Journal of Reproductive Immunology, 18 (1990) 187-- 197
Elsevier Scientific Publishers Ireland Ltd. JRI 00670
Sperm preparation affects reactivity of monoclonal antibodies Satish K. Gupta*, David L. Fulgham and Nancy J. Alexander Eastern Virginia Medical School, Section of Reproductive Immunology, Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Norfolk, Virginia(U. S. A.)
(Accepted for publication 14 May 1990)
Summary The polyacrylamide gel electrophoresis (SDS-PAGE) patterns of extracts prepared from thrice-washed human sperm were compared with extracts of swim-up and non-rise sperm. For sperm extraction sodium deoxycholate (DOC), octylphenoxypolyethoxyethanol (NP-40), dithiothreitol (DTT), lithium 3,5-diiodosalicylate (LIS), or 3-[(3-cholamidopropyl) dimethylammonio]-I-propane sulfonate (CHAPS) were used. A pellet of pooled thrice-washed semen was divided and extracted with these detergents. Similar gel electrophoresis profiles, as revealed by silver staining, were observed with the LIS, DOC, NP-40 and C H A P S extracts, whereas DTT extracts lacked the 150 and 200 kDa bands. Different thrice-washed semen pools varied in their protein pattern, probably due to the presence of sloughed germinal cells, white blood cells and sloughed cells from the reproductive tract. Swim-up sperm extracts had a different protein profile from that of thrice-washed pools but pools of swim-up sperm had a more constant protein pattern than those of thricewashed semen pellets. Monoclonal antisperm antibodies did not always recognize antigens of the same molecular weights in western blots when extracts of swim-up sperm versus thrice-washed sperm were compared. In immunoblot studies with monoclonals (produced against thrice-washed sperm, cloned five times, reactive by ELISA and sperm function tests), 4/12 reacted with 0.3070 NP-40 extracts of thricewashed sperm and 5/12 with 0.3% NP-40 extract of swim-up sperm. These findings suggest that definition of antigens by monoclonal antisperm antibodies will be influenced by the nature of sperm extract employed. *Permanent address: National Institute of Immunology, J.N.U. Campus, Shaheed Jeet Singh Marg, New Delhi 110067, India. Correspondence to: Nancy J. Alexander Ph.D, NICHD, Contraceptive DevelopmentBranch, 600 Executive Plaza North, Bethesda, MD 20892, U.S.A.
0165-0378/90/$03.50 © 1990ElsevierScientific Publishers Ireland Ltd. Published and Printed in Ireland
Key words: human sperm; SDS-PAGE electrophoresis; immunoblotting; monoclonal antibodies.
The search for sperm antigens pertinent to contraceptive vaccine development often involves the use of serum from patients with putative immunologic infertility or monoclonal antisperm antibodies. With both of these approaches, an important technique in the initial assessment of antigen characteristics is immunoblotting to define molecular weight. The antigen preparation used for such blotting techniques is an essential consideration; yet numerous preparations (pooled-washed or swim-up sperm) and extraction procedures (sodium dodecyl sulfate (Naaby-Hansen and Bjerrum, 1985), sodium deoxycholate (Hald et al., 1987; Naz et al., 1986), lithium 3,5-diiodosalicylate (Naz et al., 1986) and NP-40 (Herr and Eddy, 1980)) are commonly used. This paper compares human sperm extracts prepared with different solubilizing agents separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Monoclonal antisperm antibodies were tested with these sperm preparations by immunoblotting. Sperm prepared by the swimup method seem the most appropriate and constant for defining sperm antigens. Materials and Methods
