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Res. Immunol. 1994, 145, 533-539

0 INSTITUT PASTEURIELSEVIER

Paris 1994

Characterization of monoclonal antibodies specific for human sperm: effect of CRL-10 on acrosome reaction V. Garcia-Framis

(I), E. Martin-Lunas cl), A. Iborra (I), P. Andolz M.A. Bielsa (*I and P. Martinez (‘)(*)

(*),

(I) Institut de Biologia Fonamental i Departament de Biologia Cellular i Fisiologia, UAB. 08193 Bellaterra, Barcelona , and (2)Unidad Andrologia Bcisica, Servicio de Obstetricia y Ginecologt’a, Hospital de1Mar, Barcelona (Spain)

SUMMARY

Monoclonal antibodies to human sperm were obtained from hyperimmunized BALB/c mouse spleen cells fused with myeloma NS-1 cells. Each antibody recognized definite regions in fresh unfixed sperm : equatorial region, acrosome, postacrosome, midpiece, tail. All the antibodies were specific for sperm. We selected CRL-10 monoclonal antibody, specific for acrosome, for a detailed study. The expression of the CRL-10 antibody-bound antigen was detected in other mammalian species. When CRL-10 antibody was added prior to sperm incubation in a capacitating medium, promotion of the acrosome reaction was observed. Key-words: Sperm, Acrosome, Monoclonal recognition.

INTRODUCTION During fertilization, several surface molecules of both gametes are involved in gamete recognition. Characterization of sperm membrane antigens by means of monoclonal antibodies (rnAb) may help in the elucidation of their function. Because of the need for capacitation and acrosome reaction of sperm prior to fertilization, acrosomal antigens have been identified by several authors (Fenichel er aZ., 1990; Dubova-Mihailova et al., 1991). The acrosome reaction (AR) has been evaluated by means of specific mAb and com-

Submitted

December

3, 1993, accepted August 9, 1994.

(*) Corresponding author.

antibody;

Human, Specificity,

Gamete

pared with other staining techniques such as the triple stain (Braun ef al., 1991). Other methods have been described for the assessment of the acrosomal status, such as the ability to bind lectins (Holden and Trounson, 1991), and this has been correlated with zona-h-ee hamster egg penetration test (Salonen and Kallajoki, 1987). By using rnAb we can investigate the distribution of several antigens and the involvement of these antigens in fertilization. The purpose of this study was to induce some human sperm specific mAb and to determine the effect of these antibodies on the acrosome reaction.

V. GARCiA-FRAMIS

534 MATERIAL

Immunization

AND METHODS

and hybridoma

production

To generate anti-human sperm mAb, BALB/c mice were immunized with human sperm. Human semen with normal semen parameters was obtained from healthy donors by masturbation after 3-4 days of sexual abstinence. Isolated semen samples were used from a few controlled donors. The injections of a suspension of 6-10~ lo6 washed human sperm resuspended in 0.1 ml of PBS (phosphate-buffered saline) were emulsified in complete Freund’s adjuvant. Booster injections were given at 2-3 week intervals using incomplete Freund’s adjuvant. Animals showing the highest antibody titres by ELISA (enzyme-linked immunosorbent assay) were selected for the fusion. The final inoculation was given three days prior to fusion. On the day of fusion, mice were sacrified and immune spleen cells (8 x 108) were fused with mouse myeloma cells NS-1 (1 x 10’) using 30% polyethylene glycol (MW 1,300-1,600) (Sigma) in the presence of 10% dimethylsulphoxide. About 15 days after fusion, when the growing hybridomas showed antisperm antibody activity dectected by ELISA, cloning of the positive wells by limiting dilution was performed. The positive clones were recloned. BALB/c mice were injected intraperitoneally with the hybridoma cells to obtain ascitic fluid containing satisfactory titres of antibody. ELISA

for the selection of mAb

The procedure for ELISA employed in our laboratory was described previously (Benet-Rubinat et al., 1991). Briefly, 100 pl of washed intact human spermatozoa resuspended in PBS at a concentration of 5 x lo6 cells/ml were added to flat-bottom polystyrene plates. The plates were centrifuged for 10 min at 1,000 g and then futed with 0.25 % glutaraldehyde. (After three washings, the plates can be stored at -20°C for several months). Specific binding was avoided by incubating with 1% BSA (bovine serum

AR ELISA FACS FF

= = = =

acrosome reaction. enzyme-linked immunosorbent fluorescence-activated cell follicular fluid.

