EVIDENCE FOR THE PRESENCE OF HIGH-MANNOSE HYBRID OLIGOSACCHARIDE CHAIN(S) ON THE MOUSE ZP2 AND ZP3

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on plasma membranes of rat, mouse, hamster, and human spermatoza [Tulsiani et al. J Cell Biol 1989; 109:1257; Biol Reprod 1990; 42:843]. Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro, suggesting that the sperm enzyme may have a receptor-like role in binding to the complementary molecules (presumably mannose-containing oligosaccharide [OS] chains) on the mouse zona pellucida (ZP) glycoconjugates [Cornwall et al. Biol Reprod 1991; 44:913]. In the studies reported here, we demonstrate the pressence of high-mannose/hybrid-type OS on mouse zona components. Zona-intact eggs, prepared from superovulated mice, were radioiodinated, and the individual zona components (ZP1, ZP2, and ZP3) were isolated by electrophoresis followed by electroelution. The purified ZP components, when resolved by immobilized concanavalin A column chromatography, showed the following results: 1) Nearly all of the ZP1 applied to the immobilized lectin eluted in the column flow-through (effuent) fractions, and no radioactivity eluted with alpha-methyl mannoside, suggesting that ZP1 may not contain high-mannose/hybrid OS. 2) A significant amount of both ZP2 and ZP3 bound to the immobilized lectin, and nearly 16% and 8% of the two components, respectively, were repeatedly eluted with alpha-methyl mannoside. 3) Treatment of ZP2 and ZP3 with endoglycosidase H (an endoenzyme that cleaves high-mannose/hybrid-type OS chains from N-linked glycoproteins) or with jack bean alpha-D-mannosidase (an exo-enzyme that cleaves alpha-linked mannosyl residues) before the lectin column chromatography resulted in a decreased amount of radioactivity bound to the lectin and eluted with alpha-methyl mannoside. These results are consistent with the possibility that both ZP2 and ZP3 (but not ZP1) contain high-mannose/hybrid OS units. The role of these OS chains in sperm-egg interaction is discussed.