Immunoelectrophoretic Analysis of Seminal Plasma

Transcription

1 Immunoelectrophoretic Analysis of Seminal Plasma A. KLOPSTOCK, M.D., R. HAAS," and A. RIMON, Ph.D. SEMINAL PLASMA ( SP) is the medium secreted by the accessory sexual glands in which the spermatozoa are suspended. Rather than being an inert medium, this fluid apparently plays an active role in the reproductive process. Evidence to this effect has been offered by Rozin 6 who has shown that the suspension of spermatozoa of low motility in SP of normal sperm in creased their motility. The proteins of SP have been studied by various investigators. Gray and Huggins/ as well as Ross et ap have shown electrophoretically that SP cort. tain~ proteins of the albumin and of the alpha-, beta- and gamma-globulin type. Herman 2 has shown by means of immunoelectrophoresis (IE) that fresh SP consists of eight different proteins; however, he reported that none of these proteins could be identified with gamma globulin. In view of such contradictory findings, it was deemed important to restudy the biochemical composition of SP. Our report deals with the proteins of SP as elucidated from immunoelectrophoretic patterns. MATERIAL AND METHODS Semen was collected from outpatients of the Zamenhof Clinic, Tel-Aviv. Samples were subjected to centrifugation at 2500 rpm. for 0 min. within -5 hours after emission. The SP obtained was separated and then frozen and stored at -20 C. until needed for immunoelectrophoretic analysis. Each sample of semen was estimated for number and motility of its spermatozoa. Pure gamma globulin was prepared from commercial Human Immune From the Department of Microbiology, Tel-Aviv University, Tel-Aviv, Israel. The authors express gratitude to Eng. T. Zyss of the Zamenhof Clinic, Tel-Aviv, for her kind help in supplying the sperm samples used in this investigation, and to Dr. C. A. Joel of the Hadassa Maternity Hospital, Tel-Aviv, for valuable criticism of the manuscript. *Graduate student, Department of Microbielogy. Mr. Haas' participation in authorship represents part of an M.Sc. thesis. 50

2 VoL. 14, No.5, 196 SEMINAL PLASMA ANALYSIS 51 Globulin" by chromatography on diethylaminoethyl-cellulose column, using the method of Levy and Sober. 4 Adsorption of antigamma-globulin antibodies from anti-human serum was performed with alum (potassium aluminum sulfate) pre-loaded with pure gamma globulin; 7.5 ml. of a 1% gamma-globulin solution was mixed for lo min. with 9 ml. of a 10% alum suspension adjusted to ph 6.5. The mixture was then centrifuged, the sediment washed twice with saline, and brought to a volume of 2 ml. with more saline. One ml. of antiserum was then added to ml. of the alum preparation; the resulting mixture was left overnight under refrigeration, following which it was centrifuged and the supernatant used as antigamma-globulin-free antiserum. IE analysis made with this antiserum ensured the complete adsorption of the antigamma-globulin. IE was performed on microscope slides according to the micromethod of Scheidegger. 8 Precipitation lines were developed with rabbit anti-human serum. RESULTS t y f } }, - e p One hundred and two samples were checked for number and motility rate of the contained spermatozoa and subjected to immunoelectrophoretic study. The number of spermatozoa in the samples ranged from 1-10 million to million per ml., with a corresponding motility of 0-10% up to 80-90%. Figure 1 presents two typical immunoelectrophoretograms obtained with seminal plasma; these two are compared with the one obtained for human serum. In most instances, 7-10 different protein fractions could be observed that fell within the ranges of albumin and alpha, beta and gamma globulin (Fig. 1B). In many samples, however, the absence of one or more proteins was quite evident and in some, the lack of the gamma globulin (Fig. 1C) or the alpha and beta globulin (Fig. 1A) was striking. Since the presence of gamma globulin in SP is of great significance, it was essential to ascertain whether the SP protein fraction showing gamma-globulin electrophoretic mobility was indeed identical to serum gamma globulin. The problem was approached in two different ways: ( 1.) The IE of SP was performed using antiserum from which the antigamma globulin was adsorbed (Fig. 2). ( 2.) The IE was performed as before except that following the electrophoretic stage and prior to the diffusion stage a second hole ( B ) was bored in the agar-gel plate at a distance of 2 em. from the first one. Pure gamma *Marcus Memorial Blood Institute, Magen David Adorn, Israel.

