Histochemistry (1990) 93:485-490 Histochemist © Springer-Verlag 1990

Boar sperm membranes antigens. I. Topography of a mobile glycoprotein of the sperm cell membrane E. Ti~pfer-Petersen t, A.E. Friess 2, M. Stoffel 2, and W.B. Schill 3 t Department of Dermatology, Andrology Unit, University of Munich, Munich, Federal Republic of Germany 2 Institute of Veterinary Anatomy, University of Bern, Bern, Switzerland 3 Department of Dermatology and Andrology, University of Giessen, Giessen, Federal Republic of Germany

Accepted November 25, 1989

Summary. A monoclonal antibody, designated mAb P86/5, was generated by immunization of female Balb/c mice with a membrane vesicle fraction composed of the outer acroso- mal membrane and plasma membrane (PM-OAM). As de- termined by fluorescence microscopy and electron micros- copy P86/5 recognizes a sperm plasma membrane antigen that is restricted to the sperm head. In intact spermatozoa the P86/5-antigen is distributed over the surface of the sperm head with the exception of the rostral region. By comparing the antibody binding pattern generated at 4 ° C and 25 ° C, it could be shown that the P86/5-antigen is capa- ble to diffuse freely within the cell membrane overlying the acrosome whereas its lateral mobility is restricted to the post-acrosomal region. The P86/5-antigen had a molec- ular weight of about 78 kDa as revealed by SDS-PAGE and western blotting. The glycoprotein nature of the P86/5- antigen was established by lectin affinity chromatography.

Introduction

Since Singer and Nicolson (1972) presented the "fluid mo- saic model" of biomembranes the molecular mechanisms of many membrane-associated phenomena have been en- lightened. A bilayer of phospholipids forms the structural matrix of the membrane in which integral proteins are em- bedded both of which can diffuse laterally within the plane of the membrane. The lateral mobility of lipids and proteins is of fundamental significance for cell function. However, cells are able to overcome the randomizing effect of diffu- sion and can localize certain membrane components to spe- cific regions of the cell surface (Wolf 1983; Peterson and Russell 1985).

The spermatozoon is a highly polarized cell. Five mor- phologically distinct regions can be easily recognized by light microscopy and have been related to different sperm functions such as sperm motility, zona penetration and sperm-egg fusion (Fawcett 1975). Accordingly, the sperm surface is organized in distinct, highly specialized regions. By various surface labelling techniques the regionalization of the sperm surface has been described (reviewed by

Please send offprint requests to: Dr. Edda T6pfer-Petersen, Derma- tologische Klinik und Poliklinik der Ludwig-Maximilians-Un- iversit/it Miinchen, Frauenlobstr. 9/11, D-8000 Mfinchen 2/FRG

Koehler 1981). The introduction of monoclonal antibodies opened a new dimension to study the functional organiza- tion of the sperm surface at the level of individual mole- cules. Five plasma membrane domains corresponding to morphologically visible regions have been first described in the guinea pig (Primakoff and Myles 1983). Using a collection of monoclonal antibodies directed against the iso- lated plasma membrane at least 16 additional domains or subdomains have been demonstrated in boar spermatozoa (Saxena et al. 1986; Peterson et al. 1987) which may reflect the ability of the sperm cell to restrict certain membrane components to certain areas of the cell surface.

To facilitate the topographical and structural analysis of sperm components involved in the complex mechanism of fertilization we have produced monoclonal antibodies against membrane fractions of boar spermatozoa (T6pfer- Petersen et al. 1986, 1988). In this communication the to- pography of a mobile glycoprotein of the boar sperm cell membrane detected by a monoclonal antibody is described.

Materials and methods

Antibodies. Hybridoma antibodies directed against sperm mem- brane antigens were produced as previously described in detail (T6pfer-Petersen et al. 1986; Hinrichsen et al. 1985). A membrane vesicle fraction composed of the outer acrosomal membrane and of approximately 20% plasma membrane vesicles (PM-OAM-frac- tion), as revealed by electron microscopy, was used as immunogen (T6pfer-Petersen and Schill 1983). The immunized mouse was bleeded to obtain mouse anti-serum. The spleen was removed for hybridoma production using standard techniques (Fazekas de St. Groth and Scheidegger 1980). Hybridoma supernatants were tested for the presence of antibodies recognizing sperm antigens by the enzyme-linked-immunosorbent assay (ELISA) on microelisa plates (Dynatech-Cooke, Plochingen, FRG) precoated with the solubi- lized membrane vesicle fraction (T6pfer-Petersen et al. 1986). Fur- ther screening of sperm-antibody producing hybridoma cell lines was performed by means of indirect immunofluorescence. Cell lines exhibiting a fluorescence pattern at the sperm head were used for the production of ascites fluid in pristane-primed Balb/c mice. The monoclonal antibody immunoglobulin subclasses were determined as previosly described (Hinrichsen et al. 1985) using microelisa plates precoated with different dilutions (1:50 to 1:1000) of anti- mouse IgG 1, IgG 2a, IgG 2b, IgG 3 and IgM (Miles Laboratories GmbH, Frankfurt, FRG).

