Clin. exp. Immunol. (1976) 24, 292-299.

Human sperm antigens and antisperm antibodies III. STUDIES ON ACROSOMAL ANTIGENS K. S. K. TUNG Department ofPathology, School ofMedicine, University of New Mexico, Albuquerque, New Mexico, U.S.A. (Received 11 September 1975)

SUMMARY

Acrosome of human sperm possesses two distinct antigens that are immunogenic, and will elicit autoantibodies that are detectable by immunofluorescence (IF). The first antigen, Acl, diffuse in distribution, is probably glycoprotein in nature since it is removed by trypsin and periodate. It is readily removed from cells after incubation in acid buffer or phosphate-buffered saline (PBS), stable at 60'C and not affected by trypsin inhibitor. The second antigen, Ac2, discrete in distribution, is resistant to trypsin treatment. It remains stable after incubation in acid buffer or PBS, is unstable at 60'C and becomes more diffuse in distribution when incubated in acid buffer or trypsin inhibitor. The use of spermatozoa pretreated with acid buffer permits detection of anti-Ac2 antibody that coexists with anti-Acd antibody in the same serum sample. Both Acd and Ac2 antigens are demonstrable in spermatozoa from the ejaculate, epididymis and the testis; in spermatids and spermatocytes. Ac 1 antigen appears to show extensive cross-reaction with micro-organisms and with antigen(s) of human adrenal gland; and anti-Acl antibody is found frequently in the serum of men before vasectomy. In contrast, Ac2 antigen does not show cross-reaction with micro-organisms or tissue antigens tested; and its antibody is found mainly in the male and primarily after vasectomy. Thus, anti-Ac2 antibody may be more indicative of an immune response to sperm, and should be sought in diseases related to sperm immunity.

INTRODUCTION Antibodies to spermatozoa-specific antigens have been implicated in the pathogenesis of unexplained infertility (Rumke, 1964; Franklin & Dukes, 1964). Although IF studies on the sera of women from infertile couples have disclosed a number of antisperm antibodies (ASA), including antibodies to diffused acrosomal antigen (which we designate as Acl), equatorial antigen (E), postacrosomal antigen (PA), and antigen in the mainpiece of the tail (T) (Hjort & Hansen, 1971), no correlation has yet been demonstrable between these antibodies and infertility (Jones, Ing & Kaye, 1973; Wall et al., 1975). Recently, based on studies on vasectomy patients, additional antibodies to spermatozoa have been found, and these included antibodies to protamine of sperm nucleus (N) (Kolk, Samuel & Rumke, 1974), and to a second acrosomal (which we designate as Ac2) antigen (Tung, 1975). Unlike the other antibodies, anti-N and anti-Ac2 antibodies were found mainly after, and not before, vasectomy (Tung, 1975). This implies that these two antibodies developed as an immune response to spermatozoa itself, and might carry greater pathogenetic significance than those ASA described previously. In this paper we describe: (1) the characteristics of Acl and Ac2 antigens; (2) any cross-reaction between these antigens Correspondence: Dr Kenneth S. K. Tung, Department of Pathology, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87131, U.S.A.

