Human Sperm Binding Is Mediated by the Sialyl-Lewisx Oligosaccharide on the Zona Pellucida

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Science  23 Sep 2011:
Vol. 333, Issue 6050, pp. 1761-1764
DOI: 10.1126/science.1207438


Human fertilization begins when spermatozoa bind to the extracellular matrix coating of the oocyte, known as the zona pellucida (ZP). One spermatozoan then penetrates this matrix and fuses with the egg cell, generating a zygote. Although carbohydrate sequences on the ZP have been implicated in sperm binding, the nature of the ligand was unknown. Here, ultrasensitive mass spectrometric analyses revealed that the sialyl-Lewisx sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], a well-known selectin ligand, is the most abundant terminal sequence on the N- and O-glycans of human ZP. Sperm-ZP binding was largely inhibited by glycoconjugates terminated with sialyl-Lewisx sequences or by antibodies directed against this sequence. Thus, the sialyl-Lewisx sequence represents the major carbohydrate ligand for human sperm-egg binding.

Mammalian sperm-egg binding is primarily mediated by the interaction of an egg-binding protein (EBP) on the sperm plasma membrane with carbohydrate sequences expressed on glycoproteins of the egg’s zona pellucida (ZP) (1, 2). Evidence that carbohydrate recognition plays a major role in human gamete binding was initially obtained when the polysaccharide fucoidan was shown to potently block this interaction (3). Fucoidan also inhibited leukocyte adhesion in the vascular and lymph systems in the same concentration range in which it blocks human sperm-ZP interactions (3, 4). The binding of leukocytes to endothelial cells is mediated by C-type lectins known as selectins (5, 6). Therefore, human sperm-ZP binding was hypothesized to involve a binding specificity that overlaps with the selectins (7). However, the structures of the human ZP glycans have remained enigmatic. Furthermore, characterization of these glycans is a challenge because of the scarcity of human eggs and their microscopic size. We have addressed this challenge by employing ultrasensitive mass spectrometric methodologies for cell and tissue glycomics (8, 9). In N- and O-glycans, an antenna is defined as a branch emanating from a “core” structure (10). We have defined the structures of the majority of N-and O-glycans attached to human ZP and show that sialyl-Lewisx is the dominant antenna sequence. This epitope is present at densities that are at least two orders of magnitude higher than levels of selectin-ligand expressed on somatic cells (11).

ZP from 195 unfertilized human oocytes was isolated for glycan sequencing. Purity was assessed by proteomics analysis. Human ZP4 was identified as the top hit in the Mascot search [algorithm for mass spectral proteomic data sets (table S1)]. The other ZP glycoproteins—ZP1, ZP2, and ZP3—were third, fifth, and sixth, respectively, on the Mascot list. At second and fourth position were haptoglobin and transthyretin, which are known constituents in follicular fluid (12).

The structures of the N- and O-glycans in the ZP sample were determined by glycomics analysis (8). Glycans were analyzed by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry (MS), as well as by collisionally activated dissociation (CAD) on a MALDI-TOF-TOF instrument [tandem mass spectrometry (MS/MS)]. Mixtures of N- and O-glycans were released from tryptic digests by peptide N-glycosidase F and reductive elimination, respectively, and were permethylated before MS and MS/MS analyses.

The MALDI-TOF N-glycan fingerprint (Fig. 1) showed four families of bi-, tri- and tetra-antennary structures, three of which (shaded in yellow, green, and pink) displayed an unusually high density of sialyl-Lewisx antennae [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc]. All members of the latter three families were core fucosylated, and each was fully sialylated. Heterogeneity was confined to differences in antenna fucosylation and length. Thus, biantennary glycans carried zero, one, or two sialyl-Lewisx antennae [mass/charge ratio (m/z) 2966.6, 3140.7/3589.9/3764.0, and 3314.8/3764.0/3938.1, respectively]; triantennary glycans carried zero, one, two, or three sialyl-Lewisx antennae (m/z 3777.1, 3951.2, 4125.2/4574.8/4748.6, and 4299.3/4748.6/4922.6, respectively); and tetra-antennary glycans carried zero, one, two, three, or four sialyl-Lewisx antennae [m/z 4587.5, 4761.6, 4935.8/5384.9/5559.0, 5109.4/5559.0/5733.1 (major portion), and 5733.1 (minor portion), respectively].

Fig. 1

MALDI-TOF profiling of human ZP N- and O-glycans. MALDI-TOF mass spectrum of N-glycans of human ZP. The upper panel covers the m/z range from 2700 to 4500, and the lower, overlapping, panel spans m/z 4200 to 5800. Each panel was normalized so that the most abundant peak is 100% intensity. Structural assignments were based on compositions assigned from molecular weights, complemented by MS/MS information and the results of the neuraminidase digest (fig. S2). Where more than one structure is shown, the upper structure is the more abundant. Gray structures have antennae carrying α2-6 sialic acid. All the other glycans are exclusively α2-3 sialylated. Yellow, biantennary; green, triantennary; pink, tetra-antennary.

