glycoconjugates of human sperm surface. a study with fluorescent lectin conjugates and lens...

Ceil Biology international Reports, Vol. 9, No. 2, February 1985 151

GLYCOCONJUGATES OF HUMAN SPERM SURFACE. A STUDY WITH FLUORESCENT LECTIN CONJUGATES AND LENS CULINARIS AGGLUTI- NIN AFFINITY CHROMATOGRAPHY

M. Kallajokil, R. Malmi', I. Virtanen2 and J. Suominen'

1 Institute of Biomedicine, Department of Anatomy, Univer-

sity of Turku, Kiinamyllynkatu 10, SF-20520 Turku, Finland

'Department of Pathology, University of Helsinki, Haart- manninkatu 3, SF-00290 Helsinki, Finland

ABSTRACT

Distribution of glycocompounds in human spermatozoa was studied by using fluorecent lectin-conjugates. Con A bound predominantly to acrosomal and posterior head regions whereas RCA I bound to the acrosomal region of intact spermatozoa, stained in suspension. Other lectins used (LCA, WGA, SBA, PNA) stained the the entire sperm surface. In airdried sperm smears binding of both Con A and RCA I were identical with the staining pattern obtained with living cells whereas LCA, WGA, SBA and PNA now bound heavily into acrosomal region. As a similar staining pattern was obtained with permeabilized sperm cells, this staining is apparently due to binding to intracellular structures. The efficiency of Lens culinaris agglutinin affinity chromatography in purification of human sperm glycoproteins was tested after their external radiolabelling with the neuraminidase/galactose oxidase/sodium borohyride method. 22% of applicated radioactivity could be eluted from the column with the specific inhibitory saccharide, and most of the radio- labelled surface glycoproteins of the whole sperm lysate, were also present in the LCA affinity column eluate. LCA affinity chromatography seems thus be an effective method to enrich membrane glycoproteins of human spermatozoa,

INTRODUCTION

Mammalian spermatozoa undergo surface associated restruc- turing processes during maturation in epididymis, capacitation, acrosome reaction and sperm-egg fusion. The molecular mechanisms of these alterations are yet unclear and therefore e.g. sperm cell membrane have been widely studied with a variety of techniques. These have included morphological analysis of ultrastructure, radioactive labellinq of surface components, binding of colloidal iron hydroxide, measurement of surface charge in electric field as well as binding of antibodies and lectins

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152 Cell Biology International Reports, Vol. 9, No. 2, February 1985

(reviewed by Koehler, 1978; Friend, 1984). Lectins have succesfully been used in characterization of surface glycoproteins because of their specificity for particular saccharide moieties. Lectins are able to precipitate glycoproteins as well as to agglutinate cells and when coupled to solid carriers, lectin affinity chromatography has been used for purification of cell membrane glycoproteins (Lotan and Nicolson, 1979). Lectins can also be labelled with visible markers (fluorochromes, ferritin, gold-particles, peroxidase) and used for mapping cell surface glycocompounds and monitoring surface changes in various phenomena.

Surface characteristics of mammalian spermatozoa have been studied with lectins by several investigators (reviewed by Koehler, 1981). Early studies indicated that spermatozoa can be agglutinated with certain lectins (Nicolson and Yanagimachi, 1972; Uhlenbruck and Herman, 1972). The densities of binding sites on the spermatozoa have also been measured using radioactively labelled lectins (Edelman and Millette, 1971). Variations in the lectin binding between different species and regional differences within the same species have been observed (Millette, 1977; Nicolson et al., 1977). Since observations on human spermatozoa have been few and scattered, we have used in this work several fluorochrome-conjugated lectins to analyse lectin binding sites of human sperm. We have also examined the usefulnes of Lens culinaris agglutinin affinity chromatography in purification of human sperm surface glycoproteins.