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with antigens of other human tissue and micro-organisms; and (3) the relative incidence of these antibodies in the general population and in men after vasectomy based on an improved method for detecting anti-Ac2 antibodies. MATERIALS AND METHODS Patients. Five sera with diffuse acrosomal staining and five sera with discrete acrosomal staining were used for the immunofluorescence study that investigated the properties of Acl and Ac2 antigens. They came from patients following vasectomy and the antibody titres ranged from 1/40 to 1/60. To determine the incidence of anti-Acl and anti-Ac2 antibodies, sera were collected from twenty-nine infants under 1 year (sixteen males, thirteen females ), eight-seven children (forty-one males, forty-six females) between 1 and 10 years, fifty-eight teenagers (thirty-one males, twenty-seven females) between 11 and 20 years, thirty-eight women between 22 and 55 years, and forty-seven elderly individuals (seventeen males, thirty females). In addition, sera were collected from 114 men (aged 25-55) before their vasectomy; and from 112 of these patients, sera were again collected at 2 months, and seventy at 6-9 months. These sera were kept at - 70'C until study. Determination of ASA by indirect immunofluorescence (IF). The sperm and testicular tissues for IF studies came from men of blood group 0, and included the following: (1) ejaculated sperm were washed twice in 50 ml of phosphate-buffered saline (PBS), 0-15 M, pH 7-2, by centrifugation at 200 g, for 10 min at 4VC, smeared on clean glass slides and dried; (2) frozen sections of testis and epididymis, 5 pm in thickness, were cut in a cryostat; (3) smears of testicular cells were prepared by trimming blocks of testis in PBS; the cell suspension was washed in PBS by centrifugation at 200 g for 10 min at 4VC, and spread on glass slides by a cytocentrifuge. The tissues for (2) and (3) came from a normal man 4 hr after his accidental death. The methods for preparation of fluorescein isothiocyanate (FITC)-conjugated antisera to human IgG and for the IF study have been described in detail elsewhere (Tung, 1975). Briefly, sera were obtained from rabbits that were immunized three times with chromatographically purified normal human IgG in Freund's complete adjuvant. The rabbit IgG was purified on a DE-52 column, and the antibody was made y-chain-specific by appropriate immunoabsorption. The antisera were conjugated with FITC and the conjugated antisera were further purified on a DE-52 column by stepwise elution with increasing ionic strength. The fraction with fluorescein/protein ratio of 2 was used in this study. Sperm for IF study came from healthy donors with 0 blood group. Liquescent semen samples were washed twice by centrifugation at 200 g for 10 min at 4°C in PBS. The sperm were suspended in a small volume of PBS and were smeared on clean glass slides, air dried, fixed in absolute methanol for 30 min, again air dried, and stored at - 20'C to be used within 1 week. To detect antibody to adrenal gland, frozen sections of human adrenal gland, removed from surgery as part of a nephrectomy specimen, were used. The adrenal sections were studied unfixed, fixed in methanol or acetone each for 30 min, or fixed in 95%4 ethanol-ether (v/v = 1/1) for 10 min, then 95%4 ethanol for 20 min. To perform the IF study, tissue sections or cell smears were incubated with serum, usually diluted 1/10 with PBS, for 30 min, and washed in PBS, the tissue section or cell smear was incubated with the FITC-conjugated antiserum for 30 min. The slides were again washed in PBS and coverslipped with buffered glycerin. The cell smear or tissue section was kept moist during the entire procedure since drying was found to result in non-specific staining by the FITC-conjugated antisera alone. In order to minimize the variations inherent in the IF test, different serum samples of the same vasectomy patient were always studied in the same IF experiment using smears of sperm from the same donor. Also included are serial dilutions of a positive control. The experimental results were accepted only when the titre of the positive control fell within one tube dilution of its expected titre. The specimens were examined with a Leitz Ortholux II phase-fluorescence microscope, and photographed with the Orthomat camera, using Kodak Tri-X film. In order to detect antibody to the surface of sperm, IF staining of a suspension of viable human-ejaculated sperm was carried out. Sperm were washed three times in PBS, and resuspended at 108 cells per ml. 0-1 ml of the sperm suspension was incubated with 0-1 ml of serum with anti-Acl or anti-Ac2 antibody, each diluted 1/10 in PBS, for 30 min at 4°C. The sperm were then washed twice in PBS by centrifugation at 200 g at 40C for 10 min, and resuspended in 0-1 ml of FITCconjugated anti-human IgG. Following incubation for 30 min at 4°C, the sperm were washed twice in PBS, and examined as a wet mount by phase-ultraviolet fluorescence microscopy. Absorption of antisperm antibodies with preparations of human tissues or micro-organisms. Tissue powder of human brain, testis, adrenal gland, kidney and liver was prepared from healthy individuals within 4 hr of accidental death. The tissue was homogenized with equal volume of PBS and lyophilized. Ejaculated sperm was washed three times in PBS; and seminal plasma was obtained from the ejaculate of vasectomized patients and then lyophilized. Pure culture of micro-organisms, including Staphylococcus albicans, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Candida albicans were mixed with sodium azide, and were lyophilized. To absorb, 200 ,1 of serum was incubated with approximately 10 mg of lyophilized tissue or micro-organisms for 1 hr at 37°C, then overnight at 4°C. This was repeated twice. As a control, 200 pl of the same sera was incubated similarly in the absence of tissue or micro-organisms. Preparation of reagents. These include: (1) trypsin, 2 pg/ml in PBS adjusted to pH 8 with 0-1N NaOH (Type I, Sigma Chemicals, St Louis, Missouri); (2) sodium citrate buffer, 0-15 M, adjusted to pH 3, 5 or 7 by citric acid; (3) sodium metaperiodate (Bakers, Phillipsburg, New Jersey), 0-1 M, in PBS, pH 7-2; (4) soybean trypsin inhibitor (Type I, Sigma Chemicals, St Louis, Missouri), 1 mg/ml in PBS, pH 7-2.

