Journal of Reproductive Immunology, 19 (1991) 269--285 Elsevier Scientific Publishers Ireland Ltd.
Evaluation of human sperm-zona pellucida tight binding by presence of monoclonal antibodies to sperm antigens* M a r y C o n d o n M a h o n y a, D a v i d L. F u l g h a m a, P e t e r F. B l a c k m o r e b a n d N a n c y J. A l e x a n d e r a ~Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 855 W. Brambleton Ave., Norfolk, VA 23510 and bDepartment of Pharmacology, Eastern Virginia Medical School, Norfolk, VA 23501 (U.S.A.) (Accepted for publication 8 October 1990)
Summary Characterized WHO monoclonal antibodies (MAbs) to human sperm antigens were evaluated as to whether they inhibited sperm-zona pellucida tight binding as assessed by the hemizona assay (HZA). Of the 26 MAbs tested, only one inhibited zona binding. The whole sperm-specific MAb inhibited zona binding by 70%. The MAb also caused strong agglutination. Two procedures, Sephadex column chromatography and papain digestion, were used to determine whether agglutination or steric hindrance was a factor in the capability of MAb to inhibit zona binding. However, inhibition remained comparable to previous results. The MAb did not prevent capacitation, nor calcium influx and the resulting increase in hyperactivated motility and acrosome reaction. Since its inhibitory influence is not due to agglutination factors, steric hindrance or prevention of normal pre-fertilization maturation, the MAb may be blocking a portion of the zona binding receptor and may be useful in elucidating sperm antigens important to sperm-egg interaction. The approach used in this study allows definition of sperm surface antigens involved in zona pellucida binding. Key words: sperm antigen; zona pellucida; monoclonal antibodies; fertiliza-
tion; capacitation; calcium influx.
*This paper was presented in part at the 1990 Annual Meeting of the American Society of Andrology. Correspondence to: Nancy J. Alexander, Ph.D., National Institute of Child Health and Human Development, Center for Population Research, 600 Executive Plaza North, Bethesda, MD 20892, U.S.A.
0165-0378/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Elucidation of sperm antigens involved in the human fertilization process is important both for the development of a contraceptive vaccine and to treat infertile couples better. Characterization of monoclonal antibodies (MAbs) for their ability to block some component of the fertilization process offers one approach towards the identification of relevant sperm antigens (Anderson et al., 1987). One component of the fertilization process, the tight binding of sperm to the zona pellucida, occurs prior to zona penetration and subsequent fertilization (Hartmann and Hutchinson, 1974). Some monoclonal and polyclonal antibodies produced against non-human sperm antigens have been shown to block sperm-zona binding and, therefore, fertilization (O'Rand, 1981; Primakoff et al., 1988; Saling and Lakoski, 1985). Because sperm-zona interaction is species specific (Bedford, 1977), definition of human sperm antigens involved in zona binding will allow new approaches towards fertility control. Such assessment of human sperm-zona pellucida binding is possible using non-living oocytes (Overstreet and Hembree, 1976; Bronson et al., 1982). Recently, Burkman et al. (1988) reported a new method for evaluating human sperm-zona tight binding. With this hemizona assay (HZA), an oocyte is bisected to yield two matching zona halves or hemizona. The control hemizona provide an excellent internal control for the assay. In this study, we used the HZA to screen a panel of MAbs to human sperm antigens previously characterized in a World Health Organization (WHO) workshop (Anderson et al., 1987) for their capability to interfere with zona binding. Human sperm undergo a series of changes before fertilization can occur (Yanagimachi, 1988); antisperm antibodies that prevent sperm-zona binding may be doing so by interrupting some point in the pre-fertilization maturation of sperm (Trimmer et al., 1985; Saling, 1986). Any MAb that interfered with sperm-zona binding was also evaluated for its potential effects on capacitation, calcium influx, and the resulting increases in hyperactivated motility and the acrosome reaction. Materials and Methods
Monoclonal antibody selection A panel of MAbs to human sperm antigens supplied by a number of investigators was coded, diluted 1:10 in saline and evaluated as part of a WHO workshop (Anderson et al., 1987). The selection of the subset of these MAbs to be tested in this study for their ability to inhibit human sperm-zona pellucida tight binding was based on the results presented at that workshop.
Those MAbs that met the following criteria were included: a positive sperm agglutination (SA) titer, a positive sperm immobilization (SI) titer, or inhibition of sperm penetration of zona-free hamster oocytes (SPA) as reported by Anderson et al. (1987). Several MAbs with negative results for these assays were included.
