Immunochemical Properties of Normal and Pathologic Seminal Plasma

Transcription

1 Immunochemical Properties of Normal and Pathologic Seminal Plasma RONALD L. SEARCY, Ph.D., ROBERT G. CRAIG, M.D., and LOIS M. BERGQUIST, M.S. ALTHOUGH Landsteiner first described seminal plasma antibodies over 50 years ago, the immunochemistry of human semen remains poorly defined in health or disease. Antigenicity of human spermatozoa in seminal plasma appears determined by the accessory organs of reproduction rather than the t-estisp Moreover, human seminal plasma and saline extracts of human prostate possess identical antigenic properties. 1 Human seminal plasma from normal subjects, as well as from individuals with diseases of the prostate, have been studied in order to further elucidate qualitative and quantitative immunochemical responses. MATERIALS AND METHODS Preparation of Seminal Plasma Antiserum Semen collected from 15 clinically healthy medical students was pooled and centrifuged. After separating cells, homogeneous aliquots of seminal plasma were frozen. Rabbits were given 0.1 ml. of the pool substance intravenously twice during the first week. Doses of seminal plasma were progressively increased to 0.5 ml. during the next 4-week period. Animals were also given weekly subcutaneous injections of 1.0 ml. of a mixture, of equal parts of Freund's adjuvant and seminal-plasma pool. After 1 week of rest, animals were bled and again given 0.5 ml. of pooled seminal plasma intravenously together with 1.0 ml. of the adjuvant-seminal-plasma mixture subcutaneously. Bleedings and the latter immunization regimen were repeated for 2 additional weeks. Rabbit serum was treated with 0.1% sodium azide ( pooled and refrigerated). From the Departments of Pathology and Medicine, Los Angeles County General Hospital (Unit 2) and California College of Medicine, Los Angeles, Calif. This investigation was supported, in part, by grants from the U.S.P.H.S. (Projects A-4609; H-6479, and C-5917), Baxter Laboratories, and the Attending Staff Association of the Los Angeles County General Hospital (Unit 2), Los Angeles, Calif. Submitted for publication June 6,

2 2 SEARCY ET AL. FERTILITY & STERILITY Immunoelectrophoresis on Agar Gel Microscope slides (1 X 3 in.) were uniformly covered with melted agar ( 1.5%) dissolved in veronal buffer (ph 8.2; ionic strength, 0.05). Well!; were punched in solidified agar with a 14-gauge cannula before placing slides in a Durrum-type electrophoresis chamber. A wick of filter paper (Whatman No.1) was applied to both ends of the films, thereby establishing contact with the buffer reservoir. Following a 30-min. equilibration at 110 v and a constant current of 4.5 mamp. per slide, test seminal plasma was placed in each well. Equilibration current and potential were then maintained for 4 hr. Slides were removed and rinsed in a phosphate buffer for 10 min. Parallel longitudinal troughs were made on either side of plasma wells. Troughs were filled with seminal-plasma antiserum, humanserum antiserum (Hyland) or antiserum for lipoproteins of density < gm.jml. (Hyland). Diffusion was allowed to proceed for 72 hr. in a humidified chamber at room temperature. Electrophoresis on Cellulose Acetate After liquefaction, semen was centrifuged to separate spermatozoa. Supernatants were carefully removed and stored 48 hr. at refrigerator temperature. Strips of cellulose acetate (Oxoid No G) were impregnated with a solution of 8.8 gm. of barbitone buffer (Oxoid No. BR l1-g) dissolved in 1 L. of distilled water. Saturated strips were blotted lightly and placed in a Shandon electrophoretic cell. A 20-,uJ. aliquot of seminal plasma was applied in the middle of each strip as a streak. Electrophoresis was allowed to proceed for 3 hr. at a constant current of 0.4 mamp./cm. of width of cellulose acetate. Strips were removed, air-dried and floated on the surface of a 0.2% solution of Ponceau S (Oxoid No ) for min. When penetration appeared complete, strips were immersed in the stain for another 5 min. A clear background was obtained by rinsing in a 5% aqueous solution of glacial acetic acid, followed by distilled water. RESULTS AND DISCUSSION In order to quantitate proteinaceous material, seminal plasma from 10 subjects was treated with biuret reagent. 4 Colors produced were equivalent to protein concentrations ranging from 1.8 to 4.7 gm.%. Seminal-plasma proteins were also estimated in three specimens collected from each of 5 individuals within a 96-hr. period (Table 1). Total protein concentrations remained relatively constant during the time-interval studied. It is noteworthy that this relationship obtained, despite progressive decreases in

