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 Bouncer’s 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 Bouncer’s function in sperm binding and clarifying the molecular requirements for Bouncer’s sperm interaction partner.