ET AL. albumin) in PBS+O.OS% Tween-20 buffer for 1 h at room temperature. Supematants from hybridoma cultures were incubated for 2 h. The plates were washed three times with PBS+Tween -20, and goat anti-Ig (Fc) from mouse immunoglobulin conjugated to horseradish peroxidase was added and kept for 2 h at 37°C. The wells were washed again and the MBTHDMAB (0.8 mM 3-methyl-2-benzothiazolinone hydrazone hydrochloride ; 40 mM 3-dimethylaminobenzoic acid)-substrate, 3 mM in I-$0*, was incubated for 20 min. The reaction was stopped with 2 M sulphuric acid, and the optical density was read at 600nm by an “Anthos 2001” plate reader (Anthos Labtec Instruments). For the titration of the mAb and the cross-reactivity experiments, 1 x IO6 cells/ml (human, mouse, sheep and goat) were used to coat the plates, as this was determined to be the optimal concentration previously. The responses were expressed as ELISA titres, defined as the absorbance at 600nm of every mAb at the dilution that gives 70% of the maximal response; 1 mg/ml of homologous or heterologous mAb was used, and the protocol was the same as that described above. Control responses with no antigen and with no antibody were included in every ELISA plate. Antibody

sorter.

from ascites

The immunoglobulins obtained from the ascites were purified by affinity chromatography using protein-A-Sepharose-CL-4B (Pharmacia). The antibody peak was eluted with 0.1 M citric acid, pH 4. The class and subclass of immunoglobulin was determined by double diffusion in 1% agarose Ouchterlony plates with antibodies raised against various subclasses of imrnunoglobulins. Isoelectrofocusing Monoclonality of the antibodies was analysed by isoelectric focusing, in a “Phast-System” provided by Pharmacia. A gel with a linear gradient from 3 to 9 was used. The gel was loaded with 1 pl of the purified mAb at a concentration of 1 mg/ml. The bands were developed by the silver staining technique. The isoelectric pH range was calculated for each mAb using a lo-protein p1 calibration kit.

FITC assay.

purification

mAb PBS

= = =

fluorescein isothiocyanate-conjugated. monoclonal antibody. phosphate-buffered saline.

mAb SPECIFIC FACS (fluorescence-activated sis of blood lymphocytes

FOR HUMAN

SPERM:

cell sorter)

analy-

Peripheral blood mononuclear cells obtained from healthy human donors were isolated by gradient centrifugation on Ficoll (Bijyum, 1968). The cells were resuspended at a concentration of 20x lo6 cells/ml in 10 % rabbit serum inactivated at 56°C; 50 p.1 of the cell suspension were incubated for 30 min at 4°C with 50 ~1 of each mAb (2 mg/ml) diluted 1:200. Cells were washed three times and were incubated with 50 pl of fluorescein isothiocyanate-conjugated (FITC)-goat antimouse IgG (diluted l/50) (Sigma). Positive and negative controls were included in each experiment. Each mAb was also tested with washed human sperm (20x lo6 sperm/ml). The fluorescence analysis of the cells was performed with a FACS “Star Plus” analyser (Becton Dickinson) with a “Consort 32” data analysis. Indirect

immunofluorescence

on human sperm

Washed spermatozoa were selected by swim-up (Andolz et al., 1987) in BWW (Biggers, Whitten and Whittingham) medium (Biggers et al., 1971) for 1 h. After sperm selection, 20~10~ cells/ml were incubated with the mAb at a concentration of 1 mg/ml in suspension (l/l ; v/v) for 60 rnin at 37°C. Binding of antibody was visualized after incubation with FITC-goat anti-mouse IgG for 30 min at 37°C. Samples were analysed using a Leitz epifluorescence microscope. Indirect