3 VOL. 15, No.1, 1964 SEMINAL-PLASMA PROPERTIES 3 TABLE 1. Estimation of Seminal-Plasma-Total-Protein Concentrations in Terms of Biuret Color Reaction Total protein (gm.%) Subject First specimen Second specimen Third specimen A B C D E numbers of spermatozoa. These data support the view that testicles do not appreciably contribute to the protein spectrum of human seminal plasma,u Aliquots of pooled serum, semen, seminal plasma, and washed spermatozoa were subjected to electrophoresis on cellulose acetate. Comparison of serum and seminal-plasma electrophoretic patterns demonstrated both qualitative and quantitative differences in protein constituents (Fig. I). Staining with Ponce au S revealed at least eight distinct bands on seminal plasma electrophorograms. Other workers 3, 7, 8 have obtained at least five, and sometimes six, fractions following separation of normal seminal plasma by filter paper or Tiselius electrophoresis. Component A of normal seminal plasma moved rapidly toward the cathode and appeared to have no serum counterpart. By contrast, no fraction exhibiting a mobility of serum alpha-i globulin was discernible in seminal plasma. However, protein-staining material of seminal plasma was evident in the region usually occupied by serum alpha-2, beta, and gamma globulins. Using filter-paper electrophoresis, Raboch 7 was able to separate seminal-plasma fractions with mobilities similar to serum alpha-i, alpha-2, beta, and gamma globulins. Ross et az's utilized solubility characteristics to classify three normal seminal plasma proteins as globulins. Normal seminal plasma contains a protein-staining fraction possessing mobility consistent with that of serum albumin. The albumin-like material (Component E) is associated with two faster moving boundaries. Rapid anodal-migrating components of normal seminal plasma have been previously observed by free or filter paper electrophoresis. 7, 8 Electrophoresis of whole semen on cellulose acetate disclosed a zone at the point of origin with a marked affinity for Ponce au S. Spermatozoa isolated from the semen pool were washed repeatedly with aqueous NaCI (0.85%). Fractionation of a concentrated suspension of spermatozoa on the same medium again revealed a bright-staining zone at the origin. Comparisons of electrophoretic patterns suggest that spermatozoa may be

4 4 SEARCY ET AL. FERTILITY & STERILITY largely responsible for the uptake of Ponceau S at the point of application. It is interesting that additional protein-staining components of spermatozoa exhibited mobilities similar to serum albumin and globulins, despite thorough washing in saline. Thus, some proteinaceous material associated with Fig. 1. Electrophoretic separation of (top to bottom) pooled serum, seminal plasma, semen, and washed spermatozoa. All on cellulose acetate.

5 VOL. 15, No.1, 1964 SEMINAL-PLASMA PROPERTIES 5 seminal spermatozoa is not easily removed by saline. The antigenicity of ejaculated spermatozoa stems from seminal plasma and may be related to saline-insoluble electrophoretic components. Weil et al. ll observed that spermatozoa collected from spermatoceles lack powerful antigens associated with seminal spermatozoa. Two specimens of seminal plasma were secured one week apart from each of 4 medical students in order to evaluate intraindividual variation in electrophoretic patterns of ejaculates. Typical patterns obtained when specimens were subjected to electrophoresis and stained with Ponceau S. Furthermore, protein distributions were relatively constant in the two specimens obtained from the same individual. The major components (B, C, and D) routinely contained over 70% dye (Table 2). Only 6-10% of dye was bound to the three zones designated as Fraction E. There is some disagreement concerning quantities of albumin in seminal plasma. Reportedly, values range from 0.02 to 22.7%.6 Immunoelectrophoresis of human serum against human-serum antiserum usually produces approximately 18 precipitin lines (Fig. 2). When normal seminal plasma was treated in this manner, only four lines of precipitation were discernible. Precipitin arcs did not appear to correspond exactly with those obtained from serum (Fig. 3). Principal lines of precipitation, however, were suggestive of both albumin and globulin fractions. Hermann was also able to distinguish four immunoelectrophoretic arcs from seminal plasma diffused against human-serum antiserum. One fraction was immunologically and electrophoretically identical with serum albumin. On TABLE 2. Dye Uptake of Seminal Plasma Electrophoretically Fractionated on Cellulose Acetate Protein fractions Subject A B C D E 1 14" *Each figure refers to the percentage of total Ponceau S bound.

6 6 SEARCY ET AL. FERTILITY & STERILITY Fig. 2. Immunoelectrophoretic pattern of pooled human serum diffused against serum antiserum. Fig. 3. Immunoelectrophoretic pattern of pooled human seminal plasma diffused against serum antiserum. Fig. 4. Immunoelectrophoretic pattern of seminal plasma from normal subjects diffused against seminal-plasma antiserum. Fig. 5. Immunoelectrophoretic 'pattern of seminal plasma from subjects with prostatic hypertrophy diffused against seminal-plasma antiserum. Fig. 6. Immunoelectrophoretic pattern of seminal plasma from subject with prostatic carcinoma diffused against seminal-plasma antiserum.

