Sulphated glycoconjugates are powerful inhibitors of spermatozoa binding to the vitelline envelope...

Biol. Cell (2005) 97, 435–444 (Printed in Great Britain) Research article

Sulphated glycoconjugates arepowerful inhibitors of spermatozoabinding to the vitelline envelope inamphibian eggsMariangela Caputo, Stefania Riccio, Paolo Paglierucci, Loredana Tretola, Clementina Diglio, Rosa Carotenuto,Nadia De Marco and Chiara Campanella1

Dipartimento di Biologia Evolutiva e Comparata, Universita di Napoli Federico II, Napoli, Italy

Background information. In amphibians, the role of sulphated glycans has not been determined in sperm-atozoa–egg interaction, although they are known to be involved in other systems. In previous studies, it wasfound that, in Discoglossus pictus, a VE (vitelline envelope) glycoprotein of 63 kDa exhibits high homology toXenopus laevis gp69/gp64 and to ZP2 of mammals. gp63 and a glycoprotein of 75 kDa are both capable of bindingthe spermatozoa in in vitro assays and, having similar peptide maps and different glycosylation, are probably twoglycoforms of the same protein.

Results. In the present study, binding assays performed by treating dejellied eggs with metaperiodate suggestthat hydroxy groups of sugars are not directly involved in spermatozoa–vitelline envelope binding. Competi-tion assays between dejellied eggs and spermatozoa preincubated with dextran, dextran sulphate or fucoidanindicated that sulphated oligosaccharides have an inhibitory effect on spermatozoa binding. In similar com-petition assays, Lex (Lewisx) trisaccharide 3′-sulphate inhibited spermatozoa binding to VE in contrast with 3′-sialyl-Lex tetrasaccharide. Assays performed with gp75- or gp63-coated beads and spermatozoa treated withfucoidan or dextran sulphate indicated that sulphated oligosaccharides competitively inhibit spermatozoa bind-ing to gp75-coated beads, yet not to gp63-coated beads. Finally, solubilized VE digested with N-glycosidaseF retains the inhibitory activity in spermatozoa–VE binding assays in contrast with VE treated with α-N-acetyl-galactosaminidase.

Conclusion. It was concluded that VE sulphate groups are involved in spermatozoa binding. These groups arepresent in gp75 glycoconjugates and are probably located in O-linked glycoconjugates.

IntroductionThe molecules responsible for egg envelope–sperm-atozoa binding are glycoconjugates and have selectiveproperties with respect to spermatozoa according totheir molecular components. In mammals, O-linked

1To whom correspondence should be addressed ([email protected]).Key words: Discoglossus pictus, fertilization, fucoidan, polysaccharide chain.Abbreviations used: BCA, bicinchoninic acid; DAPI, 4,6-diamidino-2-phenylindole; DIG, digoxigenin; DSA, Datura stramonium agglutinin; GNA,Galanthus nivalis agglutinin; VE, vitelline envelope; PNGase F, N-linkedglycosidase F.

polysaccharide chains possess suitable properties forspermatozoa binding, whereas the polypeptide back-bone is capable of eliciting the spermatozoa acrosomereaction (Bleil and Wassarman, 1988). Studies onthe spermatozoa–egg interaction in sea urchin indic-ated that a jelly coat glycoconjugate, rich in fucosesulphate, is the acrosome reaction inducer (SeGall andLennarz, 1979; Vileda-Silva et al., 1999; Gunaratneet al., 2003). For a 350 kDa glycoprotein at the eggsurface/VE (vitelline envelope), O-linked sulphatedoligosaccharides bind the spermatozoa with low af-finity, followed by a high-affinity interaction with

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M. Caputo and others

the protein backbone (Dhume and Lennarz, 1995).O-glycans containing fucose and/or sulphated fucoseare involved in spermatozoa–VE binding in themollusc Unio elongatus (Focarelli and Rosati, 1995;Capone et al., 1999), in ascidians and in mammals(see Miller and Ax, 1990; Oehninger, 2001). Inter-estingly, sulphate fucose was also shown to be in-volved in the spermatozoa–zona-free hamster interac-tion (Dravland and Mortimer, 1988). In humans andU. elongatus, spermatozoa selectin-like molecules areinvolved in binding to the zona (Oehninger, 2001)or to the VE (Focarelli et al., 2003) respectively. InU. elongatus, gp273, the VE ligand, contains a Lewis-like structure with fucose as determinant (Focarelliet al., 2003).

Therefore fucose or fucose sulphate in O-linkedoligosaccharides may interact at various stages ofspermatozoa–egg recognition, at the external coatsor at the egg surface, before or after the acrosomereaction.

