Characterization of a Monoclonal Antibody to a Human Intra

BIOLOGY OF REPRODUCTION 51, 1117-1125 (1994)

Characterization of a Monoclonal Antibody to a Human Intra-Acrosomal Antigen That Inhibits Fertilizationl CLEMENT JIMENEZ, 2'3 BENOIT SION, 3 GENEVIEVE GRIZARD, 3 CHRISTINE ARTONNE, 3 JEAN-LOUIS KEMENY, 4 and DANIEL BOUCHER 3 Laboratoire de Biologie du Ddveloppement et de la Reproduction,3 Facult de Mdecine, BP 38, 63001 Clermont-FerrandCedex, France Laboratoire d'Anatomie Pathologique,4 CHRU GabrielMonipied, BP.69 63003 Clermont-FerrandCedex, France ABSTRACT Among monoclonal antibodies (mAb) selected after the immunization of mice with human ejaculated spermatozoa, mAb 19G9 (IgG,kappa) was found by immunoperoxidase staining to label most of the acrosome of human spermatozoa permeabilized with methanol-acetone. The antigen was poorly expressed on the surface of fresh ejaculated sperm, but was detectable on most viable sperm after 5-h incubation in medium containing human serum albumin (HSA) followed by 30-min incubation with the calcium ionophore A23187. This treatment resulted in acrosomal loss. Immunoelectron microscopy labeling with 19G9 mAb localized the antigen within the acrosome. Immunocytochemistry on testis sections showed that antigen was located in the round spermatids within the adluminal compartment of the seminiferous epithelium. Western blotting of sperm extract proteins showed that sperm intra-acrosomal antigen (SIAA) recognized by I9G9 mAb had a polymorphism of immunogenic peptides from 16 to 35 kDa. Most of the antigenic peptides possessed an isoelectric point of approximately 5. When spermatozoa were treated with a series of protease inhibitors, the polymorphism of immunogenic peptides was reduced, suggesting that the multiple form of the antigen was due, at least in part, to proteolytic processing. In the testis, only a single peptide band of 35 kDa was detected with mAb 19G9. Studies of human tissue specificity by Western blotting showed that the epitope recognized by I9G9 mAb was present solely in ejaculated spermatozoa and the testis. 19G9 mAb did not agglutinate or immobilize sperm but inhibited the penetration of zona-free hamster ova by human sperm.

INTRODUCTION Mammalian spermatozoa are highly differentiated cells with several microdomains. In the anterior head, the plasma membrane overlies the acrosome. Vesiculation of the outer acrosomal membrane and the overlying plasma membrane occurs during the acrosome reaction. The plasma membrane covering the postacrosomal region of the sperm head seems to have an important role, for it is here that spermegg fusion is initiated [1]. When human sperm are mixed with hamster eggs from which the zona pellucida has been removed, only sperm that have undergone the acrosome reaction can fuse with the hamster oolemma [2]. Monoclonal anti-sperm antibodies have been used as probes to identify specific fertilization-related sperm antigens. Sperm antigens have been defined in several species, including humans. A number of antigens on human mature spermatozoa [3-16] have been identified with monoclonal antibodies (mAb) that reportedly recognize antigenically distinct subsets of human spermatozoa. Some have been involved in functional aspects; for example, Isahakia and Accepted July 21, 1994. Received February 7, 1994. 'This work was supported by grants from DRED (contrat DRED n91-1497) and DRED (EA 975: Pouvoir fecondant des spermatozoides et r6le des lipides). 2 Correspondence: Clement Jimenez, Laboratoire de Biologie du Developpement et de la Reproduction, Faculte de Medecine, Place Henri Dunant, BP 38, ClermontFerrand, France. FAX: 73.27.29.73.

