Effect of sperm-associated antibodies on the dynamics of sperm movement and on the acrosome reaction of human spermatozoa

Journal o f Reproductive Immunology, 22 (1992) 59-72 Elsevier Scientific Publishers Ireland Ltd.

59

JRI 00770

Effect of sperm-associated antibodies on the dynamics of sperm movement and on the acrosome reaction of human spermatozoa R. Zouari, M. D e A l m e i d a a n d D. F e n e u x Laboratoire de Biologie de la Reproduction, Histologie-Embryologie-Cytogkn~tique, C.H.U. Bic~tre, 78 rue du G~n~ral Leclerc, 94275 Kremlin-Bicdtre Cedex (France) (Accepted for publication 10 January 1992)

Summary Anti-sperm antibodies were eluted from the sperm cell fraction of autoimmune human ejaculates and transferred onto normal motile spermatozoa. The movement and the acrosomal status of these antibody-coated spermatozoa were evaluated after incubation in a capacitating medium. The amplitude of lateral head displacement (ALH) and the straight line velocity (VSL) were analyzed using an HTM automated motility analyser. Acrosomal loss was monitored by an FITC-conjugated lectin binding technique. During the 6-h incubation in BWW-BSA medium, antibody-free and antibody-coated spermatozoa exhibited significant changes of ALH and VSL distribution that evolved differently in the two populations. The dynamics of sperm movement in control spermatozoa were apparently modified by the presence of antibodies on the sperm membrane. The low percentage of spontaneous acrosomal loss obtained in control populations, even after 20 h of incubation, was not modified by the presence of antibodies on spermatozoa. However, the same antibodies decreased the acrosomal loss induced by a calcium ionophore after 3 h of incubation in capacitating conditions. These results suggest that sperm capacitation and acrosome reaction, considered as essential for successful fertilization, can be altered by antisperm antibodies present on human ejaculated spermatozoa. Key words: autoantibodies; human spermatozoa; sperm movement," capacitalion," acrosome reaction Correspondence to." M. De Almeida, Laboratoire de Biologie de la Reproduction, HistologieEmbryologie-Cytog6n~tique, C.H.U. BicStre, 78 rue du G6n6ral Leclerc, 94275 Kremlin-Bic6tre Cedex, France. 0165-0378/92/$05.00 @ 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

60

Introduction

The presence of anti-sperm antibodies on the sperm surface may alter the fertilizing capacity of spermatozoa, as demonstrated by the behaviour of spermatozoa from men with anti-sperm autoimmunity. These antibodycoated spermatozoa have an impaired ability to penetrate human cervical mucus (Haas, 1986; Clarke, 1988) and human oocytes (Clarke et al., 1985; De Almeida et al., 1989). There has been some controversy on the possible effects of anti-sperm antibodies on sperm motility in semen (Cerasaro et al., 1985; Mathur et al., 1986; Barratt et al., 1989; Menge and Beitner, 1989). In fact, the presence of antibodies in men with low sperm motility may be an epiphenomen and not a direct cause of decreased motility. In order to investigate a direct effect of anti-sperm antibodies on sperm functions, several studies have been performed with serum antibodies passively transferred onto normal spermatozoa. These studies provided evidence that anti-sperm antibodies may affect different stages of the fertilization process, including sperm movement (Mathur et al., 1988; De Almeida et al., 1991), zona pellucida recognition and penetration (Bronson et al., 1982) as well as penetration of zona-free hamster oocytes (Bronson et al., 1981; Alexander, 1984). Some authors have also studied the movement characteristics of normal spermatozoa passively coated with antibodies obtained from seminal plasma (Mathur et al., 1988; Adeghe et al., 1989; De Almeida et al., 1991). A significant effect of these antibodies on sperm movement could not be demonstrated except by complement mediated membrane attack. As antibodies present in serum or in seminal plasma may not reflect those fixed on the surface of ejaculated spermatozoa (Bronson et al., 1987; Hellstrom et al., 1988), a more accurate picture of the effect of antibodies on sperm functions would be obtained using in vivo sperm-bound antibodies. Only these antibodies have the opportunity of altering the fertilizing ability of spermatozoa. We therefore coated normal spermatozoa with antibodies eluted from the sperm cell fraction of autoimmune ejaculates. In a previous study (De Almeida et al., 1991), we provided evidence for an effect of these sperm-eluted antibodies on the pattern of movement of normal motile spermatozoa after 1 h of incubation in non-capacitating conditions. Under these conditions sperm-eluted antibodies reduced the amplitude of lateral head displacement and increased the progressive velocity. As sperm capacitation and acrosome reaction are essential for successful fertilization, in the present study we investigated the effect of sperm-eluted antibodies on the dynamics of sperm movement as well as on the acrosomal status of spermatozoa incubated in a capacitating medium for 3-20 h.

