Upsala J Med Sci 79: 63-64, 1974 The Amount of Zinc Detected in Washed Human Spermatozoa ,JAN FRIBERG and OVE NILSSON Department of Obstetrics and Gynecology, and Znstitute of Human Anatomy, University of Uppsala, Uppsala, Sweden ABSTRACT Samples of human semen, the supernatants from washings of these samples, and the suspensions of washed sperma- tozoa, were examined for zinc in a Perkin-Elmer spectro- photometer. Almost all the zinc was found in the original semen samples and in the first washings. Zinc could not be detected in the heads of fourfold-washed spermatozoa on analysis in a wavelength dispersive X-ray spectrophoto- meter attached to a scanning electron microscope. INTRODUCTION High concentrations of zinc have been demon- strated in seminal fluid (1, 8, 9, 11) and in prostatic fluid (2, 6, 7). Mawson & Fischer (8) performed zinc analysis of the ashed infranatant after centrifugation of human ejaculates and ob- served about 2 mg zinc/g dry weight. Hall (3) used an X-ray microprobe to examine the zinc content of individual spermatozoa and found a high concentration in the heads of the sperm cells. Since unwashed spermatozoa were used, an unintentional contamination of the samples with zinc from the seminal fluid cannot be excluded. This possibility is strengthened by the finding that spermatozoa which were obtained from pa- tients with infertility problems and/or suspected prostatitis and then separated by density gradient centrifugation and analysed by atomic absorption spectrophotometry showed a mean zinc concentra- tion of 5.5 pg/lOS spermatozoa (5). T o obtain information about the content of zinc in carefully washed human spermatozoa from normal donors, analyses were performed in the present study both by atomic absorption spectrophotometry and by a wavelength dispersive X-ray spectrophotometer attached to a scanning electron microscope. was previously demonstrated by artificial insemination. Two ml of each semen sample (average 40-60 million sperm cells/ml) were spun at 1500 rpm, washed 4 times in 0.01 M phosphate-buffered saline (PBS), p H 7.4, and finally suspended in the same volume of PBS as the original sperm sample. The motility of the spermatozoa was not markedly affected by this treatment. The semen samples, the supernatants from each washing, and one lot of the fourfold-washed sperm suspension, were subjected to zinc analysis in a Perkin-Elmer spectro- photometer using the niethod described by Parker (10) and Kahnke (4). Small drops of suspended, washed spermatozoa were placed o n a stage of pure aluminium adapted for a Jeol SMU-3 scanning electron microscope. The heads of single spermatozoa were then examined for their zinc content by means of a wavelength dispersive X-ray spectrophoto- meter. The microscope was operated at 25 k V with 0.01 pA and 10 cps f o r full scale o n the recorder. The minimum detection level of zinc under the conditions used was calculated to be about 1 x gram zinc. RESULTS Zinc analyses of the sperm samples, the super- natants from each washing, and the final sperm suspensions showed that almost all the zinc was found in the original semen samples and the supernatants from the first washings. The con- centration of zinc in the final solution of four- fold-washed spermatozoa was about 9 x 10-8 mol/ ml or about 12 pg/108 spermatozoa. No zinc couId be demonstrated in the heads of individual spermatozoa when investigated by means of the dispersive X-ray spectrophotometer attached to the scanning electron microscope. This is only to be expected since the amount of zinc in a single washed spermatozoon was about 1 >: 10-13 gram. DISCUSSION The in the present study indicate that Only minute amounts Of zinc can be present in Upsala J Med Sci 79 MATERIAL AND METHODS Two masturbated ejaculates from each of 4 fertile donors were used for the present study, The fertility of the donors 64 J . Friberg and 0. Nilsson the sperm cells. The high zinc concentrations previously ascribed to spermatozoa were probably due t o contamination with zinc from the seminal fluid. The minute amounts of zinc present in human spermatozoa, according t o the results in this study, may derive from contamination by epithelial cells and cellular debris or from the carbonic anhydrase contained in the spermatozoa. The experiments do not exclude the possibility that some zinc may have leaked out from the spermatozoa during the washing procedures. The healthy donors were found to have a zinc concentration of about 12 p g / 10s spermatozoa. This figure lies in the upper range of values ob- tained from patients with infertility problems a n d / o r suspected prostatitis (mean 5.5 p g / los spermatozoa, range 0.20-12.63) (5). However, the results are sufficiently similar to permit the con- clusion that the zinc concentration of human spermatozoa is low, lying around some pg/lOs spermatozoa. ACKNOWLEDGEMENT D r H. Ulfendahl is gratefully acknowledged for the analyses by the atomic absorption spectrophotometer and Mr K. Ayaghi, Jeol Europe, for the analysis by the dis- persive X-ray spectrophotometer. This study was supported by the Swedish Medical Research Council (Project no. 12X-70) and by the Ford Foundation (Grant no. 66-405 to Professor Carl Gemzell). REFERENCES 1. Eliasson, R. 8; Lindholmer, C.: Zinc in human seminal plasma. Andrologie 3: 147, 1971. 2. Gyorkey, F., Min, K.-K'., Huff, J. A. & Gyorkey, P.: Zinc and magnesium in human prostate gland: normal, hyperplastic and neoplastic. Cancer Res 27: 1348, 1967. 3. Hall, T. A.: The microprobe analyses of zinc in mammalian sperm cells. In Optique des rayons X et microanalyse (ed. R. Castaing & P. Deschamps), pp. 679-686. J. Philibert Hermann, Paris, 1966. 4. Kahnke, M. J.: Atomic absorption spectorphotometry applied to the determination of zinc in formalinized human tissue. Atomic Absorption Newsletter 5: 7, 1966. 5. Lindholmer, C. & Eliasson, R.: Zinc and magnesium in human spermatozoa. In1 J Fert 17: 153, 1972. 6. Mackenzie, A. R., Hall, T. & Whitmore, W. F.: Zinc content of expressed human prostatic fluid. Nature 193: 72, 1962. 7. Mawson, C. A. & Fischer, M. I.: The occurrence of zinc in the human prostate gland. Can J Med Sci 30: 336, 1952. Upsala J M e d Sci 79 8 . 9. 10. 11. - Zinc and carbonic anhydrase in human semen. Biochem J 55:696, 1953. - Zinc in aspermic human semen. Nature, 177: 190, 1956. Parker, H. E.: Magnesium, calcium and zinc in animal nutrition. Atomic Absorption Newsletter 2: 23, 1963. Schirren, C., Beltermann, R., Haensch, M., Kohn, D. & Lossin, J.: Biochemische Untersuchungen am menschlichen Spermaplasma: Zinc- und Phosphohexose- Isomerase-Aktivitat. Arch Klin Exp Dermatol 218: 323, 1964. Received M a y 4, 1973 Address for reprints: Jan Friberg Department of Obstetrics and Gynecology University Hospital S-750 14 UPPSALA 14 Sweden