Alexander [11] observed mAb with immobilizing and agglutinating properties, and some antibodies are able to inhibit the binding and penetration of zona-free hamster ova by human spermatozoa in the hamster egg penetration test [12-15] or even to prevent the fertilization of human oocytes [16]. The present paper describes the characteristics of a mAb (I9G9) generated against human sperm germ cells as well as its influence on sperm-egg interaction. MATERIALS AND METHODS Production of mAbs Two 6-wk-old female Balb/c mice were immunized i.p. with 107 sperm from a healthy fertile donor that were collected after 3 days of sexual abstinence. Semen samples were centrifused at 1000 x g for 10 min. The pellet was resuspended in PBS. An aliquot containing 107 spermatozoa was emulsified with an equal volume of complete Freund's adjuvant and injected i.p. into the mice on Days 1, 30, 60, 90, 120, and 150. Immunized mice were bled to check the immune response by indirect immunocytology as described below. On Day 153, the mice received a spleen injection of 107 spermatozoa. Three days later, the spleen was removed separately from each mouse and fused with P3X63. Ag. 653 myeloma cells at a 10:1 ratio. The cell fusion was carried out for 2 min in the presence of 40% polyethylene

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glycol 1000 in Dulbecco's Modified Eagle's medium [17]. The fused cells were seeded into 96 wells of standard culture plates (cell density of 105 cells per well) and cultured in hypoxanthine/aminopterine/thymidine (HAT) selective medium [17]. During the first two weeks, almost all the unfused spleen cells and myeloma cells died. The hybridized cells were then cultured in medium without aminopterine. Clones were considered positive if they secreted an antibody reacting in an immunocytochemistry assay with both motile and immotile ejaculated sperm exposed to methanol/acetone (v/v) fixative. The positive hybridoma was recloned twice by limiting dilution to ensure that a given well contained the progeny of only one hybridoma cell. Each of the positively identified and selected hybrid cells was injected i.p. at a density of 107 cells/ml into Balb/c mice for 7-10 days. The ascites fluid was drawn from each mouse and the mAb were recovered by centrifugation at 27 000 x g for 10 min to remove cell debris. The supernatant containing a high concentration of mAb was stored at -20°C. All cultures were frozen in liquid N2 medium containing 50% (v/v) fetal calf serum and 10% (v/v) dimethyl sulfoxide. The cloned hybrid cell lines and their secreted antibodies were given numbers to identify the mouse and culture from which they derived.

treated by two protocols. First, to permeabilize the spermatozoa, they were fixed for 30 min with methanol acetone (v/v) and then washed three times in PBS by centrifugation at 400 x g. Secondly, the spermatozoa were fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) and washed three times in PBS. Before fixation they were incubated for 30 min with trypan blue in order to eliminate dead spermatozoa from the analysis. All spermatozoa then underwent the same treatment. They were air-dried on microscope slides; then cells were preincubated in PBS containing 10% normal human serum (NHS) for 30 min and washed three times in PBS. The slides were treated with 0.15% hydrogen peroxide (H 2 0 2 ) in methanol to block endogenous peroxidase. After three washes in PBS, the slides were incubated for 30 min with a 1/800 dilution of mAb I9G9 or a control mouse IgG, kappa mAb (MOPC 31; Sigma) in 2% NHS. They were again washed three times in PBS and then incubated with a polyclonal antibody raised in sheep to mouse IgG labeled with peroxidase (Amersham) 1/100 in PBS for 30 min. This was followed by a final three washes in PBS. Brown reaction product, indicating the location of antigen, was developed in 0.05 M Tris containing 0.05% diaminobenzidine and 0.03% H2 02 . Immunogold Technique Transmission Electron

Determinationof the Isotype of the mAb Selected (I9G9)

To determine the isotype of mAb I9G9 we used the mouse monoclonal isotyping kit (Amersham, Les Ulis, France) according to the manufacturer's instructions. Purificationof Immunoglobulins

Purification was done on a protein G-sepharose 4B fast flow column (Sigma, Saint Quentin Fallavier, France). The pH of the ascites fluid was adjusted to 8.0 by the addition of 1/10 volume of 1.0 M Tris (pH 8.0). The antibody solution was passed through the protein G bead column. The beads were washed with ten column volumes of 100 mM Tris (pH 8.0) and then with ten column volumes of 10 mM Tris (pH 8.0). Elution was performed with 100 mM glycine (pH 3.0) buffer. The immunoglobulin-containing fractions were identified by absorbance at 280 nm. Sperm Immunostaining

