IMMUNODETECTION OF A HUMAN CHORIONIC GONADOTROPIN-LIKE SUBSTANCE IN HUMAN SPERM

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1 FERTILITY AND STERILITY Copyright' 1977 The American Fertility Society Vol. 28, No. 11, November 1977 Printed in U.S.A. IMMUNODETECTION OF A HUMAN CHORIONIC GONADOTROPIN-LIKE SUBSTANCE IN HUMAN SPERM RICARDO H, ASCH, M,D.* EMILIO O. FERNANDEZ, M.D. CARL J. PAUERSTEIN, M.D. Center for Research and Training in Reproductive Biology and Voluntary Regulation of Fertility, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas The presence of a human chorionic gonadotropin (hcg)-like substance in human spermatozoa is reported. A highly sensitive immunocytochemical procedure was utilized (double-antibody immunofluorescence technique). Rabbit anti-hcg or rabbit anti-hcg ~subunit was used as the first antibody. A positive fluorescence reaction was found in all human specimens analyzed and in positive controls (choriocarcinoma cells). No fluorescence was detected in the other species studied (sheep, pig, goat, horse, bull, and guinea pig), nor in the negative controls. These findings open a new research area on the physiologic role of this hcg-like substance in human reproduction. Trophoblastic cells, cancer cells, and sperm share the common characteristic of invasive interactions with other cells and tissues. The mechanism underlying the invasive properties of these cells is unknown. Other similarities exist between these cell types. The synthesis of human chorionic gonadotropin (hcg) by non-endocrine, nontrophoblast, neoplastic cells has been demonstrated by immunohistochemistry in humans and other species.!' 2 The antigen is present both in the cytoplasm and on the surface of the cells. This hormone, present at high concentrations in trophoblastic cells, was also demonstrated in human non-neoplastic testicular homogenates by hcg ~-subunit radioimmunoassay. The germ cells may be the source of hcg in the testis. 3 The ~-subunit of chorionic gonadotropin was demonstrated in the spermatozoa of seven volunteers, using a fluorescein-labeled double-antibody technique. The hcg-like substance was found in Received April 29, 1977; revised June 6, 1977, and July 7, 1977; accepted July 14, *Rockefeller Foundation Postdoctoral Fellow in Reproductive Biology. To whom reprint requests should be addressed at The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Tex % to 7% of spermatozoa in all specimens analyzed. 4 These findings prompted us to seek the presence of chorionic gonadotropin in the spermatozoa of humans and other species. MATERIALS AND METHODS Semen samples were obtained from 30 human volunteers (by masturbation) without evidence of neoplastic diseases; from sheep, pigs, and goats (by electroejaculation); from horses and bulls (artificial vagina); and from guinea pigs (epididymal flushing) (three animals of each species). Fresh specimens were used when possible and others were frozen first in a vapor of liquid nitrogen at C and then stored at C until processed. Spermatozoa were separated from seminal plasma by centrifigation and washed three times with phosphate-buffered saline (PBS) (ph 7.4). Thin-layer sperm smears were prepared on glass slides and air-dried. After washing in PBS, three drops of rabbit anti-human chorionic gonadotropin (Cappel Laboratories, Inc., Downington, Pa.) at a 1:10 dilution were added to the slides for 30 minutes. The slides were immersed in PBS for 10 minutes and drained; two drops of the second antibody (fluorescein-conjugated

2 FIGs. 1 AND 2. Photomicrograph of human spermatozoa using the immunofluorescence double-antibody technique. Note the positive fluorescence reaction in different segments of some spermatozoa. Figure 1 also shows the presence of a nonimmunofluorescent cell (x400).

3 1260 COMMUNICATIONS-IN-BRIEF November 1977 immunoglobulin G fraction of goat anti-rabbit y-globulin, Cappel Laboratories, Inc., Cochranville, Pa.) at 1:1 dilution were then added. All subsequent operations were performed in a darkroom. The second antibody was incubated with the specimen for 30 minutes; the slides were again washed in PBS for 10 minutes and immediately mounted in PBS for visualization under a fluorescence microscope. Three slides were prepared for each sample. The first two slides were processed as described and the third with PBS or normal rabbit serum instead of the first antibody, as a negative control. Choriocarcinoma cells (BeWo, CCL 98, American Type Culture Collection) were used as positive controls. In some specimens, the first antibody was incubated with an excess of hcg for 30 minutes at room temperature before being added to the smear; the experiment was then continued as usual. Human erythrocytes and guinea pig spermatozoa were washed in PBS and incubated with human seminal plasma for 30 minutes at room temperature and then either immediately exposed to the double-antibody technique or washed three times after incubation with human seminal plasma and then exposed to the antibody technique. In a parallel experiment, another 15 human specimens and 2 specimens of each of the other species cited above were evaluated by the double- FIG. 3. Photomicrograph of a human sperm treated with PBS instead of the first antibody (negative control). The cell shows no immunofluorescence reaction (x400).