immunofluorescence

on human

tissues

Indirect immunofluorescence was performed with spleen, pancreas, thyroid, heart, skin, intestine and lung frozen human tissues. The 5-pm tissue sections were prepared in a cryostat and were air-dried for 1 h; 50 ~1 of the purified mAb at a concentration of 1 mg/ml were incubated for 30 min at room temperature, and after 3 washings, FITC-goat antimouse IgG (Southern Biotechnology Association, Inc., USA) diluted 1:lOO was used as a second antibody. Slides were rinsed in PBS, mounted in glycerine and read under an epifluorescence microscope. Sperm capacitation

and acrosome

staining

Sperm was selected by swim-up in Ham F-10 medium containing 30 mg/rnl of human serum albumin. A 200+1 aliquot from the sperm suspension (5-10x 106 cells/ml) was incubated with 200 ~1 of follicular fluid (FF). FF was previously diluted 1: 10 in Ham F-10 and concentrated by ultrafiltration

CRL-10

AND ACROSOME

535

REACTION

through a l,OOO-Da cut-off filter. It was inactivated for 35 min at 56°C. Alternatively, 200 pl of sperm suspension were incubated with 200 pl of mAb (specific or non-specific for sperm) diluted in FF at a concentration of 1 mg/mI. An aliquot of 200 pl of sperm was assayed in Ham F-10 medium as a control of spontaneous acrosome reaction. Then, aII the aliquots were aIlowed to capacitate for a period of 10 h in 5% CO, at 37°C. For the triple staining technique (Talbot and Chacon, 1981), an equal volume (200 ~1) of 2% Trypan blue in albumin-free Ham F-10 was added to a 200 p.1 aliquot. The tube was then incubated for 15 min at 37°C. it was diluted in 1 ml of Ham F-10 and centrifuged at 600 g for 10 min. After two washings, the supernatant was removed and the sperm in the pellet was resuspended in 2 ml of albumin-free medium and centrifuged as before. Sperm was resuspended in 200 ~1 of 3% glutaraldehyde in 3 % saline solution and it was placed for 1 h at 4°C. Sperm was washed twice and an aliquot of the sperm suspension was placed on a slide and smeared with a second glass slide. The smears were air-dried at room temperature. Spermatozoa on the slides were stained for 30 min with 0.8% brown bismark in distilled water pH 1.8. Slides were stained with 0.8% rose bengal in 0.1 M Tris buffer pH 5.3 for 10 min. A coverslip was mounted, and the spermatozoa were examined with bright-field microscopy at x 1,000. At least 400 spermatozoa were classified according to the presence of one of four patterns. These include a) light brown postacrosomal regions with pink acrosomes: live sperm with non-reacted acrosome ; b) light brown postacrosomal regions with unstained acrosomal regions: live sperm with reacted acrosome; c) dark brown postacrosomal regions with dark pink acrosomes: dead sperm with non-reacted acrosome ; d) dark brown postacrosomal regions with unstained or dark blue acrosomal regions: dead sperm with reacted acrosome.

RESULTS

Isolation

and specificity

of anti-sperm

Five hybridomas secreting mAb sperm antigens were selected. These were produced in female BALB/c mice ing in vivo tumours in their peritoneal

the antibodies

were of the IgGl

mAb to human antibodies by induc-

cavity. Ail subclass. The

purity of the mAb was checked by isoelectrofocusing, and the p1 were as follows: THY-6 6.38-

7.06; PAT-5, 6.18-6.72; ASP-3, 6.52-7.06; 12, 5.80-6.50; and CRL-10, 6.59-6.86.

NAT-

536

V. GARCjA-FRAMIS

The specificity of antibodies was tested by several techniques. To investigate the species distribution of the antigens recognized by esch mAb, goat, ram and mouse sperm were used for coating the wells in an ELISA microtitre plate. Goat sperm was the cell that reacted at the highest rate with all the mAb, except with PAT-5. Ram and mouse sperm also expressed the antigens recognized by the mAb (table I). The expression of each antigen in the human tissues was visualized on tissue slices by indirect immunofluorescence microscopy (using the purified antibodies and FlTC-rabbit anti-mouse IgG). All mAb were negative for the human tissues tested. As for reactivity with human erythrocytes, lymphocytes and monocytes, none of the five mAb showed any fluorescence staining by FACS analysis; the percentage of positive stained sperm by FACS ranged from 50 to 80% depending on the mAb used (table II).

