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Binding of antisperm isoantibodies from a woman at risk for immunologic infertility to intraacrosomal components of donor spermatozoa
Vol. 44, No.1, July 1985 Printed in U.8A.
FERTILITY AND STERIUTY Copyright c 1985 The American Fertility Society
Binding of antisperm isoantibodies from a woman at risk for immunologic infertility to intraacrosomalcomponents of donor spermatozoa
Jan Tesatik, M.D., Ph.D.*t Pavel Travnik, M.D., Ph.D.t . Lenka Mecova t Ladislav Pilka,M.D., Ph.D.t Faculty of Medicine, J. E. Purkyne University, Brno, Czechoslovakia
The poor correlation of the commonly employed assays for circulating antisperm antibodies, including sperm agglutination, immobilization, and immunofluorescence, with the presence of infertility! is probably related to the inability ofthese methods to distinguish ·the specific sperm antigenic sites to which the potentially present antibodies are directed and whose blockade mayor may not alter the sperm function. The need for an individual assessment of the specificity of the sperm-reactive antibodies in the evaluation of their contribution to reproductive failure has recently been stressed. 2 Using a screening assay for antisperm antibodies in sera from infertile women, based on an indirect immunofluorescent test, we have detected, in some cases, a strong fluorescence limited to heads of only a small fraction of the sperm population, suggesting an intracellular localization of the antibody-binding sites. This is a preliminary report of binding of naturally occurring antisperm isoantibodies from one of these suspect sera to intraacrosomal components of do-
Received August 7, 1984; revised and accepted March 28
W~
,
*Reprint requests: Dr. Jan Tesatik, Department of Histology and Embryology, Faculty of Medicine, J. E. Purkyne University, tf. Obrancd miru 10, 66243 Brno, Czechoslovakia. tDepartment of Histology and Embryology. :j:First Department of Obstetrics and Gynecology. 138
Tesaiik et aI. Communications-in-brief
nor spermatozoa as revealed by an immunoperoxidase method at the ultrastructural level. MATERIALS AND METHODS Co~mercially available immunoglobulin (Ig)G fractIOns from swine antisera against human IgG, IgA, and IgM conjugated with fluorescein isothiocyanate (FITC) or with peroxidase were used. The IgG fractions were purified by affinity chromatography and conjugated with FITC (molar ratio, FITC/protein, 2.5) or with horseradish peroxidase (molar ratio, peroxidase/Ig, 1.5) by the manufacturer (Sevac, Prague, Czechoslovakia). The investigated human sera were heated to 56°C for 30 minutes and stored at - 40°C until tested. Semen samples were obtained from fertile donors. All were judged normal by standard criteria of volume, sperm concentration, and motility. Following liquefaction at room temperature (20 minutes), 0.5 ml of each ejaculate was mixed with 2 ml ofDulbecco's phosphate-buffered saline (PBS), and spermatozoa were washed three times by repeated cycles of centrifugation (500 x g, 5 minutes) and resuspension of sperm pellets in 2 ml of PBS. For detection of passive antibody transfer at the light-microscopic level, smears were prepared from the washed spermatozoa. The smears were allowed to air-dry and were exposed to the patients' sera. One milliliter of serum was pipetted on each slide, and incubation was carried out in a
Fertility and Sterility
wet chamber with 100% humidity at room temperature for 2 hours. After a thorough washing in PBS, the smears were incubated with FITC-Iabeled anti-IgG, anti-IgA, and anti-IgM at room temperature for 2 hours. The three specific antiIgs were applied separately for each patient's serum tested. The concentration of the specific antiIg was approximately 0.2 mg/ml. The slides were then washed again and examined under a fluorescence microscope at a magnification of 500 x with the use of oil immersion. Control smears were incubated either with sera from women of proven fertility instead of the patients' sera or directly with FITC-Iabeled anti-Ig for the exclusion of nonspecific binding of both the first-layer and the second-layer antibodies as well as the presence of autoantibodies on the spermatozoa used in the test system. An attempt was made to evaluate the effect of sperm incubation for capacitation in vitro on the exposure of intracellular antigens. Motile spermatozoa were selected by "swim up" and capacitated in vitro as described in detail previously.3 Smears were prepared and processed as indicated above. For electron-microscopic localization of the sperm-bound antibodies, washed