Antennae compositions were defined by CAD-MS/MS. As shown in Fig. 2 and fig. S1, the most abundant fragment ions arose from cleavage of amino-sugar glycosidic bonds. CAD-MS/MS data from the highest molecular weight species observed in Fig. 1 (m/z 5733), together with its desialylated counterpart (m/z 4287.6, fig. S2) are shown in the upper and lower panels, respectively, of Fig. 2. These data demonstrated that m/z 5733 was a mixture of tetra-antennary glycans having one extended antenna and three or four sialyl-Lewisx moieties. The extended antenna were largely composed of the sialyl Lewisx-Lewisx sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc], although a minority of glycans had only a single fucose on this antenna (right cartoon on Fig. 2, lower panel).

Fig. 2

Partial MALDI-TOF-TOF fragment ion spectra obtained after collisional activation of the molecular ion at m/z 5733 in Fig. 1 and m/z 4288 in fig. S1 (upper and lower panels, respectively), illustrating how the fragment ions arising from loss of antennae in sialylated and desialylated samples, respectively, defined the antennae sequences. A minus refers to the loss of the designated antenna from molecular ion. Isomeric glycans were identified, which differ in fucose location (see cartoons on lower panel).

Fucose was confirmed to be 3-linked in the sialyl-Lewisx moiety through its diagnostic elimination in CAD-MS/MS experiments (Fig. 2 and fig. S1). Linkages involving sialic acid were defined by MALDI analysis of an α2-3–specific neuraminidase digest of the N-glycans (fig. S2), which showed that α2-6 sialylation was confined to the aforementioned fourth family (Fig. 1, shaded gray), which differs from the other families in having no core fucosylation. The human plasma glycome is characterized by the absence of core fucose plus high levels of α2-6 sialylation (13, 14). Therefore, we concluded that this fourth glycan family was largely derived from the follicular fluid constituents that copurify with ZP (table S1).

An extended sialyl-Lewisx-Lewisx antenna, and/or its monofucosylated counterpart, which was identified in the m/z 5733 component (Fig. 2), was additionally found in seven other members of the sialyl-Lewisx–containing families (Fig. 1, m/z 3589.9, 3764.0, 3938.1, 4574.8, 4748.6, 5384.9, and 5559.0). These extended sequences were firmly established by MS/MS analyses; examples of diagnostic fragment ions are illustrated in fig. S1C.

ZP N-glycans were also investigated to determine whether any were sulfated, a modification that is known to play a key role in selectin-mediated leukocyte trafficking (15, 16). This study was done after desialylation by using ultrasensitive MS methodologies that have been optimized for sulfoglycomics (17). Only trace levels of sulfated N-glycans were observed (fig. S3). Their compositions correspond to sulfated counterparts of the core fucosylated glycans shown in Fig. 1.

ZP-associated O-glycans were released from the glycopeptides recovered from the peptide N-glycosidase F digestion, permethylated, and analyzed by MALDI-TOF-TOF. A limited number of core 1 and core 2 O-glycans were observed (fig. S4), the latter carrying a single sialyl-Lewisx epitope. No sulfated O-glycans were detected in the MS experiments. Potential O-sulfation was also investigated by using the MECA-79 antibody, which recognizes 6-sulfated GlcNAc on extended core 1 sequences, including those terminated by 6-sulfo sialyl-Lewisx. No immunoreactivity was observed (fig. S5).

The high density of sialyl-Lewisx antennae observed on the ZP N-glycans was unusual. Examples of multivalent sialyl-Lewisx N-glycans are displayed in Fig. 3. This epitope is highly expressed in cells and tissues associated with many human cancers (18) and on orosomucoid in the sera of septic shock patients (19). However, in healthy humans, where sialyl-Lewisx plays a vital role in leukocyte trafficking, glycomics studies have suggested that fewer than 1% of the N-glycans carry sialyl-Lewisx and none has been found to carry more than one sialyl-Lewisx antenna (11). Another feature of the ZP N-glycome was the presence of extended antennae carrying an internal Lewisx sequence. This structure was found in members of all three families and was a particularly abundant constituent of the tetra-antennary family. This extended sialyl-Lewisx-Lewisx sequence has previously been found on tumor cells but not on normal somatic cells (20).

Fig. 3

Structures of the core fucosylated, multivalent sialy-Lewisx members of each of the bi-, tri-, and tetra-antennary families (shaded yellow, green, and pink, respectively) that were found on human ZP.