Human semen samples were collected after three days of sexual abstinence from normal fertile donors. The samples were liquefied for 30 min and diluted 1/5 in Biggers, Whitter and Whittingham (BWW) medium [18]. Sperm were isolated by centrifugation at 400 x g and washed three times in BWW medium. Sperm at this point in the preparation process were termed "untreated," They were induced to undergo acrosomal loss by incubation at 370C for 5 h in BWW medium containing 3% human serum albumin (HSA) followed by a 30-min incubation at 37 0C in 0.3% HSA and 10 p.M calcium ionophore A23187. The spermatozoa were

Microscopy Sperm induced to acrosome-react were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) and then incubated for 30 min at room temperature with 10% NHS in PBS to block nonspecific sites. I9G9 mAb or control mouse IgGi kappa mAb was diluted 1/100 in 2% NHS and reacted for 30 min with the spermatozoa. After washing with PBS, the spermatozoa were incubated for 30 min in a 1/5 dilution of 30-nm colloidal gold particle-conjugated goat antimouse IgG (Amersham). After washing with PBS, the spermatozoa were postfixed in 2% osmium tetroxide. The pellet was embedded in Epon 812. Sections were washed in PBS, stained for 10 min in 5% uranyl acetate, and then examined with a Philips 300 electron microscope. Immunocytochemistry of Human Testis and Epididymis

The testes and epididymides were autopsy specimens from a 42-yr-old man. They were fixed in Bouin's fluid and embedded in paraffin. Five-micrometer sections were mounted on gelatin-coated microscope slides, deparaffinized in a graded series of ethanols, and rehydrated in PBS. Sections were then treated as described above. PAGE and Electrotransfer

The sperm were washed twice with PBS. One fraction (F1) was frozen at -800 C without treatment, while the other fraction (F2) was frozen in the presence of protease inhibitors (2 mM PMSF; 20 g/ml leupeptine; 20 jig/ml pepstatine; 20 pxg/ml aprotinin). After being thawed, F1 and a

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part of F2 were sonicated and the protein extracts were recovered after centrifugation at 15 000 x g for 20 min. Proteins of the other part of F2 were precipitated by 10% trichloroacetic acid (TCA), and the pellet was washed with ethanol ether 1/1 (v/v) and spun at 15 000 x g for 20 min. Proteins were dissolved in sample buffer containing 0.0034 M SDS and 0.7 M P3-mercaptoethanol. The proteins were analyzed by SDS-PAGE according to Laemmli [19], and two-dimensional electrophoresis was performed by the procedure of O'Farrell [20]. The method of Towbin et al. [21] was used to electrotransfer. Other autopsy specimens of human tissues were exracted in the presence of protease inhibitors, and proteins were analyzed as described above. Western Blot The nitrocellulose was blocked in 10% skim milk in PBS and incubated in I9G9 mAb (1/10 000) or IgG kappa mAb (1/100) in PBS-10% skim milk overnight at 4°C. Sheep antimouse IgG peroxidase (Amersham) was used at 1/500 dilution. Antibody Binding to Oocytes To test whether there was adherence of I9G9 mAb to the oocyte membranes, zona-free hamster oocytes prepared as described below (10 per antibody) were incubated in PBS-0.1% BSA for 45 min and in 1 ml of I9G9 mAb (1/1000) in PBS or with control mouse IgGi kappa in PBS1% BSA at 37°C. Oocytes were washed three times in PBS, and a 1/100 dilution of fluorescein isothiocyanate (FITC)conjugated goat anti-mouse IgG (Sigma) was added for 1 h. Oocytes were washed three more times with PBS and placed between the slide and coverslip for observation under a Nikon (Tokyo, Japan) microscope equipped with epifluorescence.

FIG. 1. Indirect immunoperoxidase staining localization of the antigen defined by mAb 19G9 on methanol/acetone-fixed ejaculated human sperm. A) Reactivity of permeabilized spermatozoa with 19G9 mAb: about 90% of spermatozoa are labeled in the region of the acrosomal cap (x380). B) Reactivity of permeabilized spermatozoa treated with the control mouse IgG1kappa: spermatozoa are not stained (x380).

