A glycoprotein with molecular mass of 42 kDa was identified as the major component of the chicken egg-envelope, the filamentous, extracellular matrix known as the perivitelline layer. By using a DNA probe amplified with degenerative primers derived from the protein's partial amino acid sequences, a cDNA clone encoding the eggenvelope 42-kDa glycoprotein (gp42) was isolated from a hen's ovary cDNA library. The gp42 open reading frame encoded 435 amino acid residues, including a putative signal peptide of 20 amino acids. The deduced amino acid sequence of gp42 showed significant similarity to egg-envelope glycoproteins of the ZPC family of several other vertebrate species, including human ZP3, mouse ZP3, Xenopus laevis gp43 and medaka (Oryzias latipes) ZI3 (LS-F), which play important roles for sperm±egg interaction. A single N-glycosylation site present in chicken gp42 is conserved among all five of these proteins: carbohydrate analysis of gp42 revealed the presence of a complex type glycan chain at this site. N-terminal sequence analysis of the mature polypeptide suggests that C-terminal processing of the pro-protein occurs during synthesis and secretion. The 1.4-kb gp42 transcript was detected only in follicles, and was found to be accumulated in granulosa cells in a manner dependent on ovarian follicular development. Furthermore, a metabolically radio-labeled gp42 was immunopreciptated from both cell lysate and culture supernatant of the granulosa cells with specific anti-gp42 antibody, suggesting granulosa cell-specific synthesis and secretion of the glycoprotein.Keywords: zona pellucida; perivitelline; chicken egg; egg-envelope.Avian oocytes in ovarian follicles are surrounded by a layer of extracellular matrix termed perivitelline layer [1,2], which has been shown to morphologically correspond to egg-envelope of other animal species [3]. During ovulation a split occurs between the perivitelline layer and the granulosa cells, and consequently the granulosa cells and basal lamina remain in the follicle and the ovulated ovum is enveloped by perivitelline layer, indicating that the ovarian follicular perivitelline layer is the precursor of egg-envelope in avian species. An ovulated ovum, enveloped by the perivitelline layer, is engulfed by the infundibulum of oviduct, where fertilization is thought to occur [4,5]. At fertilization, cock spermatozoa penetrate the perivitelline-derived egg-envelope, followed by initiation of spermatozoa acrosome reaction. The chicken perivitelline layer separated from the ovulated ovum has been reported to retain spermatozoa receptor activity [6]. This and other data [1±5,7] suggests that the avian egg perivitelline layer is functionally analogous to the well-characterized egg-envelope matrices of mammals [8±10].The egg-envelope glycoproteins, which play important roles in the envelope-matrix formation during oocyte development and the gamete interaction during fertilization, have been extensively investigated in zona pellucida of mammals [8±13], vitelline envelope of frogs [14±...

The chicken egg-envelope is made of two major glycoprotein components, which are designated as gp97 and gp42 (after their molecular masses). To elucidate how these two components are involved in macromolecular organization of the chicken egg-envelope, the isolated egg-envelope was characterized by immunochemical and biochemical methods. The gp97 was suggested to be a homologue of mouse ZPB based on the similarities of N-terminal and internal sequences. Immunoblotting using anti-gp97 monoclonal antibodies and two-dimensional gel electrophoresis with or without mercaptoethanol treatment revealed that gp97 formed a homodimer through disulfide bonds, whereas gp42 did not. Under indirect immunofluorescence microscopy, the anti-gp97 antibody visualized indistinct, small spots on the egg-envelope, whereas the anti-gp42 antibody showed a meshwork of blurry, fibrous structures. The hole formation on the egg-envelope by in vitro sperm penetration was completely inhibited by two anti-gp97 monoclonal antibodies. Interestingly, the anti-gp97 monoclonal antibodies blocked the proteolysis not only of gp97 but also of gp42 during incubation of the egg-envelope with either sperm or the crude chicken acrosin. Taken together, these results indicate that gp97 may play pivotal roles not only in constitution of the macromolecular organization of the egg-envelope but also in triggering hydrolysis of the egg-envelope during sperm penetration.