Complementary antibody- and recombinant protein-based approaches contribute to the functional assessment of porcine sperm zona pellucida–binding proteins (#540)
Background
Sperm–zona pellucida (ZP) recognition is mediated by multiple proteins acting cooperatively within dynamic sperm membrane complexes. Despite the identification of numerous candidate ZP-binding proteins, their functional validation remains challenging due to limitations associated with experimental approaches. This study assessed the functional involvement of lactadherin (MFGE8) and a putative ZP-binding protein (presumed ZPBP) in porcine sperm–ZP binding and examined methodological factors that influence interpretation of functional assays.
Methods
Candidate boar sperm surface proteins lactadherin and presumed ZPBP were investigated by using in-house monoclonal antibodies, as well as commercially available polyclonal antibodies. Functional involvement in porcine sperm–ZP interaction was examined using antibody-mediated inhibition assays with porcine oocytes, including vehicle and isotype control. Additionally, a competitive binding approach with corresponding recombinant proteins was used. For each experimental group, at least 60 oocytes (n ≥ 60) were evaluated across a minimum of three independent biological replicates.
Results
Competitive binding with recombinant lactadherin significantly inhibited sperm–ZP binding. In contrast, antibody-mediated inhibition assays using both commercial and in-house anti-lactadherin antibodies did not consistently inhibit the sperm binding, highlighting the importance of epitope accessibility, and optimization of antibody concentration. Antibody blocking experiments identified putative ZPBP as a more promising candidate ZP-binding protein.
Conclusion
This study provides evidence for the involvement of lactadherin and a presumed ZPBP in porcine sperm–ZP binding. It further highlights key methodological considerations, including the importance of species-specific antibodies, optimized antibody concentrations, appropriate controls, and the use of recombinant proteins as complementary tools, to improve the reproducibility and interpretation of functional sperm–ZP binding studies.
Acknowledgement: Study funded by the Ministry of Education, Youth and Sports of the Czech Republic under the INTER-EXCELLENCE II Programme, INTER-ACTION Subprogramme (LUAUS25072), the Internal Grant Agency of the CULS Prague (SV25-2-21230), and institutional support from IBT CAS (RVO: 86652036).
