Histochemical and structural characterization of egg extra-cellular matrix in bufonid toads,Bufo bufoandBufotes balearicus: Molecular diversity versus morphological uniformity

Abstract

The extra-cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta-elimination PAS) and lectin-histochemical (Con A, WGA, Succinyl-WGA, PNA, RCA-1, DBA, SBA, AAA, UEA-I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J1 and J2). The fibers of J2 are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N-linked. In B. bufo there are also residuals of mannose and/or glucose and N-acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J1 layer of both species presents sialosulfoglycoproteins, mostly O-linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo, whereas the opposite is seen in the amount of fucosylated residuals. The J2 layer is similar in composition to J1 but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J2 . Several microorganisms, in particular blue algae, are observed in the J2 layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types.

Comparative glycopattern analysis of mucins in the Brunner's glands of the guinea-pig and the house mouse (Rodentia)

“…(2) incubation in the lectin with the addition of the appropriate inhibitory sugar (concentrations are in Table 1); (3) binding to the colonic glands of the mouse or to the egg envelope of the toad Bufo bufo, the mucins of which are demonstrated to be labelled by the tested lectins (Liquori et al, 2012;Mentino et al, 2014).…”

“…Details for the lectins employed, their concentrations, their sugar specificities and references are summarized in Table 1. Lectin protocols followed Mentino et al (2014) and Mastrodonato et al (2013). Sections were incubated for 1 h at room temperature with the lectin solution in HEPES and subsequently rinsed in the same buffer and mounted in Fluoromount (Sigma-Aldrich).…”

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

“…The histochemical approach is useful in localizing the sites in which glycans are expressed and to evidence differences in both expression and functionality among cells, tissues, organs, developmental stages and species. (e.g., Mastrodonato et al, ; Mentino et al, ; Scillitani et al, ).…”

“…The histochemical approach is useful in localizing the sites in which glycans are expressed and to evidence differences in both expression and functionality among cells, tissues, organs, developmental stages and species. (e.g.,Mastrodonato et al, 2014;Mentino et al, 2014;Scillitani et al, 2012).Different methods and interpretation of results renders comparison among studies difficult. Traditional evaluation of the intensity of staining in both bright and fluorescent light consists in a semiquantitative analysis in which observers judge the level of staining by assigning progressive numbers.…”

Comparative histochemical analysis of intestinal glycoconjugates in the blunthead pufferfish Sphoeroides pachygaster and grey triggerfish Balistes capriscus (Teleostei: Tetraodontiformes)

“…After 1 h incubation at room temperature in the appropriate lectin solution in 4‐(2‐hydroxyethyl)‐1‐piperazine‐ethane‐sulphonic acid (HEPES), sections were rinsed in the same buffer and mounted in Fluoromount (Sigma‐Aldrich) for observation. Three different methods were performed as controls for lectin labelling: (a) incubation with lectin‐free HEPES, (2) incubation with lectin plus an inhibitory sugar (types and concentrations given in Table ), and (c) binding to secreting epithelia of the duodenum of the mouse or guinea‐pig, as well as to the egg extracellular matrix of the toad Bufo bufo , the mucins of which have been previously demonstrated to be labelled by the tested lectins (Mentino et al ., ; Scillitani & Mentino, ).…”