LOCALIZATION OF PENULTIMATE CARBOHYDRATE RESIDUES IN ZONA-PELLUCIDA AND ACROSOMES BY MEANS OF LECTIN CYTOCHEMISTRY AND ENZYMATIC TREATMENTS

Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalAc) respectively. Ga lactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of termina l residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penulti mate and terminal Gal/GalNAc residues. The areas selected for the demo nstration of the method included rat zona pellucida and acrosomes of r at spermatids, which contain abundant glycoproteins with terminal Gal/ GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. Afte r galactose oxidase treatment, terminal Gal/GalNAc residues are oxidiz ed, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/SBA has the following eff ects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins p reterminal Gal/GalNAc residues; and (iii) binding of the lectins to th e sugar residues. Acrosomes were reactive to PNA and SBA. No LFA react ivity was detected, thus indicating the absence of terminal sialic aci d residues. Therefore, no labelling was observed after both galactose oxidase-PNA/SBA and galactose oxidase-neuraminidase-PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neurami nidase and PNA/SBA cytochemistry is a useful technique for the demonst ration of penultimate carbohydrate residues with affinity for these le ctins.