Determination of sperm surface specificity The specificity of the MAb to the sperm surface was determined by a modified sperm mixed agglutination reaction (sperm MAR) (Coombs et al., 1956; Jager et ai., 1978). Amino functionalized polystyrene beads (diameter 5 ~ n ) (Polysciences, Warrington, PA) were activated with 8°70 glutaraldehyde in phosphate buffered saline, (PBS) pH 7.2 for 4--6 h at room temperature. One aliquot of beads was coated with mouse IgG (whole molecule, Pierce, Rockford, IL) and the other aliquot coated with mouse IgM (Organon, Teknida-Cappel, West Chester, PA). Each set of beads was blocked with ethanolamine and bovine serum albumin (BSA). After washing, the beads were stored in PBS with BSA, sodium azide and glycerol at 4°C until use.
Semen specimens obtained from healthy proven fertile donors were washed by centrifugation to remove seminal plasma. The motile fraction was collected by swim-up from the pellet for one hour into Ham's F-10 medium (Gibco, Grand Island, NY) supplemented with 3.5070 human serum albumin (HSA). The concentration of the motile fraction was adjusted to two million motile sperm/ml and incubated with the MAb added at the appropriate dilution for one hour at 37°C and 5070 C O 2 in air. The dilution was determined by the agglutination titer of the antibody as assessed by the microtray agglutination test (Friberg, 1974). When the antibody did not cause agglutination, a 1:10 dilution was used. Agglutinating antibodies were added at a dilution that resulted in less than 10070 motile agglutination. After incubation, antibody coated sperm were mixed on a microscope slide with rabbit anti-mouse Ig G,A,M (Zymed, San Francisco, CA) and the appropriate mouse Ig coated beads. The percentage of motile sperm coated with beads was determined after 5 min and 30 min incubation.
The hemizona assay: initial screening The HZA, as it is used for clinical testing, has been described in detail elsewhere (Burkman et al., 1988; Franken et al., 1989). H u m a n oocytes were obtained from cadaver tissues or excised ovarian tissue and were stored until use at 4°C in a solution of 1.5 M magnesium chloride with 0.1 °70 polyvinylpyrrolidone (MW 36,000) and 40 mM Hepes buffer to stabilize the pH at 7.0 (Yanagamachi et al., 1979; Yoshimatsu et al., 1988). Some oocytes were stored at - 70°C in a 2.0-M DMSO solution in PBS (Franken et al., 1989).
One day prior to use, the salt-stored or frozen oocytes were rinsed copiously in Biggers, Whitten and Whittingham (BWW) medium supplemented with 0.35070 BSA (Biggers et al., 1971). The oocytes were cut in half using Narishige micromanipulators (Narishige, Tokyo, Japan) mounted on a phasecontrast microscope (Nikon, Garden City, NY). The ooplasm was removed by pipetting each hemizona through a finely pulled pipette. Each pair of hemizona were stored overnight at 4 °C in a droplet of medium under mineral oil. Swim-up sperm were incubated with each MAb in the same manner as the sperm MAR. One hemizona of the pair was added to a 100-pl drop of the sperm with antibody, while the other hemizona of the pair was placed in a control drop of sperm to which no antibody had been added. After a 4-h coincubation, each hemizona was rinsed in fresh medium to dislodge loosely attached sperm, and the number of sperm tightly bound to the outer surface of the zona pellucida counted. The hemizona index (HZI) was calculated as follows: Number of sperm bound in the presence of antibody x
Number of sperm bound in the absence of antibody
The hemizona assay: additional evaluation Once the initial screen on the panel of MAb was completed, those antibodies that inhibited tight binding by at least 5007o were evaluated further. To determine that the inhibition was sperm specific and not caused by some other factor in the ascites fluid, the H Z A was repeated with one change. After incubation of swim-up sperm with the MAb, the sperm were washed by centrifugation. The supernatant was removed and replaced with fresh medium. The concentration of sperm was readjusted to two million motile sperm/ml and the H Z A repeated. In addition, for any antisperm MAb that still inhibited zona binding after washing to remove excess antibody, the H Z A was repeated using isotype matched non-sperm specific (as determined by the modified sperm MAR) mouse ascites (Organon Teknika-Cappel) added as control to swim-up sperm at a concentration equal to that used for the antisperm MAb. Sephadex G-200 column chromatography The optimal concentration of MAb to be added later to the column bed was determined by trial and error using the agglutination titer as a guide. When the amount of antibody was too great, sperm agglutinated in the column bed or in the collected fractions. When the concentration was too low, sperm were not coated with antibody.