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RESULTS Immunofluorescence observations The possible existence of two acrosomal antigens detectable by IF on human sperm was first suggested by their difference in distribution. While both antigens were located anterior to the equatorial region ofthe sperm, Acd was diffuse (Fig. 1), and Ac2 was speckled (Fig. 2B) in appearance. The discrete distribution of Ac2 was particularly prominent in some sperm smears where the antigens had been partially detached from the sperm head, but remained on what appeared to be a membrane (Fig. 2A, C and D). In contrast, Acl antigen was always confined to the anterior region of the sperm acrosome. The different staining patterns of sera with anti-Acl and anti-Ac2 antibodies were consistent, and remained so in five studies on sperm from different donors. Both antigens were sperm and not spermcoating antigen (Weil, 1965) specific since (1) anti-Acl or anti-Ac2 antiserum did not stain surface of viable sperm in suspension, and (2) these antigens appeared during spermatogenesis since they were detectable in epididymal sperm, testicular sperm, spermatids (Fig. 3) and cells that appeared to be spermatocytes.

'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......

2

|~~~~~~~~~~~~~~~~~~~~~~~~-- IN% I FIG. 1. Immunofluorescence photomicrograph showing the staining pattern of (a) Acl antibody, and the loss of stainable Acl antigen in spermatozoa pretreated with (b) periodate or (d) trypsin. (c) Sperm incubated in PBS for 15 min.

Although IF clearly differentiated Acl from Ac2 in most instances, this was nevertheless a subjective test. Therefore, additional differences of the two antigens were investigated.

Biochemical analysis Solubility in PBS. To compare the solubility of Acl and Ac2 antigens in PBS, aliquots of washed ejaculated sperm at 17O per ml of PBS were kept at 4VC. After intervals ranging from 2 to 55 hr the sperm were smeared on slides, fixed in methanol and studied for the presence of acrosomal antigens by IF. The result (Fig. 4), which was based on the percentage of positive sperm in 200 counted, showed that Acl antigen in PBS disappeared within 12 hr, whereas many sperm retained Ac2 antigens after 50 hr. Effect of trypsin. Sperm smears were fixed in methanol for 30 min, dried, incubated in trypsin for 15 min, washed in PBS, and studied for the presence of Acl and Ac2 antigens by IF. Control slides were incubated in PBS without trypsin. While Acl was completely removed by trypsin treatment (Fig. 1, T), Ac2 was not affected.

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L_>a~~~~ FIG. 2. Immunofluorescence photomicrograph showing the speckled staining pattern of anti-Ac2 antibody. The antigen has discrete distribution over the acrosomal region (b), and is readily dislodged from the sperm (a, c and d) but appears to remain attached to a membrane (a and c).