Reagents Sodium deoxycholate (DOC), octylphenoxypolyethoxyethanol (NP-40), dithiothreitol (DTT), aprotinin and phenylmethyl sulfonyl fluoride (PMSF) were obtained from Sigma Chemical Company, St. Louis, MO. Lithium 3,5diiodosalicylate (LIS) was purchased from Eastman Kodak Company, Rochester, NY and 3-[(3-cholamidopropyl) dimethylammonio]-l-propane sulfonate (CHAPS) from Serva, Heidelberg, F.R.G. Human sperm Semen ejaculates obtained from normal male donors or from men attending an infertility clinic (The Jones Institute for Reproductive Medicine, Norfolk) were allowed to liquefy at room temperature. No sample contained elevated levels of white blood cells upon inspection. Sperm were collected by centrifugation at 400 x g for 15 min, washed three times in Tris-buffered saline (TBS; 10 mM Tris, 145 mM NaCI, pH 7.4), pooled from several donors, and stored at - 70°C before use (thrice-washed sperm). Alternately, after liquefaction, 2 ml of semen was carefully placed beneath BWW
medium (Biggers et al., 1971) and motile sperm were allowed to swim up for a 60-rain incubation period at 37°C. Motile sperm in BWW medium were pooled, counted with a Cell Soft Automated Semen Analyzer (CRYO Resources Ltd., New York, NY), washed three times in TBS and stored at 70°C (swim-up sperm). Sperm which did not swim up in BWW were also pooled, counted, washed three times in TBS and stored at - 70°C (non-rise sperm). -
Extraction of human sperm antigens Antigens were extracted from pools of thrice-washed semen pellets, swimup sperm and non-rise sperm by the following methods: LIS The l-ml volume of the thawed sperm (5 X 108 sperm) was doubled by addition of 0.6 M LIS in TBS, to which 1 mM (final concentration) PMSF was added as a protease inhibitor. The suspension was incubated for 1 h at room temperature with continuous end-to-end mixing. DOC Extraction procedure was the same as for LIS except that 30 mM DOC was used instead of LIS. NP-40 m The procedure was the same as described above for the LIS preparation except that a 0.6°7o (0.3°7o final concentration) or 1°70 v/v concentration of NP-40 was used. CHAPS - - For CHAPS solubilization the sperm (5 x l0 s) were extracted with 5 mM CHAPS in TBS, containing 10 m l U / m l aprotinin, for 1 h at room temperature by continuous end-to-end mixing. DTT - - The volume of the thawed sperm (5 x los) was doubled by the addition of 0.8 mM DTT in TBS, to which 1 mM (final concentration) PMSF was added. The cell suspension was sonicated with a micro ultrasonic cell disruptor (Kontes, Vineland, N J) at 4 w power output for four 20-s intervals. During sonication the cell suspension was kept on ice, and a 2-min cooling period between sonications prevented overheating. After the extraction, various suspensions were centrifuged for 30 min at 20,000 x g at 4°C. The supernatants from each preparation were collected, pooled separately and dialyzed extensively against TBS in a 3500 molecular weight exclusion dialysis membrane at 4°C. -
Monoclonal antibodies Mouse hybrid cell clones secreting monoclonal anti-human sperm antibody were developed by fusion of splenocytes obtained from BALB/c mice that had been immunized with human sperm or sperm-extracted antigens with SP2/0-Agl.4 mouse myeloma, as described elsewhere (Gupta and Talwar, 1980). Briefly, female BALB/c mice (Charles River Labs, Wilmington, MA) were immunized subcutaneously with human sperm (lsahakia and Alexander, 1984) or human sperm antigens (50/~g/animal) extracted by NP-
40 or LIS in complete Freund's adjuvant, followed by an intraperitoneal booster injection in incomplete Freund's adjuvant on day 21. After 3 to 4 weeks, mice with the highest titers of antisperm antibodies (determined by ELISA) were given intravenous/intraperitoneal injections for 3 consecutive days of human sperm antigen (100 tag protein/injection) in TBS, 4 days prior to fusion, and used as spleen donors. Spleen cells were fused with SP2/ 0Agl.4 mouse myeloma in 2:1 ratio with 50°7..'0 polyethylene glycol (PEG, molecular weight 1500). After fusion, cells were cultured in 96-well plates in selection medium containing RPMI-1640 (Gibco Laboratories, Grand Island, NY), 20°70 fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), and HAT (hypoxanthine, aminopterin, thymidine; Boehringer Mannheim, GmbH, F.R.G.). Ten to 14 days after hybridization, supernatant fluids from all the wells were screened in ELISA for antisperm antibodies. Positive hybrid cells were expanded and subcloned at least twice under limiting dilution culture conditions (0.5 ceil/well).
