Glycopattern analysis and structure of the egg extra-cellular matrix in the Apennine yellow-bellied toad, <i>Bombina pachypus (Anura: Bombinatoridae)</i>

Abstract

We studied the glycopatterns and ultrastructure of the extra-cellular matrix (ECM) of the egg of the Apennine yellow-bellied toad <i>Bombina pachypus</i>, by light and electron microscopy in order to determine structure, chemical composition and function. Histochemical techniques in light microscopy included PAS and Alcian Blue pH 2.5 and 1.0, performed also after <i>b</i>-elimination. Lectin-binding was tested with nine lectins (AAA, ConA, DBA, HPA, LTA, PNA, SBA, UEA-I, WGA). An inner fertilization envelope (FE) and five jelly layers (J1&#8211;J5) were observed, differing in histochemical staining, lectin binding and ultrastructure. Most glycans were O-linked, with many glucosamylated and fucosylated residues. The fertilization envelope presented a perivitelline space and a fertilization layer, with mostly neutral glycans. The jelly layers consisted of fibers and granules, whose number and orientation differed between layers. Fibers were densely packed in J₁ and J₄ layers, whereas a looser arrangement was observed in the other layers. Jelly-layer glycans were mostly acidic and particularly abundant in the J₁ and J₄ layers. In the J₁, J₂ and J₅ layers, neutral, N-linked glycans were also observed. Mannosylated and/or glucosylated as well as galactosyl/galactosaminylated residues were more abundant in the outer layers. Many microorganisms were observed in the J₅ layer. We believe that, apart from their functions in the fertilization process, acidic and fucosylated glycans could act as a barrier against pathogen penetration. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 2, pp. 306&#8211;316)

Keywords

• amphibia
• Bombina pachypus
• egg jelly
• lectin
• electron microscopy