Isolation, physicochemical properties, and macromolecular composition of zona pellucida from porcine oocytes

Abstract

Oocytes released en masse from pig ovaries were isolated in large quantities by using sieving techniques. The isolated oocytes were gently homogenized, and the largely intact zona pellucida "ghosts" were purified by using sieving techniques. Sufficient amounts of zonae were recovered to permit, for the first time, adequate characterization of the zona pellucida in chemical, physical, and macromolecular terms. The isolated zonae were greater than 93% pure as determined by chemical, enzymatic, and microscopic criteria. The zonae were completely solubilized by a variety of conditions that do not break covalent bonds. The extent of solubilization was a function of pH, ionic strength, temperature, and the presence of various solubilizing agents such as detergents and urea. Chemically, the zonae were composed predominantly of protein (71%) and carbohydrate (19%). After acid hydrolysis of the zonae, no unusual amounts or types of amino acids were detected. The monosaccharides present after hydrolysis were those typically found in animal glycoproteins (Fuc, Man, Gal, GalNAc, and GlcNAc). Sialic acid in glycosidic linkage and sulfate and phosphate esters were present and were considered to be true constituents of the zona pellucida. Other substances detected, but considered contaminants rather than true constituents, included fatty acids (esterified and free) and uronic acids. The binding by several fluorescein-conjugated plant lectins to the in situ zona pellucida was determined by using light microscopy. The binding of the lectins to the zona pellucida was not uniform, indicating that the carbohydrate moieties of the zona pellucida were asymmetrically distributed. The zona pellucida was composed of at least three macromolecules as indicated by immunodiffusion and sodium dodecyl sulfate gel electrophoresis experiments. Determination of the number of macromolecules composing the zona pellucida was compromised by the aggregation and/or microheterogeneity of its constituent macromolecules. We conclude that the zona pellucida is composed of several glycoprotein macromolecules; interaction of these macromolecules to form supramolecular complexes and the integral zona pellucida is dependent on noncovalent forces.