Sperm PenetrationAssay The hamster egg penetration test was performed according to the protocol described by Yanagimachi [22]. Ova and attached cumulus cells were recovered from superovulted golden hamsters and treated with hyaluronidase (1 mg/ml) and trypsin (0.1%) in BWW to disperse the cumulus cells and remove zona pellucida, respectively. Zonafree eggs (20-30) were transferred to 200-,l aliquots containing 2 x 106 BWW/HSA-treated spermatozoa. One hour before the test, either I9G9 mAb or IgG, kappa mouse mAb or control medium was added to the BWW/HSA-treated spermatozoa. After insemination, the eggs were washed in BWW, mounted under cover glass, and examined at 400 x magnification under a phase-contrast microscope. Decondensing sperm heads having an attached or closely associated sperm tail in the cytoplasm and the spermatozoa bound per egg were counted.

StatisticalMethods Means were expressed with standard error of the mean (+ SEM). Chi-square test was used to compare proportions, and multifactorial variance analysis was performed for sperm binding to oocytes. RESULTS Five weeks after cell fusions, more than 400 independently derived clones were produced from two mice immunized with human sperm. From these clones, we selected mAb I9G9, which proved to be an IgG, kappa molecule and which stained the acrosomal cap region of the sperm head (Fig. 1A). This staining was not seen when a control mouse IgG, kappa mAb was substituted for mAb I9G9 (Fig. B).

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FIG. 2. Immunohistochemical localization of the antigen defined by mAb 19G9 in human testis and cauda epididymis. ST, seminiferous tubule; IT, intertubular space; Lu, cauda epididymis lumen; Ep, epithelial cells of cauda epididymis. A) Cross section of seminiferous tubules reacted with the 19G9 mAb (x200). B) At higher magnification (x1000), both crescent-shaped (arrow) and smaller granular reaction products (arrowhead) are observed in cohorts of similar-stage germ cells within a single seminiferous tubule. C) Tissue section treated with the control mouse IgGlkappa shows no staining (x80). D) Tissue of cauda epididymis treated with 19G9 mAb: spermatozoa contained within the lumen are stained (x380).

Localization of the Epitope Defined by the mAb Selected (I9G9) (Figs. 1-3) ImmunohisIn testis and cauda epididymis (Fig. 2). tochemical examination of paraffin-embedded human testis exposed to mAb I9G9 revealed binding to adluminal spermatids within the seminiferous tubules (Fig. 2A). Within round spermatids, immunostaining was observed in crescent-shaped structures and in smaller ovoid granules (Fig. 2B). Basal spermatogonia, Sertoli cells, spermatocytes, and cells within the testicular interstitium had no immunoreactivity. In cauda epididymis, mAb I9G9 stained spermatozoa in the lumen (Fig. 2D). At a higher magnification, it was clear that only acrosomal caps were stained (data not shown). Control sections of human testis incubated with control mouse IgGikappa mAb showed no immunoreaction product (Fig. 2C). On ejaculatedspermatozoa (Figs. 1 and 3). The I9G9 mAb stained the acrosomal cap of more than 90% of methanol/acetone-fixed ejaculated human spermatozoa (Fig. 1A). In samples containing spermatozoa fixed with glutaraldehyde, mAb I9G9 reacted with 14 + 3% of untreated spermatozoa, 29 ± 8% of sperm exposed to capacitating con-

ditions, and 56 + 14% of sperm induced to undergo acrosomal loss after exposure to calcium ionophore (mean + SEM of four separate experiments). Immunoelectron microscopy labeling with mAb I9G9 and colloidal gold-conjugated second antibody evidenced no binding if I9G9 antibody to acrosome-intact sperm (Fig. 3A); it also showed the corresponding epitope to be located in the matrix of the principal part of the acrosome (Fig. 3B). In completely acrosome-reacted sperm, labeling was along the inner acrosomal membrane (Fig. 3C). Incubation with control mouse IgGlkappa mAb showed no staining under any conditions (data not shown). Biochemical Characterizationof the Antigen Recognized by mAb 19G9 The biochemical characteristics of the antigen were studied by Western blotting of one-and two-dimensional electrophoresis gels of sperm homogenates. The pattern of immunoreactive sperm proteins observed in Western blots of a 12.5% acrylamide one-dimensional SDS-PAGE gel gave resolution of at least 12 distinct peptide bands (Fig. 4a) with a range from 16 to 35 kDa. Control Western blots incubated