The hemizona assay was employed to determine the effect of sialyl-Lewisx terminated glycoconjugates on sperm-ZP binding (21). In this assay, nonliving human eggs were bisected by surgical manipulation, generating two equivalent hemispheres of ZP (hemizona). This test allows for an internally controlled comparison of sperm binding to a matching zona surface. Compared with the untreated controls, the number of sperm bound to the hemizona was significantly decreased after treatment with sialyl-Lewisx-BSA (bovine serum albumin) at concentrations of 0.5 μM (P = 3.3 × 10–4), 1 μM (P = 9.4 × 10–6), 1.5 μM (P = 3.7 × 10–8), and 2.0 μM (P = 1.2 × 10–7). The sialyl-Lewisx oligosaccharide also significantly decreased binding at concentrations of 100 μM (P = 1.9 × 10–3), 200 μM (P = 3.2 × 10–5), and 500 μM (P = 6.9 × 10–9) (Fig. 4A and fig. S6). Except for sialyl-N-acetyllactosamine oligosaccharide at the highest concentration, no significant inhibition was observed with Lewisx, sialyl-N-acetyllactosamine oligosaccharide, or BSA conjugates of these sequences (Fig. 4A and fig. S6). Fluorescently labeled sialyl-Lewisx-BSA was bound to the head of capacitated spermatozoa (Fig. 4B) but not to the hemizona (fig. S7). In contrast, Lewisx-BSA, sialyl-N-acetyllactosamine-BSA, and BSA were not bound to the sperm head (fig. S8). These treatments did not affect the acrosomal status and motility of spermatozoa (fig. S9). Consistently, antibody to sialyl-Lewisx (Fig. 4C), but not antibody to Lewisx (fig. S5), was bound strongly to ZP and suppressed sperm-ZP binding dose-dependently (Fig. 4D and fig. S10). To confirm the importance of sialylation, the binding of fluorescence-labeled native and desialylated solubilized ZP to human spermatozoa were compared. Desialylation significantly reduced the binding of solubilized ZP to capacitated spermatozoa (Fig. 4E).

Fig. 4

Sialyl-Lewisx is involved in sperm-ZP binding. (A) Comparison of hemizona binding index (HZI) of capacitated spermatozoa incubated in the presence of sialyl-Lewisx (SLEX), Lewisx (LEX), and sialyl-N-acetyllactosamine (SLN) oligosaccharide or their BSA conjugates with medium alone (control). Each point represents the mean ± SEM of the results of 10 hemizona assays. *, P < 0.05 when compared with the corresponding untreated control. †, P < 0.05 when compared with the desialylated counterpart. (B) Representative fluorescent images of capacitated spermatozoa incubated with Alexa Fluor-594 labeled sialyl-Lewisx-BSA. N = 5 separate experiments. (C) Immunostaining of sialyl-Lewisx sequences on hemizona. Matching hemizona were incubated with antibody to sialyl-Lewisx (CSLEX1), antibody to 6-sulfo lacNAc sequences on extended core 1 O-glycans (MECA-79), antibodies to Lewisx, or preabsorbed antibodies. N = 5 separate experiments. (D) Effect of CSLEX1 and antibody to Lewisx on sperm-ZP binding in hemizona binding assays. Matching hemizona were incubated with the antibodies or preabsorbed antibody as outlined in the supporting online material. N = 5 hemizona binding assays using five oocytes and five different sperm samples. *, P < 0.05 when compared with the control. (E) (Left) Effect of desialylation of solubilized ZP (1 μg/ml) on sperm-ZP binding in hemizona binding assays. N = 5 hemizona binding assays using five oocytes and five different sperm samples. (Right) Representative photographs of the binding of native or desialylated ZP to spermatozoa.

Previous studies indicated that antibodies directed against sialyl-Lewisa, sialyl-Lewisx, and Lewisb epitopes react with human ZP (22, 23). The antibody to Lewisb blocked human sperm-ZP binding in the hemizona assay (22). Erythroagglutinating phytohemagglutin also binds to human ZP, indicating that bisecting type N-glycans are expressed on this matrix (24). However, the current results have established that only the sialyl-Lewisx antigen is expressed at physico-chemically confirmable levels on ZP. Based on assessments of signal-to-noise ratio for detected molecular and fragment ions, it was estimated that other antigens must be substantially less than 1% of the glycome. The biophysical analyses here confirm the expression of carbohydrate sequences. Antibodies and lectins are useful for detecting carbohydrate ligands once their existence is confirmed, but they would not detect the multivalent presentations of sialyl-Lewisx and sialyl-Lewisx-Lewisx sequences.

Little is known about how human spermatozoa bind to eggs. The work described here provides insight into the key binding interactions that are essential for natural human fertilization, supporting the hypothesis that human gamete binding primarily involves the participation of the selectin ligand sialyl-Lewisx. Because human spermatozoa do not express selectins (25), the major egg-binding protein is very likely a lectin with a binding specificity that overlaps with the selectins.

Supporting Online Material

Materials and Methods

Figs. S1 to S10

Table S1

References (2635)

References and Notes

  1. Acknowledgments: This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC), grant BBF0083091 (to A.D., H.R.M., and S.M.H.); a Royal Society International Joint Project Award (to A.D. and K-H.K.); the Breeden-Adams Foundation and a Life Sciences Mission Enhancement Reproductive Biology Program funded by the state of Missouri (to G.F.C.); and the University Research Committee, University of Hong Kong (to P.C.N. and W.S.B.Y). The proteomics and sulfoglycomics data were acquired at the National Research Program for Genomic Medicine (NRPGM) Core Facilities for Proteomics and Glycomics funded by the Taiwan National Science Council (NSC99-3112-B-001-025). An intellectual property disclosure has been filed with the University of Missouri that covers technology described in this paper.
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