The extracellular matrix surrounding avian oocytes, called the perivitelline membrane (PL), consists of at least two major glycoproteins, ZP3 and ZP1. Our previous study using Japanese quail had demonstrated that the PL obtained from the preovulatory follicles was incubated in vitro with spermatozoa, and perforations were observed. This result indicated that the PL might contain a constituent that possesses activity to initiate the acrosome reaction (AR) in quail. In order to elaborate upon our previous findings, we evaluated the effects of ZP3 and ZP1 on the induction of sperm AR in Japanese quail. Ejaculated sperm were incubated with or without the purified PL glycoprotein, and their acrosome status was determined based on the presence or absence of the acrosome. Treatment of spermatozoa with increasing doses of the purified monomeric ZP1 led to a concentration-dependent stimulation of AR. The purified dimeric ZP1 had similar effect. Moreover, we found that the ZP1-induced AR was significantly blocked by the digestion of the PL protein with PNGaseF. In contrast, the addition of purified ZP3 failed to induce AR at any doses tested. These results indicate that N-linked glycans on ZP1 play an important role in triggering the AR in Japanese quail.

The extracellular matrix surrounding avian oocytes, referred to as the perivitelline membrane (PL), exhibits a three-dimensional network of fibrils between granulosa cells and the oocyte. We previously reported that one of its components, ZPC, is synthesized in granulosa cells that are specifically incorporated into the PL; this incorporation might be mediated by a specific interaction with ZPB1, another PL constituent, which is synthesized in the liver. In order to extend our previous findings, we established an expression system for quail ZPB1 using a mammalian cell line, and several ZPB1 mutants lacking the zona pellucida (ZP) domain or the glutamine-rich repeat region were produced. Western blot analysis of the immunoprecipitated materials with anti-ZPC antiserum indicated that ZPB1 was coimmunoprecipitated with the antiserum in the presence of ZPC. Ligand blotting also revealed the specific binding of ZPC and ZPB1 and indicated that the binding of these two components might be mediated via an ionic interaction. An analysis using recombinant ZPB1 demonstrated that the ZPB1 lacking the ZP domain did not bind to ZPC, whereas the mutant missing the glutamine-rich repeat region retained its capacity for binding. Furthermore, although the ZPB1 lacking the N-terminal half of the ZP domain was able to bind to ZPC, the deletion of the C-terminal half completely abolished ZPB1 binding to ZPC. These results suggested that the C-terminal half of the ZP domain of ZPB1 contains a binding site for ZPC, and that it appears to be involved in insoluble PL fibril formation in the quail ovary.

The extracellular matrix surrounding the oocyte before ovulation is called the perivitelline membrane (PL) in avian species. We have previously reported that one of its components, ZPC, is produced in ovarian granulosa cells by the stimulation of follicle-stimulating hormone and testosterone. Another component, ZP1, is synthesized in the liver and might be transported to the surface of the oocyte of the follicles. These glycoproteins are assembled to form a three-dimensional network of coarse fibers between the granulosa cells and the oocyte. In the present study, we have evaluated the involvement of the interaction of ZPC and ZP1 in the formation of the PL of Japanese quail. By measuring the incorporation of tritium-labeled proteins into the PL, we have found that tritium-labeled ZPC is specifically incorporated into the PL. Whole-mount autoradiographic analysis of the PL has also revealed the incorporation of the secreted ZPC into the isolated PL. To study which component in the PL is responsible for the specific incorporation of ZPC, PL lysates were incubated with the conditioned medium of the granulosa cells and were immunoprecipitated with anti-ZPC antiserum. Western blot analysis of the immunoprecipitated materials indicated that the 175-kDa and 97-kDa ZP1 forms were co-immunoprecipitated with anti-ZPC antiserum. These results demonstrate that ZPC secreted from the granulosa cells specifically binds with ZP1, and that the phenomenon might be involved in insoluble PL fiber formation in quail ovary.

The extracellular matrix surrounding the oocyte before ovulation is called perivitelline membrane (PL) in avian species. We previously reported that one of its components, ZPC, is produced in the ovarian granulosa cells by the stimulation of follicle-stimulating hormone and testosterone. Another component, ZP+, is synthesized in the liver, and might be transported to surface of the oocyte in the follicles. These glycoproteins are assembled to form a three-dimensional network of coarse fiber between the granulosa cells and the oocyte. In order to address the mode of PL formation, we investigated the progressive changes in contents of ZP+ and ZPC in PL during the follicular development in quail ovary. Western blot analysis using specific antisera indicated that ZP+ band was first appeared as 31 kDa in molecular mass when the granulosa layer was isolated from the fourth largest follicle, and the intensity of the band was dramatically increased after the follicle matured to the third largest one. On the other hand, immunoreactive ZPC appeared as early as in the granulosa layer obtained from the small yellow follicles (SYF), and the intensity of the immunoreactive band increased progressively during follicular development. The immunohistochemical analysis indicated that the immunoreactive ZP+ and ZPC were not detected on the sections of follicular wall of the very small stroma-embedded white follicles. The immunoreactive ZPC was detected in the PL of SYF sections, whereas no detectable ZP+ was seen in the SYF sections. These results demonstrated that the accumulation of ZP+ was not synchronized to that of ZPC in the PL during follicular development. Alternatively, the accumulation of ZPC in the PL was preceded to that of ZP+ during follicular development in quail ovary.

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.