Table I. Reactivity

of anti-human sperm mAb with human sperm and sperm from other species. Sperm from:

mAb

human

ram

goat

mouse

THY-6 PAT-5 ASP-3 NAT-12 CRL-10

1.04 0.700 0.665 0.630 0.600

0.450 0.550 0.297 0.166 0.420

0.630 0.400 0.450 0.315 0.470

0.480 0.160 0.200 0.300 0.450

localization by five mAb.

of anti-

The values

(A,)

are obtained

Table II. Immunofluorescent gens recognized mAb

by ELISA.

Labelled cell ~1 (%)

Immunofluorescence on washed human spermatozoa c2)

50 82 65 51 55

Equatorial region Postacrosome Acrosome/equatorial region/tail Acrosome/(postacrosome) Midpiece

ASP-3 PAT-5 THY-6 (XL-10 NAT-12 (I) Results t2) Isolated

obtained by FACS semen samples.

analysis.

ET AL.

Anti-I-ILA class II antibody was used as a positive control, and supematant from NS-1 myeloma was used as negative control.

Immunolocalization

of antigens on spermatozoa

Unfixed spermatozoa were incubated with each purified mAb, and several head staining patterns were found (acrosome, equatorial region, postacrosome, midpiece, tail). Figure 1 shows THY-6 with a multiple staining pattern (acrosome, equatorial region, tail) and CRL-10, with fluorescence in the acrosome, spreading to the postacrosome ; this antibody is used in further functional studies. When the plasma membrane was damaged, either by methanol or by acetone fixation, none of the mAb stained the cell as observed by immunofluorescence. No fluorescence was observed with a non-specific mAb such as anti-ribonuclease mAb.

Acrosome

staining

Experiments for the assessment of the acrosome reaction were performed with CRL-10 mAb, which showed acrosome staining. In the percentage of acrosome-reacted cells, only the live reacted sperm was considered (true acrosome reaction). The acrosome reaction (AR) was substantially increased by the presence of (XL-10 mAb. The other mAb showed similar values of AR as did control or mAb non-specific for sperm (table III). The experiments were performed in the presence of FF, (1: 10, v/v) which induces AR (4 % of acrosome-reacted cells versus 1.5 %).

DISCUSSION Antisera, as well as mAb raised to isolated acrosomal membranes have been applied to the recognition, topographical situation and functional analysis of some membrane antigens involved in the fertilization events (Topfer-Petersen et al., 1986). Monoclonal antibodies stain specifically some of the regions on sperm, showing a surface staining or an intracellular localiza-

mAb SPECIFIC

FOR HUMAN

Fig. 1. Immunofluorescent

SPERM:

Ham-F1 O/no mAb Follicular fluid/no mAb Anti-ribonuclease mAb Anti-acrosome mAb (CRL- 10)

AND ACROSOME

localization of antigens recognized by (XL-10 in non-capacited human sperm.

Table III. Effect of CRL-10 mAb on AFL Treatment

CRL-10

Percentage of AR (n = 3) 1.5 f 4k 5.08 + 28.5 +

0.70 2.27 1.53 2.94

Results are given in percentages of live sperm with reacted acrosome (mean * SD).

tion. Kallajoki and Suominen (1984) describe an mAb that detects an intra-acrosomal localization on acetone-fixed sperm, but no staining is obtained with living, intact spermatozoa. The mAb described in the present paper stain surface antigens in untreated, intact membranes. The epitopes recognized by these mAb are destroyed after exposure to some fixatives such as acetone or methanol, and no fluorescence is observed after fixation.

REACTION

(a) and THY-6

537

@) mAb

The percentage of mAb-stained cells was analysed by FACS. The antibodies specific for some regions, such as the postacrosome, provided a high percentage of positive cells (82 %), whereas the antibodies binding the acrosome, midpiece or equatorial region showed a lower percentage of positive cells (50-59 %). From our experience, other mAb obtained in our laboratory, specific for the tail, stained higher numbers of cells (70-92 %). This is in agreement with other authors (Villarroya and Sholler, 1987), who detect fluorescence in several regions of sperm and establish better labelling in individual ejaculates for the entire tail (90100%) as compared with other regions: acrosome (12-56 %), midpiece (9-35 %) or equatorial region (8-35 %). Monoclonal antibodies to acrosome antigens have been used to detect abnormal acrosomes and may be considered as good markers of acrosome morphology (Moore et al., 1987; Peknicova and Moos, 1990). The mouse mAb GB24, that reacts with a human acrosomal sperm antigen after the acrosome reaction induced by different

