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

The macromolecular composition of zona pellucida (ZP) isolated from hu man 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 specim ens obtained after in vitro fertilization were removed from pools of h eavily fragmented embryos and inseminated oocytes that failed to ferti lize. For unfertilized oocytes, two major bands with an apparent molec ular weight of, respectively, 96 and 76-54 kDa were observed after 1-D -SDS-PAGE and silver staining. When ZP were isolated from fragmented e mbryos, the electrophoretic pattern showed a marked attenuation of the 96-kDa band. Silver-stained 2-D-SDS-PAGE analysis of ZP components fr om 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 mar ked heterogeneity. Fertilization-associated changes were apparent in t he electrophoretic pattern. ZP1 (Mr = 92-86 kDa, pI = 5.0-5.8) display ed 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 contri buted to a zona block to polysperma. (C) 1995 Wiley-Liss, Inc.