61

Material and Methods

Source and preparation of sperm eluted samples Eight autoimmune ejaculates from spontaneously infertile men were selected according to sperm concentration ( > 5 x 10 7 cells/ml), motility (>40% progressive motility) and proportion of antibody-coated spermatozoa ( > 70%, as measured by the direct immunobead test) in order to elute the antibodies fixed on the sperm cells. Within 60 rain of collection, the semen samples were centrifuged at 600 × g for 10 min and the sperm cells washed twice with phosphate buffered saline (PBS). Sperm pellets were resuspended in 1-2 ml of 0.1 M glycine-HC1 buffer at pH 2.8 and incubated for 15 min at room temperature. After a 5-min centrifugation at 12 000 × g the supernatants were neutralised with Tris 3 M, dialysed against PBS and filtered through 0.22 t~m Millex GV sterile filters (Millipore, Polylabo, France). All eluted samples, when tested by the indirect immunobead test (IBT), had high levels of antibodies. In a previous experiment (not presented here), these samples were tested separately on the same spermatozoa. As the effects on sperm movement and on induced acrosomal loss were similar, the eight positive samples were put together in two pools in order to provide sufficient material for the different experiments reported here. The samples used as controls were prepared by pooling eluted material from 3-4 normal ejaculates with negative IBT results for IgG and IgA. In order to establish the adequacy of these controls, in one experiment the antibody activity of the tested autoirnmune eluted sample was absorbed by means of isospecific anti-human IgA and IgG immunobeads (Biorad, France). Five milligrams of the anti-IgG immunobeads were washed twice in PBS, resuspended in 300/zl of the auto-immune eluted sample and incubated at 37°C for 60 min with gentle shaking. After 2 min centrifugation at 12 000 × g, the supernatant was recovered and the same procedure was applied using anti-IgA immunobeads. The antibody activity became undetectable in this immunodepleted sample which was tested as control in parallel with the usual antibody-free sperm-eluted sample.

Sperm preparation and antibody transfer Semen samples were collected by masturbation from 6 normal healthy donors and allowed to liquefy for 30-60 min. After 2 washings (10 min at 600 x g) in BWW medium supplemented with 5 mg/ml bovine serum albumin (BSA; fraction V, Sigma Chimie, France), the sperm pellet was gently overlayed with 0.5-1 ml of BWW-BSA and incubated at 37°C in an atmosphere of 5°,/0 CO2 in air for 60 rain to achieve a swim-up migration of highly motile spermatozoa. The upper layer, containing more than 90%

62

motile sperm cells, was carefully recovered and the final concentration of the migrated cell suspensions was adjusted to 2 × 107/ml. Four hundred microliters of this suspension were added to 800 #1 of antibody-free and antibody-positive sperm-eluted samples and incubated for 60 min at 37°C. Under these experimental conditions over 90% of migrated spermatozoa were coated with antibodies without apparent agglutination following their incubation in the autoimmune sperm-eluted samples. Sperm suspensions were washed twice (5 min at 600 × g), resuspended in BWWBSA and incubated at 37°C under 5% CO2 in air to promote in vitro capacitation. lmmunobead test

Twenty microliters of each sperm suspension were washed twice with BWW-BSA and resuspended in 50 /~1 of medium. Ten microliters of this suspension were placed on a glass slide, mixed with the same volume of isospecific anti-IgA or anti-IgG immunobeads prepared as previously described (De Almeida et al., 1986) and covered with a 22-mm coverslip. After 10 min incubation in a moist chamber at room temperature, the percentage of motile spermatozoa coated with the immunobeads was scored and the site of immunobead binding was recorded. Measurement o f movement parameters