538

V. GARCiA-FRAMIS

methods, is localized on the inner acrosomal membrane (FCnichel et al., 1989, 1990). Some mAb are used for evaluating the acrosomal reaction in human sperm (Herr et al., 1990; Parinaud et ul., 1993). Some inhibitory effects of anti-sperm mAb have been described with the use of some functional probes, such as sperm penetration of zona-free hamster eggs. The incubation of mAb for acrosome antigens may also inhibit the AR and impair or induce the penetration through the oocyte. In our results we observed a promotion of the AR in an antibody that stained the acrosome: CRL-10. The distribution of this antigen after capacitation has a spot-like arrangement. This induction of acrosome reaction seems to be specific for CRL-10, since other mAb non-specific for sperm have no effect on acrosome. Other mAb specific for acrosome showed an increase in the percentage of acrosome reaction after antibody-sperm incubation in a concentration-dependent manner (results not shown). This role in the induction of the AR has also been described by Aarons et al., (1992) in capacitated mouse sperm: an mAb, J-23, specific for a sperm antigen, induces AR in a concentration-dependent manner; these results are in agreement with the effect produced by the mAb CRL- 10. Our data indicate that, after capacitation, the antigen recognized by CRL-10 mAb still has head localization. The rearrangement of these antigens over the sperm head might induce AR in a manner similar to that obtained by the aggregation of zona-binding components on sperm (Macek et al., 1991). Interestingly, the antigen bound to CRL-10 might play a role in egg penetration, as this antibody shows a high inhibitory effect on mouse fertilization: 87 and 100% inhibition at several antibody concentrations (0.4 mg/ml and 0.9 mg/ml, respectively) (Garcia-Framis et al., 1994).

Acknowledgements This research was supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS 8810891; 89/0225).

ET AL.

Caract&isation d’anticorps monoclonaw sp6cifiques du sperme humain : effet de l’anticorps (XL-10 sur la &action de l’acrosome

Des anticorps monoclonaux anti-sperme humain ont CtC obtenus g partir de cellules spltniques fusionnkes avec des cellules de myklome NS-1. Chaque anticorps reconnait une rCgion dtfinie du

spermatozoi’de frais, mobile : rCgion Cquatoriale, acrosome, post-acrosome, pibce moyenne, queue. Tous les anticorps

sont spkcifiques

du sperme. En

vue d’une ttude d&We. nous avons sClectionnC l’anticorps monoclonal CRL-10, spCcifique de l’acrosome. L’anticorps CRL-10 1iC ?i l’antigbne a ttt mis en tvidence chez d’autres espbces de mammiferes.

Quand l’anticorps

avant l’incubation

du sperme

(XL-10

est ajoutC

dans un milieu

adkquat, on observe une stimulation de I’acrosome.

de la &action

Mots-cl& : Sperme, Acrosome, Anticorps monoclonal ; Homme, SpCcificitC, Reconnaissance des gambtes.

References Aarons, D., Battle, T., Boettger-Tong,H. & Pokier, G.R. (1992). Role of monoclonalantibody J-23 in inducing acrosomereactionsin capacitated mousespermatozoa.J. Reprod Fertil., 96, 49-59. Andolz, P., Bielsa,M.A., Genesca,A., Benet, J. & Egozcue, J., (1987). Improvement of sperm quality in abnormalsemensamplesusing a modified swim-up procedure.Hum. Reprod., 2, 99-101. Benet-Rubinat,J.M., Martinez, P., Lepp, W.A., Egozcue, J., Andolz, P. & Bielsa, M.A., (1991). Detection of induced anti-sperm antibodies by an improved enzyme-linked immunosorbentassay.ht. J. Fertil., 36, 48-56.

