THE CHICKEN HOMOLOG OF ZONA-PELLUCIDA PROTEIN-3 IS SYNTHESIZED BY GRANULOSA-CELLS

Oocyte development within avian ovarian follicles is an intricate proc ess involving yolk deposition and the formation of extraoocytic matric es. Of these, the perivitelline membrane (pvm) not only plays a role i n sperm binding but also provides mechanical support for the large ooc yte's journey through the oviduct after ovulation. To date we have foc used on the mechanisms for uptake of yolk precursors into oocytes of t he chicken; now we extend our studies to a detailed analysis of the pv m. In the course of characterization of its major components, we obtai ned partial protein sequences; comparison with the GenBank database re vealed that one of the pvm proteins is the homologue of mammalian zona pellucida glycoprotein 3 (ZP3), a key component in sperm binding. Fol lowing a nomenclature based on gene structure, the protein is referred to as chicken ZPC (ch-ZPC). The chicken protein (444 residues) and mu rine ZP3 (424 residues) are highly conserved, with 41% of the amino ac ids identical. As shown by Northern blot analysis, the avian ZPC gene is expressed exclusively in the granulosa cells surrounding the oocyte , in contrast to murine ZP3, which is synthesized by the oocyte. Upon reaching a size larger than 1.5 mm in diameter, follicles accumulate c hZPC in highly polarized fashion, i.e., in the space intercalated betw een the oocyte and the granulosa cells, as revealed by immunohistochem istry of follicle sections. ChZPC synthesis and secretion by granulosa cells was demonstrated directly by metabolic labeling and immunopreci pitation from the culture medium of granulosa cell sheets isolated ex vivo from follicles. Immunoblot analysis and glycosidase treatment of chZPC from preovulatory and freshly ovulated oocytes, as well as laid eggs, revealed that the primary product undergoes a two-step decrease in size from follicle to laid egg that is unlikely to be due to modifi cation of the carbohydrate moiety.