In the amphibian Xenopus laevis, the VE gp69/gp64is a homologue to components of the ZPA family(Tian et al., 1997a, b) such as the mouse zona pellu-cida ZP2, which is capable of binding the sperma-tozoa by a secondary binding (Bleil and Wassarman,1988). gp69/gp64 display adhesive properties withrespect to the spermatozoa (Tian et al., 1997a, b).According to Vo and Hedrick (2000), in additionto gp69/gp64, gp41, a component of the ZPC fam-ily, has spermatozoa-binding activity. The gp69 andgp64 glycoproteins are two glycoforms of the samepolypeptide and have approximately the same num-ber of N-linked oligosaccharide chains, but differ inthe extent of O-glycosylation. The gp69/gp64 bind-ing motif may reside in a 27 amino acid domain wherepotential O-glycosylation sites are present (Tian et al.,1997a, b, 1999). In Bufo, VE carbohydrate moietiesappear to be involved in binding to the fertilizingspermatozoa (Omata and Katagiri, 1996). Yet, in am-phibians, the role of terminal fucose and sulphatedglycans in spermatozoa–VE binding remains to beestablished.

In Discoglossus pictus, fucose was found in glycocon-jugates selectively located at the plasma membraneof the dimple, the site of spermatozoa entry, thepresence of fucose being associated with spermato-zoa binding for the first time (Denis-Donini andCampanella, 1977). ‘In vitro’ assays later showedthat these fucose-rich glycoconjugates (Gualtieri and

Andreuccetti, 1996) are capable of specifically bind-ing the spermatozoa (Maturi et al., 1998; Talevi andCampanella, 1988). In D. pictus VE, gp75 and gp63display adhesive properties with respect to sperma-tozoa. Indeed, competition assays and peptide mapsshowed that gp63 and gp75 interact with sperma-tozoa in D. pictus VE and that they may representtwo glycoforms of the same protein (Caputo et al.,2001; Infante et al., 2004). Consistently, the two gpshave differences in lectin affinity, with only gp63cross-linking Ulex europaeus agglutinin I. This find-ing suggests that the terminal gp63’s fucose residuesrecognized by this lectin plan an important role inbinding the spermatozoa.

In D. pictus, binding of spermatozoa to VE occursonly after the acrosome reaction (A23187-exposedspermatozoa; Gualtieri and Andreuccetti, 1996;Caputo et al., 2001). Binding occurs all over the egg,although it is more abundant in the animal than inthe vegetal half of the egg (Infante et al., 2004). The63 kDa glycoprotein cDNA (DpZP2) was sequencedand cloned (Vaccaro et al., 2001) and shown to ex-hibit high homology to X. laevis gp69/gp64 and toZP2 of mammals.

In the present study, we investigated whether, inD. pictus VE, fucose and/or sulphate groups areinvolved in spermatozoa–VE binding and whetherthey are present in gp63 or gp75, using competitionassays. Surprisingly, we found that sulphates by them-selves have a major role in spermatozoa–VE binding,irrespective of whether they are borne by fucose, dex-tran or Lex oligosaccharides. They appeared to belocated in the gp75, where fucose is absent, probablyin O-linked oligosaccharide chains.

ResultsMetaperiodate pretreatment and binding assayDejellied eggs, i.e. deprived of jelly layers J1, J2, J3and the jelly plug, were treated with sodium meta-periodate and incubated with acrosome-reacted sper-matozoa. After washing to remove the unboundspermatozoa, the eggs were fixed and stained withDAPI (4,6-diamidino-2-phenylindole). Comparisonof the number of eggs having bound spermatozoain the treated eggs versus untreated eggs showedthat binding of spermatozoa was not affected bythe treatment (results not shown). Therefore it ap-pears that hydroxy groups are not directly involvedin spermatozoa–VE binding.

436 C© Portland Press 2005 | www.biolcell.org

Sulphates in amphibian fertilization Research article

Competition assays between dejellied eggs andfucoidan-, dextran sulphate- or dextran-treatedspermatozoaHaving undergone the acrosome reaction after expos-ure to the calcium ionophore A23187, spermatozoawere incubated with fucoidan and then added to de-jellied eggs. The results indicated a dose-dependentcompetition of fucoidan on spermatozoa–VE bind-ing. As shown in Figure 1(a), for untreated sper-matozoa, a high percentage of eggs having boundspermatozoa occurred, whereas the binding percent-age obtained with fucoidan-treated spermatozoa de-creased by approx. 20% already at the fucoidan con-centration of 0.005 mg/ml. There were practically noeggs with bound spermatozoa at the concentration of5 mg/ml. Figure 1(b) is an example of an egg hav-ing bound spermatozoa, when spermatozoa were notexposed to fucoidan, and Figure 1(c) shows an egg ex-posed to spermatozoa treated with 5 mg/ml fucoidan.Figure 1(d) depicts a spermatozoon in the VE. Theplasma membrane and the outer acrosome membraneare in the process of vesiculation and only the outer-most acrosome component is present in the acrosomecap (see Campanella et al., 1997).

To investigate whether the inhibitory effect of fuc-oidan was due to fucose or SO4

2− groups, the acro-some-reacted spermatozoa were treated with dextranor dextran sulphate and then incubated with dejel-lied eggs. Binding percentages are shown in Figure 2.For dextran-treated spermatozoa, a high percentage ofeggs with spermatozoa bound to VE along the wholerange of concentrations was observed. In contrast,the percentage of eggs with bound spermatozoa inthe dextran sulphate-treated spermatozoa assays was35% lower at the concentration of 0.0005 mg/ml andabolished at 0.5 mg/ml (Figure 2).

Competition assays between spermatozoa andfucoidan- or dextran sulphate-treated eggsSince, in the competition assays, the competing sug-ars cannot be completely washed away from the sper-matozoa, further experiments were needed to deter-minewhether, inourexperiments, receptivemoleculesinteracting with sugars are located on the sperma-tozoa or on eggs. To this end, dejellied eggs wereexposed to fucoidan or dextran sulphate and thenrinsed before spermatozoa–egg incubation. No sper-matozoa binding inhibition was found (Figure 3),indicating that the above-mentioned SO4

2− groups

Figure 1 Competition assays with fucoidan(a) Spermatozoa–VE binding significantly decreases in a fuc-

oidan dose-dependent way. Grey bar, spermatozoa unex-

posed to fucoidan; white bar, fucoidan-exposed spermato-

zoa. Bars represent S.D. (b) Egg in an experiment where

eggs were incubated with spermatozoa unexposed to fuc-

oidan and (c) Egg in an experiment where eggs were in-

cubated with spermatozoa treated with 5 mg/ml fucoidan.

In (b), spermatozoa are bound to the VE surrounding the egg,

whereas, in (c), the spermatozoa are absent. x = 14. (d) Ul-

trathin section of an inseminated egg, where a spermato-

zoon is shown in the innermost jelly coat (J1) and in the VE.

The spermatozoon is surrounded by its inner acrosome mem-

brane (IAM) and the acrosome is empty except for some of

its outermost components (small arrow). The large arrow in-

dicates vesicles derived from vesiculation of the spermato-

zoon plasma membrane and outer acrosome membrane. x =14 000.

are located on the VE and that fucoidan and dex-tran sulphate interact with receptive molecules onthe spermatozoa.



Figure 2 Competition assays with dextran anddextran sulphateSpermatozoa treatment with dextran (white bars) before the

addition of dejellied eggs results in a high percentage of eggs

with bound spermatozoa over the entire range of concentra-

tions tested. In contrast, on spermatozoa exposure to dextran

sulphate (grey bars), the percentage of eggs with bound sper-

matozoa decreased in a dose-dependent way. Bars represent

S.D.

Figure 3 Binding assay between spermatozoa andfucoidan/dextran sulphate-pretreated eggsWhite bar, fucoidan; grey bar, dextran sulphate. Egg pretreat-

ment with these glycans does not interfere with spermatozoa

binding to VE. Bars represent S.D.

Competition assays between dejellied eggs andLex trisaccharide 3′-sulphate- or 3′-sialyl-Lex

tetrasaccharide-treated spermatozoaCompetition binding assays with Lex trisaccharide3′-sulphate and 3′-sialyl-Lex tetrasaccharide sugars

Figure 4 Competition assays with Lex trisaccharide3′-sulphate and 3′-sialyl-Lex tetrasaccharide(A) When spermatozoa were exposed to Lex trisaccharide

3′-sulphate, spermatozoa binding to VE was inhibited in a

dose-dependent manner. In contrast, (B) spermatozoa pre-

treatment with 3′-sialyl-Lex tetrasaccharide did not, in prac-

tice, change the percentage of eggs with bound spermatozoa

in the control assays. Grey bars, control binding assay with

untreated spermatozoa; white bar, pretreatment assays.

were performed by incubating spermatozoa withone of these oligosaccharides before exposure to de-jellied eggs. Lex trisaccharide 3′-sulphate inhibitedspermatozoa–VE binding by approx. 60 and 100%at sugar concentrations 0.05 mg/ml and 0.2 mg/mlrespectively (Figure 4a). Conversely, 3′-sialyl-Lex tet-rasaccharide was not inhibitory at the concentrationsused (Figure 4b), confirming the role of SO4

2− groupsin spermatozoa–VE binding in contrast with CO2H−groups.



