Mol. Hum. Reprod. Advance Access published online on May 3, 2007
Molecular Human Reproduction, doi:10.1093/molehr/gam030
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Multiple proteins present in purified porcine sperm apical plasma membranes interact with the zona pellucida of the oocyte
1 Department of Biochemistry and Cell Biology, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands 2 Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK 3 School of Chemical Sciences, The University of Birmingham, UK 4 Department of Farm Animal Health, Graduate School of Animal Health, Utrecht University, Utrecht, The Netherlands
5 Correspondence address. E-mail: b.m.gadella{at}vet.uu.nl
An important step in fertilization is the recognition and primary binding of the sperm cell to the zona pellucida (ZP). Primary ZP binding proteins are located at the apical plasma membrane of the sperm head. In order to exclusively study primary zona binding proteins, plasma membranes of sperm heads were isolated, highly purified and subsequently solubilized with a mild or a strong solubilization procedure. Native, highly purified ZP ghosts were used as the binding substrate for solubilized sperm plasma membrane proteins, and a proteomic approach was employed to identify ZP binding proteins. Two-dimensional gel electrophoresis of ZP fragments with bound sperm proteins showed very reproducibly 24 sperm protein spots to be associated to the zona ghosts after mild plasma membrane solubilization whereas only three protein spots were detected after strong plasma membrane solubilization. This indicates the involvement of multiple sperm proteins in ZP binding. The three persistently bound proteins were identified by a tandem mass spectrometry as isoforms of AQN-3 and probably represent the main sperm protein involved in ZP binding. P47, fertilin ß and peroxiredoxin 5 were also conclusively identified. None of the identified proteins has a known acrosomal origin, which further indicated that there was no sample contamination with secondary ZP binding proteins from the acrosomal matrix. In this study, we showed and identified multiple zona binding proteins involved in primary sperm–zona binding. Although we were not able to identify all of the proteins involved, this is a first step in understanding the event of primary sperm–zona interactions and the relevance of this for fertilization is discussed.
Key Words: sperm zona binding/plasma membrane/spermadhesin/fertilin/lactadherin
Submitted on February 18, 2007; resubmitted on March 19, 2007; accepted on March 23, 2007.
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