Evaluation of ZP2 domains of functional importance with antisera against synthetic ZP2 peptides

Abstract

The mouse zona pellucida protein ZP2 plays an important role in the process of fertilization by mediating secondary sperm binding to mammalian oocytes. ZP2 primary structures are highly conserved as revealed by cDNA cloning. The aim of the study was to identify ZP2 domains of functional relevance. Antisera were raised against synthetic peptides that are either conserved in the structure of ZP2 from different mammalian species (AS ZP2-20) or present in the human ZP2 but not in the mouse ZP2 amino acid sequence (AS ZP2-26). Antibody binding to zona pellucida proteins was assessed by assaying the antisera with human hemizonae. Using human zonae pellucidae, we demonstrated that anti-ZP2 common antibodies and anti-ZP2 human peptide antibodies react with human zona pellucida antigens. For the first time, ZP2 domains of functional relevance for human sperm-oocyte interaction could be identified applying the competitive hemizona assay. Antiserum AS ZP2-20 significantly inhibited binding of spermatozoa to test hemizonae, whereas treatment of hemizonae with AS ZP2-26 did not influence sperm-oocyte interaction. These results show that antibodies against synthetic ZP2 peptides react with ZP2 protein and that AS ZP2-20 identifies a linear ZP2 epitope that is of possible functional importance for sperm-oocyte interaction.

New Insights into the Mechanisms of Fertilization: Comparison of the Fertilization Steps, Composition, and Structure of the Zona Pellucida Between Horses and Pigs1

“…6h). The specificity of ZPB/ZP4 and ZPC/ZP3 antibodies was verified previously in equine [42] and porcine [46] species. The specificity of ZPA/ZP2 and ZP1 antibodies was verified by Western blot (Supplemental Fig.…”

“…The oocytes were then incubated overnight at 48C with polyclonal antibodies at the following dilutions in the blocking solution: rabbit anti-ZPA/ZP2 diluted 1:200, chicken anti-ZPB/ZP4 diluted 1:200, chicken anti-ZPC/ZP3 diluted 1:500, or rabbit anti-ZP1 diluted 1:100. The anti-ZPA/ZP2 antibody is directed against a common ZPA/ZP2 synthetic peptide sequence [42] and was kindly provided by Drs. E. and K.-D. Hinsch (Center of Dermatology and Andrology, Justus Liebig University, Giessen, Germany).…”

Development of mouse-specific contraceptive vaccines: infertility in mice immunized with peptide and polyepitope antigens

“…Polyepitope vaccines that simultaneously target species-specific regions of multiple proteins with similar structural or functional importance, such as all three zona pellucida proteins ZP1, 2 and 3, might induce more effective contraception. This approach is supported by our demonstration that contraceptive effectiveness is increased when the ZP1 and ZP3 are used in combination and that contraceptive mouse-specific ZP2 peptides are likely to exist (Hinsch et al 1998, Sun et al 1999. Thirdly, nonantibody immune responses, such as cytotoxic T-cell lymphocytes (CTLs), may generate immunocontraceptive effects and contraception after immunization with the ZP3 peptide has been reported to require both T-cell and antibody responses (Lou & Borillo 2003).…”

Can mammalian mothers influence the sex of their offspring peri-conceptually?

“…The work was done with metaphase II oocytes, and whether this stage of development is sufficiently mature to allow an influence on the ZP in mice is unknown. However, as noted above, there is some evidence that the ZP2 in mice functions differently to that in other species (Hinsch et al 1998). Therefore, a more parsimonious explanation for these results could be that no selection was taking place at the ZP.…”