To isolate the IgG fraction, ascites fluid (500 ~tl) was applied on a Mono Q (HR5/5) column (Pharmacia, Freiburg, FRG) and

486

eluted with a sodium chloride gradient (0-0.5 M NaC1 in 20 m M A492 Tris-HC1 pH 7.4; Burchiel et al. 1984). Column fractions were ana- lyzed by means of ELISA on microelisa plates precoated with the ~.5. solubilized membrane fraction. Antibody-containing fractions were pooled and concentrated by ammonium sulfate precipitation. The precipitate was solubilized and dialyzed against PBS resulting in a mAb P86/5 stock solution (8.6 mg/ml). The antibody was further

1.0 tested by means of ELISA. Briefly, washed spermatozoa of differ- ent species (104 to 10 v cells/well) were immobilized on microelisa plates and sequentially incubated with the monoclonal antibody (1:500; overnight at 4°C in PBS, 0.05% BSA, 0.05% Tween 20) and peroxidase-conjugated anti-mouse IgG (1 : 1000, 60 min, 37 ° C o.5 in PBS containing 0.05% BSA, 0.05% Tween 20). Peroxidase activ- ity was demonstrated with o-phenylendiamine (Sigma, Deisenho- fen, FRG) in the presence of hydrogen peroxide as recently de- scribed in detail (T6pfer-Petersen et al. 1986).

Alternatively, the PM-OAM membrane fraction or seminal plasma (0.04 gg to 5 p.g protein/well) were immobilized on microel- isa plates and treated as described above.

Indirect immunoJluorescence. Freshly ejaculated boar spermatozoa were centrifuged through 4% BSA in PBS to remove seminal plas- ma, smeared on clean glass slides and fixed in absolute methanol (15 min). Smears were incubated with the isolated P86/5 IgG frac- tion (1:500 in PBS, overnight 4 ° C), washed in PBS-BSA (0.5% BSA) and reacted with rabbit anti-mouse IgG (1 : 500 in PBS, 1 h, 37 ° C; Paesel, Frankfurt, FRG). Following additional washing steps the antibody-treated smears were incubated with FITC-conju- gated sheep anti-rabbit IgG (1:16 in PBS, 1 h, 37 ° C; Miles, Frank- furt, FRG), washed and examined with a Zeiss microscope equipped with epifluorescence optics. For control the incubation step with the monoclonal antibody was replaced by incubation with 0-ascites fluid.

Alternatively, sperm were examined live. Washed spermatozoa (105 cells/ml) were sequentially incubated with P86/5 IgG (1 : 500 in PBS) for 30 min and following the washing procedure by centrif- ugation through 4% BSA with rabbit anti-mouse IgG (1:500 in PBS) for additional 30 min. To remove unbound antibody sperma- tozoa were centrifuged again through 4% BSA. All these steps were carried out at 4 ° C. One aliquot of the cells were fixed in 2% formaldehyde in PBS (15 min) followed by washing in PBS containing 0.2 M glycine to remove unreacted aldehyde. Another aliquot of spermatozoa was warmed up to 25 ° C for 10 min and afterwards fixed in the aldehyde. Spermatozoa were then placed on clean glas slides treated with FITC-conjugated anti-rabbit IgG and examined as described above.

Electron microscopy. Washed spermatozoa were fixed in diluted Karnovsky fixative (1.25% glutaraldehyde, t % paraformaldehyde in 0.1 M cacodylate buffer) and subsequently washed in PBS con- taining 0.2 M glycine to remove excessive aldehyde. Fixed sperma- tozoa were sequentially incubated in suspension with mAb P86/5 (1 : 500 in PBS-BSA overnight at 4 ° C) followed by careful washing in PBS-BSA and rabbit anti-mouse IgG (1:500 in PBS-BSA, 90 min, 37 ° C; Paesel, Frankfurt, FRG). The antibody binding was detected with protein-A colloidal gold (60 min, 25 ° C) and prepared for ultrathin section and specimen preparation in toto as previously described in detail (T6pfer-Petersen et al. 1985).