SDS-polyacrylamide gel electrophoresis SDS-PAGE was carried out in 1.5-mm thick, 16 x 18 cm slab gels (Hoefer Scientific Instruments Apparatus, San Francisco, CA) as described by Laemmli (1970). Briefly, discontinuous gels composed of a running gel (10070 or 15°70 polyacrylamide; pH 8.8) and a stacking gel (3°70 polyacrylamide; pH 6.8), both with 0.1070 SDS, were prepared. Samples containing human sperm extracts prepared by various procedures (50--70 tag) were diluted 1:1 with reducing sample buffer (20°7.0 glycerol, 3070 SDS, 12.5°70 0.5 M Tris--HCl pH 6.8, 5% 2-mercaptoethanol and 0.5070 bromophenol blue) and boiled for 5 min. The electrophoresis buffer chamber contained 25 mM Tris base, 0.19 M glycine and 0. ! 070 SDS. Electrophoresis was performed at 30 milliamps (constant current) at room temperature with a circulating cold water cooling system. Known prestained standards (BRL, Gaithersburg, MD) consisting of myosin, phosphorylase-B, bovine serum albumin (BSA), ovalbumin, a-chymotrypsinogen,/3-1actoglobin, lysozyme, bovine trypsin inhibitor and insulin with apparent molecular weights of 200, 97, 68, 43, 25, 18, 14, 6 and 3 kDa were also run. After electrophoresis was complete, the gel was fixed with gentle rocking overnight in methanol and water (1:1) and then developed with a silver stain (Wray et al., 1981 ).
lmrnunoblotting Proteins from the gels were electrophoretically transferred to 0.22 /am nitrocellulose membrane (BioRad Laboratories, Richmond, CA) by the blotting procedure of Towbin et al. (1979). In brief, the electrophoretic transfer of proteins took place at 150 milliamps constant current in transfer buffer (25 mM Tris, pH 8.4, 192 mM glycine, 20°70 v/v methanol) overnight at room
temperature. Once transferred, the outermost lane of sperm antigens was developed with amido black (0.5070 w/v) in acetic acid, methanol and water (10:45:45) using the solvent as a destainer. Thereafter, non-specific binding sites were blocked by incubating the nitrocellulose membrane for 2 h at room temperature with TBS containing 3070 BSA. The nitrocellulose membrane was then washed with TBS containing 0.1070 Tween-20 (TBS-Tween) to remove excess BSA and was allowed to react with different monoclonal antibodies in a miniblotter-45 (lmmunetics, Cambridge, MA) for 8 h at room temperature or overnight at 4°C. After incubation, the nitrocellulose membrane was washed with TBS-Tween and incubated with a 1:300 dilution of peroxidase-labeled rabbit anti-mouse immunoglobulins (Dako Corp., Santa Barbara, CA) for 2 h at room temperature. Excess conjugate was removed by washing several times with TBS-Tween. The enzyme bound to the nitrocellulose membrane was visualized by adding freshly prepared substrate solution (30 mg of a-chloronaphthol (Bio-Rad Laboratories, Richmond, CA) dissolved in 10 ml methanol and diluted 1:4 with TBS, to which hydrogen peroxide was added at a final concentration of 0.06070). The reaction was terminated by washing the nitrocellulose membrane with distilled water, followed by 10 min of incubation with stopping solution (0.1070 NAN3). The nitrocellulose blot was rewashed, stored in distilled water, and photographed. Results
The solubilized components derived from various sperm preparations were analyzed by SDS-PAGE with 10070 separating gels. The silver-stained gels of extracts prepared from human sperm by various procedures are shown in Fig. I. Extracts prepared from pooled, thrice-washed human semen by LIS (lane A), DOC (lane B), C H A P S (lane D) and 0.3070 NP-40 (lane E) had almost identical profiles. The profile obtained by the DTT method (lane C) was different from that of LIS DOC, C H A P S and NP-40 extracts (lanes A, B, D and E, respectively) as evidenced by the absence of high molecular weight bands of 150 and 200 kDa. Use of Triton X-100 and SDS as solubilizing agents also revealed similar patterns to that of LIS, DOC, C H A P S and NP-40 (data not shown). Although the type of reagent used for extraction may have some affect on the protein pattern (e.g. DTT differs from the others), greater variation was observed when different pools of thrice-washed semen were used. Comparison of lane E with lanes F and G reveals that different pools exhibit some similar bands but many differences. When we compared different pools of swim-up sperm (Fig. 2), we observe that their protein profiles were much