spermatozoa or those incubated for in vitro capacitation were resuspended in PBS to an approximate cell count of 10 to 20 x 106/m l. One milliliter of the living sperm suspension was then incubated with 1 ml of the patient's serum at room temperature for 2 hours. After three cycles of centrifugation at 1000 x g for 5 minutes and resuspension in PBS, the sperm were pelleted again; and 2 ml of peroxidase-conjugated anti-Ig diluted to a final concentration of 0.2 mg specific antibody/ml PBS was added. Incubation was carried out at room tempe.:ature for 2 hours. After washing in PBS, the sperm were fixed in 0.1 M cacodylate-buffered 3%' glutaraldehyde for 30 minutes, washed in PBS, and incubated under continuous agitation in medium consisting of 5 mg of 3,3' -diaminobenzidine tetrachloride (DAB; Sigma Chemical Company, St. Louis, MO) as peroxidase substrate, 0.1 ml of 1% H 2 0 2 , and 10 ml of 0.05 M Tris HCI buffer (pH 7.5) for 30 minutes. Specimens were then washed in PBS, fixed with 1% OS04 in 0.1 M cacodylate buffer, embedded in Durcupan (Fluka AG, Buchs, Switzerland), sectioned with the use of an Ultrotome III LKB ultramicrotome (LKB-Produkter AB, Bromma, Sweden), and examined in a Tesla BS 500 electron microscope (Tesla, Bmo, CzechoVol. 44, No.1, July 1985
slovakia). Appropriate control incubations with sera of fertile women and with specific anti-Igs alone were carried out.
RESULTS A 28-year-old childless woman with a 6-year history of unexplained infertility was investigated. Screening assays for circulating antisperm antibodies in the patient's serum with the use of modified microagglutination4 and immobilization 5 tests were negative. However, strong fluorescence restricted to heads of - 10% of spermatozoa was found after successive exposure of smears of spermatozoa from three donors to the patient's serum and fluorescein-conjugated anti-IgG. The reactions with both anti-IgA and anti-IgM were negative. All control incubations also gave negative results. After incubation of spermatozoa for capacitation in vitro, the occurrence of antibodybinding sperm was increased approximately twice in comparison with nonincubated spermatozoa. The electron-microscopic examination of donor spermatozoa exposed to the patient's serum and treated with peroxidase-conjugated anti-IgG did not reveal any binding of antibodies to intact sperm cells, with the exception of occasional labeling of small areas in the sperm head postacrosomal region (Fig. 1A). On the other hand, spermatozoa having lost their acrosomes showed a massive binding of antibodies to the inner acrosomal membrane (Fig.1B). In contrast to spermatozoa with intact acrosomes, t4e acrosome-lost spermatozoa often showed a significant labeling of the remaining intact portions of the plasma membrane in the equatorial and postacrosomal regions of the sperm head. N~ither the plasma membrane of the sperm tail nor the nuclear membrane was labeled, even when the latter was partially denuded and thus apparently exposed to the patient's serum (Fig. 1B). Interestingly enough, when spermatozoa just undergoing an acrosome reaction were examined, the site of antibody/antigen interaction could be localized not only on the inner acrosomal membrane; the reaction product was associated with membrane vesicles formed during the acrosome reaction and with remnants of the acrosomal content as well (Fig. 1C). No reaction product was ever observed on either intact or acrosome-lost spermatozoa incubated with control sera or directly with peroxidase-conjugated anti-IgG. The proportion of laTesatik et al. Communications-in-brief
139
Figure 1 (A), Head of an intact spermatozoon showing the absence of DAB reaction product except for small patches on the postacrosomal region plasma membrane (arrowheads); acrosome (arrows) (x 20,000). (B), Head of an acrosome-lost spermatozoon with large amounts of DAB reaction product attached to the inner acrosomal membrane (a) . Persisting remnants ofthe plasma membrane are also labeled in the equatorial and postacrosomal regions (arrows). No reaction product is present either on the tail midpiece (t) or on remnants of the plasma membrane of the anterior acrosomal region (p) . Note the absence oflabel on the partially denuded nuclear membrane (arrowhead) (x 20,000). (C), Head of a spermatozoon in the course of acrosome reaction. In addition to DAB reaction product on the inner acrosomal membrane (a), it is also associated with membrane vesicles (arrows) originating during the acrosome reaction and with remnants of the acrosomal content (arrowheads) (x 20,000).