Sperm movement analysis was performed before the incubation period in BWW-BSA (TO) and after 3 h (T3) and 6 h (T6) incubation in the same medium, by using an HTM-2030 motility analyzer (Hamilton-Thorn Research, Danvers, MA). A 6-tzl drop of sperm preparation was deposited on a Makler chamber (10/~m depth) that was loaded into the analyser at 37°C. Different fields of the same preparation were selected, giving a measurement of 100-200 cells. Two drops of the same sample were examined and the final result was expressed as the mean of the two measurements. The settings used during the analysis were: frames acquired, 20; frame rate, 25/s; minimum contrast, 8; minimum size, 2; low size gate, 0.4; high size gate, 1.6; low intensity gate, 0.4; high intensity gate, 1.6. The validity of this set up was repeatedly verified by using the 'playback' option that permitted the differentiation of motile spermatozoa from non-motile ones or from debris. The movement parameters studied were the straight line velocity (VSL) expressed in ~m/s and the amplitude of lateral head displacement (ALH) expressed in ~m. Distributions rather than mean values were determined for each parameter as values averaged over the entire population do not appreciate the evolution of subpopulations of cells in the same sperm sample. Acrosomal assessment

Spontaneous acrosome reaction was assessed in antibody-free and

63

antibody-coated spermatozoa at TO, T3, T6 and T20 of incubation period in BWW-BSA. Acrosome reaction was induced in spermatozoa preincubated 3 h in BWW-BSA to promote capacitation by adding divalent calcium ionophore (free acid Ionomycin; Calbiochem) dissolved in DMSO at 10 ~M final concentration for 60 min. The proportion of spontaneous and ionophore-induced acrosome reactions were evaluated by using the FITC-conjugated lectin staining technique described by Cross et al. (1986). Briefly, sperm suspensions were incubated with the supravital stain Hoechst 33258 (H258, Sigma) to label dead sperm with disrupted membranes and then washed through a solution of Polyvinylpyrrolidone-40 (PVP-40, Sigma) in PBS to remove excess H258. The sperm pellets were fixed and permeabilised in ethanol, dried onto a microscope slide, labeled with FITC conjugated Lens culinaris Agglutinin (FITC-LCA, Sigma) and finally mounted in a Glycerol-PBS solution (Citifluor, London, U.K.). The sperm cells were examined with an Olympus BH2 microscope equipped for epifluorescence. Acrosomal status was assessed only in live sperm populations (that excluded H258). Cells with uniform fluorescent acrosome were scored as acrosome intact. Those showing no fluorescence or only a fluorescence of the equatorial segment were scored as acrosome reacted. At least 200 spermatozoa were examined for each determination. Statistical analysis The Chi-square test was used to compare the sperm distribution of VSL and A L H and the percentage of induced acrosome reaction between control and test samples. Results

Immunobead test results Immunobead testing of donors' spermatozoa incubated with the pooled immune samples showed 70-100°/'0 of sperm cells coated with both IgA and IgG antibodies in the two pools; immunobeads were localized mainly over the head with pool I and over the whole of the spermatozoon with pool II. Time dependent effect o f anti-sperm antibodies on sperm movement In order to evaluate the effect of antibody-binding on the dynamics of human sperm movement associated with capacitation, normal motile spermatozoa were coated with sperm-eluted antibodies and incubated for 6 h in capacitating conditions. Two experiments were performed using two different pools of sperm-eluted antibodies as described in Materials and Methods. In each experiment the same pool was tested with the spermatozoa of two different donors. One pool of antibody-free eluted samples was used