more similar than those o f different pools of thrice-washed semen (compare Figs. 1 and 2). Since swim-up sperm were exposed to BWW medium containing BSA, it was necessary to determine if any protein bands were the result of substances adhering to the sperm from the medium. Thrice-washed semen was exposed to B W W medium for the same duration and at the same temperature as
required to obtain swim-up sperm. S D S - P A G E analysis o f a pool o f thricewashed semen incubated with or without B W W medium revealed similar patterns (data not shown).
The binding pattern o f 12 monoclonal antibodies to two different sperm extracts (A = 0.3°7,0 NP-40 extraction o f thrice-washed semen, B = 0.3070
lib im m
18--'-~ 14 ~ ~-"r Mr (kD)
~..~: [ A
...... r B
Fig. I. SDS-PAGE analysis (10% gel); reduced conditions of human sperm proteins extracted by xarious methods as revealed by silver staining. Lanes A, B, C, D and E received extracts from thrice-washed sperm prepared by the LIS, DOC, DTT, C H A P S and 0.3°o NP-40 methods, respectively. Lanes F and G recei,,ed 0.3% NP-40 exlracts, of different pools of thrice-washed sperm• All lanes received 50,ug protein. Weight markers for lanes A to E are left and for lanes F and G are right.
,~i/ilD , I s U D
Fig. 2. SDS-PAGE (10w0 gel) of human swim-up sperm proteins extracted with 0.3°~0 NP-40 from three pools, 50 ~g protein per lane.
NP-40 extraction of swim-up sperm) is shown in Fig. 3. With the thricewashed semen preparation, there was a non-specific reaction at about 10 kDa. Although all of these monoclonals reacted by ELISA, only four reacted to thrice-washed semen (MA 7, 10, 15, 49) and five reacted to swimup (MA 7, 10, 15, 36 and 40). As has been amply shown in the literature, not all monoclonals react in western blotting. Monoclonal antibodies MA 7, 10 and 15 showed different reactivity patterns with antigens prepared from thrice washed semen as compared to swim-up sperm. MA 36 and 40 recognized high molecular weight antigens (> 43 kDa from the swim-up sperm preparation and not the thrice-washed semen extract, on the other hand, monoclonal antibody MA 49 failed to show any reactivity with swim-up sperm. Distinctive antigens are recognized depending on the antigen preparation used for blotting. Interestingly, the antigen preparation used for the
a b c d e f g h i I kl
Fig. 3. Western immunoblotting of the monoclonal binding pattern to human ~perm preparations. Extracts, prepared from thrice-washed sperm b) 0.3°"o NP-40 (lane A), s~im-up sperm b.~ 0.30~0 NP-40 (lane B), were electropboresed on a 15% SDS gel under reduced conditions. ]-~el~e monoclonals: a. MA7; b, MA-10; c, MA-15; d, MA-35; e, MA-36; f, MA-40; g, MA-42; h, MA-43; i, MA-45; j, MA-44; k, MA-47; l, MA-49 were analyzed with each preparation. The immunoglobulin classes are IG, for MA-7, 15, 35, 36, 40,42, 43, 44, 45, 47, 49 and IgM for MA-10.
initial immunization prior to monoclonal antibody production was not a major factor. MA 7, i0 and 15 were from mice immunized with washed human semen yet MA 7 and l0 reacted with swim-up sperm. MA 35, 36 and 40 were produced from mice immunized with a LIS extract of thrice-washed semen and yet 36 and 40 reacted with swim-up sperm. MA 42, 43, 44, 45, 47 and 49 were prepared from swim-up sperm extracted with NP-40 yet MA 49 did not react with swim-up sperm but did react with thrice washed semen.