beled spermatozoa was similar to that assessed in the immunofluorescent test. DISCUSSION
This report demonstrates that naturally occurring ·antisperm isoantibodies from human sera can be directed to antigens located within the acrosome of human spermatozoa. The antibody binding described in these experiments apparently involves both dead spermatozoa and those undergoing or having undergone an acrosome reaction, the two conditions exposing intraacrosomal antigens which are sequestered by an impermeable plasma membrane in living acrosome-intact spermatozoa. The significant extension of the antibody-binding area of the sperm head postacro140
Tesatik et al. Communications-in-brief
somal region plasma membrane after the acrosome loss or a spontaneous acrosome reaction may reflect changes in antigenic properties of this area of the sperm surface because of sperm death or the acrosome reaction. Naturally occurring antibodies to intracellular components of sperm are relatively ubiquitous. 6 They may cross-react with bacterial antigens and probably play no role in infertility in most cases. Antigenic sites within the acrosome or on acrosomal membranes, however, are exposed to antibodies potentially present at the site of fertilization before spermatozoa are able to penetrate the egg. Hence, the participation of antibodies against the intraacrosomal antigens in immunologic infertility should be taken into account. Furthermore, the possibility that the antibodies Fertility and Sterility
detected on the acrosomal region of spermatozoa with the use of the common fluorescent antibody techniques are in fact bound to some intraacrosomal components and not to the sperm surface should always be borne in mind, especially where only a subpopulation of sperm is stained. How spurious conclusions can be made, assuming that these procedures detect molecules only on the sperm surface, has recently been pointed out. 7 The immunofluorescent test was performed in this study only as a preliminary screening assay for antisperm antibodies because of its relatively high sensitivity and simplicity. Electron microscopy with the use of labeled anti-Ig antibodies is the most efficient method for determination of the regional specificity of antibody/antigen interaction and in the precise localization of sperm intracellular antibody-binding sites. The use of in vitro capacitation, allowing additional spermatozoa to expose their intraacrosomal antigens during a spontaneous acrosome reaction, and comparing the proportion of antibody-bound sperm with a fresh sample with the use of an immunofluorescent test would be a simple screening assay for acrosome-directed antibodies. The electronmicroscopic immunoperoxidase technique, as a more specific method, could be used with suspect sera to exclude false-positive cases and those with antibodies to sperm intracellular antigens other than intraacrosomal. From our results, it is not quite clear whether the antigenic sites on the acrosomal membrane include the membrane's integral proteins or whether they are merely components of the acro-
Vol. 44, No.1, July 1985
somal content bound to the membrane surface. Labeling ofthe acrosomal content in spermatozoa undergoing an acrosome reaction suggests that these antigens may be primarily located in the acrosomal matrix and remain associated with the acrosomal membrane for a certain time period after an artifactual acrosome loss or after a spontaneous acrosome reaction. Studies are in progress aimed at characterization of these antigens and evaluation of the effect of their blockade on sperm/egg interaction in vitro. REFERENCES 1. Jones WR: Immunologic infertility-fact or fiction? Fertil
Steril 33:577, 1980 2. Bronson R, Cooper G, Rosenfeld D: Sperm antibodies: their role in infertility. Fertil Steril 42:171, 1984 3. Tesatik J, Kopecny V, Dvorak M: Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro. Fertil Steril 41:919, 1984 4. Franklin RR, Dukes CD: Antispermatozoal antibody and unexplained infertility. Am J Obstet Gynecol 89:682, 1964 5. Isojima S, Li TS, Ashitaka Y: Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained sterility. Am J Obstet GynecollOl:677, 1968 6. Tung KSK, Cooke WD Jr, McCarty TA, Robitaille P: Human sperm antigens and antisperm antibodies. II. Age-related incidence of antisperm antibodies. Clin Exp Immunol 25:73, 1976 7. Jones R, Brown CR, Cran DG, Gaunt SJ: Surface and internal antigens of rat spermatozoa distinguished using monoclonal antibodies. Gamete Res 8:255, 1983
