Localization of penultimate carbohydrate residues in zona pellucida and acrosomes by means of lectin cytochemistry and enzymatic treatments |
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Authors: | M Aviles M T Castells J A MARTINEZ-Menarguez I Abascal J Ballesta |
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Institution: | (1) Section of Histology and General Embryology, Department of Cell Biology, Medical School, University of Murcia, Murcia, Spain |
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Abstract: | Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc)
respectively. Galactose 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 terminal 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
penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida
and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was
labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity
to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/ SBA has the following effects:
(i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the
lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to
PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid 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, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration
of penultimate carbohydrate residues with affinity for these lectins.
This revised version was published online in November 2006 with corrections to the Cover Date. |
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