EXPERIMENT

1

Sperm 1

-==

£

TO

T3

T6

<40 40-80 >80

<40 40-80 >80 VSL(tun/s) T3

<40 40-80 >80

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EXPERIMENT

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2

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<40 40-80 >80

Sperm 4

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40 30 20

m " <40 40-80 >80

65

as control in both experiments. Movement parameters were analyzed just before the incubation period (TO) and after 3 h (T3) and 6 h (T6) of incubation and the results illustrated by distributions of VSL and A L H values in three classes: < 4 0 prrds, 40-80 prn/s and >80/zrn/s for VSL; <4/~m, 4 - 8 /~m and > 8/~m for ALH. At TO, following antibody-labeling and before the incubation under capacitating conditions, the presence of antibodies on the sperm surface already induced some changes in sperm movement parameters, as expected from previous results (De Almeida et al., 1991). The proportion of fast moving spermatozoa (VSL > 80 pm/s) was decreased in sperm populations 2 and 4 in the presence of antibodies from pools I and II (Fig. 1). The values of A L H were diversely affected by the same antibodies in sperms 1 and 3 (Fig. 2). The ratio of spermatozoa with A L H < 8/zm was increased in sperm I, whereas in sperm 3 there was a higher percentage of spermatozoa with A L H > 4 #m. During the 6-h incubation in BWW-BSA, antibody-free spermatozoa exhibited significant changes of A L H and VSL that varied from one sperm to another, as reported by others (Morales et al., 1988; Robertson et al., 1988; Mack et al., 1989). In our experimental conditions, VSL values changed to a significant increase of the proportion of spermatozoa moving at 40-80 #m/s (sperms 1, 2 and 3) or over 80/~rn/s (sperm 4). These changes occurred at T3 with sperms 1, 3 and 4 and at T6 with sperm 2. With regard to ALH values, there was an evolution towards an increased percentage of spermatozoa with A L H > 4 pm at T3 in three out of the four sperm populations (sperms 2, 3 and 4). With sperm 1, we observed a decreased proportion of spermatozoa with A L H > 8 #m. In the presence of antibodies, the distribution of the analysed parameters altered from the controls, resulting in the prevalence of different sperm classes at the end of the incubation period. Antibody binding to sperm 1 resulted in an increase of the slowest spermatozoa at the expense of those moving over 80 pro/s, whereas the same antibodies on sperm 2 caused a shift of sperm cells from class 2 (40-80/,m/s) to class 3 ( > 80/~m/s) when com-

Fig. 1. Temporal changes in VSL (straight line velocity) distribution of migrated spermatozoa exposed for 60 min to sperm-eluted samples with (11, test) or without (1"1, control) antibodies, washed (TO) and incubated in BWW/BSA medium for 3 h (T3) and 6 h (T6) at 37°C under 5% CO 2 in air. Two experiments were performed using two pools of sperm-eluted antibodies (pool I in experiment 1, pool I1 in experiment 2) and one pool of antibody-free samples. Each pool was tested with two different sperm populations. *P < 0.05, **P < 0.01, ***P < 0.001 using X2 test. a,b: significant difference between TO and T3, respectively, in control and test sample; c,d: significant difference between T3 and T6, respectively, in control and test sample; e,f,g: significant difference between control and test sample at TO, T3 and T6, respectively.

66

EXPERIMENT

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ALH(Iam) EXPERIMENT

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,

67

pared to VSL distribution in the respective controls at T6 (Fig. 1). Antibodies apparently did not affect the VSL values either in sperm 3 or in sperm 4, in spite of the initial (TO) effect registered in sperm 4. The changes observed in the distribution of ALH values in antibody-coated spermatozoa (Fig. 2) resulted in a decrease of the narrowest sperm head trajectories ( < 4 txm) in favour of one of the two other classes in the four sperm populations tested. This change became significantly different from the control at T3 with sperm 4 and at T6 with sperm 1, 2 and 3. We may summarize these results by saying that during the 6-h incubation in BWW-BSA, antibody-free spermatozoa exhibited significant changes of ALH and VSL distribution that differed from one sperm population to another, as expected. Antibody binding to the same spermatozoa interfered with these dynamic changes, mainly at T6, resulting in increased percentages of spermatozoa with large ALH. VSL values were diversely affected by the antibodies. Time-dependent effect of anti-sperm antibodies on the acrosome reaction Throughout the experiments described above, the acrosomal status was assessed at different times of the incubation period in order to test the ability of antibody-coated spermatozoa to undergo an acrosome reaction as an indicator of successful capacitation. The low percentages of spontaneous acrosomal loss observed in antibody-free sperm suspensions after 3 h (2.2 ± 0.6), 6 h (4.4 4- 2.8) and 20 h (6.9 4- 3.1) of incubation in capacitating conditions were not modified by the preincubation of the same sperm suspensions with sperm eluted antibodies (Table 1). We therefore investigated the effect of the same antibodies on the ionophore induced acrosome reaction (Fig. 3). Ionomycin was added for 60 min at 10 #M to antibody-free and antibodycoated sperm populations preincubated for 3 h at 37°C under 5% CO2 in air to promote capacitation. Six experiments were done using the sperm of five different donors (same donor in experiment A and D) and two pools of sperm-eluted antibodies (pool I in experiments A, B and C; pool II in experiments D, E and F).

