Distinct features in fish Bouncer proteins determine sperm-egg compatibility

All sexually reproducing organisms depend on fertilization to survive as species. Despite the importance of fertilization, the mechanisms that drive sperm-egg compatibility are poorly understood. In fish, the egg protein Bouncer is necessary for fertilization and is species-specific between medaka and zebrafish. Here, we investigate whether Bouncer is generally species-specific in fish and identify features mediating its medaka/zebrafish specificity. In vitro fertilization experiments using zebrafish and medaka show that Bouncer is not a general specificity factor. Instead, its homologs exhibit wide compatibility with sperm, in line with the pervasive purifying selection that dominates Bouncers evolution. We further uncover specific features of Bouncer-- distinct amino acid residues and N-glycosylation patterns--that differentially influence the function of medaka and zebrafish Bouncer homologs and contribute to medaka/zebrafish specificity. This work reveals important themes central to understanding Bouncers function in sperm binding and clarifying the molecular requirements for Bouncers sperm interaction partner.