MATERIALS AND METHODS

Lectins. Both fluorescein isothiocyanate (FITC)- and tet- ramethylrhodamine isothiocyanate (TRITC)-conjugated lectins were purchased from Vector laboratories (Eurlingame, CA). The lectins used and their carbohydrate specifi- cities (Goldstein and Hayes, 1978) are listed below: Concanavalin A (Con A) D-Man, D-Glc Lens culinaris (LCA) D-Man, D-Glc Tritium vulgaris (LJGA) D-GlcNAc, sialic acids Glycine maximum (SBA) D-GalNAc Arachis hypogaea (PNA) D-Gal-B-(l-3)-D-GalNAc Ricinus communis 120 (RCA I) D-Gal The terminal hexoses (underlined) were purchased from Sigma Chemical Co. (St. Louis, MO) and used in concentration of 0.2M to inhibit the lectin binding.

Preparation of spermatozoa. Semen samples were obtained from several healthly donors, and only samples with normal semen characteristics (sperm concentration over 50 milj/ml, and at least 50% of spermatozoa with normal

Cell Biology International Reports, Vol. 9, No. 2, February 1985 153

progressive motility and normal morphology) were used. For lectin labelling, sperm samples were washed three times in NaCl-P buffer (140 mM NaCl, 1OmM sodium phos- phate, pH 7.2) by low speed centrifucations. Finally 1 milj. intact spermatozoa were suspended into diluted lectin solutions.

For radioactive surface,labelling spermatozoa were sepa- rated from other cellular particles in semen by using a linear 15-75:; Percoll (Pharmacia, Uppsala, Sweden) gradients on 95% Percoll bottom layer and sentrifucation at 1500 g for 45 min as described by Lessley and Garner (1983). Sperm fractions were washed three more times with NaCl-P buffer and final sediment was suspended into NaCl-P buffer supplemented with Ca++and Mg++ to a concentration of 300 milj/ml. In the final specimen over 90% of cells were alive as shown by the trypan blue exclusion test.

Lectin staininq and microscopy. To reveal the surface binding, intact or paraformaldehyde fixed spermatozoa were incubated for 15 min at O°C in suspension with lectins (100-200 pg/ml) To reveal intracellular lectin binding sites, stainings were performed in suspension, with spermatozoa fixed in paraformaldehyde and permeabilized in 0.1% Nonidet NP-40 for 15 min (BDH, England; Laurila et ai., 1978). After 2 washes sperms were smeared on glass slides and unfixed cells were fixed in 3.5% paraformaldehyde in NaCl-P buffer for 15 min at RT. Experiments were done also by using samples smeared on slides, airdried and fixed in paraformaldehyde, and thereafter incubated for 15 min with lectin dilut- ions. Slides were washed three times in NaCl-P buffer, mounted with either glyserol-Verona1 buffer (l:l, pH 8.4) or buffered nicetamide (RCA, PNA, SBA) and examined in Leiz-Dialux 20 fluorescence microscope equipped for epi- illumination and filters for FITC and TRITC-fluorescence.

Radioactive surface labellinq and electrophoretic analysis. To radiolabel surface glycoproteins of human spermatozoa the method of Gahmberg and Hakomori (1973) was used. 600 milj spermatozoa in 2 ml of NaCl-P buffer supplemented with Ca++ and Mg++ were incubated with 20 U galactose oxidase (Sigma Chemical Co, St Louis, MO) and 25 mU neuraminidase (from Vibrio Cholerae, Behringwerke AG, Marburg-Lahn, F.R.G.) for 30 min at 37OC. After 2 washes with NaCl-P buffer the cells were suspended into NaCl-P buffer and 5 mCi NaB3Hq (Radiochemical Centre Ltd., Amershamn, England) was added and cells were left at RT for 30 min. After three washes with NaCl-P buffer cells’were suspended to a final concentration of 100 milj/ml in solubilization buffer: 1%

Cell Biology International Reports, Vol. 9, No. 2, February 1985

Fig. 1. Human spermatozoa stained in suspension with FITC-Con A (a), TRITC-LCA (c) and FITC-WGA (e) show surface binding of the lectins. Con A stain mostly the surface overlying the acrosorne and the posterior part of the head whereas LCA and WGA were bound evenly to the entire sperm surface. After airdrying and paraformadehyde fixation Con A binding pattern remained similar, but more intense (b) and LCA (d) and WGA (f) v!ere bound more brightly to the acrosomal region. Bar represents 10pm. 950 x.

