A new method for preparation of pure zonae pellucidae in large quantities from porcine ovaries

Abstract A new method for preparation of large quantities of zona substance in pure form from porcine ovaries has been described. In addition to the previously reported techniques of glass wool treatment and sieving by saran meshes, the following three significant improvements have been introduced: disruption of ovaries by an electric machine equipped with two multi-needled disks, which resulted in a considerably accelerated recovery of follicular oocytes with a high yield; low-speed centrifugation at 170 × g for 15 s was found to be an obligatory step to eliminate light particulate material from the crude oocyte suspension; use of 50% sucrose solution in discontinuous density gradient centrifugation permitted complete separation of homogeneous samples at two stages of the final preparation of zona-encased oocytes or of oocyte-free zonae. Microscopic examination revealed no contaminating components in the zona preparation. With this method 93 mg of lyophilized zona preparation were obtained from 24 553 porcine ovaries. Analysis of a solubilized zona substance by Sephacryl S-200 column chromatography showed the presence of two major glycoproteins which could not be separated completely from each other. By analysis of the two components with SDS-PAGE, only a single, but broad, band of glycoprotein was found, indicating the successful isolation of a major component(s) from porcine zonae.