A number of studies have shown that the sperm population in an ejaculate is heterogeneous in oligosaccharides (Koehler, 1981), proteins (Hjort et al., 1982), lipids (Elias et al., 1979), surface charges (Yanagimachi et al., 1972), antigens (Villarroya and Scholler, 1986), physiological state (Burkman, 1984, 1986) and contaminating cells (Olsen and Shields, 1984), so we used a pool of ejaculates from several donors for comparison of the extraction procedures. The LIS, DOC, C H A P S and NP-40 methods produced nearly identical protein bands. The DTT method consistently resulted in the lack of the 150 and 200 kDa bands, perhaps due to the action of sonication a n d / o r the treatment with DTT. This extraction procedure, however, does retain relevant antigens, since these antigens will induce preferential lymphocyte adherence inhibition in vasectomized men compared to intact controls (Anderson et al., 1982). Although detergent extraction can affect the number and molecular weights of proteins, a more important consideration is the sperm pool itself. Pools of thrice-washed semen extracted by the same method exhibited marked differences. The swim-up extracts of different sperm pools were more similar to one another. When we compared different pools of swim-up sperm, we observed that their protein profiles were much more similar than those of different pools of thrice-washed semen (compare Figs. 1 and 2). The differences between different pools of thrice-washed semen (lanes E, F and G) point to (a) the importance of using a single pool for any experimental series and (b) to the difficulty of comparing gels from one laboratory with those of another. Even though the semen samples used did not contain elevated white cell counts, we suggest the heterogeneity of thrice-washed semen pools is most likely due to a composite of motile and non-motile sperm and also a population of round cells, including germ cells and white blood cells. A significant number (approximately 106) of viable leukocytes are present in ejaculates of normal men (Olsen and Shields, 1984). To better define the antigens of white cells and sperm, reconsitution experiments with varying ratios of white cells of various types could be performed. Swim-up sperm were all motile and likely to be those sperm associated with fertilization. Not only were bands present in extracts of swim-up sperm that were not present in the thrice-washed sperm, but some of the common bands were present in different proportions. These bands in the swim-up preparations were probably not because of proteins in the medium adhering to the sperm surface since extracted sperm incubated with and without BSA yielded similar patterns. Since all sperm were extracted at similar sperm concentrations and the electrophoresis was performed on similar
protein concentrations, we suggest that the amounts of certain antigens are dissimilar. Moreover, monoclonal antibodies produced from mice immunized with NP-40 extracts of thrice-washed semen did demonstrate differences in the binding pattern in western blots depending on whether the extract employed was prepared from thrice-washed or swim-up sperm. That the sperm antigen preparations used in gel electrophoresis can affect results can partially explain the finding in the WHO Workshop where only a few antisperm monoclonal antibodies reacted by immunoblot (Anderson et al., 1987). Furthermore, blots made from different pools of thrice-washed semen would be expected to vary significantly, thus making inter-laboratory comparisons difficult unless adequate information on sperm preparation is provided. We suggest that swim-up sperm rather than thrice-washed are far more appropriate for studies of sperm antigens associated with motile sperm. Finally, batch-to-batch variation can be expected in sperm pools and, therefore, a large pool must be collected before any experiments are begun.
Acknowledgements This work was supported by NIH grant HD23096-02 and an agreement between the National Institute of Immunology, New Delhi, India and the U.S. Agency for International Development (A.I.D.). The views expressed by the authors do not necessarily reflect the views of A.I.D. Travel support from NIH for S.K. Gupta is gratefully acknowledged. The authors thank M.L. Mountain for his technical help.
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