7 VOL. 15, No.1, 1964 SEMINAL-PLASMA PROPERTIES 7 the other hand, the presence of serum globulins in seminal plasma could not be as clearly defined. Total cholesterol1o in seminal plasma from 10 clinically healthy subjects ranged from 60 to 90 mg.%. Inasmuch as a majority of cholesterol in serum is bound to beta globulins, attempts were made to separate this lipid-bearing fraction from seminal plasma. Immunoelectrophoresis of seminal plasma against antiserum specific for serum lipoproteins of density < gm.jml. yielded no precipitin arcs. Therefore, lipoproteins of seminal plasma appear antigenic ally distinct for those of serum. When viewed under ultraviolet illumination, lipid-bearing molecules were evident on cellulose acetate strips containing seminal plasma stained with Protoporphyrin IX. Previous studies indicate that Protoporphyrin IX is an extremely sensitive fluorescent stain for detecting serum lipoproteins.9 Seminal plasma from 30 normal subjects, 5 patients with prostatic hypertrophy, and one patient with prostatic carcinoma were subjected to immunoelectrophoresis against seminal-plasma antiserum. Normal seminal plasma routinely yielded seven sharp precipitin arcs (Fig. 4), whereas pooled serum failed to react with seminal-plasma antiserum. Specimens obtained from the group of patients with prostatic hypertrophy showed a diminished electrophoretic mo bili ty (Fig. 5). Several arcs of the normal seminal-plasma-precipitin spectrum were absent from the prostatic carcinoma pattern (Fig. 6). Moreover, this pathologic seminal plasma produced a precipitin line indicating the presence of an antigen with rapid mobility which was absent in normal specimens. By the Ouchterlony geldiffusion technic, Flocks et al. 2 were unable to obtain evidence for a specific antigen in prostatic-cancer-tissue extracts as contrasted with hypertrophied or normal prostatic tissue. SUMMARY Protein constituents of normal seminal plasma electrophoretically separated on cellulose acetate showed little intraindividual variation. Ponce au S-stained electrophoretic patterns of seminal plasma were qualitatively and quantitatively different from those of serum. Washed spermatozoa subjected to electrophoresis exhibited patterns indicating the presence of saline-insoluble proteins similar to those of seminal plasma. Immunoelectrophoresis of normal seminal plasma against human-serum antiserum produced only four precipitin arcs. At least seven lines of precipitation resulted from immunoelectrophoresis of normal seminal plasma against human seminal-plasma antiserum prepared in rabbits. Seminal

8 8 SEARCY ET AL. FERTILITY & STERILITY plasmas from patients with diseases of the prostate produced abnormal patterns. Pooled human serum did not react with seminal-plasma antiserum. Several protein components of seminal plasma seem to be immunochemically distinct from those of serum. Los Angeles County General Hospital Unit N. State St. Los Angeles 88, Calif. REFERENCES 1. FLOCKS, R. H., BANDHAUR, K., PATEL, C., and BEGLEY, B. J. Studies on spermagglutinating antibodies in antihuman prostate sera. ]. Urol. 87:475, FLOCKS, R. H., URICH, V. C., PATEL, C. A., and OPITZ, J. M. Studies on the antigenic properties of prostatic tissue. J. Urol. 84: 134, HERMANN, G. Studies on spermagglutinating antibodies in antihuman prostate sera. CUn. Chim. Acta 4: 116, 19.5'9. 4. KINGSLEY, G. R. A rapid method for the separation of serum albumin and globulin ]. Biol. Chem. 188:731, LANDSTEINER, K. Zur kenntnis der spezifisch auf blutkorperchen wirkenden sera. Zentralbl. Bakt. 25:546, MANN, T. The Biochemistry of Semen. New York RABOCH, J. Electrophoresis of seminal plasma proteins. Internat. ]. Pertil. 6:31, Ross, V., MILLER, E. G., JR., MOORE, D. H., and SIKORSKI, H. Elecrophoretic patterns of seminal plasma from some "abnormal" human semens. Froc. Soc. Exper. BioI. & Med. 54:179, SEARCY, R. L., and BERGQUIST, L. M. Lipoprotein Chemistry in Health and Disease. Thomas, Springfield, Ill., SEARCY, R. L., BERGQUIST, L. M., and JUNG, R. C. Rapid ultramicro estimation of serum total cholesterol. J. Lipid Res. 1:349, WElL, A. J., HERMAN, J. R., GOLDBERG, A. S., and RODENBURG, J. M. Immunological and chemical studies of spermatocele fluid. ]. Urol.85:665, WElL, A. J., and RODENBURG, J. M. Immunological differentiation of human testicular (spermatocele) and seminal spermatozoa. Froc. Soc. Exper. BioI. & Med. 105:43, 1960.