Characterization of the acidic N-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach

Oocyte maturation is a prerequisite for successful fertilization. Growing evidence suggests that not only the oocyte but also the surrounding zona pellucida has to undergo maturational changes. In the pig, two-dimensional electrophoretic analysis demonstrated an acidic shift of the zona pellucida glycoproteins of about 1.5–2.0 pH units during the maturation process. These findings were corroborated by histological studies that indicated the synthesis of acidic glycoconjugates in the cumulus cells and an increased occurrence of acidic glycans in the zona pellucida after oocyte maturation. In order to provide structural data on prepuberal zona pellucida N-glycosylation, N-glycans were released from prepuberal zona pellucida glycoproteins by N-glycosidase F and studied by mass spectrometry before and after desialylation and treatment with endo-β-galactosidase. Our results verified the presence of high-mannose-type Man5GlcNAc2 compounds as well as diantennary N-glycans as major neutral species, whereas sialylated diantennary and triantennary species constituted the dominant non-sulfated acidic sugar chains. The major acidic N-glycans of prepuberal animals, however, represented mono-sulfated diantennary, triantennary and tetraantennary oligosaccharides carrying, in part, N-acetyllactosamine repeating units as well as additional Neu5Ac or Neu5Gc residues. Glycans comprising more than one sulfate residue were not detected. In contrast to the literature data on zona pellucida glycoprotein-N-glycans of cyclic animals, our data thus reveal a lower degree in glycan sulfation of the prepuberal zona pellucida.

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