At the start of the experiment, a 0.5 by 15 cm column was packed with swollen and degassed Sephadex G-200 particles (Pharmacia, Piscataway, NJ). After settling, the column was first rinsed with Ham's F-10 medium and then with H a m ' s F-10 supplemented with 3.5°70 HSA just prior to use. The MAb at the appropriate dilution was placed at the top on the column. Immediately after it entered the column, it was followed with Ham's F-10 supplemented with HSA to move the antibody into the column bed. Finally, swim-up sperm were added again followed by the medium. Fractions of motile sperm which did not exhibit motile agglutination were pooled, centrifuged and the concentration was adjusted with Ham's F-10 with 3.5070 HSA to 2 x 106 motile sperm/ml for the HZA. For a control, swim-up sperm were applied to a column to which no antibody had been added and prepared for use in the H Z A in the same manner as for the experimental group. Sperm from each group were washed with PBS and placed on glass spots of tefloncoated slides. To confirm the presence of antibody on the surface of the sperm, these slides were air-dried, methanol fixed and subsequently stained with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG,A,M (Zymed).
Isolation of lgG Fab fragments Use of Fab fragments with the H Z A was employed to answer any lingering questions regarding the effects of agglutination. In addition, it provided information as to whether inhibition of zona binding was a result of steric hindrance of the MAb or the binding to a specific zona binding antigen on the surface of the sperm. Any agglutinating MAbs that inhibited zona binding were papain digested to collect the Fab fragments. Immobilized papain was added to the MAb according to manufacturer's guidelines (Pierce, Rockford, IL). The reaction was stopped after 5 h by centrifugation to remove the immobilized papain. A Protein G column (Gaithersburg, MA) was used to collect the Fab fragments in the void fraction; the Fc fragment and undigested antibody bound to the column. The collected Fab fragments were dialyzed against PBS and lyophilized to concentrate. Polyacrylamide gel electrophoresis was performed to assess the recovered samples for adequate enzyme digestion. In order to insure that the recovered Fab fragments maintained their ability to bind to sperm without effecting sperm agglutination, sperm agglutination tests and indirect IF assays were performed utilizing the recovered Fab fragments. Finally the Fab fragments were incubated with swim-up sperm for 1 h, washed to remove excess fragments and used in the HZA.
Capacitation/chlortetracycline staining Capacitation was evaluated with a flourescence probe method using chlor-
IgG~, sperm were treated to induce the acrosome reaction by incubation with calcium ionophore. Both the control and experimental groups were capacitated for 3 h and then treated with a 10-/aM calcium ionophore A23187 in dimethyl sulfoxide for 30 min. After treatment, sperm were washed three times by centrifugation with PBS and placed on teflon coated spot slides (Roboz, Washington, D.C.) at a concentration of five million sperm/ml. Slides were air dried, fixed for 10 min in methanol and frozen at - 7 0 ° C until use. In addition to CTC staining, we used a monoclonal antibody, T6, to differentiate acrosome intact sperm from those that were reacted (Ochs et al., 1986). Acrosome-reacted sperm treated with T6 will exhibit only a bar across the equatorial segment. The entire cap will be stained in sperm that are not acrosome-reacted. Silver enhanced colloidal gold staining was completed according to the manufacturer's guidelines (Janssen, Olen, Belgium). Non-specific binding was blocked with 0.8°7o BSA, 0.1°70 gelatin (Janssen), 2 mM sodium azide and 5°7o normal rabbit serum in PBS, pH 7.4. The specimens were next incubated with the primary antibody, T6 (Humagen, Charlottesville, VA), washed and then incubated with biotinylated rabbit anti-mouse total IgG, IgA, IgM (Zymed, San Francisco, CA), each for 1 h. The specimens were stained with colloidal gold (5 nm) conjugated to streptavidin (Auroprobe, Janssen) for 1 h. After washing with PBS and double distilled water, silver amplification was completed for 15 min with IntenSEM (Janssen). Acrosomal status was assessed at 400 X or 1000 X (oil) magnification. A positive reaction was seen as a dark brown to deep black reaction product.