Competition assays with fucoidan- and dextransulphate-treated spermatozoa on beads-gp63and/or beads-gp75Since both gp63 and gp75 of VE specifically bind thespermatozoa (Infante et al., 2004), we supposed thatsulphated sugars are present in one or both of theglycoproteins. Accordingly, we performed assays ongp63 and gp75 immobilized on polystyrene beadsutilizing spermatozoa pretreated with fucoidan ordextran sulphate.

Electroeluted gp63 and gp75 were adsorbed onbeads. To estimate the amount of bound glycopro-tein/bead, adsorption was checked by boiling thebeads in sample buffer and the released proteinwas analysed by SDS/PAGE in each binding exper-iment. By knowing the concentration of the gly-coprotein in the eluate before and after adsorptionand the number of 350 µm beads/vol., the amount ofbound glycoprotein/bead was estimated to be in therange of 0.0075 µg (i.e. 7.2×1010 molecules) and of2 µg/cm2. Commercial BSA was adsorbed on beadsand used as control in spermatozoa-binding experi-ments.

Spermatozoa were preincubated with fucoidan ordextran sulphate and exposed to gp63 beads (Fig-ure 5). The binding of fucoidan- and dextran sul-phate-incubated spermatozoa in the range of sugarconcentrations used was almost the same as the bind-ing of the unincubated spermatozoa (60, 58 and 65%respectively). Spermatozoa treated with soluble gp63(0.5 mg/ml) underwent significantly lower adhesionto gp63 beads (20% eggs with bound spermatozoa).Similar assays were performed with gp75 beads (Fig-ure 5a). Binding of spermatozoa to gp75 beads wassignificantly affected by treating the spermatozoawith fucoidan or dextran: it decreased to 30–25%with respect to control untreated spermatozoa (64%binding; Figures 5a and 5b). Only 23% of the beadshad bound spermatozoa when the latter were incub-ated with gp75 (0.5 mg/ml).

Competition assays with solubilized VEs treatedwith PNGase F (N-linked glycosidase F) orα-N-GalNAcase (α-N-acetylgalactosaminidase)Preliminary experiments showed that addition of 40or 20 µg of solubilized VEs per 200 µl of reactedspermatozoa suspension in a final volume of 1 mlproduced an inhibition of the order of 70–60%. Toinvestigate the involvement of VE’s N-linked sugar

Figure 5 Competition assays between the spermatozoaexposed to fucoidan or dextran sulphate and gp63beads/gp75 beads(a) 0, untreated spermatozoa. The concentrations of fucoidan

(1, 5 and 10 mg/ml) and dextran sulphate (0.5, 1 and 2 mg/ml)

used for spermatozoa preincubations gave practically identi-

cal results and therefore they were pooled together in a single

bar. Data indicate that both sulphated glycans do not interfere

upon spermatozoa adhesion to gp63 beads, whereas they

exert a strong inhibition on the binding of spermatozoa–gp75

beads. (b, c) gp75 beads exposed to untreated spermatozoa:

spermatozoa adhere to the beads. (d) gp75 beads exposed

to fucoidan-incubated spermatozoa: the spermatozoa do not

bind to the beads. Bead size is 350–400 µm.

chains in spermatozoa binding, solubilized VEs wereexposed to increasing concentrations of PNGase F.Previous work showed that lectin blots of solubilizedVEs clearly cross-linked GNA (Galanthus nivalis ag-glutinin), a Man α1–3 Man-specific lectin, detectingN-linked sugars (Caputo et al., 2001). In the presentstudy, when solubilized VEs were exposed to PNGaseF in the ratio 1 unit/4 µg of VEs, no cross-linkingoccurred with GNA, in contrast with untreated VE



Figure 6 Enzyme-treated VEs(A) Competition assays using solubilized VEs digested by PN-

Gase F or α-N-GalNAcase. 0, untreated spermatozoa; 1, sper-

matozoa treated with soluble VEs; 2, enzyme-digested VEs.

In the PNGase F assay, the inhibitory action of solubilized

VE in spermatozoa–VE binding (38% binding) is not removed

by the digestion of VE by the enzyme (35% binding). In the

α-N-GalNAcase assay, the inhibitory action of solubilized VE

in spermatozoa–VE binding (13% binding) is removed by the

enzyme (85% binding). (B) Lectin blot of solubilized VEs. a,

untreated VEs; b, PNGase F-treated VEs. In the GNA blot,

the PNGase F-treated VEs lose their cross-reactivity to the

lectin, whereas in the DSA blot, the cross-reactivity is partially

retained by some of the VE glycoprotein.

blots (Figure 6). In addition, VE blots were sim-ilarly exposed to the Galβ1–4GlcNAc-specific DSA(Datura stramonium agglutinin). In this case, the lectincross-linking of VE glycoproteins was only partiallyeliminated by the PNGase F treatment (Figure 6).In parallel competition trials, reacted spermatozoa

suspensions were incubated with 40 µg of VEs pre-viously treated with PNGase F (1 unit/4 µg of VEs)and then used in spermatozoa–VE competition assaysover dejellied eggs. The results show that PNGase Ftreatment did not eliminate the competing ability ofsoluble VEs on spermatozoa–VE binding (Figure 6).In contrast, in the spermatozoa–VE competition assaywhere α-N-GalNAcase was used for VE digestion, anenzyme/VE ratio as low as 0.03 unit/1 µg was suffi-cient for abolishing VE inhibitory action. In these ex-periments, the proportion of eggs having bound sper-matozoa was 85%, whereas the proportion with un-treated VEs was 12% (Figure 6). α-N-GalNAcase iscapable of cleaving terminal α1–α3-linked GalNAcfrom glycoconjugates as well as GalNAc-Ser/Thr i.e.it acts on GalNAc O-linked to the peptide backbone(see Schachter and Brockhausen, 1992). Although wecannot discriminate between the two actions of theenzyme, overall the results of PNGase F and α-N-GalNAcase indicate that determinants for bindingare not located in N-linked oligosaccharide chains,but rather on O-linked chains.

DiscussionCompetition assays performed on dejellied eggs oron immobilized gp63 and gp75 indicate that VE-sulphated sugars are essential for the occurrenceof spermatozoa–VE binding in D. pictus. Indeed,competition trials utilizing three kinds of sulphatedsugars (fucoidan, dextran sulphate and Lex trisacchar-ide 3′-sulphate) showed a dose-dependent inhibitionof VE–spermatozoa binding. Inhibition did not occurif unsulphated dextran or 3′-sialyl-Lex tetrasaccharidewas used. 3′-Sialyl-Lex tetrasaccharide/Lex trisacchar-ide 3′-sulphate competition assays, in addition, in-dicated that sulphate-negative charges specifically acton the interaction in contrast with negative chargescarried by carboxylic groups. Moreover, these ex-periments indicated that the presence of fucose didnot affect competitively spermatozoa–VE binding.Consistently, for D. pictus, previous studies (Infanteet al., 2004) showed no cross-reactivity with anti-U. elongatus gp273 antibodies. The latter recognizeglycans containing a Lewis-like structure with fucoseas the determinant (Focarelli et al., 2003). Moreover,metaperiodate trials support the conclusion that, inspermatozoa–VE binding, sugar radicals such as hy-droxy groups are not implicated.



VE digestion by PNGase F and by α-N-GalNAcasesuggests that the sugars carrying sulphate groups arelocated in O-linked chains, as shown both in the seaurchin (Dhume and Lennarz, 1995) and in U. elong-atus (Capone et al., 1999). In the amphibian Cynopspyrrhogaster, heparan sulphate-like molecules locatedon acrosome-reacted spermatozoa are responsible forspermatozoa binding to the uterine envelope of thisspecies. A protein of approx. 70 kDa purified from theenvelope appears to bind to the heparan sulphate-likemolecules expressed on spermatozoa (Nakai et al.,1999). In contrast, in the present study, sulphates ap-peared to be part of the VE glycoconjugates, as sug-gested by binding assays with eggs pretreated withsulphated sugars. In particular, competition assays us-ing immobilized gp63 or gp75 strongly suggest thatSO4

2− groups are present on gp75 glycoconjugates,whereas gp63, its fucose-containing glycoform, hasno SO4

2− groups involved in spermatozoa binding.In most cases (see Miller and Ax, 1990; Oehninger,

2001; Focarelli et al., 2003), in spermatozoa–egg in-teraction, fucose has been found to have a dominantrole, in particular in its sulphated form. However,in some cases, as in rabbit spermatozoa–zona interac-tion, sulphate groups are the determinants carried bythecarbohydratemoietywhichare important forbind-ing to occur (O’Rand et al., 1988). In human, por-cine and mouse spermatozoa–zona interaction, acro-sin, a spermatozoa acrosome enzyme, appears to bindto sulphated polysaccharides of the zona pellucida(Moreno et al., 1998; Howes and Jones, 2002). Thisbinding is inhibited by sulphated but not by de-sulphated polymers (Williams and Jones, 1990).Moreover, in bovine reproductive biology, sulphateglycoconjugates not containing fucose are knownto modulate spermatozoa adhesion to the oviductalepithelium (Talevi and Gualtieri, 2001). In the seaurchin Strongylocentrotus purpuratus, sulphates play acritical role in determining the binding activity ofVE on spermatozoa. Desulphated fucoidan has noactivity, but resulphation of fucoidan restores the in-hibition of bindin-induced agglutination (De Angelisand Glabe, 1987). In the same species, a two-stepmodel was hypothesized for spermatozoa–egg surfaceinteraction (Dhume and Lennarz, 1995), i.e. (i) anionic weak binding involving sulphate groups of O-linked oligosaccharide chains, part of the 350 kDaglycoprotein, and (ii) a high-affinity binding in-volving one or more domains of the 350 kDa poly-

peptide chain in the second step. Moreover, sulphategroups appear to modulate the activity of some oli-gosaccharide chains in VE–spermatozoa interaction,since binding efficiency is proportional to the pres-ence of sulphate groups (Dhume and Lennarz, 1995).In D. pictus, gp75 and gp63 are the two glycopro-teins displaying adhesive properties to spermatozoa,but only one, gp75, contains sulphate groups, onthe basis of indirect yet compelling evidence in thepresent study. It may be postulated that, similar tothe sea urchin, multistep binding may occur in thissystem, where sulphate groups in gp75 oligosacchar-ide chains provide initial adhesion, and gp63 andgp75 protein backbones provide second binding. InS. purpuratus jelly coats, sulphate positions 2-O- and 4-O- in the saccharide chains of the sulphated α-L-fucandetermine spermatozoa cell recognition (Vileda-Silvaet al., 1999) and later, in embryos, dermatan sulphatebearing high amounts of O-4- and O-6-disulphatedgalactosamine units are essential for development tooccur (Vileda-Silva et al., 2001). In D. pictus VE,further studies are required to determine whetherspecific positions of sulphate groups are similarlyneeded in gp75 sugar chains for adhering to a sper-matozoa.

Materials and methodsAnimals and gametesAdult male and female D. pictus were injected in the dorsallymphatic sac with 200–250 units of Profasi HP (Serono, Rome,Italy) in Amphibian Ringer (111 mM NaCl, 1.3 mM CaCl2,2 mM KCl, 0.8 mM MgSO4 and 5 mM Hepes, pH 7.8). Uterineeggs were obtained 18 h after hormone injection. The semen(spermatozoa and seminal fluid) was collected from the seminalvesicles (see Caputo et al., 2001) 24 h after hormone treatment.Standard control insemination was performed by the addition ofa drop of semen to eggs immersed in 1/10 Ringer.

Preparation of VEEggs were first dejellied in dejellying buffer (100 mM NaCl,5 mM dithioerythritol and 50 mM Tris/HCl, pH 8.5). VEswere then manually removed and transferred into HB (25 mMHepes, pH 7.5, containing 900 mM glycerol, 0.02 mM NaN3,1 mM ATP, 1 mM dithiothreitol, 5 mM EGTA, 2 mM N-p-tosyl-arginine methyl ester, 5 mg/ml soya-bean trypsin inhib-itor, 5 µg/ml aprotinin and 10 µM E64). After extensive rinsingand brief centrifugations at low speed, VEs were exposed to0.6 M KI for a few min to remove the innermost jelly layerJ1 (see Caputo et al., 2001) and, after extensive rinsing in 1/10Ringer, dissolved in 2% (w/v) SDS by heating at 95◦C for at least2 min. After centrifugation at 14000 g for 10 min, the sampleswere processed for electrophoresis or binding assays.



Protein determination, SDS/PAGE and gel stainingProtein concentration of the samples was determined with theBCA (bicinchoninic acid) or Micro BCA Protein Assay Reagent(Pierce, Rockford, IL, U.S.A.); 15 µg of the VE-soluble proteinswas applied on each lane of the gels. Samples were prepared forSDS/PAGE by the addition of sample buffer and heating at 40◦Cfor a few minutes. Protein samples were analysed by SDS/PAGEusing Laemmli’s Tris/glycine buffer system. The running gelcontained 10% (w/v) acrylamide. Molecular-mass standards werethe following: 200, 116, 97.5, 66.2, 45, 31 and 21.5 kDa (Bio-Rad, Hercules, CA, U.S.A.). After fixation in methanol/aceticacid, gels were stained with either Coomassie Blue or silver.