FIG. 3. Immunofluorescence photomicrograph demonstrating testicular localization of Acl (a and c) and Ac2 (b and d), antigens in presumed spermatocytes (a, arrows), spermatids (a, b and c) and sperm (d) in testicular section (a) and cytocentrifuged smears of testicular cell suspension (b, c and d).

Effect of trypsin inhibitor. Sperm smears, prefixed in methanol for 30 min and dried were incubated in soybean trypsin inhibitor, washed in PBS, and studied for Acl and Ac2 antigens. Following this treatment, the distribution of Ac2 became diffused and concentrated in the anterior rim of the acrosomal apparatus, whereas Ac2 showed no alteration. Ejfrct of periodate. Sperm smears, pre-fixed in methanol for 30 min and dried, were incubated in periodate for 15 min, washed in PBS, and studies by IF for Acl and Ac2 antigens. While periodate

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clearly removed Acl from the sperm (Fig. lb), it affected Ac2 only slightly. Since Acl was sensitive to both trypsin and periodate, it was tentatively considered to have both protein and polysaccharides in its antigenic determinants. The nature of Ac2, on the other hand, remained obscure. Effect of heat. Sperm smears, fixed in methanol for 30 min, were incubated in PBS that had been equilibrated at 60'C or 80'C in a waterbath. 15 min later the slides were studies for acrosomal antigens by IF. Acd appeared to be more heat stable, since it remained stainable after incubation at 60'C, whereas Ac2 was not. Effect of different pH. To evaluate the effect of pH on the acrosomal antigens, sperm smears that had been fixed for 30 min in methanol were incubated in citrate buffer of pH 3, 5 or 7 for 15 min, washed in PBS and studied by IF. Acl was lost from the sperm after incubation in pH of either 3 or 5, whereas staining of Ac2 antigen actually became intensified and changed from speckled to a diffuse distribution (Fig. 5). Since the differential effects of acid pH on the acrosomal antigens was clear cut and consistent, it was further evaluated as a method (the acid pH test) for differentiating anti-Acl from anti-Ac2 antibodies. 100

800

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FIG. 4. ACd (o) is more soluble than Ac2 (e) in PBS since more sperm retained Ac2 after incubation in PBS. Aliquots of sperm in PBS were incubated at 4VC. At different time intervals, the sperm were smeared, fixed in methanol, and stained by antisera to Acl and Ac2 at dilutions two tubes from the titre.

Differentiating anti-AcI from anti-Ac2 antibodies by the acid pH test Two sera with clear-cut anti-Acl antibodies were mixed in the three combinations shown in Table 1. The mixtures were then used to stain sperm smears that were pretreated in pH 3 or pH 7 and fixed in methanol. The result showed that the acid pH test: (1) consistently differentiated the two anti-acrosomal antibodies; and (2) could detect anti-Ac2 antibody that coexisted with anti-Acl antibody in the same serum. Next, the acid pH test was used to evaluate the natural incidence of anti-Acl and anti-Ac2 antibodies in males and females of different ages, and in men before and after vasectomy.

Age-related incidence of anti-Acl and anti-Ac2 antibodies in human As shown in Fig. 6, many differences were apparent: (1) anti-Acl antibodies were more commonly found than anti-Ac2 antibodies; (2) the age-related incidence of the two antibodies differed, thus antiAcl antibodies appeared early in childhood, with a peak incidence of over 80% between the ages of 1 and 10 years, thereafter declined, whereas the incidence of anti-Ac2 antibodies rose gradually with age; and (3) while anti-Acl antibodies were evenly distributed between the sexes, a higher incidence of anti-Ac2 antibody was found in the male.

Hunman sperm antigens and antisperm antibodies

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FIG. 5. Immunofluorescence photomicrograph showing the difference in stainable Acl antigen (a, b and c) and Ac2 antigen (d, e and f) after incubation of methanol fixed sperm smears in pH 7 (a and d), pH 5 (b and e) and pH 3 (c and f). Note diffuse and intensified Ac2 staining after acid pH (e and f).