Cell Biology International Reports, Vol. 9, No. ,2, February 1985 155

sodium deoxycholate (DOC) and 5 mM sodium azide in 10 mM Tris-HCl pH 8.4, together with 100 U/ml aprotinin (Bayer, F.R.G.) and 1.25 mM 4-aminobenzamidin (Merck, Darmstadt, F.R.G.) as inhibitors of proteolysis. The suspension was incubated for 30 min at O°C with stirring and centrifuged 30000 g for 30 min. The supernatant was applicated on to Lens culinaris agglutinin-Sepharose 48 column (Pharmacia, Uppsala, Sweden) with 2 ml bed volume equilibrated with 0.5% DOC in 10 mM Tris-HCl pH 8.4 and 5 mM NaN3. After the application the column was washed with equilibration buffer and before elution 50 mM NaCl was added into washing buffer. Elution was made with 20% (w/v) h-methylmannoside in washing buffer (Hayman and Grumpton, 1972). 2 ml fractions were collected and 50 ul samples were counted for radioactivity with lOm1 scintillation fluid (ACS, Amershamn, Arlington Heights, IL) in LKB- Wallac 1210 Ultrobeta liquid scintillation counter. A sample from the original extract, unbound fraction and eluted fraction were dialysed against water overnight to remove DOC and then lyophilized. The labelled glycoprotein were analyzed with polyacrylamide gel electrophoresis in the presence of sodiumdodecylsulphate (Laemmli, 1970). Gels were processed for fluorography as described by Pulleyblank and Booth (1981).

RESULTS

When human spermatozoa were labelled with fluorescent lectin-conjugates a quite variable labelling fashion was found, especially when labelling was performed in suspension before fixation. This is probably due to a large amount of morphologically abnormal spermatozoa, present even in normal human semen samples. Therefore, we only observed spermatozoa with normal morphology. The results on staining experiments with cells in suspension or on airdrieed specimens were distinctly different. When paraformaldehyde prefixed spermatozoa were stained in suspension, LCA, SBA, PNA and WGA were bound in a proportion of the spermatozoa predominantly to the acrosomal regions. If the cells were permeabilized with Nonidet NP-40 detergent after paraformaldehyde fixation, -1ectins were bound strongly to the acrosomal regions in all spermatozoa, resembling thus the binding pattern of airdried and paraformaldehyde fixed specimens. After airdrying and paraformaldehyde fixation the lectins seemed to bind more intensely to the acrosomal region than when stained in suspension after Pf fixation.

Con A. When human spermatozoa were labelled with FITC- conjugated Con A in suspension the sperm surfaces were brightly stained. However, the plasma membrane overlying


Fig. 2. Lectin binding to intact human spermatozoa stained with FITC-SBA (a), FITC-PNA (c) and TRITC-RCA I (e). SBA and PNA showed a weak binding to the entire sperm surface. RCA I VJ~S also bound to the whole sperm surface with the acrosomal cap region most intensely stained. In airdried and paraformaldehyde fixed specimen SBA (b) and PNA (d) were bound predominantly to the acrosomal region whereas RCA I binding remained similar as in suspension. Bar represents 10pm. 950 x.

Cell Biology lnternationat Reports, Vol. 9, No. 2, February 1985

acrosome and neck region were more intensely fluorescent than the postnuclear sheet area (Fig. la). Staining of airdried smears gave a similar binding pattern, but was more intense (Fig. lb).

LCA. Following the labelling of human spermatozoa with TRITC-conjugated LCA in suspension the whole sperm surface membrane was fluorescent (Fig. lc). In addition, in airdried specimen the acrosomal region was intensely stained (Fig. Id).