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FIG. 4. Coomassie blue-stained one-dimensional SDS-polyacrylamide gel (B) and immunoblot (A) using 19G9 mAb on proteins extracted from human sperm. Molecular weight standards x10 -3 . a) Pattern of immunoreactivity of proteins extracted from human sperm without prior treatment with protease inhibitors. b) Pattern of immunoreactivity of proteins extracted in the presence of protease inhibitors. c) Pattern of immunoreactivity of proteins extracted in the presence of protease inhibitors and precipitated by TCA.

with control mouse IgGkappa mAb showed no immunoreactive proteins (data not shown). In the presence of protease inhibitors, the polymorphism of immunoreactivity was lower, particularly when proteins were precipitated by TCA (Fig. 4, b and c). On Western blots of two-dimensional gel electrophoresis performed on proteins extracted in the presence of protease inhibitors, mAb I9G9 reacted with a series of peptide spots with isoelectric points from 5.5 to 4.8 (Fig. 5). Study of tissue specificity by Western blotting of 18 human tissue extracts including psoas muscle, kidney, liver, spleen, seminal vesicle, prostate, deferent duct, efferent duct, heart, lung, pancreas, adrenal, testis, and stomach showed no bands except in the testis, in which a band of 35 kDa immunoreacted with I9G9 (Fig. 6n). In Figure 6, only 14 tissues are shown (hypophysis, tongue, thyroid, and parathyroid are not included).

FIG. 3. Immunogold detection of 19G9 binding to sperm by transmission electron microscopy. A) Spermatozoa with intact acrosome did not show any binding of the antibody (x21,120). B) and C) Spermatozoa that had undergone the acrosome reaction exhibited binding of antibody to acrosomal contents and possibly to the inner acrosomal membrane (x21,120).

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FIG. 5. Coomassie blue-stained two-dimensional gel (A) and immunoblot (B) using 19G9 mAb on proteins extracted from human sperm in the presence of protease inhibitors; mAb 19G9 reacted with a series of peptide spots with isoelectric points from 4.8 to 5.5. Molecular weight standards - 3. x10

Sperm PenetrationAssay I9G9 mAb was tested for its ability to modify binding to and penetration of zona-free hamster oocytes by human spermatozoa. Five independent experiments were carried out with frozen ejaculates from five donors and fresh ejaculates from six other donors. Table 1 summarizes the results with frozen ejaculates. Examination of the hamster eggs 3 h after insemination in vitro revealed that, compared to the control medium, control mouse IgG,kappa mAb did not modify the rate of fertilization but significantly decreased (p < 0.05) the number of spermatozoa bound per egg. In contrast, each concentration of mAb I9G9 decreased the rate of fertilization (p < 0.006) and sperm attachment (p < 0.05). Compared to the medium containing 500 Lg/ml of

FIG 6. Coomassie blue-stained one-dimensional SDS-polyacrylamide gel (A) and immunoblot (B) using 19G9 mAb of proteins extracted from a) human sperm, b) psoas muscle, c) kidney, d) liver, e) spleen, f) seminal vesicle, g) prostate, h) deferent duct, i) efferent duct, j) heart, k) lung, I) pancreas, m) adrenal, n) testis, o) stomach. Arrow indicates the position of with 19G9 mAb in testis. the single band of proteins that - immunoreacted . Molecular weight standards x 10

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MONOCLONAL ANTIBODY TO A HUMAN INTRA-ACROSOMAL ANTIGEN TABLE 1. Effect of 19G9 mAb on sperm penetration of hamster eggs and on sperm binding to the hamster eggs: results with frozen ejaculates.* No. of eggs examined Controls Medium IgG1kappa (500 jg/ml) 19G9 mAb 125 pg/ml 250 jLg/ml 500 tig/ml

117 129

117 127 140

Percentage of eggs penetrated 22.9 -+3.3 15.0 + 3.4

8.5 ± 2.0" 7.8 + 2.4a 2.1 -+0.9' b"

No. of sperm bound/egg

TABLE 2. Effect of 19G9 mAb on sperm peneration of hamster eggs and on sperm binding to the hamster eggs: results with fresh ejaculates.* No. of eggs examined

