Oocytic expression of zona pellucida protein ZP4 in Japanese quail (Coturnix japonica).

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

Zona pellucida protein ZP2 is expressed in the oocyte of Japanese quail (Coturnix japonica).

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.

Changes in post-translational modifications of prolactin during development and reproductive cycles in the chicken.

Changes in proportion of glycosylated prolactin in the anterior pituitary glands of chickens were assessed using one- and two-dimensional western blotting analysis during the perihatch stage of embryos and reproductive cycles. Multiple isoforms of prolactin were detected by one-dimensional analysis and glycosylated (G) and non-glycosylated (NG) isoforms were identified by N-glycosidase and neuraminidase treatment. Increases of ratio of G to NG isoforms were observed in both embryonic stages and reproductive cycles by the one-dimensional analysis. Although a similar tendency of increase of proportion of G prolactin was obtained, different values of proportion were observed between one-dimensional and two-dimensional analysis. Since two-dimensional analysis may better resolve isoforms differing slightly in molecular size of G prolactin, the results from two-dimensional analysis may reflect the actual proportion of prolactin isoforms. Furthermore, isoforms differing in isoelectric points were detected after N-glycosidase and neuraminidase treatment. These results indicate that prolactin may also be additionally post-translationally modified such as by phosphorylation. Thus function and biological activity of prolactin were, at least in part, regulated by post-translational modification in the various physiological stages.

Molecular characterization of egg envelope glycoprotein ZPD in the ovary of Japanese quail (Coturnix japonica).

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.

Incorporation of ZP1 into perivitelline membrane after in vivo treatment with exogenous ZP1 in Japanese quail (Coturnix japonica).

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.