WGA. The labelling pattern with FITC-WGA in suspension was quite similar to LCA: the whole sperm surface was heavily labelled (Fig. le). In airdried smears expecially the acrosomal area was brightly fluorescent but other parts of the spermatozoa were also stained (Fig. If).

SBA. Spermatozoa stained with FITC-SBA in suspension showed a weak, even fluorescence over the whole spermatozoa (Fig. Za). After airdrying of the spermatozoa before paraformaldehyde fixation and lectin staining, the acrosomal region became more intensely fluorescent (Fig. Zb).

PNA. When sperm suspension was treated with FITC-PNA the surface labelling covers weakly the whole spermatozoa (Fig. 2~). In airdried specimen the acrosomal region was very brightly fluorescent and the other parts of the spermatozoa gave only a negligible fluorescence (Fig. 2d).

RCA I. -- TRITC-RCA I labelled the whole human sperm surface when stained in suspension. The fluorescent pattern was patchy and the plasma membrane above the acrosome was more intence that other parts of the sperm cell (Fig. 2e). On sperm smears the lectin binding pattern remained similar but more even and the acrosome region was more intensely stained (Fig. 2f).

Binding of the lectins used could ne inhibited by pre- incubating the lectin solutions with appropriate inhibitory sugars before staining, with the exeption of WGA, binding of which was essentially decreased.

Surface labelling and LCA-Sepharose 48 affinity chromatography. From the total radioactivity applicated into LCA- column, 22% could be eluted with the inhibiting sugar. The unbound fraction consisted 68% of the total activity and the recovery was calculated to be 90% of the total radioactivity. The eluation profile of LCA-column is shown in Fig. 3. The pattern of DOC soluble glycoproteins (gp) after their external tritiation and SDS-PAGE analysis in shown in Fig. 4 lane a. The fluorography revealed


SAMPLE

20000 - I/ LCA

15000-

E x

u F!

m I I 2

10000 - k-z k

< z

0 L 12 20 28 36 LB 52 60

VOLUME (ml)

Fig. 3. Elution profile of Lens culinaris agglutinin affinity chromatography of tritiated and DOC-solubilized human sperm surface components. Column was washed and equilibrated in DOC-buffer as described in materials and methods. 20% a-methyl-mannoside in DOC-buffer was added at the point indicated. 2 ml fractions were collected and radioactivity was counted from 50 ~1 samples. A part of the applicated sample, unbound fraction and eluted fraction were pooled, dialyzed and lyophilized for SDS-PAGE analysis and fluorography.

5 major gp bands with Mr 120,000, 105,000, 68,000, 36,000 and 24,000 daltons. In addition, there were several minor bands: a 40,000 gp and faintly visible bands with Mr- values of 86,000-78,000, 50,000-45,000, 26,000 and 20,000-18,000 daltons. All except the 40,000 gp were visible also in the unbound fraction of LCA-column (Fig. 4 lane b). Most of the glycoproteins in the crude extract were present also in fraction eluted with a-methylmannoside (Fig. 4 lane c). The bands with 68,000, 50,000-45,000, 26,000 and 24,000 were not present in the eluate.

DISCUSSION

Regional differences in the surface binding of some lectins were observed in this study. Con A was bound more avidly to acrosomal and neck regions than other parts of the sperm surface and RCA I was mainly localized in the acrosomal area. Studies with other mammalian spermatozoa have revealed specialized areas of sperm plasma-


Fio. 4. SDS-PAGE (7-1576 acrylamide slab gel) pattern of human sperm surface glycoproteins fractionated by Lens culinaris agglutinin affinity chromatography. Lane 1 DOC-solubilized human sperm surface glycoproteins; line 2 unoound fraction; fraction.

lane 3 by alfa-methylmannoside eluted Standard proteins for molecular weight determi-

nation on the left.