TABLE 1. Detection of anti-Acl and anti-Ac2 antibodies by the acid pH test

Titre of antisperm antibodies in four studies No. 1

Antiserum combinations

pH 7

aAcl*+aAc2 aAcl+aAcl aAc2-+ aAc2

1/80 1/80 1/80 < 1/10 1/40 1/40

pH 3

No. 2 pH 7

No. 3

pH 3

pH 7

1/160 1/80 1/80 < 1/10 1/80 1/40

* aAcl = anti-Acl antibody; aAc2

pH 3

1/80 1/80 1/80 < 1/10 1/40 1/20 =

No. 4 pH 7

pH 3

1/80 1/80 1/160 < 1/10 1/40 1/40

anti-Ac2 antibody.

The incidence ofanti-acrosomal antibody in men after zasectomry Following vasectomy, men developed an increasing incidence of both antibodies to Acl and Ac2 antigens at a comparable rate (Fig. 7).

Cross-reaction between acrosomal antigens and micro-organisms and human tissue antigens By absorption studies, Acl but not Ac2 antigen was found to cross-react with many micro-organisms, including Staphylococcus aureus, Staphylococcus albus, Streptococcus haemolyticus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans. Similarly, anti-Acl antibody was

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readily absorbed by extracts of human adrenal gland, while anti-Ac2 antibody was not. The crossreactive antigen(s) in the human adrenal gland was not visualized in the IF study with anti-Acl antibody. Both anti-Ac and anti-Ac2 antibodies were absorbed by sperm and by homogenates of the testis but not by homogenates of other tissues. BrO 601 60

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Age groups

FIG. 7 FIG. 6 in male (o or II) and antibodies (U or anti-Ac2 C1) 0) and FIG. 6. Age-related incidence of anti-Acl (O or female(- or *). Age groups: (A) < 1 year; (B) 1-10 years; (C) 11-20 years; (D) 22-55 years; (E) 70-85 years. FIG. 7. Incidence of anti-Acl (o) and anti-Ac2 (o) antibodies in men, before, at 2 months, and at 6-9 months after vasectomy.

TABLE 2. Differences between Acl and Ac2 antigens in human sperm

Sperm antigens Studies Acl

Ac2

Speckled, dislodged readily from cells No change Decreases Not readily Became intensified and diffuse

Immunofluorescence pattern

Diffuse

Trypsin, 2 pg/ml, pH 8, 15 min* Periodate 0 1 M, 15 min* Solubility in PBS pH 3-5, 15 min*

Disappears Disappears Readily Disappeared

600C, 15 min* Trypsin inhibitor, 1 mg/ml, 15 min*

No change No change

Disappeared Became intensified and

Human adrenal gland absorption

Cross-reacts Cross-reacts

diffuse No cross-reaction No cross-reaction

Micro-organism absorptiont

* Smears of sperm fixed in methanol, 30 min, and dried; incubated with appropriate reaction mixtures, rinsed in PBS; incubated with antibodies to Acl or Ac2; then with FITC anti-human Ig. t See text.

DISCUSSION The present study has demonstrated that anti-acrosomal antibody heretofore detected by IF (Hjort & Hansen, 1971; Jones et al., 1973; Wall et al., 1975) was in fact heterogeneous, and represented antibodies to at least one or both of two distinct acrosomal antigens: Acl and Ac2. Since the characteristics of Acl and Ac2 are different in so many respects (these are summarized in Table 2), they are undoubtedly