Tesafik et aI. Communications-in-brief
141
FERTILITY AND STERIUTY Copyright c 1985 The American Fertility Society
Binding of antisperm isoantibodies from a woman at risk for immunologic infertility to intraacrosomalcomponents of donor spermatozoa
Jan Tesatik, M.D., Ph.D.*t Pavel Travnik, M.D., Ph.D.t . Lenka Mecova t Ladislav Pilka,M.D., Ph.D.t Faculty of Medicine, J. E. Purkyne University, Brno, Czechoslovakia
The poor correlation of the commonly employed assays for circulating antisperm antibodies, including sperm agglutination, immobilization, and immunofluorescence, with the presence of infertility! is probably related to the inability ofthese methods to distinguish ·the specific sperm antigenic sites to which the potentially present antibodies are directed and whose blockade mayor may not alter the sperm function. The need for an individual assessment of the specificity of the sperm-reactive antibodies in the evaluation of their contribution to reproductive failure has recently been stressed. 2 Using a screening assay for antisperm antibodies in sera from infertile women, based on an indirect immunofluorescent test, we have detected, in some cases, a strong fluorescence limited to heads of only a small fraction of the sperm population, suggesting an intracellular localization of the antibody-binding sites. This is a preliminary report of binding of naturally occurring antisperm isoantibodies from one of these suspect sera to intraacrosomal components of do-
Received August 7, 1984; revised and accepted March 28
W~
,
*Reprint requests: Dr. Jan Tesatik, Department of Histology and Embryology, Faculty of Medicine, J. E. Purkyne University, tf. Obrancd miru 10, 66243 Brno, Czechoslovakia. tDepartment of Histology and Embryology. :j:First Department of Obstetrics and Gynecology. 138
Tesaiik et aI. Communications-in-brief
nor spermatozoa as revealed by an immunoperoxidase method at the ultrastructural level. MATERIALS AND METHODS Co~mercially available immunoglobulin (Ig)G fractIOns from swine antisera against human IgG, IgA, and IgM conjugated with fluorescein isothiocyanate (FITC) or with peroxidase were used. The IgG fractions were purified by affinity chromatography and conjugated with FITC (molar ratio, FITC/protein, 2.5) or with horseradish peroxidase (molar ratio, peroxidase/Ig, 1.5) by the manufacturer (Sevac, Prague, Czechoslovakia). The investigated human sera were heated to 56°C for 30 minutes and stored at - 40°C until tested. Semen samples were obtained from fertile donors. All were judged normal by standard criteria of volume, sperm concentration, and motility. Following liquefaction at room temperature (20 minutes), 0.5 ml of each ejaculate was mixed with 2 ml ofDulbecco's phosphate-buffered saline (PBS), and spermatozoa were washed three times by repeated cycles of centrifugation (500 x g, 5 minutes) and resuspension of sperm pellets in 2 ml of PBS. For detection of passive antibody transfer at the light-microscopic level, smears were prepared from the washed spermatozoa. The smears were allowed to air-dry and were exposed to the patients' sera. One milliliter of serum was pipetted on each slide, and incubation was carried out in a
Fertility and Sterility
wet chamber with 100% humidity at room temperature for 2 hours. After a thorough washing in PBS, the smears were incubated with FITC-Iabeled anti-IgG, anti-IgA, and anti-IgM at room temperature for 2 hours. The three specific antiIgs were applied separately for each patient's serum tested. The concentration of the specific antiIg was approximately 0.2 mg/ml. The slides were then washed again and examined under a fluorescence microscope at a magnification of 500 x with the use of oil immersion. Control smears were incubated either with sera from women of proven fertility instead of the patients' sera or directly with FITC-Iabeled anti-Ig for the exclusion of nonspecific binding of both the first-layer and the second-layer antibodies as well as the presence of autoantibodies on the spermatozoa used in the test system. An attempt was made to evaluate the effect of sperm incubation for capacitation in vitro on the exposure of intracellular antigens. Motile spermatozoa were selected by "swim up" and capacitated in vitro as described in detail previously.3 Smears were prepared and processed as indicated above. For electron-microscopic localization of the sperm-bound antibodies, washed spermatozoa or those incubated for in vitro capacitation were resuspended in PBS