membrane: distinct cell surface domains have been found with electron microscopical methods (Friend, 1984), binding of monoclonal antibodies (Myles et al., 1981; Schmell et al., 1982) and lectins (Schwarz and Koehler, 1979; Nicolson et al., 1977; Edelman and Millette, 1971) and probes for lipids (Bearer and Friend, 1982). Schtvarz and Koehler (1979) found that Con A and lrJGA bound intensely over the acrosomal area of guinea pig spermatozoa and SBA bound mainly only over the acrosomes. Binding of ferritin-conjugated lectins to rabbit spermatozoa have also been studied (Nicolson et al., 1977). Con A have been found to bind predominantly to sperm head and mid-piece regions and RCA I to acrosomal area. UGA reacted weakly with surface of ejaculated rabbit sperm. Russel et al. (1983) have studied boar sperm surface and they found most of the lectins (Con A, SBA, RCA, PNA) to label the acrosomal region of sperm plasma membrane. All the lectins except PNA bound also to other parts of spermatozoa with less intensity. These studies and our observations indicate that vari- ation in lectin binding between different mammalian


species exist. Some of the differences noted might also be due to different pretreatments of spermatozoa before lectin staining.

Human sperm surface is known to have binding sites for RCA I and WGA independently of the bloodgroup of the sperm donor. This uas verified by the work of Uhlenbruck and Herman (1972), who also noticed that Helix pomatia agglutinin only agglutinated spermatozoa of bloodgroup A donors. Our results confirm the expression of RCA I and EGA binding sites on human spermatozoa. Recently Virtanen

et al. (1984) have studied the binding of several lectins to human spermatozoa. Result presented in this report suggest that bright acrosonal staining obtained with FITC-SEA and FITC-PNA represent intracellular lectin binding instead of surface binding. The surface binding reported with LCA, Con A and WGA is in agreement with the present results.

All the lectins used in this study displayed a distinct binding pattern to human spermatozoa. The staining of intact spermatozoa in suspension however, differed essentially from that of airdried specimens, as the acrosomal region was more intensely stained in airdried sperm smears. Also the prefixation with paraformaldehyde increased the number of acrosome-positive sperm cells. As strong acrosomal staining could be seen in paraformaldehyde fixed and detergent treated cells, the acrosomal staining most probably represents staining of intracellular structures. Due to these facts we consider that the lectin staining of intact cells in suspension most likely represents the true surface binding pattern, because large lectin molecules cannot penetrate through intact plasma membrane. Unlike cultured cells (Laurila et al., 1978) the paraformaldehyde fixation alone seem partially to permeabilize the sperm plasma membrane. The sperm acrosome is knobJn to be derived from Golgi apparatus and it contains hydrolytic enzymes, many of which are glycoproteins (Gould and Bernstein, 1977). Lectins giving the acrosomal type of staininq in this study also typically label the Golgi apparatus in other cell types (Virtanen et al., 1980). The results presented here are in a agreement with observations on lectin binding to mature and developing acrosomes in frozen sections of human testis (Malmi et al., submitted). We conclude that paraformaldehyde fixation and airdrying of spermatozoa on slides may disrupt the sperm plasma membrane and exposes acrosomal and other intracellular glycoconjugates to lectin binding, and thus giving false positive surface staining results.


Ejaculated spermatozoa are known to carry sperm coating antigens, which are firmly bound to sperm surface and which cannot be washed away in routine washing procedures. Some of the lectin staining on spermatozoa may be caused by these absorbed components, secreted by acces- sory sex glands. The negative charge of ram sperm surface increases during the passage through the epidymis, which is caused by the addition of sialic acid groups to the sperm surface. These sialic acid groups were uneffec- ted by routine washing procedures indicating that they

were firmly attached to the plasma membrane (Holt, 1980). A similar change in surface charge of human spermatozoa during epididymal maturation has been found (Bedford et al., 1973). As WGA binds N-acetylglucosamine and sialic acids, the strong uniform WGA- binding to human spermatozoa may be partly due to sialylation of sperm surface glycocompounds in epidymis.