6.3 + 2.0 3.9 + 1.2a

Controls Medium IgGl kappa 125 Lg/ml 250 iLg/ml 500 RIg/ml

4.7 + 1.9" 2.9 + 1.1abc 2.4 + 1.0ab,

19G9 mAb 125 ikg/ml 250 jIg/ml 500 AIg/ml

*Five experiments were done with five ejaculates from five donors. Zonapellucida-free hamster oocytes were incubated for 3 h with capacitated human sperm pretreated with purified mAb 19G9 or control IgG,kappa at the concentrations indicated. For each experiment, the number of penetrations and sperm bound per oocyte were counted (mean SEM). aSignificantly different from medium control. bSignificantly different from IgGlkappa control (500 jIg/ml). CSignificantly different from 19G9 (125 ig/ml). dSignificantly different from 19G9 (250 jLg/ml).

control IgGlkappa mAb, mAb I9G9 decreased the rate of fertilization at a concentration of 500 ,ag/ml (p < 0.0001) and the number of spermatozoa bound per egg at concentrations of 250 and 500 gxg/ml (p < 0.05). The lowest rate of fertilization was obtained with 500 ,ug/ml of mAb I9G9, and the smallest number of spermatozoa bound per egg was seen with 250 and 500 ,ug/ml. Table 2, summarizes the results with fresh ejaculates. Compared to the control medium, control mouse IgGkappa decreased the rate of fertilization (p < 0.0001) at a concentration of 500 ,ag/ml. Each concentration of mAb I9G9 decreased the rate of fertilization (p < 0.0001). Compared to medium containing 500 ,ug/ml of control IgGkappa mAb, I9G9 decreased the rate of fertilization at a concentration of 500 jzg/ml (p < 0.0001). Compared also to medium containing 125 ,ug/ml and 250 ,ug/ml of control IgGlkappa mAb, mAb I9G9 decreased the rate of fertilization (p < 0.0001). Antibody Binding to Oocytes

I9G9 mAb did not react with hamster oocytes (data not shown). DISCUSSION

Monoclonal antibody I9G9 reacted only with round spermatids and cells of subsequent stages of spermiogenesis on testis sections and was localized within the acrosome at the ultrastructural level. The sperm intra-acrosomal antigen (SIAA) that this mAb recognizes is expressed on the surface of acrosome-reacted sperm. On Western blots of human spermatozoa extracts, this mAb identified a series of peptides with molecular masses ranging from 16 to 35 kDa. To data, no sperm sample tested, either by immunocytochemistry (n = 50) or by Western blotting (n = 20)

has failed to react with I9G9 mAb, indicating that SIAA is

Percentage of eggs penetrated

No. of sperm bound/egg

164

33.4 + 5.8

2.5 + 0.3

159 156 165

34.7 + 8.9 33.6 + 6.5 21.9 + 7.9 "b

3.3 -+0.7 3.9 + 1.0 2.9 + 0.5

158 180

18.3 + 4.8ab 16.5 + 4.6bC

3.0 + 0.8 3.8 + 0.7

10.2 + 3 .2bcdf

1.8 + 0.4

150

a

*Six experiments were done with six ejaculates from six donors. Zona-pellucida-free hamster oocytes were incubated for 3 h with capacitated human sperm pretreated with purified mAb 19G9 or control IgGlkappa at the concentrations indicated. For each experiment, the number of penetrations and sperm bound per oocyte were counted (mean - SEM). 'Significantly different from medium control. bSignificantly different from IgGlkappa control (125 pig/ml). CSignificantly different from IgG kappa control (250 jig/ml). dSignificantly different from IgGlkappa control (500 Rig/ml). "Significantly different from 19G9 (125 Aig/ml). fSignificantly different from 19G9 (250 g/ml).

conserved in the human population. The multiple forms of the peptides that are identified by Western blotting with mAb I9G9 may be the consequence of post-translational modification, proteolytic processing of the proteins within the acrosome, multiple gene products, or several of these possibilities acting in concert. The decrease in the polymorphism of immunogenic peptides in proteins extracted in the presence of inhibitor proteases clearly demonstrated that this polymorphism was due, at least in part, to proteolytic processing. Furthermore, Western blotting of human testis extracts evidenced a single band of 35 kDa. This finding indicates that post-testicular modifications occur in the genital male tract, or possibly after ejaculation. Similar modifications have been observed for some proteins of the acrosomal region in mouse sperm during epididymal transit [23]. Similar results were obtained with acrogranin, which appeared as a single protein band of 67 kDa in guinea pig testis and consisted of four bands in epididymal sperm extracts of 62, 51, 39, and 22 kDa [24]. It has been hypothesized that these acrogranin variants result from proteolytic processes that develop during epididymal maturation [24]. A sperm intra-acrosomal antigen, SP-10, has been characterized [12, 25]. On Western blots, the mAb (MHS-10) directed against this antigen identified a series of peptides with molecular masses ranging from 18 to 34 kDa, with a polymorphism of immunogenic peptides closely similar to that we obtained in this molecular mass range. Whether or not mAb I9G9 recognizes the same antigen has not been clearly demonstrated. SP-10 heterogeneity results from endoproteolytic processes [26], but multiple forms of SP-10 from 18 to 34 kDA have been observed in extracts collected in a cocktail of protease inhibitors [26], in SDS extracts of