Fig. 2. Temporal changes in ALH (amplitude of lateral head displacement) distribution of migrated spermatozoa exposed for 60 min to sperm-eluted samples with (1, test) or without (r-I, control) antibodies, washed (TO) and incubated in BWW/BSA medium for 3 h (T3) and 6 h (T6) at 37°C under 5% CO, in air. Two experiments were performed using two pools of sperm-eluted antibodies (pool 1 in experiment !, pool II in experiment 2) and one pool of antibody-free samples. Each pool was tested with two different sperm populations. *P < 0.05, **P < 0.01, ***P < 0.001 using x 2 test, a,b: significant difference between TO and T3, respectively, in control and test sample; c,d: significant difference between T3 and T6, respectively, in control and test sample; e,f,g: significant difference between control and test sample at TO, T3 and T6, respectively.

68 TABLE 1 Time-dependent acrosome reaction response of antibody-free and antibody-coated spermatozoa. Incubation time in B W W (h)

Acrosome reaction (%)a Control

Test

0 3 6 20

2.3 2.2 4.4 6.9

3.3 2.2 3.0 6.5

q± ± ±

0.9 0.6 2.8 3.1

q± ± +

1.9 1.3 0.8 5.8

aMigrated spermatozoa from healthy donors were exposed for 1 h to sperm-eluted samples with (Test) or without (Control) antibodies and then washed in BWW/BSA medium before being incubated in this medium for 3 h, 6 h and 20 h at 37°C under 5% C O 2 in air. Values are the mean 4- S.D. from 4 experiments done with different sperm populations and two pools of sperm-eluted antibodies.

The rate of induced acrosomal loss, which varied from 14% to 53% in control spermatozoa was decreased to 3.5-26% after antibody-binding. This decreasing effect reached statistical significance in experiments A, B and D where antibody-free spermatozoa showed the highest percentages of induced acrosomal loss (32.5-53%). When the same spermatozoa were tested with the two pools (exp. A and D) a clear decrease of the percentage of acrosomal loss was observed in both experiments.

50

1

30

~

Control Test

0 A

B

C

D

E

F

Experiment Fig. 3. Ionophore-induced acrosome reaction response of antibody-free and antibody bound spermatozoa preincubated for 3 h at 37°C under 5% CO 2 in air. Ionomycin was added for 60 min at 10/~M. Six experiments were done using the sperm of five different donors (same donor in experiment A and D) and two pools of sperm-eluted antibodies (pool I in experiments A, B and C; pool II in experiments D, E and F). ***P < 0.001 vs. control using X2 test.

69

Immunoabsorption experiment In one experiment, an aliquot of an antibody-positive sperm-eluted sample was incubated with anti-IgG and anti-IgA immunobeads, before testing for sperm movement and ionophore induced acrosome reaction. The immunodepleted sample lost its ability to modify the tested sperm functions. There was no notable difference in the movement parameters and the percentage of acrosome reaction between spermatozoa incubated with the control antibody-negative sample (used through the experiments) and those incubated with the absorbed antibody-positive sample (results not shown). Discussion

Before acquisition of fertilizing ability, the plasma membrane of mammalian spermatozoa must undergo a variety of subtle changes known as capacitation. These plasma membrane changes are associated with an increased permeability to calcium present in the extracellular environment (Singh et al., 1978) leading to modifications of the sperm motility pattern and to the induction of the acrosome reaction in the immediate vicinity of the oocyte. These modifications of membrane ion conductance may vary in relation to the functional state of spermatozoa, resulting in appreciable heterogeneity within the sperm population and also between different sperm populations. This heterogeneity has been demonstrated for movement characteristics (Mack et al., 1989) and for the acrosome reaction (Byrd and Wolf, 1986; Mortimer et al., 1989). The presence of anti-sperm antibodies on the sperm surface may prevent the surface modifications during capacitation and thereby impair the capacitation-related changes of sperm motion and acrosomal state. In this study we investigated the functional effects of two pools of spermeluted antibodies on the sperm movement as well as on the acrosomal loss of normal motile spermatozoa incubated under capacitating conditions. Swim-up migrated spermatozoa obtained from different donors were incubated for 1 h in sperm-eluted samples, washed and further incubated for 3 h to 20 h in BWW-BSA. Just before this incubation period, sperm movement was already modified by antibody-binding as was described earlier after 1 h of incubation in a non-capacitating medium (De Almeida et al., 1991). Under these conditions the majority of sperm-eluted samples exhibited increased VSL and decreased A L H mean values. In the present study, the percentage of fast moving spermatozoa (VSL > 80 #rn/s) was significantly decreased by antibodies in 2 out of 4 sperm populations tested, whereas ALH values were diversely affected by the same antibodies in sperm populations 1 and 3. During capacitation, several temporal changes were observed in the distributions of VSL and A L H values in control spermatozoa, as