Biggers,J.D., Whitten, W.K. & Whittinham, D.G., (1971), The culture of mouseembryosin vitro, in “Methods in mammalian embryology”, (Daniel, J.C.), (pp. 86106). W.H. Freemanand Co San Francisco. Bijyum, A. (1968), Isolation of mononuclear cells and granulocytesfrom humanblood. Sand. J. Clin. Lab. Invest., 21, (suppl.97), 77-89. Braun, J., Hirsch, Y., Krause,W. & Zieglesr, A., (1991), Evaluation of the acrosomereactionusing monoclonal antibodiesagainstdifferent acrosomalantigens. Comparisonwith the triple stain technique.Intemat. J. Andrology, 14, 431-436. Dubova-Mihailova, Mollova, M., Ivanova, M., Kehayou, I. KcKyurchiev, S., (1991), Identification and characterization of humanacrosomalantigendefined by a monoclonalantibody with blocking effect on in vitro fertilization. J. Reprod. Immunol., 19, 251-268.

mAb

SPECIFIC

FOR

HUMAN

SPERM:

Ftnichel, P., Hsi, B.L., Farahifar. D., Donzeau, M., Bartier-Delpech, D. & Yeh, C.J.G., (1989). Evaluation of the human sperm acrosome reaction using a monoclonal antibody, GB24, and fluorescence-activated cell sorter. J. Reprod. Fertif., 87, 699-706. Fenichel, P., Gottfried, D., Grivaux. C., Cervoni, F., Donzeau, M. & Hsi, B.L. (1990). Localization and characterization of the acrosomal antigen recognized by GB24 on human spermatozoa. Mol. Reprod. Develop., 27, 173-178. Garcia-Frank, V., Martorell, R., Marquez, C., Benet, J., Andolz, P. & Martinez, P., (1994), Inhibition by antisperm monoclonal antibodies of the penetration of zona-free hamster oocytes by human spermatozoa. Imtnunol. Cell Biol., 72, 1-6. Herr, J.C., Flickinger, C.J., Homyk, M., Klotz, K. & John, E., (1990), Biochemical and morphological characterization of the intra-acrosomal antigen SP-10 from human sperm. Biof. Reprod., 42, 181-193. Holden, C.A. & Trounson, A.O., (1991), Staining of the inner acrosomal membrane of human spermatozoa with concanavalin A lectin as an indicator of potential egg penetration ability. Fertil. Steril., 56, 967-974. Kallajoki, M. & Suominem, J., (1984), An acrosomal antigen of human spermatozoa and spermatogenic cells characterized with a monoclonal antibody. Int. J. Andrology, 7, 283-296. Macek, M.B., Mpe~ L.C. & Shur, B.H. (1991). Aggregation of P-1,4-galactosyltransferase on mouse serum induces the acrosome reaction. Develop. Biol., 147.440444.

CRL-10

AND

ACROSOME

REACTION

Moore, H.D.M., Smith, C.A., Hartman, T.D. & Bye, A.P. (1987). Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody. Gamete Rex 17. 245-259. Parinaud, J., Labal, B., Vieitez. G., Richoilley, G. & Grand Jean, H.. (1993). Comparison between fluorescent peanut agglutinin lectin and GB24 antibody techniques for the assessment of acrosomal status. Hum. Reprod., 8, 16851688. Peknicova, J. & Moos, J., (1.990), Monoclonal antibodies against boar acrosomal antigens labelling undamaged acrosomes of spermatozoa in immunofluorescence test. Andrologia. 22, 427-435. Salonen, I. & Kallajoki, M., (1987), Monoclonal antibody against human sperm acrosome inhibits sperm penetration of zona-free hamster eggs. Int. J. Andrology, 10, 731-739. Talbot, P. & Chacon, R.S., (1981), A triple-stain technique for evaluating normal acrosome reaction of human sperm. J. Exp. Zool., 215, 201-208. TBpfer-Petersen. E., Auerbeck, J., Weiss, A., Friess, A.E. & Scholl, W.B., (1986), The sperm acrosome: immunological analysis using specific polyclonal and monoclonal antibodies directed against one outer acrosomal membrane of boar spermatozoa. Andrologia. 18, 237-251. Villarroya, S. & Scholler, R., (1986), Regional heterogeneity of human spermatozoa detected with monoclonal antibodies, J. Reprod. Fertil.. 76. 435-447.