Lectin blot detection on nitrocelluloseProteins subjected to SDS/PAGE were electrophoretically trans-ferred on to nitrocellulose sheets by the method of Towbin et al.(1979) overnight at 180 mA (for more details, see Infanteet al., 2004). DIG (digoxigenin)-labelled DSA and GNA werepart of a DIG glycan differentiation kit (Roche, Mannheim,Germany) and were utilized according to the manufacturer’s in-structions. Positive controls were run for the DIG-labelled lect-ins according to the DIG glycan differentiation kit, i.e. fetuinfor DSA.

ElectroelutionVE glycoproteins (gp63 and gp75) were separated by SDS/PAGEand visualized by staining with 0.3 M CuCl2. The area of theselected bands was cut out and the gel slices were electroelutedwith 25 mM Tris/HCl (pH 8.3) containing 1.44 mM glycine.Electroeluted proteins were dialysed at 4◦C against distilledwater and stored at –20◦C after concentration in a speed vacuumconcentrator Savant. The protein content and purity of eachsample were checked by SDS/PAGE and/or the BCA proteinassay method.

Metaperiodate-binding assaysAfter dejellying, groups of approx. 10–15 eggs were exposedto sodium metaperiodate (25, 50 and 100 mM) in Ringer at18–20◦C for 15 min. Five assays for each metaperiodate con-centration were performed. In some experiments, after meta-periodate treatment, eggs were further incubated with sodiumborohydride (20 mM) to stabilize the transformation of oligosac-charide hydroxy groups into aldehyde groups. Treated eggs werethen used for the spermatozoa–egg binding assays.

Spermatozoa–egg binding and competition assaysSpermatozoa suspensions as recovered from the seminal vesiclewere diluted in 1/10 Ringer to provoke complete unravellingof the spermatozoa bundles and spermatozoa ejection from thebundles. Spermatozoa are motile for approx. 14 s (see Maturiet al., 1998). The spermatozoa suspension was then treated with0.25 µM A23187 (Sigma) for 2 min, because previous workshowed that dejellied, unfertilized eggs bind to the spermatozoaif spermatozoa are pretreated with this Ca2+ ionophore to induceacrosome reaction (Gualtieri and Andreuccetti, 1996; Caputoet al., 2001) and then washed with 1/10 Ringer. As a generalcontrol for testing the binding ability of the spermatozoa to VE,groups of fully dejellied eggs were added to calcium ionophore-

treated spermatozoa and incubated for 15 min at room tempera-ture (22◦C) under gentle rocking. The spermatozoa-exposed eggswere then washed to separate the unbound spermatozoa fromthose bound to the eggs (see Caputo et al., 2001). The sampleswere either directly observed under a compound microscopeor, after fixation in 95% (v/v) ethanol, stained with DAPI forcounting spermatozoa head attachment to the VEs under a ZeissAxioskop microscope equipped with a Progress 3800 colourvideo camera and KS300 image analysis software.

The binding assay (see Maturi et al., 1998) considers the factthat, when spermatozoa come out of the bundles, they may getentangled because of their length (2.33 mm), and the result-ing suspension of spermatozoa is, therefore, rather uneven. As aconsequence, it cannot be established whether more bound sper-matozoa (or spermatozoa-bound beads, for the experiment in thenext section) means a greater ability of the VE to bind the sper-matozoa or more spermatozoa available for the egg. Therefore wemeasured successful spermatozoa collision (binding) with eggsunder gentle rocking in terms of percentage of eggs with sper-matozoa, without considering the number of spermatozoa/egg.

In competition assays, acrosome-reacted spermatozoa weretreated with fucoidan at the concentrations of 0.005, 0.05, 0.5,1 and 5 mg/ml and with dextran or dextran sulphate at theconcentrations of 0.0005, 0.005, 0.05 and 0.5 mg/ml. Altern-atively, spermatozoa were pretreated with Lex trisaccharide 3′-sulphate at the concentrations of 0.01, 0.05 and 0.2 mg/ml orwith 3′-sialyl-Lex tetrasaccharide at 0.025 and 0.25 mg/ml. Thespermatozoa suspension was then incubated with groups of 10–15 eggs for 15 min to allow spermatozoa binding under gentlerocking.

In a further set of experiments, groups of approx. 10–15 eggs were treated with fucoidan at the concentrations of 5and 10 mg/ml or dextran sulphate at the concentrations of0.005 and 0.5 mg/ml, washed to remove competing sugars andmixed with acrosome-reacted spermatozoa. A minimum of threeassays was performed for each sugar dilution used. The eggs werestained with DAPI for counting spermatozoa at the VE surface.