4 Vol. 28, No. 11 COMMUNICATIONS-IN -BRIEF 1261 antibody technique, but using as a first antibody rabbit anti-hcg,8-subunit (H971675, generously supplied by Dr. Vernon Stevens, Ohio State University, Columbus, Ohio) undiluted or at a dilution of 1:10. Finally, eight human semen specimens, selected at random from our population, were processed in a,8-subunit hcg radioimmunoassay (RIA kit, Serono Laboratories, Inc., Boston, Mass.). RESULTS Spermatozoa demonstrating positive fluorescence were found in all human specimens. The fluorescence was detected in different segments of the spermatozoa (acrosome, postacrosomal sheath, posterior ring, middle piece, and/or the rest of the tail) (Figs. 1 and 2). The proportion of labeled spermatozoa was significantly higher than that reported previously4 and was found in as many as 80% of the sperm cells. None of the other species studied exhibited positive fluorescence. No fluorescence was noted in any sample incubated with PBS or rabbit serum instead of the first antibody (Fig. 3). Fluorescence was never found after incubating the first antibody with an excess of hcg. Fluorescence was demonstrated on the surface of human erythrocytes and guinea pig spermatozoa after incubation with seminal plasma. However, if the test cells were washed three times in PBS prior to exposure to the double-antibody technique, fluorescence was never present. All of the human specimens in which the first antibody used was rabbit anti-hcg,8-subunit demonstrated a positive fluorescence reaction with the same characteristics as described above, whereas the human negative controls (with PBS or normal rabbit serum) and all of the cited species remained unlabeled. The results of this more specific immunohistochemical technique strongly support our findings. All human semen samples evaluated by radioimmunoassay were positive for the,8-subunit of hcg. DISCUSSION We conjectured that four possible explanations existed for the presence of positive fluorescence in human spermatozoa: nonspecific binding of the second antibody, nonspecific cross-reaction of the first antibody with a protein antigen other than gonadotropin, cross-reaction with luteinizing hormone (LH), and presence of an hcg-like substance in human spermatozoa. Nonspecific binding of the second antibody was excluded by using PBS or normal rabbit serum in place of the first antibody. The absence of fluorescence after incubating the first antibody with an excess of hcg suggested that the positive fluorescence reactions were specific for gonadotropin. Although LH is present in human seminal plasma,5, 6 the possibility that LH was bound nonspecifically to the cell surface and crossreacted with the first antibody was ruled out by the demonstration that the surface fluorescence induced in erythrocytes and guinea pig spermatozoa was reversed by washing. There have been no reports of specific LH receptor sites on spermatozoa, although such sites have been demonstrated in the interstitial cell compartment of the testis7-9 and in the apical cytoplasm of Sertoli cells associated with late spermatids Finally, the demonstration of,8-subunit ofhcg in all eight human specimens assayed by radioimmunoassay and in those in which the first antibody of the immunocytochemical technique was anti-hcg,8-subunit strengthens our conclusions that the observed fluorescence is specific for hcg. For all of these reasons we conclude that the positive fluorescence reaction we observed in human sperm cells is due to the presence of an immune hcg-like substance in the spermatozoa. The presence of this substance in human spermatozoa raises interesting biologic speculations. Research in this area will provide new information on the physiologic role-of hcg in human reproduction and may well open new approaches to conception control in humans. Acknowledgment. The authors are grateful to Dr. Duane C. Kraemer, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station, Tex., for assistance in the collection of semen samples from different species. REFERENCES 1. McManus LM, Naughton MA, Martinez-Hernandez A: Human chorionic gonadotropin in human neoplastic cells. Cancer Res 36:3476, Acevedo HF, Slifkin M, Pouchet GR, Rakhshan M: Human chorionic gonadotropin in cancer cells. I. Identification in in vitro and in vivo cancer cell systems. In Proceedings of the Third International Symposium on Detection of Prev Cancer, Edited by HE Nieburgs. New York, Marcel Dekker Inc, 1977

5 1262 COMMUNICATIONS-IN -BRIEF November Braustein GD, Rasor J, Wade ME: Presence in normal human testes of a chorionic-gonadotropin-like substance distinct from human luteinizing hormone. N Engl J Med 293:1339, Acevedo HF, Slifkin M, Pouchet GR, Rakhshan M: Identification of the f3-subunit of choriogonadotropin in human spermatozoa. In The Testis in Normal and Infertile Men, Edited by P Troen, HR Nankin. New York, Raven Press, 1977, p Sheth AR, Shah GV, Mugatwala PP: Levels ofluteinizing hormone in semen offertile and infertile men and possible significance of luteinizing hormone in sperm metabolism. Fertil Steril 27:933, De Aloysio D, Busacchi P, Bolelli GF, Vecchi F, Flamingni C: Radioimmunological assay of testosterone and gonadotropins in plasma and seminal fluid of healthy subjects and patients with dysspermia. Acta Eur Fertil 5:317, Castro AE, Alonso A, Mancini RE: Localization offolliclestimulating and luteinizing hormones in the rat testis using immunohistological tests. J Endocrinol 52:129, Catt KJ, Tsuruhara T, Mendelson C, Ketelslegers J-M, Dufau ML: Gonadotropin binding and activation of the interstitial cells of the testis. In Hormone Binding and Target Cell Activation in the Testis, Edited by ML Dufau, AR Means. New York, Plenum Press, 1974, Chap 1 9. Zeleznik AJ, Midgley AR Jr: In vitro binding and autoradiographic localization of human chorionic gonadotropin and follicle stimulating hormone in rat testes during development. In Hormone Binding and Target Cell Activation in the Testis, Edited by ML Dufau, AR Means. New York, Plenum Press, 1974, Chap Burgos MH, Sacerdote FL, Vitale-Calpe R, Bari D: Ultrastructural and chemical effects of LH upon the seminiferous tubule. In The Human Testis, Edited by E Rosemberg, CA Paulsen. New York, Plenum Press, 1970, p Setchell BP: Secretions of the testis and epididymis. J Reprod Fertil 37:165, 1974