Most of the cell surface proteins are qlycosylated (Gahmberg, 1981). Lectin affinity cromatography has widely been used in characterization of membrane qlycoproteins. Lens culinaris agglutin is commonly used in such studies as it oives a rich qlycoprotein yield (Hayman and Crumpton, 1972). In order to detect qlycoproteins we radio-labelled terminal qalctose and N-acetyl-galactosamine residues in the external surface of human spermatozoa. Neuraminidase pretreatment increased labelling intensity 2-3 fold, without effecting the migration of labelled qlycoproteins in SDS-PAGE (Kallajoki et al., unpublished observation). After solubilization of labelled surface components, the efficiency of LCA-affinity chromatoqraphy in further purification was tested. The electrophoretic pattern of labelled human sperm surface components differed from that of Young and Goodman (1980). They used lactoperoxidase catalyzed iodination of human sperm surface proteins and SDS-PAGE analysis of labelled proteins. They found 5 componets of MW 92, 72, 46, 30 and 20 K daltons. The amount of labelled components was higher with the technique used by us as e.g. two high MW band with Mr-values of 120,000 and 105,000 daltons were not detected by iodination method. The differences may be explained by the various labelling techniques used. Also the solublizing capacity of non-ionic detergent Triton X-100 used by Young and Goodman often fails to dissociate interactions between proteins, and integral membrane proteins, which are stable in non-ionic detergents may be dissosiated by DOC (Helenius et al., 1979). Our experiments confirm that LCA-affinity chromatography is effective in enriching also human sperm surface qlycoproteins. As FITC-LCA was shown to label intensely the whole surface of intact spermatozoa, the LCA eluate


probably represents the majority of qlycoproteins of human sperm surface.

In this paper we have described the binding of some lectins to human spermatozoa and the use of LCA-affinity

chromatography in purification. It remains to be eluci- dated whether lectins also are useful in monitoring physioloqical events related to capacitation and acrosome reaction.

ACKNOl.jLEDGEMENTS

The skilful technical assistance of Mrs. Pirkko Rauha- mgki, Mrs. Raija Andersgn and Mrs. Marja Ovaska is gratefully acknowledged.

REFERENCES

Bearer, E.L., Friend, D.S. (1982) Modification of anionic lipid domains preceeding membrane fusion in guinea pig sperm. Journal of Cell Biology 92: 604.-615.

Bedford, J.M., Calvin, H., Cooper, 6.k. (1973) The maturation of spermatozoa in the human epididynis. Journal of Reproduction and Fertility, Supplement I-8: 199-213.

Friend, D.S. (1984) Merlbrane organization and differenti- ation in guinea pig spermatozoon. In: J.Van Bler!;om and P.M. Motta (eds.). Ultrastructure of Reproduction. Ch. 7, pp 75-85. Martinus Nijhoff, Publishers, Boston, The Hauge, Dordrecht, Lancaster.

Gahmberg, C.G., Hakomori, S. (1973) External labelling of cell surface galactose and galactosamine in glycplipid and glycoprotein of human erythrocytes. Journal of Biological Chemistry 248: 4311-4317.

Gahmberg, C.G. (1981) Membrane qlycoproteins and glycoli- pids: structure, localization and function of the carbohydrate. In: J.B. Finean and R.H. Mitchell (eds.). Membrane Structure. Ch 4, pp. 127-101. Elsevier/North- Holland Biomedical Press, Amsterdam, New York, Oxford.

Gould, S.F., Bernstein, M.H. (1977) Cytochenistry of spermatozoa. In: E.S.E. Hafez (ed.). Techniques of Human Andrology. Ch. 3, pp. 189-202. North-Holland Publishing Company, Amsterdam, New York, Oxford.


Goldstein, I.J., Hayes, C.E. (1978) The lectins: carbohydrate binding proteins of plants and animals. Ad- vances in Carbohydrate Chemistry and Biochemistry 35: 128-341.

Hayman, M. J., Crumpton, M.J. (1972) Isolation of glycoproteins from plasmamembrane using Lens culinaris phy- tohemagglutinin. Biochemical and Biophysical Research Communications 47: 923-930.

