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

Abstract

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.

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

“…2B). This pattern is very different from that for the other ZP glycoproteins in quail, in that immunoreactive ZP3 appears as early as in the PL obtained from the SYF, and the ZP1 and ZPD accumulate thereafter (Pan et al 2001, Sato et al 2009). In mice, Epifano et al (1995) investigated the accumulation of ZP1, ZP2, and ZP3 transcripts in oocyte lysates during oogenesis using an RNase protection assay.…”

“…A recent study has revealed that another minor constituent, ZPD, was produced and secreted from the granulosa cells in Japanese quail (Sato et al 2009). In the present study, we clearly demonstrated that both the mRNA for ZP2 and the ZP2 protein are present in the oocytes of immature follicles in the quail ovary.…”

“…In avian species, three glycoproteins have been identified as the component of PL, ZP1, ZP3, and ZPD in both quail (Pan et al 2001, Sasanami et al 2003b, Sato et al 2009) and chickens (Waclawek et al 1998, Takeuchi et al 1999, Bausek et al 2000, Okumura et al 2004. These glycoproteins coordinately assemble into the fibrous matrix on the surface of the oocyte , and one of its major components, ZP1, has the ability to induce the sperm acrosome reaction in Japanese quail (Sasanami et al 2007).…”

Egg Envelope Glycoproteins ZP1 and ZP3 Mediate Sperm-Egg Interaction in the Japanese Quail

“…To observe the effect of anti-ZPs antibodies, the pvm was added into a tube containing either anti-ZP1 raised against the synthetic N-terminal peptide of ZP1 (Ohtsuki et al, 2004), anti-ZP2 raised against bacterially expressed ZP2 ( Kinoshita et al, 2010), anti-ZP3 raised against purified ZP3 from the pvm (Sasanami et al, 2002), anti-ZP4 raised against bacterially expressed ZP4 (Serizawa et al, 2011), anti-ZPD raised against bacterially expressed ZPD (Sato et al, 2009) or normal rabbit serum diluted 1:200 with HBSS, and sperm suspension was then added at 1×10 7 sperm/ml. We previously demonstrated that sperm binding to the pvm could be evaluated by inhibiting sperm AR in the presence of pertussis toxin (PTX, Calbiochem, La Jolla, CA, USA) , therefore 2 μg/ml PTX was added to the reaction mixture during the sperm-pvm incubation to analyze the sperm-pvm binding.…”

“…In avian species, pvm is composed of at least five glycoproteins (ZP1, ZP2, ZP3, ZP4, and ZPD) in Japanese quail (Pan et al, 2001;Sasanami et al, 2003;Sato et al, 2009;Kinoshita et al, 2010;Serizawa et al, 2011) and 6 glycoproteins (ZP1, ZP2, ZP3, ZP4, ZPD, and ZPAX) in chicken (Smith et al, 2005). ZP1 and ZP3 have been identified as major constituents in the pvm of both species.…”