Antigen characterization. SDS-electrophoresis and immunoprint- ing: Spermatozoa were washed three times in PBS and extracted with SDS-extraction buffer (0.01 Tris-HC1, pH 8.8, 10% glucose, 0.01% SDS, 2 m M phenylmethylsulfonyl fluoride; PMSF) for 60min at 37 ° C. After centrifugation (60min at 50000×g) the clear supernatant was either immediately or after reduction in 40 m M dithiotreitol (15 min, 100 ° C) subjected to a SDS-polyacryl- amide slab gel (10% polyacrylamide; T6pfer-Petersen et al. 1986). Proteins were then transferred electrophoretically onto nitrocellu- lose according to the method of Towbin et al. (t 979) using a Biorad transblot cell (in 25 m M Tris, 192 m M glycine, 20% methanol, 0.1% SDS, pH 8.3, at 200 mA, overnight). The blotted nitrocellu-

15;~' ~ ' ~ ' ~ '

-log, 1 protein concentration -log number Of spermatozoa

Fig. 1. Characterization of the monoclonal antibody by ELISA. a Antibody binding of mAb P86/5 (i : 500), to immobilized PM- OAM membrane fraction ( e - e - e ) , to immobilized boar seminal plasma (o-o-o) , control with mouse IgG instead of mAb P86/5 to immobilized PM-OAM membrane fraction (lx-zx~). b Antibody binding of mAb P86/5 to immobilized boar spermatozoa ( e - e - e ) , to immobilized human, goat, sheep, bull and stallion spermatozoa (O O O)

lose sheets were blocked by incubation in saturation buffer (10 m M Tris-HC1, pH 7.4, 500 m M sodium chloride, 3% BSA, 0.1% merth- iolate) supplemented with 10% normal sheep serum (Sigma, Dei- senhofen, FRG) for about 6 h. Individual strips were incubated with P86/5-IgG (1 : 500 in saturation buffer, overnight, 4 ° C) and thoroughly washed (three times, 10 min) in washing buffer (PBS containing 0.05% ovalbumin, 0.1% merthiolate, 0.05% Tween 20). Incubation with peroxidase-conjugated anti-mouse IgG in goat (Biorad, Munich, FRG) in saturation buffer (3 h, at room tempera- ture) was followed by washing the treated strips with washing buffer (twice, 10 rain). After incubating the strips in 0.1 M Tris- HC1, pH 7.5 for 30 min the peroxidase activity was demonstrated with 3.3'diamino benzidine (0.5 mg/ml in 0.1 M Tris-HC1, pH 7.5). The reaction was stopped by rinsing the strips with water.

Lectin-affinity chromatography: Spermatozoa were washed three times in PBS and extracted overnight at 4 °C in buffer A (25 m M Tris-HC1, pH 7.4, 0.5 M sodium chloride, 1% sodium cho- late, 2 m M PMSF). The sperm extract (5-10 mg protein) was sepa- rated on a column with Con A-sepharose (6 ml, Pharmacia, Frei- burg, FRG), which was prewashed with t0 column volumes of buffer A containing 1% BSA and 0 .2M or-methyl mannoside (Fluka, Buchs, CH) and then equilibrated with buffer B (25 m M Tris-HC1, pH 7.4, 0.5 M sodium chloride, 1% sodium cholate, 1 m M magnesium chloride, calcium chloride and mangan chloride, 2 m M PMSF). The Con A-binding fraction was released from the column with 0.2 M or-methyl mannoside in buffer B. The column fractions were tested for antibody-binding by means of ELISA. Aliquots (20 ~tl) of each fraction were immobilized on microelisa plates and treated as described above.