Sperm motion characterisitics Swim-up sperm, collected as previously described, was adjusted to two million/ml and incubated for 1 h at 37 °C and 5o7o CO 2 in air with the MAb, S19, or the IgG r Sperm motion was evaluated with the HTM-2030 Motility Analyzer (Hamilton-Thorn Research, Danvers, MA). Each control or experimental sperm sample was loaded by capillary action into a flat glass tube (0.2 m m deep) (Vitro Dynamics, Rockaway, N J). Each tube was transferred to the HTM-2030 where it was maintained at 37°C for 2 min prior to the start of data acquisition. Data collection was completed on randomly selected fields along the length of the flat tube until at least 100 motile sperm were analyzed. The pertinent settings used during the HTM assessment were: analysis duration of 0.67 s ( = 20 frames); minimum contrast = 7; minimum size = 6, "slow cells" were accepted as motile (slow gate = 5/am/s); H T M magnification factor = 2.13; low size gate = 0.4; high size gate = 1.6; low inten-
sity gate = 0.4; high intensity gate = 1.6. At the outset of each experiment, we verified that the settings permitted accurate differentiation of motile sperm versus non-motile sperm or debris by utilizing the "playback" option. During playback, the motions of sperm in the previous field were replayed: a red dot was located over the head of all motile sperm for each frame and a blue dot was positioned over the head of non-motile spermatozoa. When errors were detected, the settings were adjusted until the problem was corrected.
Hyperactivation To differentiate hyperactivated sperm from those that were not hyperactivated, the following settings were utilized in the automatic SORT program for the HTM-2030, as previously determined by Burkman (1990): track speed or curvilinear velocity (VCL, the velocity derived from all 20 head positions) = 100--500 /am/s; linearity (VSL/VCL, a measure of the straightness of the trajectory) = 0m65; maximal lateral head displacement (ALH, a measure of the side-to-side movement of the head) = 7.5m100; progressive velocity (VSL, the velocity based on the first and last head positions only) = 0--500/am/s; path velocity (VAP, five point running average) = 10--500/am/s. As sperm tracks were evaluated, any spermatozoon that had been selected as hyperactivated but was actually stuck by the tail to the capillary tube and not free swimming was excluded from the evaluation.
Statistical evaluation Data were statistically evaluated using either Student's t-test or two-way analysis of variance.
The initial screen for inhibition of sperm-zona pellucida tight binding as determined by the H Z A is presented in Table 1. Of the 26 antisperm MAbs tested, 6 inhibited tight binding by at least 50°70. The sperm MAR assay indicated high levels of antibody binding sperm in 4 of the 6 inhibiting MAbs. The inhibitory effect of the 6 MAbs was re-evaluated with the H Z A after washing to remove unbound antibody and other components of the mouse ascites (Table 2). The monoclonal antibody, S19, an antibody reacting with the whole sperm (data not shown), maintained its inhibitory effect after this procedure. It also caused a high degree of interference with sperm penetration of zona-free hamster oocytes (Anderson et al., 1987). The monoclonal antibody, S19, was compared with isotype matched nonsperm specific mouse ascites of the isotype, IgG~, at the concentration of S19
TABLE 1 C o m p a r i s o n o f h e m i z o n a a s s a y results w i t h o t h e r f u n c t i o n a l assays f o r a p a n e l o f W H O m o n o c l o n a l s (MAbs). F u n c t i o n a l assays MAb
Non-inhibitors o f zona binding" S03
S05 S06 S07
G3 M M
85.4 98.3 112.5
S09 S14 $20 $31
M M GI G2
4+ + -
109,6 83,8 88,2 91.5
$36 $37 $38 S39 $41
G1 G1 G1 G1 GI
-/-/+/+ +/+ -/-
+ + -
101.7 50.2 70.4 84.5 72.0
$42 $43 $44 $45
GI M M M
52.9 68.8 67.3 69.5
Inhibitors o f zona binding ~ S19 S05 $34 $58
GI G2a M G
+ + +
29.5 35.5 28.5 39.5
aThe results o f s p e r m a g g l u t i n a t i o n (SA), s p e r m i m m o b i l i z a t i o n (SI) a n d the h a m s t e r egg p e n e t r a t i o n test ( H E P T ) w e r e r e p o r t e d b y A n d e r s o n et al. (1987) as p a r t o f the W H O w o r k s h o p o n s p e r m antigens. b H Z A : T h e h e m i z o n a a s s a y - t w o to f o u r pairs o f H Z w e r e assessed f o r e a c h M A b . cNon-inhibiting: H Z I > 50. dInhibiting: H Z I < 50.
