The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian oocytes, is composed of at least three glycoproteins. Our previous studies have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver and is transported to the ovary by blood circulation. In this study, we report the isolation of cDNA encoding quail ZP2 and its expression in the female bird. By RNase protection assay and in situ hybridization, we demonstrate that ZP2 transcripts are restricted to the oocytes of small white follicles (SWF). The expression level of ZP2 decreased dramatically during follicular development, and the highest expression was observed in the SWF. Western blot and immunohistochemical analyses using the specific antibody against ZP2 indicate that the 80 kDa protein is the authentic ZP2, and the immunoreactive ZP2 protein is also present in the oocytes. Moreover, ultrastructural analysis demonstrated that the immunoreactive ZP2 localizes to the zona radiata, the perivitelline space, and the oocyte cytoplasm in the SWF. By means of western blot analysis and immunofluorescence microscopy, we detected a possible interaction of the recombinant ZP2 with ZP3 and that this interaction might lead to the formation of amorphous structure on the cell surface. These results demonstrate for the first time that the avian ZP gene is expressed in the oocyte, and that the ZP2 protein in the oocyte might play a role for the PL formation in the immature follicles of the ovary.

The egg envelope surrounding avian oocytes exhibits a three-dimensional network of coarse fibers between the granulosa cells and the oocyte. Our previous studies have demonstrated that one of the matrix's components, ZP3, is synthesized in the ovarian granulosa cells. Another component, ZP1, which is critically involved in triggering the sperm acrosome reaction, is synthesized in the liver. We have previously isolated cDNAs encoding quail ZP3 and ZP1, and we now report the isolation of cDNA encoding quail ZPD. By RNase protection assay and in situ hybridization, we have demonstrated that ZPD transcripts are restricted to the granulosa cells of preovulatory follicles. The expression level of ZPD increased progressively during follicular development, and the highest expression was observed in the largest follicles. Western blot analyses using the specific antibody against ZPD indicate that the 40 kDa protein is the authentic ZPD, and the contents of ZPD protein also increased during follicular development. Moreover, we found that the addition of FSH to the culture media enhances the ZPD secretion in the cultured granulosa cells. Two-dimensional gel electrophoresis revealed the presence of several ZPD isoforms with different pI values ranging from 5.5 to 7. Immunohistochemical analyses indicate that the materials recognized with anti-quail ZPD antibody were accumulated in the egg envelope of large yellow follicles. These results demonstrate the presence of ZPD protein in the egg envelope, and that the amount of ZPD in the egg envelope as well as the mRNA in the cells increases at the latter stages of folliculogenesis.

1The avian perivitelline layer, an extracellular matrix homologous to the zona pellucida 2 of mammalian oocytes, is composed mainly by zona pellucida gene family glycoproteins. 3Our previous studies in Japanese quail have demonstrated that the matrix's components, 4 ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is 5 synthesized in the liver. Recently, we demonstrated that another minor constituent, ZP2 is 6 produced in the oocytes of the immature follicles. In the present study, we report the 7 isolation of cDNA encoding quail ZP4 and its expression and origin in the female birds. 8By RNase protection assay and in situ hybridization, we demonstrated that ZP4 transcripts 9were transcribed in the oocytes of small white follicles. The expression level of ZP4 10 decreased dramatically during follicular development, and the highest expression was 11 observed in the small white follicles. Western blot analysis using the specific antibody 12 against ZP4 indicated that the immunoreactive 58.2 kDa protein was present in the lysates 13 of the small white follicles. These results demonstrate for the first time that the avian ZP4 14 is expressed in the oocyte, and that the expression pattern of the gene is similar to that of 15 ZP2. 1617

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The avian vitelline membrane in laid eggs consists of three layers: the outer layer; the continuous membrane; and the inner layer [1]. The outer layer, which is composed of a varying number of sublayers of latticed fine fibrils, is formed in the infundibulum part of the oviduct [2]. The continuous membrane, which is a very thin granular membrane, is also formed in the infundibulum [2]. The inner layer, a 3D network of coarse fibers, is found between the granulosa cells and the oocyte in follicles before ovulation and is called the perivitelline membrane (PL) [3]. The PL is a homologue of the egg envelope in other vertebrates, the zona pellucida in mammals, the vitelline membrane in amphibians and the chorion in teleosts. These egg In birds, the egg envelope surrounding the oocyte prior to ovulation is called the perivitelline membrane and it plays important roles in fertilization. In a previous study we demonstrated that one of the components of the perivitelline membrane, ZP3, which is secreted from the ovarian granulosa cells, specifically interacts with ZP1, another constituent that is synthesized in the liver of Japanese quail. In the present study, we investigated whether ZP1 injected exogenously into the blood possesses the ability to reconstruct the perivitelline membrane of Japanese quail. When ZP1 purified from the serum of laying quail was injected into other female birds, the signal of this exogenous ZP1 was detected in the perivitelline membrane. In addition, we revealed, by means of ligand blot analysis, that serum ZP1 interacts with both ZP1 and ZP3 of the perivitelline membrane. By contrast, when ZP1 derived from the perivitelline membrane was administered, it failed to become incorporated into the perivitelline membrane. Interestingly, serum ZP1 recovered from other Galliformes, including chicken and guinea fowl, could be incorporated into the quail perivitelline membrane, but the degree of interaction between quail ZP3 and ZP1 of the vitelline membrane of laid eggs from chicken and guinea fowl appeared to be weak. These results demonstrate that exogenous ZP1 purified from the serum, but not ZP1 from the perivitelline membrane, can become incorporated into the perivitelline membrane upon injection into other types of female birds. To our knowledge, this is the first demonstration that the egg envelope component, when exogenously administered to animals, can reconstruct the egg envelope in vivo.Abbreviations CBB, Coomassie Brilliant Blue; DIG, digoxigenin; PL, perivitelline membrane; PVDF, poly(vinylidene difluoride); ZP, zona pellucida.