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

Abstract

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.

Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica)

“…Although we did not pursue the discrepancy of the results between the dot blot assay and far-western blotting, we assume that the interaction of the 45 kDa acrosin with the PVM is not simply mediated via a single molecule but supported by a complex of PVM proteins. Actually, we previously reported that the interaction of ZP1 and ZP3 (Ohtsuki et al 2004, Sasanami et al 2006 as well as that of ZP2 and ZP3 (Kinoshita et al 2010) play a role in the formation of the PVM during follicular development in quail. Moreover, we found that the sperm acrosin contains disulfide-bonded threedimensional arrangement with a modification of sugar moiety in the molecule, though a role of these structures for the sperm-egg interaction in fertilization remains to be studied (Fig.…”

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

“…Although the cause of this phenomenon is not clear, there is a possibility that the ZP proteins are responsible. ZP2 is expressed in the oocyte of immature follicles and exists in the pvm as a minor component in Japanese quail (Kinoshita et al, 2010). ZP2 in the chicken also accumulates in the egg envelope of immature oocytes but it stays behind in the germinal disk region even after the follicle grows to the largest (Nishio et al, 2014).…”

“…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.…”

Sperm-Egg Interaction during Fertilization in Birds

“…In mice, it was reported that ZP2 also supports the sperm binding (Bleil et al, 1988), and the structural change of the ZP2 as a result of limited digestion by the cortical granule protease protects the polyspermic fertilization (Burkart et al, 2012). In birds, ZP2 is expressed in the oocyte of immature follicles and exists in the pvm as a minor component (Kinoshita et al, 2010). Very recently, Nishio and colleagues demonstrated that ZP2 in the chicken accumulates in the egg envelope of immature oocytes and remains in the germinal disk region of the mature egg (Nishio et al, 2014).…”