used in the previous experiments (2.2/~g/ml) (Table 3). Inhibition was still greater than 50%. Two methods were incorporated into the HZA to reassess zona binding in the absence of the agglutinating properties. First was the use of column chromatography, previously employed clinically to collect unagglutinated sperm from antisperm antibody-positive patient semen specimens (Kiser et al., 1987). Here a similar procedure was employed to collect motile unagglutin-
TABLE 2 Comparison of sperm-zona pellucida tight binding in the presence of antisperm monoclonal antibodies before and after treatment to remove excess antibody. Each monoclonal was added at a dilution that resulted in minimal or no motile agglutination. Hemizona index Monoclonal
(n = 4)
S04 $34 $58 $61 $65
(n (n (n (n (n
35.5 28.5 39.5 47.7 49.2
81.9 98.9 82.1 98.2 65.3
= = = = =
4) 3) 2) 4) 4)
ated but antibody-coated sperm for evaluation with the HZA. Since S19 strongly agglutinates sperm, the Sephadex G-200 column was used. The sperm collected were unagglutinated but coated with antibody as determined by indirect immunofluorescence. Their use in the HZA is presented in Table 4. Inhibition of sperm-zona binding after this treatment was maintained. Papain digestion of S19 yielded Fab fragments as determined by a decrease in banding at the 150 kDa level and the occurrence of a 50 kDa band with gel electrophoresis. The results of the HZA with Fab fragments indicated the inhibition was not due to agglutination or to steric hindrance but to epitope specificity (Table 4). Two-way analysis of variance indicated a significant decrease in spermzona tight binding in the presence of the MAb, S19, with this inhibition being consistent over all of the treatments (i.e. initial screening, against isotype matched ascites, papain digestion, etc.) (P < 0.001).
TABLE 3 Comparison of sperm-zona pellucida tight binding in the presence of an antisperm monoclonal antibody compared to non-specific isotype matched mouse ascites. S 19 was added at a dilution that resulted in minimal or no motile agglutination. Both S19 and IgG~ ascites were diluted to a concentration of 2.2 #g/ml. Hemizona index Monoclonal
Control sperm antibody
without IgG~ ascites
with IgG~ ascites
(n = 4)
Comparison of sperrn-zona pellucida tight binding in the presence of a sperm agglutinating monoclonal antibody before and after treatment to remove agglutinating properties. Hemizona index Monoclonal antibody
S19(n = 4)
•S19 was added at a dilution that resulted in minimal or no motile agglutination.
The results of CTC staining to assess the effects of MAb on capacitation are presented in Table 5. CTC staining differentiates pre-capacitated from capacitated and acrosome-reacted sperm. No difference was noted at either time point in the sperm patterns between the control and experimental groups. The addition of H f f to capacitated human sperm resulted in an immediate uptake of calcium that is observed in elevated levels of intracellular calcium (Blackmore et al., 1990). In the two donor specimens evaluated here, there were similar increases in intracellular calcium in the presence of both S19 and the control IgG 1 (Fig. 1). The intensity of response in each control and treated sample was comparable to the increase noted in capacitated sperm treated with H f f in the absence of either antibody (data not shown). No significant difference was noted in the percentage of sperm that reacted with acrosome in the presence of $19 when sperm were induced to acrosome-react (Fig. 2). The percentage of acrosome-reacted sperm was comparable for the control (56o70) and experimental (52o70) groups after the
Chlortetracycline staining to assess prefertilization maturation in the presence of the monoclonal antibody, S19. Time
Assessment of prefertilization stage (x ± S.D.)
°70 Acrosome reacted
23 ± 6 12 ± 8
76 ± 6 83 ± 3
1 -+ 1 5 _+ 4
1 ± 1 1 ± 1
90±3 94 ± 3
9±3 5 -+ 2
lh +IgG + S19
ITime is number of hours after the addition of either the monoclonal antibody, S19 or the isotype matched IgG I control ascites.
"" 400 + IgG 1 c o n t r o l L)
200 0 o'J 0
TIME ( S E C ) Fig. 1. Assessment of the ability of human follicular fluid to induce calcium influx in capacitated sperm treated with the monoclonal antibody, S 19.
