Introduction The eutherian mammalian zona pellucida (ZP) is composed of three sulphated glycoproteins: ZPA (or ZP2), ZPB (or ZP1) and ZPC (or ZP3), each of which is encoded by a gene that is highly conserved among species (Harris et al., 1994). Although the protein components of the zona pellucida glycoproteins are highly homologous among species, interspecific differences in the sugar components of the N- and O-linked oligosaccharides are evident, and these may relate to species specificity of sperm-zona pellucida binding where this occurs. The importance of the oligosaccharides for sperm binding is well established and supported by the finding that addition of various saccharides and lectins to the in vitro fertilization environment can prevent sperm-zona pellucida binding and fertilization (Shalgi et al., 1986; Cornwall et al., 1991; Mori et al., 1993; Tulsiani et al., 1997). In mice, the O-linked oligosaccharides of ZPC facilitate primary sperm binding (Florman and Wassarman, 1985), either through a terminal α-galactose (α-Gal) (Bleil and Wassarman, 1988) or N-acetylglucosamine (GlcNAc) (Miller et al., 1992). In pigs, an N-linked oligosaccharide attached to Asn220 of ZPB in a ZPB–ZPC heterocomplex appears to be the primary binding site for spermatozoa (Kudo et al., 1998; Yurewicz et al., 1998) while, in humans, sperm–zona pellucida binding may be mediated through a selectin-like ligand on the zona pellucida which recognizes fucose (Fuc)- rich oligosaccharides (Patankar et al., 1993; Clark et al., 1996; Oehninger et al., 1998). Removal of terminal sialic acid residues (desialylation) from both the human zona pellucida and sperm surface results in an increase in sperm binding (Lassalle and Testart, 1994; Banjeree and Chowdhury, 1997; Ozgur et al., 1998). Lectin histochemistry has been used to identify the saccharides and their distribution within eutherian zonae pellucidae (Nicolson et al., 1975; Skutelsky et al., 1994). Observational studies on the zonae pellucidae of rats (Shalgi et al., 1991), humans (Bar-Shira Maymon et al., 1994) and river buffalo (Parillo et al., 1998) have demonstrated interspecific variation in the intensity of lectin-binding patterns and the distribution of various saccharide components. The zonae pellucidae of marsupials appear to be thinner and more readily solubilized by proteases than those of eutherians (Bedford, 1991, 1996; Bedford and Breed, 1994). The three genes encoding the zona pellucida proteins in Journal of Reproduction and Fertility (2000) 119, 111–120 Interspecific variation of zona pellucida glycoconjugates in several species of marsupial J. A. Chapman 1 , O. W. Wiebkin 2 and W. G. Breed 1 * Departments of 1 Anatomical Sciences and 2 Medicine, University of Adelaide, South Australia 5005, Australia The zona pellucida glycoconjugate content of several marsupial species was investigated using differential lectin histochemistry. Ovaries from fat-tailed dunnarts, a southern brown bandicoot, grey short-tailed opossums, brushtail possums, ringtail possums, koalas and eastern grey kangaroos were fixed, embedded in paraffin wax, sectioned and stained with ten fluorescein isothiocyanate-conjugated lectins. Sections were also incubated with either neuraminidase or saponified, respectively, before incubation with the lectins to identify saccharide residues masked by sialic acids or O- acetyl groups on sialic acids. The zonae pellucidae surrounding the oocytes of the marsupials demonstrated interspecific variation in glycoconjugate content, with mannose-containing glycoconjugates exhibiting the greatest variation. Some of the zona pellucida glycoconjugates of all species, except those of the opossums, were masked by sialic acid with an increase in fluorescence with lectins from Arachis hypogea (PNA), and Glycine max (SBA), after desialylation. The disaccharide β- galactose(1-4)N-acetyl-D-glucosamine appeared to be conformationally masked by O- acetyl groups of sialic acids in the zonae pellucidae of all species, with an increase in fluorescence with the lectin from Erythrina cristagalli (ECA), after saponification. Similar intensity and localization of β-(1-4)-N-acetyl-D-glucosamine, as shown by staining of the lectin from Triticum vulgaris (WGA), to the inner and outer regions of the zona pellucida, were found to those reported in eutherian species. WGA fluorescence became uniform throughout the zonae pellucidae after saponification, indicating differential O- acetylation of sialic acids on the internal compartment of the zonae pellucidae. © 2000 Journals of Reproduction and Fertility Ltd 0022–4251/2000 *Correspondence. Revised manuscript received 19 November 1999.