3 52 KLOPSTOCK ET AL. FERTILITY & STERILITY A B c Fig. 1. IE of representative specimens of SP as compared with human serum (HS). Rabbit anti-human serum was used for immuno-diffusion. Protein fractions for A, B, and C are discussed in the text. (Specimen number is indicated on the right hand side.) globulin was introduced into the second hole, the channel was filled with antiserum and diffusion was allowed to take place (Fig. ). Figure 2 shows no precipitation line characteristic for gamma globulin, but Fig. confirms that SP gamma globulin is identical with serum gammaglobulin. Once the presence of gamma globulin in SP was confirmed, it was of interest to determine whether any correlation existed between it and the normality or pathema of the spermatozoa. As seen from Table 1, no such correlation was detected. DISCUSSION Knowledge concerning the mechanism by which SP influences the spermatozoa is still rather scanty. Possibly an immunological process is involved.

4 VoL. 14, No.5, SEMINAL PLASMA ANALYSIS 2 Fig. 2. IE of SP against whole anti-human serum (top channel) and against antiserum from which the antigamma-globulin was adsorbed (bottom channel). Fig.. Reaction of identity between SP gamma globulin and serum gamma globulin. TABLE 1. Presence of Gamma Globulin in SP Correlated with the Number and Motility of the Spermatozoa Gamma-globulin d I,,- PreHent Ab.~ent No. spermatozoa (millions per mi.) < Motility rate (in %) < s e - l. Rumke and Hellinga7 have demonstrated the existence of autoantibodies against spermatozoa in sterile males, which could account also for the results of several other investigators, e.g., Rozin. 6 On the other hand, the findings of Herman 2 that gamma globulin is not detectable in SP would tend to contradict this view. Results of the present study indicate that gamma globulin may indeed be

5 54 KLOPSTOCK ET AL. FERTILITY & STERILITY present in SP. Moreover, since no correlation was found between the presence of gamma globulin and the physiologic state of the sperm, it is reasonable to assume that gamma globulin is a normal component of most SP. If so, then an immunological mechanism may well be involved in the pathogenesis of sterility and it is not even necessary to postulate that antibodies against spermatozoa can only penetrate into the SP through a lesion in the seminal duct which enables contact between semen and blood plasma. Noteworthy also is the inconstancy in the protein composition, i.e., the quite-random absence of one or more protein components from the SP (as shown by immunoelectrophoretograms). Whether or not this phenomenon has any clinical or biological significance remains to be seen. Of further note, Joel et al., studying the electrophoretic mobility of human spermatozoa, did not find a significant difference between normal and pathologic specimens. SUMMARY Immunoelectrophoretic analysis was carried out on 102 sperm samples, both normal and pathologic. Most sperm plasmas were found to contain a gamma globulin identical with serum gamma globulin. No correlation was observed between the physiological state of the sperm and the presence or absence of gamma globulin in the seminal plasma. Tel-Aviv University 155 Herzl St. Tel-Aviv, Israel REFERENCES 1. GRAY, S., and HuGGINS, C. Electrophoretic analysis of human semen. Proc. Soc. Exper. Biol. & Med. 50:51, HERMAN, G. Immunoelectrophoretische untersuchungen ammenschlichen Spermaplasma. Clin. Chim. Acta. 4:116, JoEL, C. A., KATSCHALSKY, A., KEDEM, 0., and STERNBERG, N. Electrophoretic measurements of normal and pathological human spermatozoa. 2 fascicolo speciale ( 1951) degli Annali di Ostetricia & Ginecologia edito a cura del centro Sterilita Coniugale di Milano. 4. LEVY, H. B., and SoBER, H. A. A simple chromatographic method for preparation of gamma globulin. Proc. Soc. Exper. Biol. & Med. 10:250, Ross, V., MooRE, D. H., and MILLER, E. G. Proteins of human seminal plasma. ]. Biol. Chem. 144:661, RoziN, S. Studies on seminal plasma: I. The role of seminal plasma in motility of spermatozoa. Fertil. & Steril. 11:218, RuMKE, P., and HELLINGA, G. Autoantibodies against spermatozoa in sterile men. ]. Clin. Path. 2:51, ScHEIDEGGER, J. J. Une micromethode d'immunoelectrophorese. Int. Arch. Allergy 7:109, 1955.