80O ..t O e3
[r Swim up Sperm
4hr Capacitated Sperm
Calcium I0nophore Treated Sperm
Fig. 2. Comparison of acrosomal status of sperm, spontaneously reacted and with calcium ionophore induction, in the presence of the monoclonal antibody S 19.
classes that were non-sperm specific, as determined by the modified sperm MAR, were used in the H Z A at a similar concentration to that of the MAbs used in this study (Brucker et al., personal communication). No noticeable difference in binding was noted, a finding that suggests that isotype or subclass differences do not affect sperm-zona binding in this assay by non-specific means. Antibodies that agglutinate sperm result in decreased fertility by interfering with a number of reproductive functions (Alexander, 1984; Menge et al., 1984). Therefore, any inhibition of zona binding noted in the initial screen of agglutinating monoclonals could be a result of the agglutinating properties of the antibody. The influence of agglutinating antibodies in serum has correlated well with interference of sperm fertilizing capacity as determined by the zona-free hamster penetration assay (Dor et al., 1981). However, whether this effect was a result of the masking of important sperm antigenic sites or due to the agglutinating properties of the antibody was not determined. Sperm function was improved in the SPA once agglutination was minimized, even though antibodies were still present on the sperm surface (Kiser et al., 1987; Fulgham and Alexander, 1988). Both Sephadex column chromatography and papain digestion allowed assessment of possible inhibitory effects of agglutination. Bivalent antibodies can exhibit non-specific actions due to agglutination (Metz, 1972). For any agglutinating monoclonal antibody that inhibited zona binding in the inital screen, papain digestion was completed to yield a univalent Fab fragment incapable of agglutination for use in the HZA. Use of Fab fragments also permitted evaluation of the possibility that the MAb was sterically hindering sperm-zona binding (Menge and Black, 1979). Maintenance of the inhibitory effects of S19 on zona binding after these two treatments suggests that the MAb may be covering a portion of the receptor involved in zona pellucida binding. Another source of inhibition might be to prevent sperm maturation required prior to fertilization (Trimmer et al., 1985; Saling, 1986). It is therefore imperative to evaluate the potential inhibitory effects of a MAb on the other points in the pre-fertilization process when assessing sperm-zona binding. Results of CTC staining suggest that S19 did not affect the capacitation capacity. No difference was noted in the percentages of pre-capaciated, capacitated or acrosome-reacted sperm. However, by the end of the 1-h swim-up sperm preparation, when the sperm were first exposed to the antibody, few sperm remained pre-capacitated. While the treatment with antibody did not cause sperm to return to the pre-capacitated state, this particular assessment of capacitation may not be sensitive enough to detect less noticeable effects. Calcium influx is a prerequisite for both hyperactivation (Fraser, 1977) and the acrosome reaction (Thomas and Meizel, 1988). Here we initiated a
transient uptake of calcium with Hff. No difference was noted in the intensity of the reaction in either the control or experimental group, suggesting that the antibody was not affecting pre-fertilization maturation by blocking calcium channels. Calcium ionophore triggered the reaction to its expected levels as determined for these particular proven fertile donors. In addition, antibody treated sperm did not begin to undergo spontaneous acrosome reactions. This is somewhat in disaggreement with previous results obtained from the completion of the SPA (Anderson et al., 1987). This assay evaluates the ability of sperm to acrosome-react spontaneously over time. The results of that study indicated that in the presence of S19, penetration was inhibited, suggesting that the sperm were prevented from acrosome-reacting as capacitation occurred. However, the SPA assesses many other events, i.e., sperm-egg fusion and decondensation of nuclear material other than the ability to acrosome-react spontaneously over time. Inhibition of one of these other events may have resulted in the inhibition noted by the SPA. Some sperm motion parameters decreased slightly in the presence of S19. However, these results were not significantly different from those obtained in the presence of non-sperm specific isotype matched control ascites. While the possibility cannot be ruled out, the high degree of inhibition of zona binding noted for S19 (70%) did not appear to be due to its effect on sperm motion parameters. However, no difference was noted for the percentage of sperm with hyperactivated motility. While this increase in hyperactivation has not been shown to be necessary for the initial binding, it may be important to provide the thrust to penetrate the zona pellucida (Katz et al., 1986). Assessment of the pre-fertilization event may also provide information regarding the successful completion of capacitation (Gwatkin and Anderson, 1969; Yanagimachi, 1969). Our evaluations indicate that in the presence of S19, sperm followed normal patterns of pre-fertilization development such that they were competent to interact with the zona pellucida. That sperm in the presence of S19 were prevented from binding suggests that S19 may be specific for a portion of the zona binding receptor. It may ultimately be useful to elucidate the antigen(s) involved in human sperm-zona pellucida interaction. Taken in conjunction with other functional assays of reproductive physiology, the hemizona assay is a valuable method to aid in the selection of antigens important to the fertilization process.
Acknowledgements This work was partially supported by the Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School, under
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