent of the opper wire used for the inubation (Table 1). X-Ray Miroanalysis Air-dried spermatozoa from two of the semen speimens that had been inubated alone and with opper were analyzed in the head, midpiee, and arosome regions for 9 elements. The opper level in all 3 regions in eah sample was diretly related to the surfae area of opper wire used for inubation, as was the zin level in the midpiee (Figs. 2 to 4). A fall in magnesium onentration was also apparent in the nuleus and arosome regions, while inonsistent but signifiant hanges were found in the levels of the other elements following opper treatment. FFCT OF Cu, Zn, AND Cd IONS ON HUMAN SPRM Motility Ten-miroliter aliquots of 20, 2, and 0.2 mm admium hloride, opper sulfate, and zin hloride ions (Analar grade, BDH Ltd., United King- 232 Battersby et al. Toxiity and uptake of heavy metals o ::- I:: " " :b Mg P s CI JrI " Co Zn Figure 3 lemental distribution in the midpiee region of spermatozoa following 2-hour inubation with metalli opper. Vertial bars, standard deviation (SD). Asterisk, SD greater than the mean. Triangle, P < for Student's t-test. Open olumns, ontrol; hathed olumns, 50. sq mm opper; stippled olumns, sq mm opper; rosshathed olumns, sq mm opper; seond open olumns, sq mm opper. dom) were eah added to 100 /J-I of semen, produing final solutions of 2, 0.2, and 0.02 mm. Ten miroliters of distilled water was added to 100 /J-I of semen, to at as a ontrol. The solutions were inubated at 20 C for 12 hours and the perentage of motile sperm in eah solution was estimated at intervals over this period (Figs. 5 to 7). In all solutions ontaining the heavy metal ions, and at all onentrations, motility fell sharply during the 1st hour, ompared with the ontrol. In 0.2 and 2 mm solutions of zin and admium ions there was a reovery in the perentage of motile sperm almost to ontrol levels after 2 hours. Cop >. L 1J iii u 0 u :g (])..Q.! W Figure 4 lemental distribution in arosome region of human spermatozoa following 2-hour inubation with metalli opper. Vertial bars, standard deviation (SD). Asterisk, SD greater than the mean. Triangle, P < for Student's t-test. Open olumns, ontrol; hathed olumns, sq mm opper; stippled olumns, sq mm opper. ::: 6 2 a p Fertility and Sterility

4 a. II).Q! 4 :a * Inubation time(h) Figure 5 ffet of 0.02 mm solutions of opper, zin, and admium salts on the motility of human spermatozoa from one semen speimen. Signifiane of differenes not determined. Closed squares, ontrol; open irles, zin; losed irles, opper; open squares, admium. per was generally the most toxi of the metals at all onentrations, exept for the large derease in motility produed by the 2 mm admium hloride solution. The sperm inubated in 2 mm zin hloride solution were hyperative throughout the 12-hour period, whqe those inubated with admium (2 mm) also showed inreased veloity and amplitude of movement during the first 3 hours. X-Ray Miroanalysis Air-dried spermatozoa were prepared from semen samples inubated in 2 mm solution of metal ions and from ontrol sperm. The ontrol, oppertreated, and zin-treated sperm were analyzed for 9 elements in the nuleus and midpiee regions with the use of the solid-state detetor, while nulear and midpiee opper and zin levels were measured in sperm that had been inubated with admium. Cadmium was also measured in these sperm with the use of th rystal spetrometers. The pentaerithritol (PT) rystal was employed, at a beam urrent of 0.1 f.la and with a 40-seond ounting time. Inubation of sperm with opper or zin ions aused signifiant inreases in. these elements in both nuleus and midpiee regions ij; 50 a. II).Q! :a 40 * ' o Inubation time (h) Figure 6 ffet of 0.2 mm solutions of opper, zin, and admium salts on the motility of human spermatozoa from one semen speimen. Signifiane of differenes not determined. Closed squares, ontrol; open irles, zin; losed irles, opper; open squares, admium Q! is 30 * o Inubation time (h) Figure 7 ffet of 2 mm solutions of opper, zin, and admium salts on the motility of human spermatozoa from one semen speimen. Signifiane of differenes not determined. Closed squares, ontrol; open irles, zin; losed irles, opper; open squares, admium. Battersby et al. Toxiity.and uptake of heavy metals 233