Helenius, A., McCaslin, D.R., Fries, E., Tanford, C. (1979) Properties of detergents. Methods in Enzymology 56: 734-749.

Holt, W.V. (1980) Surface bound sialic acid on ram and bull spermatozoa: deposition during epididymal transit and stability during washing. Biology of Reproduction 23: 847-857.

Koehler, J.H. (1978) The mammalian sperm surface: studies with specific labelling techniques. International Reviews of Cytology 54: 73-108.

Koehler, J.H. (1981) Lectins as probes of the spermatozoon. Archives of Andrology 6: 197-217.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227: 680-685.

Laurila, P., Virtanen, I., Wartiovaara, J., Stenman, s. (1978) Fluorescent antibodies and lectins stain intracellular structures in fixed cells treated with non- ionic detergent. Journal of Histochemistry and Cyto- chemistry 26: 251-256.

Lessley, B.A., Garner, D.L. (1983) Isolation of motile spermatozoa by density gradient centrifugation in Percoll. Gamete Research 7: 49-61.

Lotan, R., Nicolson, G.L. (1979) Purification of cell membrane glycoproteins by lectin affinity chromatography- Biochimica et Biophysics Acta 559: 329-376.

Malmi, R., Kallajoki, M., Suominen, J. Distribution of glycoconjugates in human testis. A histochemical study using fluorescent lectin conjugates. International Journal of Andrology (submitted for publication).

Millette, C.F. (1977) Distribution and mobility of lectin binding sites on mammalian spermatozoa. In: M. Edidin and M.H. Johnsson (eds.). Immunobiology of Gametes.


PP. 51-71- Gambridge University Press, London, New York 1 Melbourne.

Myles, D.P., Primakoff, P., Bellve, A.R. (1981) Surface domains of guinea pig sperm defined with monoclonal antibodies. Cell 23: 433-439.

Nicolson, G.L., Yanagimachi, R. (1972) Terminal sacchari- des on sperm plasma membranes: identification by specific agglutinins. Science 177: 276-279.

Nicolson, G.L., Usui, N., Yanagimachi, R., Yanagimachi, H Smith, J.R. (1977) Lectin binding sites on the piksma membrane of rabbit spermatozoa. Changes in

surface receptors during epididymal maturation and ejaculation. Journal of Cell Biology 74: 950-962.

Pulleyblank, D.E., Booth, G.M. (1981) Improved methods for fluorographic detection of weak beta-emitting radioisotopes in the agarose and acrylamide gel electrophoresis media. Journal of Biomedical and Biophys-

ical Methods 4: 339-346.

Schmell, E.D., Yuan, L.C., Gulyas, B.J., August, J.T. (1982) Identification of mammalian sperm surface antigens. I. Production of monoclonal anti-mouse sperm antibodies. Fertility and Sterility 37: 249-257.

Schwarz, M., Koehler, J.K. (1979) Alterations of lectin

binding to guinea pig spermatozoa accompanying in vitro capacitation and acrosome reaction. Biology of Repro- duction 21: 1295-1307.

Uhlenbruck, G., Hermann, W.P. (1972) Agglutination of normal, coated and enzyme-treated human spermatozoa with heterophile agglutinins. VOX Sanguinis 23: 444- 451.

Virtanen, I., Ekblom, P., Laurila, P. (1980) Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells. Journal of Cell Biology 85: 429-434.

Uirtanen, I., Badley, R.A., Paasiwuo, R., Lehto, V.-P. (1984) Distinct cytoskeletal domains revealed in sperm cells. Journal of Cell Biology 99: 1083-1091.

Young, L.G., Goodman, S.A. (1980) Characterization of human sperm cell surface components. Biology of Rep- roduction 23: 826-835.

Received: 12th November 1984. Accepted: 22nd November 1984

glycoconjugates of human sperm surface. a study with fluorescent lectin conjugates and lens...

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