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testes, and in sperm collected by ejaculation directly into SDS. In contrast, we observed no SIAA heterogeneity in testes extracts, in which only a single band of 35 kDA was detected. I9G9 mAb inhibits sperm-egg interaction in the hamster egg penetration assay. The block is not due to agglutination or immobilization of the sperm. A number of sperm antigens have been reported to regulate specific steps of the fertilization mechanism in different species, including humans [15, 27-29]. SIAA was associated with the inner acrosomal membrances of acrosome-reacted sperm (as shown by immunoelectron microscopy labeling) and was a testis-specific antigen localized within the spermatids and cells of subsequent stages of spermiogenesis. Many mAb have been raised against acrosomal components [30-32]. Some antibodies that label acrosome-intact spermatozoa may bind to the outer acrosome membranes or acrosomal matrix. After the acrosomereaction these antigens disappear [30, 31, 33-36]. Other mAb label only acrosome-reacted spermatozoa and may recognize antigens on the outside of the inner acrosome membrane. After the acrosome reaction or exposure of the inner acrosome membrane, binding activity appears [14, 3739]. I9G9 mAb might function as a probe system for scoring the acrosome reaction and make it possible to analyze viable samples and concomitantly assess other sperm functions. Furthermore, SIAA is a sperm-specific antigen, not found in somatic tissues (Western blot studies of 18 human tissue extracts detected SIAA solely on testis and ejaculated spermatozoa), and it is stage-specific. However, we studied tissue from only one inidividual and cannot positively claim that tissue specificity will be found in the population at large. In our study SIAA was immunohistologically localized within round spermatids and all cells of subsequent phases of spermiogenesis. I9G9 mAb might therefore function as a probe system for diagnosing the immature germ cells in semen, as has been shown with MHS-10 mAb [40]. The WHO task force on contraception vaccines identified six sperm mAbs as primary vaccine candidates on the basis of three criteria: 1) reactivity with human testicular germ cells and abundant surface antigens on mature spermatozoa, 2) no, or very little, cross-reactivity with somatic cells, and 3) inhibition of at least one sperm function assay [29]. Because of the intra-acrosomal localization of SIAA it is not an abundant surface antigen. Nevertheless, Herr et al. [13] assumed that the remodeling of the sperm head membrane that accompanies the acrosome reaction opens the possibility that constituents of the acrosome, although sequestered from the immune system in intact sperm, should not be dismissed as candidates for a contraceptive vaccine without examination of their fate following the acrosome reaction. Further studies, including molecular cloning of cDNA corresponding to SIAA for studies by Northern blot, are needed to determine whether SIAA is specific for testicular germ cells.

I9G9 mAb cross-reacted with a variety of species including mouse sperm (data not shown); this means it may be possible to employ experimental animal models to test the use of antigenic determinants recognized by this mAb as birth control vaccines. In conclusion, we characterized an SAA that was associated with the inner acrosomal membrane after the spermatozoa had undergone acrosome reaction. This antigen was testis-specific, was localized within the spermatids and cells of subsequent stages of spermiogenesis, and might undergo maturation in the epididymis. Furthermore, I9G9 mAb defines a sperm antigen with a putative role in the fertilization process and might function as a probe system for diagnosing the immature germ cells in semen and scoring the acrosome reaction. It would be of interest to study the expression of the I9G9 antigen on sperm from men with unexplained infertility. ACKNOWLEDGMENTS We thank Ms. C. Cohendy for tping the manuscript and Mr. J. Watts for linguistic assistance.

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