Composition of Human Zona PeUucida as Revealed

The macromolecular composition of zona pellucida (ZP) isolated from human oocytes and embryos was characterized by oneand two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (I-Dand 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 96kDa band. Silver-stained 2-D-SDS-PAGE analysis of ZP components from unfertilized oocytes revealed the presence of four protein trains: ZP1 ( M r = 92-80 kDa, pl = 4.9-5.9), ZP2 ( M r = 66-58 kDa, pl = 5.0-6.0), Z P ~ H (high) ( M r = 72-58 kDa, pl = 35-5.1), and Z P ~ L (low) ( M r = 62-54 kDa, pl = 3.5-5.1). The human ZP3 family ( Z P ~ H and Z P ~ L ) showed marked heterogeneity. Fertilization-associated changes were apparent in the electrophoretic pattern. ZP1 ( M r = 92-86 kDa, pl = 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. Q 1995 Wiley-Liss, Inc.

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