R e s u l t s

The m o n o c l o n a l an t ibody unde r cons idera t ion , genera ted by i m m u n i z a t i o n o f female Ba lb /c mice wi th the P M - O A M frac t ion is o f the IgG-1 subclass and is t e rmed m A b P86/5. I t cou ld be shown by means o f E L I S A tha t P86/5 reacts wi th the P M - O A M m e m b r a n e f rac t ion used as i m m u n o g e n (Fig. 1 a) and with washed s p e r m a t o z o a (Fig. 1 b) bu t no t wi th seminal p l a sma (Fig. I a). P86/5 displays no crossreac- t ivi ty wi th human , bull, goat , sheep, and s tal l ion spe rmato-

Fig. 2a. Immunofluorescent localization of P86/5 antigen on meth- anol fixed boar spermatozoa, b Control experiments after incuba- tion of spermatozoa with 0-ascites fluid. No fluorescence is seen; approx, x 5100

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Antigen charcterization

To identify the antigen recognized by the mAb P86/5 a SDS-extract of washed spermatozoa was separated on a SDS-polyacrylamide gel (10%) and then transferred to ni- trocellulose. Without reduction of the sperm components prior to electrophoresis, P86/5 recognized a prominent band at approximately 78 kDa and two weak bands at 52 and 45 kDa (Fig. 7 a). After reduction no immunoreaction with the antigen was detected (Fig. 7b). The specificity of the binding was controlled by replacing the monoclonal anti- body by a mouse IgG-fraction (Fig. 7 c).

The glycoprotein nature of the P86/5-antigen was estab- lished by lectin affinity chromatography. A sperm extract obtained by treatment of washed spermatoza with 1% sodi- um cholate was subjected to a Con A-Sepharose column and the eluate was screened for antibody binding by means of ELISA (not shown). The immunoreactive antigen was completely retained on the column and was at first released with the ~-methyl mannoside, indicating that P86/5 recog- nizes a sperm glycoprotein.

zoa as revealed by indirect immunofluorescence and ELISA (Fig. 1 b), suggesting that P86/5 recognizes a boar sperm specific antigenic site.

Antigen localization

Immunofluorescent localization of the P86/5 antigen on boar spermatozoa is shown in Fig. 2. When methanol-fixed boar spermatozoa were labelled with mAb P86/5 spermato- zoa displayed a mostly uniform fluorescence pattern exclu- sively at the sperm head (Fig. 2a). The fluorescence staining at the rostral region, however, appeared to be less circums- cript and less intensive indicating that the density of the P86/5-antigen may be reduced at that region. Midpiece and tail showed no fluorescence labelling. In control smears where the monoclonal antibody was replaced by 0-ascites fluid spermatozoa show no fluorescence (Fig. 2b). TEM examination of the specimen preparation in toto revealed that P86/5 recognizes a sperm plasma membrane protein that is restricted to the sperm head. In intact spermatozoa the surface of the sperm head shows a more or less uniform gold labelling (Fig. 3 a) with the exception of the rostral crescent-like area, which is avoid of any significant labelling (Fig. 3 a, b). These findings could be confirmed by ultrathin sections through different planes of the sperm head as indi- cated in Fig. 4.

The section plan B-B and C-C reveals a nearly uniform labelling of the plasma membrane over the postacrosomal region (Fig. 5a) and the acrosomal region respectively (Fig. 5 b) but no labelling of the rostral bulge can be seen (**). This is also confirmed by the section plan A-A in Fig. 5 c where the rostral end is unlabelled.

Unfixed spermatozoa incubated at 4 ° C exhibit a more or less uniform fluorescence over the entire sperm head as it is seen with methanol-fixed spermatozoa (Fig. 6a). However, warming up of the spermatozoa to 25°C after coupling the mAb P86/5 at 4 ° C leads to a significant clus- tering of the antigen in the plasma membrane overlying the acrosome and the equatorial segment. The fluorescence pattern of the post-acrosomal plasma membrane remains unaltered (Fig. 6b).

Discussion

The ability of the spermatozoon to regionalize certain mem- brane components has been established during the last few years by the use of monoclonal antibodies (Primakoff and Myles 1983; Peterson and Russell 1987). In this paper some features of the highly organized sperm surface could be shown with a monoclonal antibody directed against a mo- bile glycoprotein of the plasma membrane.

The monoclonal antibody (P86/5) recognizes predomi- nantly a sperm antigen with a molecular weight of about 78 kDa and to some extent two proteins with 52 and 45 kDa. Since, under reducing conditions all three bands failed to react with the monoclonal antibody it can be con- cluded that the low molecular weight bands represent hy- drolysis products of the prominent 78 kDa component which is sensible to reduction. A simple method to get evi- dence of the glycoprotein nature of an antigen is the analyti- cal lectin-affinity chromatography. By this method the P86/ 5-antigen binds completely to Con A. The binding could be inhibited by the competitive carbohydrate, in this case by ~-methyl mannoside indicating the specificity of the in- teraction. The Con A-binding ability characterizes a glyco- protein whose carbohydrate side chains are at least partly N-glycosidically bound to the polypeptide chain.