5 r Table 2. lemental Composition (% Dry Weight) of Control, Copper-Treated, and Zin-Treated Spermatozoa Nuleus Region Na Mg P Control (± 1.16)a (± 0.13) (± 1.31) Inubated +2 mm CuS (± 0.85) (± 0.14) (± 1.1) Inubated + 2 mm ZnCl b (± 0.73) (± 0.14) (± 1.04) afigures in parenthesis represent ± 1 standard deviation. bp < '8 Cl K Ca Cu Zn (± 0.36) (± 0.35) (± 0.84) (± 0.24) (± 0.07) (± 0.18) b 0.24 (± 0.22) (± 0.36) (± 0.66) (± 0.19) (± 0.08) (± 0.12) b b (± 0.31) (± 0.32) (± 0.46) (± 0.16) (± 0.06) (± 0.12) (Tables 2 and 3). Cadmium treatment aused a signifiant redution in nulear zin (P < 0.01), the metal itself being present in both nulear and midpiee regions, while it was not detetable in ontrols (Table 4). DISCUSSION The toxi effet of high onentrations of opper to human sperm is well doumented,6, 7, 11 but relatively few studies have measured the fators diretly involved in the deleterious effets of this metal in vitro.7 In the present study the spermatoxi effet of opper was demonstrated, as reported by others; and" in addition, it was shown that this ation was related to the surfae area of opper used for the inubation, over the range of onentrations that are usually employed for use in intrauterine devies. 4 The toxi effet of the metal was also related to uptake of opper by semen and more espeially by all regions of sperm ells (Figs. 2 to 4). Uptake of opper by human sperm has been previously demonstrated,7 but it was of partiular interest in the present investigation that in ontradition to the studies of Maynard et ai.,7 who found a displaement of zin by opper, zin levels in the midpiee region of sperm ells were inreased upon opper inubation. Moreover, this inrease in zin was diretly related to the surfae area of opper used for inubation and to the opper levels within the same regions of the sperm (Fig. 3). It has been suggested that uptake of zin by spermatozoa has a deleterious effet on their motility17 and that a fator or fators present in prostati fluid prevent this inrease. It is possible, therefore, that this failitation of zin uptake by opper is a fator in the inativation of sperm. The large fall in magnesium levels may also be highly signifiant, sine the hydrolysis of adenosine triphosphate (ATP), whih is dependent on Mg + + -dependent ATPase is the major fator responsible for the initiation and propagation of the flagellar wave. The derease in the zin onentration of whole semen aompanying opper inubation (Fig. 1) an only be explained if the metal is removed by the opper wire. The uptake of zin by the metalli opper in the present study therefore suggests that an eletrolyti proess is present when metalli opper is inubated in semen. It has been indiated 5 that in the opper dissolution proess by biologi materials, the disulfide bond is ritial in the oxidation of opper to upri ions. Copper appeared to be the most toxi of the three heavy metals in solution, as might be expeted from previous results. 18 The toxiity of the highest admium onentration was also partiularly notieable, with this level of the metal ausing depression of nulear zin onentrations. Toxiity of admium to sperm has been previously noted. 13, 18 It was evident with admium and partiularly zin that following the initial depression of sperm motility, the perentage of motile sperm was inreased, the high onentration of zin almost restoring viability to ontrol levels (Fig. 7). It is possible that admium and zin had a differ- Table 3. lemental Composition (% Dry Weight) of Control, Copper-Treated, and Zin-Treated Spermatozoa Midpiee Region Na Mg P 8 Cl K Ca Cu Zn Control (± 0.81)a (± 0.16) (± 0.8) (± 0.38) (± 0.22) (± 0.72) (± 0.56) (± 0.08) (± 0.18) Inubated + 2 mm CUS b b b 0.42 (± 1.24) (± 0.28) (± 0.64) (± 1.14) (± 0.25) (± 2.14) (± 0.61) (± 1.3) (± 2.57) Inubated + 2 mm ZnCl b b b (± 0.88) (± 0.24) (± 0.6) (± 0.3) (± 0.53) (± 0.74) (± 0.34) (± 0.22) (± 0.31) afigures in parenthesis represent ± 1 standard deviation. bp < Battersby et ai. Toxiity and uptake of heavy metals Fertility and Sterility