to an approximate cell count of 10 to 20 x 106/m l. One milliliter of the living sperm suspension was then incubated with 1 ml of the patient's serum at room temperature for 2 hours. After three cycles of centrifugation at 1000 x g for 5 minutes and resuspension in PBS, the sperm were pelleted again; and 2 ml of peroxidase-conjugated anti-Ig diluted to a final concentration of 0.2 mg specific antibody/ml PBS was added. Incubation was carried out at room tempe.:ature for 2 hours. After washing in PBS, the sperm were fixed in 0.1 M cacodylate-buffered 3%' glutaraldehyde for 30 minutes, washed in PBS, and incubated under continuous agitation in medium consisting of 5 mg of 3,3' -diaminobenzidine tetrachloride (DAB; Sigma Chemical Company, St. Louis, MO) as peroxidase substrate, 0.1 ml of 1% H 2 0 2 , and 10 ml of 0.05 M Tris HCI buffer (pH 7.5) for 30 minutes. Specimens were then washed in PBS, fixed with 1% OS04 in 0.1 M cacodylate buffer, embedded in Durcupan (Fluka AG, Buchs, Switzerland), sectioned with the use of an Ultrotome III LKB ultramicrotome (LKB-Produkter AB, Bromma, Sweden), and examined in a Tesla BS 500 electron microscope (Tesla, Bmo, CzechoVol. 44, No.1, July 1985
slovakia). Appropriate control incubations with sera of fertile women and with specific anti-Igs alone were carried out.
RESULTS A 28-year-old childless woman with a 6-year history of unexplained infertility was investigated. Screening assays for circulating antisperm antibodies in the patient's serum with the use of modified microagglutination4 and immobilization 5 tests were negative. However, strong fluorescence restricted to heads of - 10% of spermatozoa was found after successive exposure of smears of spermatozoa from three donors to the patient's serum and fluorescein-conjugated anti-IgG. The reactions with both anti-IgA and anti-IgM were negative. All control incubations also gave negative results. After incubation of spermatozoa for capacitation in vitro, the occurrence of antibodybinding sperm was increased approximately twice in comparison with nonincubated spermatozoa. The electron-microscopic examination of donor spermatozoa exposed to the patient's serum and treated with peroxidase-conjugated anti-IgG did not reveal any binding of antibodies to intact sperm cells, with the exception of occasional labeling of small areas in the sperm head postacrosomal region (Fig. 1A). On the other hand, spermatozoa having lost their acrosomes showed a massive binding of antibodies to the inner acrosomal membrane (Fig.1B). In contrast to spermatozoa with intact acrosomes, t4e acrosome-lost spermatozoa often showed a significant labeling of the remaining intact portions of the plasma membrane in the equatorial and postacrosomal regions of the sperm head. N~ither the plasma membrane of the sperm tail nor the nuclear membrane was labeled, even when the latter was partially denuded and thus apparently exposed to the patient's serum (Fig. 1B). Interestingly enough, when spermatozoa just undergoing an acrosome reaction were examined, the site of antibody/antigen interaction could be localized not only on the inner acrosomal membrane; the reaction product was associated with membrane vesicles formed during the acrosome reaction and with remnants of the acrosomal content as well (Fig. 1C). No reaction product was ever observed on either intact or acrosome-lost spermatozoa incubated with control sera or directly with peroxidase-conjugated anti-IgG. The proportion of laTesatik et al. Communications-in-brief
139
Figure 1 (A), Head of an intact spermatozoon showing the absence of DAB reaction product except for small patches on the postacrosomal region plasma membrane (arrowheads); acrosome (arrows) (x 20,000). (B), Head of an acrosome-lost spermatozoon with large amounts of DAB reaction product attached to the inner acrosomal membrane (a) . Persisting remnants ofthe plasma membrane are also labeled in the equatorial and postacrosomal regions (arrows). No reaction product is present either on the tail midpiece (t) or on remnants of the plasma membrane of the anterior acrosomal region (p) . Note the absence oflabel on the partially denuded nuclear membrane (arrowhead) (x 20,000). (C), Head of a spermatozoon in the course of acrosome reaction. In addition to DAB reaction product on the inner acrosomal membrane (a), it is also associated with membrane vesicles (arrows) originating during the acrosome reaction and with remnants of the acrosomal content (arrowheads) (x 20,000).
