Zona Pellucida Domain of ZPB1 Controls Specific Binding of ZPB1 and ZPC in Japanese Quail <i>(Coturnix japonica)</i>

Abstract

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.

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

“…Consistent with our previous report (Sasanami et al 2006), quail ZP1 expressed in the cultured Chinese hamster ovary (CHO)-K1 cells was secreted from the cells during the culture, and the immunoreactive 97 kDa band was seen in the conditioned medium of the transfected cells (Fig. 7 panel A, lane 3).…”

“…As was mentioned earlier, the PL in quail as well as in chicken is constructed mainly by ZP1 and ZP3, and these components specifically bind to form the PL fiber , Sasanami et al 2006, Okumura et al 2007a, 2007b, Kinoshita et al 2008. In fact, in vitro incubation of ZP1 with ZP3 spontaneously produced fibrous aggregates, which were visible under optical microscopy (Okumura et al 2007b).…”

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

“…This is very likely the case, since the pI value of ZP1 is around 8-9, whereas that of ZPD as well as ZP3 ranges from 5 to 7 (data not shown). This statement is also supported by our previous finding that the radiolabeled ZP3 protein failed to visualize the ZPD band when the total PL extract was detected by ligand blot analysis , Sasanami et al 2006. The question of whether such interaction between ZPD and ZP1 actually occurs during the PL formation remains to be resolved, and the elucidation of the role of ZPD protein in the formation of the insoluble PL fiber must await future analysis.…”

Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica)

“…The signals were detected with HRP-conjugated antimouse IgG (Cappel) as a secondary antibody. The immunoblot for ZP1 and ZP3 was performed as described previously (Sasanami et al 2002(Sasanami et al , 2006. For ligand blot analysis, a PVDF strip electrotransferred with pvm lysate or purified ZP1 was incubated with [ 3 H]-labeled ZP3 as described previously (Ohtsuki et al 2004).…”