For ultrastructural observation, fertilized eggs were fixed at 2,6, 10 and 20 min after insemination in 2.5% (v/v) glutaraldehydein 0.2 M phosphate buffer as described previously (Talevi andCampanella, 1988). Dehydration in graded ethanol at 4◦C wasfollowed by embedding in Epon 812. Thin sections were cut withdiamond knives, stained with uranyl acetate and lead citrate andexamined in a Philips EM-300 microscope at the CISME (CentroInterdipartimentale di Servizio per la Microscopia Elettronica)of the University of Naples Federico II.

Spermatozoa–bead binding and competition assaysThe electroeluted glycoproteins as well as commercial BSA wereadsorbed on polystyrene beads of 350–400 µm (Polysciences,Warrington, PA, U.S.A.). Adsorption on the beads was per-formed as suggested by the supplier, utilizing 0.1 M boratebuffer (pH 8.5) and blocking in 1% BSA. In the assay, sperma-tozoa were smeared on microslides, further diluted in 10%(v/v) Ringer and treated with Ca2+ ionophore. After rinsing in1/10 Ringer, approx. 100 glycoprotein-coated beads were re-leased on the spermatozoa and incubated for 15 min in Petridishes under gentle rocking.

For competition assays, reacted spermatozoa were preincub-ated with 0.5 mg/ml of eluted gp63 or gp75 in 1/10 Ringer,



with fucoidan at the concentration 1, 5 and 10 mg/ml or dextransulphate at the concentration of 0.5, 1 and 2 mg/ml for 15 min.After a 10-fold dilution with 1/10 Ringer, the spermatozoa-binding assay was performed by mixing the treated spermatozoasuspension with groups of approx. 100 gp63 or gp75 beadsand maintaining for 15 min under gentle rocking. After sev-eral rinses in 1/10 Ringer, beads with bound spermatozoa weredirectly observed under a compound microscope to count sper-matozoa attachment or stained with DAPI and observed undera UV microscope.

PNGase F and α-N-GalNAcase assaysN-linked sugar chains were released from solubilized VEs onincubation with PNGase F (glycopeptidase F) from Flavobac-terium meningosepticum (Sigma) in the ratio of 5 units of PNGaseF/20 µg of VE. Incubation was performed in 500 mM sodiumphosphate, 12.5 mM EDTA and 5 M glycerol (pH 7.2) for 16 hat 37◦C. In control experiments, the VE was incubated in anenzyme boiled at 100◦C. The enzyme/µg of solubilized VE ratiomentioned above was calibrated by exposing increasing concen-trations of VE to fixed unit numbers of enzymes, blotting thedigested VE and exposing the blot to GNA or DSA. The ratio5 units of PNGase F/40 µl VE ratio was optimal for elimin-ating Man α1–α3 Man of N-linked sugars and it was used forspermatozoa–VE competition assays. In these assays, 200 µl ofreacted spermatozoa suspensions was incubated with the PN-Gase F-exposed envelopes. The suspension was further dilutedto 1 ml and used in spermatozoa–VE competition assays overdejellied eggs.

α-N-GalNAcase (from chicken liver; Sigma) cleaves ter-minal α1–α3-linked GalNAc from glycoconjugates as well asGalNAc-Ser/Thr. The incubation of VEs with the enzyme wasperformed overnight at 37◦C in 0.1 M phosphate–citrate buffer(pH 3.6). In competition assays, 200 µl of reacted spermatozoasuspensions was incubated with the enzyme-exposed envelopes(0.03 unit of α-N-GalNAcase/1 µg of solubilized VE). The pHof the α-N-GalNAcase-exposed VE suspension was neutralizedbefore adding to spermatozoa suspension. The suspension wasfurther diluted to 1 ml and used in competition assays betweenspermatozoa and dejellied eggs. In control experiments, VEswere incubated in 100◦C-boiled enzyme.

AcknowledgementsWe thank Professors F. Rosati and R. Focarelli forhelpful discussions, Dr M. Walkers for a critical read-ing of this paper, Mr G. Falcone for image elabora-tion, A. De Filippis and L. Iannone for help in thelaboratory. C.C. was supported by MIUR (Ministerodell’Istruzione, dell’Universita e della Ricerca Scien-tifica; Programmi di Ricerca Scientifica di RilevanteInteresse Nazionale 1999).

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Received 2 August 2004; accepted 16 September 2004

Published as Immediate Publication 29 April 2005, DOI 10.1042/BC20040092


Sulphated glycoconjugates are powerful inhibitors of spermatozoa binding to the vitelline envelope...

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