beled spermatozoa was similar to that assessed in the immunofluorescent test. DISCUSSION
This report demonstrates that naturally occurring ·antisperm isoantibodies from human sera can be directed to antigens located within the acrosome of human spermatozoa. The antibody binding described in these experiments apparently involves both dead spermatozoa and those undergoing or having undergone an acrosome reaction, the two conditions exposing intraacrosomal antigens which are sequestered by an impermeable plasma membrane in living acrosome-intact spermatozoa. The significant extension of the antibody-binding area of the sperm head postacro140
Tesatik et al. Communications-in-brief
somal region plasma membrane after the acrosome loss or a spontaneous acrosome reaction may reflect changes in antigenic properties of this area of the sperm surface because of sperm death or the acrosome reaction. Naturally occurring antibodies to intracellular components of sperm are relatively ubiquitous. 6 They may cross-react with bacterial antigens and probably play no role in infertility in most cases. Antigenic sites within the acrosome or on acrosomal membranes, however, are exposed to antibodies potentially present at the site of fertilization before spermatozoa are able to penetrate the egg. Hence, the participation of antibodies against the intraacrosomal antigens in immunologic infertility should be taken into account. Furthermore, the possibility that the antibodies Fertility and Sterility
detected on the acrosomal region of spermatozoa with the use of the common fluorescent antibody techniques are in fact bound to some intraacrosomal components and not to the sperm surface should always be borne in mind, especially where only a subpopulation of sperm is stained. How spurious conclusions can be made, assuming that these procedures detect molecules only on the sperm surface, has recently been pointed out. 7 The immunofluorescent test was performed in this study only as a preliminary screening assay for antisperm antibodies because of its relatively high sensitivity and simplicity. Electron microscopy with the use of labeled anti-Ig antibodies is the most efficient method for determination of the regional specificity of antibody/antigen interaction and in the precise localization of sperm intracellular antibody-binding sites. The use of in vitro capacitation, allowing additional spermatozoa to expose their intraacrosomal antigens during a spontaneous acrosome reaction, and comparing the proportion of antibody-bound sperm with a fresh sample with the use of an immunofluorescent test would be a simple screening assay for acrosome-directed antibodies. The electronmicroscopic immunoperoxidase technique, as a more specific method, could be used with suspect sera to exclude false-positive cases and those with antibodies to sperm intracellular antigens other than intraacrosomal. From our results, it is not quite clear whether the antigenic sites on the acrosomal membrane include the membrane's integral proteins or whether they are merely components of the acro-
Vol. 44, No.1, July 1985
somal content bound to the membrane surface. Labeling ofthe acrosomal content in spermatozoa undergoing an acrosome reaction suggests that these antigens may be primarily located in the acrosomal matrix and remain associated with the acrosomal membrane for a certain time period after an artifactual acrosome loss or after a spontaneous acrosome reaction. Studies are in progress aimed at characterization of these antigens and evaluation of the effect of their blockade on sperm/egg interaction in vitro. REFERENCES 1. Jones WR: Immunologic infertility-fact or fiction? Fertil
Steril 33:577, 1980 2. Bronson R, Cooper G, Rosenfeld D: Sperm antibodies: their role in infertility. Fertil Steril 42:171, 1984 3. Tesatik J, Kopecny V, Dvorak M: Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro. Fertil Steril 41:919, 1984 4. Franklin RR, Dukes CD: Antispermatozoal antibody and unexplained infertility. Am J Obstet Gynecol 89:682, 1964 5. Isojima S, Li TS, Ashitaka Y: Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained sterility. Am J Obstet GynecollOl:677, 1968 6. Tung KSK, Cooke WD Jr, McCarty TA, Robitaille P: Human sperm antigens and antisperm antibodies. II. Age-related incidence of antisperm antibodies. Clin Exp Immunol 25:73, 1976 7. Jones R, Brown CR, Cran DG, Gaunt SJ: Surface and internal antigens of rat spermatozoa distinguished using monoclonal antibodies. Gamete Res 8:255, 1983
Tesafik et aI. Communications-in-brief
141