Identification of Affinity-enriched Sperm and Oocyte Plasma Membrane Proteins and Biological Relevance of Potential Candidates in In-vitro Fertilization
Sperm/oocyte membrane interaction in mammalian fertilization remains poorly understood. The IZUMO1 protein and its receptor is the only sperm/oocyte interaction pair known to date that is necessary for gamete fusion in mice. The current study aims to find potential candidate proteins for sperm/oocyte plasma membrane interaction in a porcine model. First, sperm and oocyte plasma membrane proteins with affinity for proteins of the plasma membrane of their partner gamete were separated by affinity chromatography. Second, the eluted proteins were identified by two different LC/MS-MS approaches using bioinformatic tools. Third, the biological role of selected candidate proteins was evaluated in in vitro fertilization (IVF) trials employing commercially available antibodies to selected proteins. Antibody binding to its antigen would be predicted to reduce fertilization if that protein antigen was involved in gamete interaction. The proteins consistently present across replicates with unique peptides were included in the bioinformatic analyses used for selection of proteins evaluated in the IVF trials. Approximately 100 oocyte plasma membrane proteins with affinity for the sperm, and 100 sperm plasma membrane proteins with affinity for the oocyte were identified with the two MS approaches. Some of these proteins were already suspected in involvement in gamete recognition and some met the threshold for affinity compared with nonspecific controls but did not exhibit a particularly strong affinity for the complementary gamete. In addition, a library of porcine oocyte membrane proteins was generated containing more than 200 proteins. In the IVF experiments, antibodies targeting oocyte proteins lipoprotein E (ApoE) and complement protein 6 (C6) significantly decreased the fertilization rate, suggesting that ApoE and C6 have a relevant role in the sperm/oocyte plasma membrane interaction. This study complements the knowledge from previous analyses on gamete interactions by using a molecular-affinity-driven process to identify sperm/oocyte plasma membrane interacting components.