Glycosylated Chicken ZP2 Accumulates in the Egg Coat of Immature Oocytes and Remains Localized to the Germinal Disc Region of Mature Eggs

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of similar to 200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs.