COMPOSITION OF HUMAN ZONA-PELLUCIDA AS REVEALED BY SDS-PAGE AFTER SILVER STAINING

The macromolecular composition of zona pellucida (ZP) isolated from human oocytes and embryos was characterized by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D- and 2-D-SDS-PAGE) under reducing conditions after silver staining. ZP specimens obtained after in vitro fertilization were removed from pools of heavily fragmented embryos and inseminated oocytes that failed to fertilize. For unfertilized oocytes, two major bands with an apparent molecular weight of, respectively, 96 and 76-54 kDa were observed after 1-D-SDS-PAGE and silver staining. When ZP were isolated from fragmented embryos, the electrophoretic pattern showed a marked attenuation of the 96-kDa band. Silver-stained 2-D-SDS-PAGE analysis of ZP components from unfertilized oocytes revealed the presence of four protein trains: ZP1 (Mr = 92-80 kDa, pI = 4.9-5.9), ZP2 (Mr = 66-58 kDa, pI = 5.0-6.0), ZP3H (high) (Mr = 72-58 kDa, pI = 3.5-5.1), and ZP3L (low) (Mr = 62-54 kDa, pI = 3.5-5.1). The human ZP3 family (ZP3H and ZP3L) showed marked heterogeneity. Fertilization-associated changes were apparent in the electrophoretic pattern. ZP1 (Mr = 92-86 kDa, pI = 5.0-5.8) displayed a dramatic decrease in intensity, and a new component had migrated to a position similar to that of ZP2. This modification may have been responsible for one aspect of the zona reaction, and could have contributed to a zona block to polysperma. (C) 1995 Wiley-Liss, Inc.