6 Table 4. Cadmium, Zin, and Copper Conentrations (% Dry Weight) in Air-Dried Sperm Treated with 2 mm CdCl 2 Nuleus Cd Cu Zn Control Inubated + 2 mm CdCl 2 (± 0.07)a (± 0.18) b (± 0.07) (± 0.08) Midpiee Cd Cu Zn Control Inubated + 2 mm CdCl 2 (± 0.08) (± 0.18) (± 0.12) (± 0.27) afigures in parenthesis represent ± 1 standard deviation. bp < ent mode of ation to opper, as they appeared to stimulate the motility apparatus of the spermatozoa diretly. Sperm inubated with zin and admium showed an inrease in flagellar beat frequeny, amplitude of the flagellar wave, and speed, although their progression was not always in a forward diretion. Zin has previously been demonstrated to stimulate dog epididymal sperm,13 and both Cd + + and Zn + + are able to diretly affet the flagellar ontratile proteins of sea urhin sperm.23 These studies onfirm the toxi effets of heavy metals to human sperm in vitro, with opper being a partiularly powerful spermatoxi agent. The motility and omposition of human sperm in ervial muus obtained from women using opper intrauterine devies and ontaining levels of the metal found in the normal uterine milieu are the subjet of a separate. report. Aknowledgments. The authors are grateful to the Tenovus Organisation for providing failities for the study. The assistane of the staff at the Fertility Clini, University Hospital of Wales, Cardiff, is espeially aknowledged. RFRNCS 1. MaLeod J: Sulfhydryl groups in relation to the metabolism and motility of human spermatozoa. J Gen Physiol 34;705, White IG: The toxiity of heavy metals to mammalian spermatozoa. Aust J xp BioI 33:359, Zipper J, Tatum HJ, Pastene L, Medel M, Rivera M: Metalli opper as an intrauterine ontraeptive adjunt to the "T" devie. Am J Obstet GyneoI105:1274, Zipper J, Tatum HJ, Medel M, Pastene L, Rivera M: Contraeption through the use of intrauterine metals. I. Copper as an adjunt to the 'T' devie. Am J Obstet Gyneol 109:771, Oster GK: Chemial reation of the opper intrauterine devie. Fertil Steril 23:18, Ullmann G, Hammerstein J: Inhibition of sperm motility in vitro by opper wire. Contraeption 6:71, Maynard PV, lstein M, Chandler JA: The effet of opper on the distribution of elements in human spermatozoa. J Reprod Fertil 43:41, lstein M, Ferrer K: The effet of a opper-releasing intrauterine devie on sperm penetration in human ervial muus in vitro. J Reprod Fertil 32:109, Rush F, lstein M: The effet of inubating a opper releasing intrauterine devie on sperm penetration and the physial properties of ervial muus. J Obstet Gynaeol Br Commonw 81:483, Makler A, Zinder 0: The effet of opper on spermatozoal motility and viability evaluated objetively with the aid of the multiple-exposure photography method. Am J Obstet Gyneol 138:156, Jeht W, Bernstein GS: The influene of opper on the motility of human spermatozoa. Contraeption 7:38, Mawson CA, Fisher MI: The ourrene of zin in the human prostate gland. Can J Med Si 30:336, Saito S, Bush 1M, Whitmore WF: ffets of ertain metals and helating agents on rat and dog epididymal sperma" tozoan motility. Fertil Steril18:517, MaKenzie AR, Hall T, Whitmore WF: The zin ontent of expressed prostate fluid. Nature 193:72, Fuji T: Notes on the presene of zin in nuleoli and in sperm middle-piee in some marine forms. Ann Zool Jpn 27:115, Gunn SA, Gould TC: The role of zin in the posttestiular ation of monohlorhydrin.. Pro So xp BioI 141:639, Lindholmer C, liasson R: In vitro release and uptake of zin and magnesium by human spermatozoa. Int J Fertil 19:56, Holland MK, White IG: Heavy metals and spermatozoa. I. Inhibition of the motility and metabolism of spermatozoa by metals related to opper. Fertil Steril34:483, Parizek J: Sterilization of the male by admium salts. J Reprod Fertil 1:294, Gunn SA, Gould TC, Anderson WAD: Zin protetion against admium injury to rat testis. Arh Pathol 71:274, Hall T: The miroprobe assay of hemial elements. In physial tehniques in biomedial researh. Seond dition. New York, Aademi Press, 1971, p Chandler JA: A method for preparing absolute standards for quantitative alibration and measurement of setion thikness with X-ray miroanalysis of biologial thin speimens in MMA. J Miros 106:291, Morisawa M, Mohri H: Heavy metals and spermatozoan motility. II. Turbidity hanges indued by divalent ations and adenosinetriphosphate in sea urhin sperm flagella. xp Cell Res 83:87,1974 Battersby et ai. Toxiity and uptake of heavy metals 235