70

reported in studies on human sperm capacitation in vitro (Morales et al., 1988; Robertson et al., 1988). The dynamics of these changes were apparently modified by the presence of antibodies on the sperm membrane. In the majority of the sperms, the distribution of A L H values changed towards an increased lateral motion of the sperm head in both antibody-free and antibody-coated populations. However, the maximal changes occurred later (6 h) in antibody-coated spermatozoa compared with control spermatozoa (3 h). This could be related to a delayed development of capacitation. The changes in control VSL values that varied from one sperm population to another were not significantly modified in the two sperm populations incubated with antibodies from pool II. In sperms 1 and 2 antibodies from pool I induced opposite changes in the percentage of rapid moving cells at T6. These results suggest that anti-sperm membrane specific antibodies may interfere with the dynamics of capacitation as expressed by temporal changes in movement characteristics. If so, the same antibodies might be expected to influence the initiation of the acrosome reaction. In rodents, antibody binding to specific antigenic determinants on the sperm plasma membrane was shown to alter their redistribution and so to prevent the occurrence of the acrosome reaction (Tung et al., 1980; Saling, 1986; Marquant Le Guienne et al., 1986). In humans, the few published reports are rather conflicting. The acrosomal status of normal spermatozoa exposed to serum antibodies was either not changed (Bronson et al., 1989) or diversely affected by these antisperm antibodies (Lansford et al., 1990). In the present study, we found no significant effect of sperm eluted antibodies on spontaneous acrosomal loss which was very low in antibody-free spermatozoa (6.9 ± 3.1 at 20 h of incubation). A calcium ionophore was used in order to test the ability of antibody-coated spermatozoa to undergo induced acrosomal loss. The application of 10/~M of Ionomycin after 3 h of capacitation resulted in 15-53% acrosome reaction in control spermatozoa. These percentages were decreased by sperm-associated antibodies, but the extent of this decrease was variable from one experiment to another. Significance was reached in the experiments showing the highest percentages of induced acrosomal loss in control spermatozoa. Apparently, the intensity of this decreasing effect was dependent to a greater extent on the sperm tested than on the pool of antibodies used. This antibody blockage of the acrosome reaction induced by the calcium ionophore suggests that membrane permeability to external C a 2÷ is affected by antibody binding. In sea urchin spermatozoa, a monoclonal antibody directed against a protein involved in Ca 2÷ channel opening was shown to inhibit the egg jelly-induced acrosome reaction and associated Ca 2÷ influx (Trimmer et al., 1985). The polyclonal antibodies used in this study were directed against different domains of the human sperm surface. Some of these antibodies may bind to an epitope critical for ion conductance,

71

although the results presented here suggest a non-specific stabilizing effect linked to the presence of large immunoglobulin molecules on the sperm membrane surface. Both mechanisms may in fact result in decreased permeability to external Ca 2÷ directly related to changes in flagellar motion and impaired acrosome reaction. This hypothesis is strengthened by a recent publication on impaired Ca 2÷ uptake in human spermatozoa in the presence of anti-sperm antibodies (Mahony et al., 1991). Some observations emphasize that serum antibodies can have opposing actions on sperm functions depending upon the class of antibodies and the distribution of the specific antigen(s) on the plasma membrane (Bronson et al., 1989). The results presented here do not allow us to argue on these two points as several sperm-eluted antibody samples were put together in two pools, both containing IgA and IgG antibodies localised over the entire membrane (pool II) or mainly over the sperm head (pool I). In any case, previous experiments done with isolated sperm-eluted samples (De Almeida et al., 1991) did not show any correlation between antibody class or localization, as detected by immunobeads and effects on sperm movement. The data presented here suggest that capacitation-related changes of human sperm motion and ionophore-induced acrosome reaction can be modified by sperm membrane specific antibodies eluted from the ejaculated spermatozoa of some infertile men. Further studies are necessary to determine the precise mechanism of these effects, mainly at the level of membrane ionic exchanges and their importance in sperm-oocyte interactions.

Acknowledgement This research was supported by INSERM CJF 88010.

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