Sunday, October 16, 2011

The Sperm’s Sweet Tooth

     All mammalian eggs are surrounded by a thick extracellular coat called the zona pellucida (ZP). To fertilize an egg, sperm must bind to the ZP, penetrate through it, and fuse with the egg’s plasma membrane ( 1, 2). Binding of sperm to this outer coat occurs in a species-restricted manner, suggesting that ZP elements to which sperm bind may differ among mammalian species, and may be attributable in part to oligosaccharides ( 1, 2). On page 1761 of this issue, Pang et al. ( 3) report that binding of human sperm to eggs is attributable to an abundance of a sequence of sugar molecules called sialyl-Lewisx (SLeX) at the ends of oligosaccharides of ZP glycoproteins. These terminal sequences are known to function in the adhesion of other cells, including blood and tumor cells ( 4).
      The ZP consists of only a few glycoproteins that possess oligosaccharides (or glycans) covalently linked to the amino acids asparagine (N-linked) and/or serine and threonine(O-linked). For example, ZP of mouse
and human eggs consists of three (ZP1 to ZP3) and four (ZP1 to ZP4) glycoproteins, respectively ( 5). ZP glycoproteins are closely related to those constituting the vitelline coat of bird, frog, and fish eggs and have regions of polypeptide in common, such as a ZP domain ( 5, 6), suggesting that they are derived from a common ancestral gene. SLeX sequences consist of four sugar moieties—sialic acid, galactose, fucose, and N-acetylgucosamine— with sialic acid as the terminal sugar. Pang et al. identifi ed bi-, tri-, and tetra-antennary branched families—polymeric chains of sugars that extend off the main chain— by using ultrasensitive mass spectrometric (MS) analysis of ZP isolated from unfertilized human eggs (see the fi gure). The MS data suggest that SLeX sequences are present as both N- and O-linked oligosaccharides, at densities at least two orders of magnitude higher than on somatic cells. Furthermore, extended polymers of concatamerized SLeX sequences were found, especially on the tetraantennary family of oligosaccharides. This
structure is normally found on tumor cells, not on somatic cells ( 7).
      Involvement of SLeX sequences in sperm binding was inferred from the inhibitory effect of SLeX [alone or coupled to bovine serum albumin (BSA)] on the binding of sperm to isolated ZP in vitro (up to 60% inhibition). The oligosaccharide LeX, which lacks a terminal sialic acid, had little or no effect (alone or coupled to BSA) on binding of sperm to isolated ZP. Sperm binding to ZP that were treated with an antibody
against SLeX was inhibited by morethan 60%, whereas binding of sperm to ZP treated with an antibody against LeX was unaffected. These fi ndings indicate that the presence of a terminal sialic acid is important
for binding of sperm to SLeX. This was confi rmed by removal of sialic acid from solubilized ZP, which then exhibited substantially reduced binding to sperm as compared with native solubilized ZP. Sialic acid is a ligand
for binding of several types of animal viruses to cells ( 4). Collectively, these observations suggest that SLeX on the ZP could serve as a ligand for sperm binding during fertilization in humans and that sialic acid is a critical determinant for binding.
       Earlier studies suggested a role for oligosaccharides in binding of mammalian sperm to the ZP. O-linked oligosaccharides purifi ed from mouse ZP3 ( 8), blood group I–related oligosaccharides ( 9), fucosylated oligosaccharides ( 10), and LeX-containing oligosaccharides ( 11) all bind to mouse sperm and inhibit their binding to eggs at low concentrations in vitro. Even various monosaccharides, disaccharides, and fucoidan (a polysaccharide consisting primarily of sulfated fucose) block binding of rat, hamster, guinea pig, and human sperm to homologous eggs. Therefore, binding of mammalian sperm to eggs may be analogous to several other binding events. These include the binding of bacteria animal viruses, parasites, and other pathogens to their cellular hosts, binding of bacteria to plants and of pollen to the plant stigma, binding of amphibian and marine sperm to eggs, and sexual agglutination in yeast. All of these events are considered to be mediated by glycans ( 6).
      The results reported by Pang et al. raise a number of questions. The functional analyses were carried out with SLeX from total human ZP, not with SLeX from individually purified ZP glycoproteins. It is thus unclear whether sperm bind to all human ZP glycoproteins (ZP1 to ZP4) containing SLeX or whether binding is restricted to one or more of them. In this context, the fi nding that SLeX that is covalently linked to BSA is orders of magnitude more effective than SLeX alone as an inhibitor of sperm binding to eggs suggests that the oligosaccharide’s orientation may be affected by the polypeptide to which it is linked ( 10, 12). Indeed, the binding of mouse sperm to egg ZP oligosaccharides is infl uenced by the polypeptide to which they are linked ( 13). Because Pang et al. conclude that the effectiveness of SLeX in sperm binding depends on the presence of a terminal sialic acid, it is uncertain whether the monosaccharide’s negative charge is responsible for binding. It is likely that SLeX does not inhibit binding of mouse sperm to eggs because of the negative charge introduced by sialic acid ( 10). Whether SLeX can block the binding of other species of mammalian sperm to homologous eggs also remains unanswered; this would bear on the issue of species specifi city during mammalian fertilization.
      Another issue concerns the nature of the egg-binding proteins on human sperm that recognize and bind to SLeX. Perhaps derivatives of SLeX could be used as effective probes to tag the proteins. Even in the well studied case of the binding of mouse sperm to the egg’s ZP, the nature of the egg-binding proteins remains contentious ( 14). Despite these lingering issues, the study by Pang et al. should stimulate considerable interest in the molecular basis of sperm-egg interaction in humans and may ultimately lead to development of new contraceptives.

1. R. Yanagimachi, in Physiology of Reproduction, E. Knobil, J. D. Neill, Eds. (Raven, New York, 1994), vol. 1, pp. 189–318.
2. H. M. Florman, T. Ducibella, in Physiology of Reproduction,J. D. Neill, Ed. (Academic Press, New York, 2006), vol. 1, pp. 55–112.
3. P.-C. Pang et al., Science 333, 1761 (2011); 10.1126/ science.1207438.
4. A. Varki et al., Essentials of Glycobiology (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, ed. 2, 2008).
5. P. M. Wassarman, J. Biol. Chem. 283, 24285 (2008).
6. L. Jovine, C. C. Darie, E. S. Litscher, P. M. Wassarman, Annu. Rev. Biochem. 74, 83 (2005). 
7. M. Fukuda et al., J. Biol. Chem. 260, 12957 (1985).
8. H. M. Florman, P. M. Wassarman, Cell 41, 313 (1985).
9. E. S. Litscher et al., Biochemistry 34, 4662 (1995).
10. C. L. Kerr, W. F. Hanna, J. H. Shaper, W. W. Wright, Biol. Reprod. 71, 770 (2004).
11. D. S. Johnston et al., J. Biol. Chem. 273, 1888 (1998).
12. J. P. Carver, S. W. Michnick, A. Imberty, D. A. Cumming, Ciba Found. Symp. 145, 6, discussion 18 (1989).
13. L. Han et al., Cell 143, 404 (2010).
14. B. D. Shur, Int. J. Dev. Biol. 52, 703 (2008).



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