The immunoelectron microscopy using the protein A- gold method (pAg) allows more precise insight into the distribution of the P86/5-sperm glycoprotein. Particularly the specimen preparation in toto offers the possibility of showing the topography of the corresponding antigen over the entire surface of the spermatozoon. In intact spermato- zoa P86/5 is restricted to the plasma membrane of the sperm head. Never could its localization be demonstrated in the region of the midpiece and the tail, neither by immunoelec- tron microscopy nor by indirect immunofluorescence.

A first indication that P86/5 recognizes a mobile protein of the plasma membrane was provided by immunofluores- cence. In some cases proteins are able to diffuse laterally within the plane of the membrane and lateral migration of mobile membrane proteins may be induced by ligand binding (0liver and Berlin 1982). By comparing the anti- body binding pattern generated at 4°C and at 25 ° C, at

488

Fig. 3a. Specimen preparation in toto. Intact spermatozoa show an uniform gold labelling for P86/5 antigen with the exception of the rostral crescent-like region (**). ×23000. b Enlargement of the rostral region indicated by the rectangle in Fig. 3 a. The plasma membrane is devoid of any significant labelling, x46000

higher temperature a significant increase of the granular appearance, particularly at the acrosomal part of the sperm head, has been observed; at low temperature the lateral diffusion of membrane components was reduced. The tem- perature-induced patching of the P86/5-antigen observed at the acrosomal region is caused by the capping effect of the bivalent antibody and indicates an integral mem- brane protein capable to diffuse freely within the cell mem- brane overlying the acrosome (Nicolson and Yanagimachi 1974). The lateral mobility appears to be restricted to the peri-acrosomal plasma membrane which is characterized by its high fluidity referring to its function to undergo the acrosome reaction. Under the same conditions the post-

acrosomal plasma membrane shows low fluidity and re- stricted mobility of its membrane components (Friend 1982). The reduced mobility of the P86/5-antigen within the post-acrosomal plasma membrane supports the concept of different membrane properties of specific regions of the sperm surface. However, it is remarkable that the P86/5 an- tigen could not be detected at the rostral region of the sperm head. It seems that the antigen, although freely dif- fusing in the peri-acrosomal plasma membrane, is prevented to enter the rostral surface domain. A similar phenomenon has been observed in the guinea pig by Myles and coworkers (1984). There, the PT-1 antigen has been shown to freely diffuse within the posterior tail region even though it did

C

• 7 ¸

A

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Fig. 4. In the schematic drawing of the sperm head the different section planes shown in Fig. 5a - c are indicated. The labelling of the plasma membrane is more or less uniform with the exception of the rostra1 end (*). x 36000

4

Fig. 6 a. Unfixed sperm, incubated at 4 ° C for P86/5 antigen reveals an unique staining pat tern similar to Fig. 2a. b Unfixed sperm labelled at 4 ° C for P86/5 antigen and heated up to 25 ° C. Cluster- ing of the fluorescence labelled antigen is visible over the acrosome and equatorial segment. The post acrosomal region remains unal- tered; approx, x 5100

490

78--

52--

4 5 ~

a b c

Fig. 7. Western blot of sperm antigens after SDS-PAGE. Lane a: unreduced sperm antigens, reacted with mAb P86/5; lane b: re- duced sperm antigens, reacted with mAb P86/5; lane c: unreduced sperm antigens, reacted with mouse lgG instead of mAb p86/5. The molecular weights are given in kDa

not leave this surface domain. The authors discuss a barrier in the membrane of still unknown nature that prevents the migration of the antigen onto other domains. A barrier to diffusion at the rostral region of the sperm head may also be postulated, inhibiting the mobile glycoprotein to leave its innate domain.

Peripheral cytoplasmic components (Nicolson 1982) and the sub-membranous cytoskeleton (Gratzer 1981 ; Vir- tanen et al. 1984) may control the mobility of proteins and glycoproteins in the lipid bilayer. Additionally, the tendency of certain lipid components to associate into structurally distinct microdomains of differing fluidity (Klausner et al. 1980) may also affect the mobility of membrane proteins and glycoproteins in order to mainta in the high structural organization of the sperm surface. It is likely that the re- gionalization of the sperm plasma membrane is related to the important physiological events during fertilization.