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distinct antigens. Although we know that these antigens reside in the region of the acrosome of mature as well as developing sperm and spermatids, and are not on the sperm surface, we have no information regarding their precise localization. The IF findings suggest that Ac2 is less firmly held within the acrosomal apparatus since it is readily dislodged when sperm is smeared on a glass slide. Likewise, the exact nature of the two antigens is unknown. Although preliminary data suggest that Acl is a glycoprotein or glycopeptide, full information on their chemical nature will have to await the isolation of the antigens, a process that may be assisted by the specific antibodies. The acid pH test was particularly useful in: (1) studying weak antibodies which rarely stain sperm in a clear-cut manner; and (2) detecting anti-Ac2 antibody that coexisted with an anti-Acl antibody in the same serum sample. Furthermore, the acid pH test enables us to discriminate the existence of the two acrosomal antigens without relying solely on their IF pattern. It is of interest that both acid pH and trypsin inhibitor altered the distribution of Ac2 antigens from discrete to diffuse. If Ac2 is ultimately shown to be bound to either the outer or the inner acrosomal membrane, this observation may represent one example of modulation of lateral movement of acrosomal membrane a~ssciated macromolecules (Frye & Edidin, 1970). The practical value in differentiating the two acrosomal antibodies relates to the potential pathogenetic significance of these antibodies in human diseases. Studies in the general population indicates that the natural incidence of the two acrosomal antibodies differ greatly. The early and the frequent appearance of anti-Acl antibody most certainly represents immune response of children to the exogenous antigens that happen to cross-react with the Acl antigen. This possibility is substantiated, though not proven, by the extensive cross-reaction demonstrable between Acl and many micro-organisms. In contrast, antiAc2 antibody is found only rarely, and is therefore more sperm specific. On the other hand, the incidence of antibody to both of the acrosomal antigens is found to increase following vasectomy, when men presumably become exposed to sperm. Therefore, it can be concluded that anti-Acl can be readily elicited by both ubiquitous antigens in the environment and sperm, while anti-Ac2 antibody more specifically reflects an immune response to sperm. It follows that anti-Ac2 but not anti-Acl antibody should be studied in clinical conditions that are putatively related to sperm immunity, as in unexplained

infertility. The author thanks Mr Donald Cooke, Jr and Ms Teresita McCarty for their excellent technical assistance. This study was supported by NIH contract number HD-3-2717, and in part by NIH grant number HD-0-7591.

REFERENCES FRANKLIN, R.R. & DuKEs, C.D. (1964) Antispermatozoal antigen detected on swollen spermheads. Clin. exp. antibody and unexplained infertility. Amer. J. Obstet. Immunol. 16, 63. Gynec. 89, 6. RUMKE, P. (1964) Spermagglutinating auto-antibodies. FRYE, L.D. & EDIDIN, M. (1970) The rapid intermixing of Their relation to male infertility. Proceedings of the 2nd cell surface antigens after formation of mouse-human International Congress on Endocrinology, London, p. 906. heterokaryons. J. Cell Sci. 7, 319. Excerpta Medica, Amsterdam. HJORT, T. & HANSEN, K.B. (1971) Immunofluorescent TUNG, K.S.K. (1975) Human sperm antigens and antisperm studies on human spermatozoa. I. The detection of antibodies. I. Studies on vasectomy patients. Clin. exp. different spermatozoal antibodies and their occurrence in Immunol. 20, 93. normal and infertile women. Clin. exp. Immunol. 8, 9. WALL, J.R., HARRISON, R.F., STEDRENSKA, J. & LESSOF, JoNES, W.R., ING, R.M. & KAYE, M.D. (1973) A comparison M.H. (1975) Antibodies against spermatozoa in infertile of screening tests for anti-sperm activity in the serum of women with poorly invading spermatozoa on postcoital infertile women. J. Reprod. Fertil. 32, 357. tests. Amer. J. Obstet. Gynec. 121, 198. KOLK, A.H.J., SAMUEL, T. & RCJMKE, P. (1974) Autoantigens WEIL, A.J. (1965) The spermatozoa-coating antigen (SCA). of human spermatozoa. I. Solubilization of a new autoAnn. N.Y. Acad. Sci. 124, 267.

Human sperm antigens and antisperm antibodies. III. Studies on acrosomal antigens.

Acrosome of human sperm possesses two distinct antigens that are immunogenic, and will elicit autoantibodies that are detectable by immunofluorescence...
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