Structural analysis of oligosaccharide‐alditols released by reductive β‐elimination from the jelly coats of the anuran Bufo arenarum

Abstract

Amphibian egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they are transported toward the cloaca. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O-linked carbohydrate chains of the jelly coat surrounding the eggs of Bufo arenarum were released by alkaline borohydride treatment. Fractionation of the mixture of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography, ion-exchange chromatography and high-performance liquid chromatography using an amino-bonded silica column, afforded 11 fractions. The primary structures of these O-glycans were determined by one-dimensional and two-dimensional 1 H-NMR spectroscopy in conjunction with matrix-assisted laser-desorption-ionizationϪtime-of-flight mass spectrometry. 11 oligosaccharide structures, possessing a core consisting of Galβ1→3GalNAc-ol with or without branching through a GlcNAc residue linked β1→6 to the GalNAc residue (core type 2 or core type 1, respectively) are described. These oligosaccharide-alditols with these types of cores have been identified previously in mammalian mucins or in oviducal amphibian jellies. These glycans contain blood group determinants such as H, A or Cad antigens.Keywords : amphibian egg jelly coats; Bufo arenarum; NMR; oligosaccharide.Mature eggs of most animal species are surrounded by extra-space, which separates the envelope from the egg plasma memcellular coats or vestments such as the vitelline envelope, the brane. Jelly coat layers are synthesized by specific regions of zona pellucida, and the jelly coat through which fertilizing the oviduct (the long pars recta convoluta portion of the oviduct) sperm have to penetrate before fusing with the egg plasma mem-and are sequentially deposited as the egg is transported toward brane. Fertilization is a fundamentally significant event, which the cloaca. Interest in these oviductally derived investments has has been intensely studied. A number of studies have shown that centered on their obligatory participation in the early events of the specific recognition between gametes is mediated by surface-fertilization. Oocytes whose jelly coat has been removed, or that located macromolecules. Much of the experimental evidence have not travelled down the oviduct are not fertilized when inavailable points to a major role for carbohydrates in sperm-egg seminated [6]. The particular properties of the jelly which make interaction. Carbohydrate moieties present within the zona pellu-its presence essential to fertilization are largely unknown. Howcida and corresponding carbohydrate-binding proteins on the ever, jelly layers become a blocking site to sperm penetration sperm surface [1] regulate this interaction. Specific carbohydrate upon hydration. Thus modifications of the anuran egg extracelmoieties mediate sperm-zona pellucida interaction in mice [2], lular matrix are fundamental processes that regulate sper...

Acquisition of species‐specific O‐linked carbohydrate chains from oviducal mucins inRana arvalis

“…By comparison with the NMR spectrum of compound 100‐H, the strong downfield shift of Gal III H‐1 (Δδ = +0.173 p.p.m.) suggests the existence of the Fuc(α1,2)Gal(β1,3)[Fuc(α1,2)]Gal(β1,3) sequence, already observed in the oviducal mucin of Xenopus laevis [8], R. dalmatina [16] and Bufo arenarum [17]. The presence of such a sequence was confirmed by the set of Gal II H‐3 and H‐4 resonances observed at δ = 4.173 and 4.018 p.p.m., respectively, and the downfield shifts observed for Gal III H‐2 (Δδ = +0.067 p.p.m.)…”

“…All the collected fractions were desalted by gel permeation on a Bio‐Gel P2 column (55 × 2 cm), and lyophilized [20]. The different steps of the purification are explained in detail in many previous papers [12–17].…”

“…Previous studies have shown a species‐specific structural diversity in carbohydrate chains of amphibian egg jelly coats [8–19]. Consequently, these carbohydrate chains represent new phenotypic markers of amphibian species and from a biological point of view, can be involved in species‐specific interaction gametes or in the specific host–parasite interaction [20].…”

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

“…When compared to Bombina pachypus , which we studied previously (Scillitani et al, ), B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types as evidenced by lectin histochemistry. Similarly, a survey of the available literature about ECM composition in bufonid toads (Arranz et al, ; Coppin et al, ; Farias et al, ; Kawai and Anno, ; Morelle et al, ; Omata, ; Reddy and Seshadri, ) indicates that in the two species we studied there are the main residuals found in other species, such as galactose, N ‐acetylgalactosamine, N ‐acetylglucosamine, sialic acid, fucose, and mannose. Besides, patterns of lectin binding allow to discriminate the two species between them and could be even used as a tool for egg identification.…”

“…It has been suggested that N ‐linked glycans, in particular lactosamine chains, have a structural function, orientate regulatory molecules, and are involved in water retention (Scillitani et al, ). Anyway, lactosaminylated residuals in O ‐linked chains cannot be excluded, as their presence in egg jelly was reported (Coppin et al, ; Morelle et al, ). As lectin binding is observed with RCA‐1 but not with PNA, galactose in saccharidic chains is probably linked to N ‐acetylglucosamine, not to N ‐acetylgalactosamine.…”