In the accompanying paper the reorganization of the mobile glycoprotein within the fluid periacrosomal plasma membrane during capacitation is described.

Acknowledgements . We thank Mrs. A. Scharf and Mrs. C. Ebener von Eschenbach for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft Schi 86/7-6 und To 114/1-1.

References

Burchiel (1984) Rapid and efficient purification of mouse mono- clonal antibodies from ascites fluid using high performance liq- uid chromatography. J Immunol Methods 69:33-42

Cowan AE, Myles DG, Koppel DE (1987) Lateral diffusion of the PH-20 Protein on guinea pig sperm: evidence that barriers

to diffusion maintain plasma membrane domains in mamma- lian sperm. J Cell Biol 104: 917-923

Fawcett DW (1975) The mammlian spermatozoon. Dev Biol 44: 394436

Fazekas de St. Groth S, Scheidegger D (1980) Production of mono- clonal antibodies: strategy and tactics. J Immunol Methods 35:1 21

Friend DS (1982) Plasma membrane diversity in a highly polarized cell. J Cell Biol 93:243-249

Gratzer WB (1981) The red cell membrane and its cytoskeleton. Biochem J 198 : 1-8

Hinrichsen-Kohane AC, Hinrichsen MJ, Schill W-B (1985) Analy- sis of antigen expression on human spermatozoa by means of monoclonal antibodies. Fertil Steril 43:279-285

Klausner RD, Kleinfeld AM, Hoover RL, Karnovsky MJ (1980) Lipid domains in membranes. J Biol Chem 71 : 49-59

Koehler JK (1981) Lectins as probes of the spermatozoon surface. Arch Androl 6:197-217

Myles DE, Primakoff P, Koppel DE (1984) A localized surface protein of guinea pig sperm exhibits free diffusion in its domain. J Cell Biol 98 : 1905-1909

Nicolson GL (1982) The mammalian sperm membrane. In: Amann RP, Seidel FE (eds) Prospecta for sexing mammalian sperm. Colorado Ass Univ Press, Colorado, pp 5-16

Nicolson GL, Yanagimachi R (1974) Mobility and restriction of mobility of plasma membrane lectin-binding components. Science 184:1294-1296

Oliver JM, Berlin RD (1982) Mechanisms that regulate the struc- tural and functional architecture of cell surfaces, lnt Rev Cytol 74:55-93

Peterson RN, Russell LD (1985) The mammalian spermatozoon: a model for the study of regional specificity in plasma mem- brane organization and function. Tissue Cell 17:769-791

Peterson RN, Gillot M, Hunt W, Russell LD (1987) Organization of the boar spermatozoan plasma membrane: evidence for sepa- rate domains (sub-domains) of integral membrane proteins in the plasma membrane overlying the principal segment of the acrosome. J Cell Sci 88:343 349

Primakoff P, Myles DG (1983) A map of guinea pig sperm surface constructed with monoclonal antibodies. Dev Biol 98:417-428

Saxena NK, Russell LD, Saxena N, Peterson RN (1986) Immunof- luorescence antigen localization on boar sperm plasma mem- brane. Monoclonal antibodies reveal apparent new domains and apparent redistribution of surface antigens during matura- tion and at ejaculation. Anat Rec 214:238-252

Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure of cell membranes. Science 175:720-731

T6pfer-Petersen E, Schill W-B (1983) Characterization of lectin receptors isolated from the outer acrosomal membrane of boar spermatozoa. Int J Androl 6 : 375 392

T6pfer-Petersen E, Friess AE, Schill W-B (1988) The acrosome reaction in boar spermatozoa. Human Reprod 3 : 31%326

T6pfer-Petersen E, Auerbeck J, Weiss A, Friess AE, Schill W-B (1986) The sperm acrosome: immunological analysis using spe- cific polyclonal and monoclonal antibodies directed against the outer acrosomal membrane of boar spermatozoa. Andrologia 18 : 232251

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamid gels to nitrocellulose sheets: procedure and some applications. Proc Nat Acad Sci 76:4350-4353

Wolf DE (1983) The plasma membrane in early embrogenesis. In: Johnsom ME (ed) Development of mammals. Elsevier Science Publ, vol 5, pp 187-207

Virtanen 1, Badley RA, Paasivuo R, Lehto V-P (1984) Distinct cytoskeleton domains revealed in sperm cell. J Cell Biol 99:1083-1091