Uncovering the Role of IZUMO Family Members in Human Fertilization

ABSTRACT This study investigated IZUMO1, IZUMO2, and IZUMO4 expression, localization, and participation in sperm‐oocyte interaction, combining standard biochemical (Western immunoblotting and fluorescence immunocytochemistry) and functional (CPA, HZA, SPA) with computational biology approaches (bioinformatics, protein's 3D‐structure modelling). Human IZUMO1, IZUMO2, and IZUMO4 transcripts were found to be testis‐enriched (HPA), and expressed since puberty (MeDAS). IZUMO2 and IZUMO4 transcripts levels were lower ( p < 0.05) in teratozoospermic men (GEO). Human protein forms of ~40 kDa (IZUMO1), ~23 KDa (IZUMO2) and ~25 KDa (IZUMO4) were specifically immunodetected in sperm extracts. IZUMO proteins were immunolocalized in the acrosomal region of acrosome‐intact and acrosome‐reacted cells. Sperm pre‐incubation with anti‐IZUMO antibodies resulted in lower CPA rates using anti‐IZUMO4 antibodies, lower HZA rates using anti‐IZUMO1, anti‐IZUMO2, or anti‐IZUMO4 antibodies, and lower SPA rates using anti‐IZUMO1 or anti‐IZUMO4 antibodies ( p < 0.05). IZUMO1 and IZUMO4 were immunodetected in mouse sperm, and antibodies also blocked homologous fertilization. Protein modeling using AlphaFold identified potential antigenic regions recognized by the antibodies used in biological assays, which impaired IZUMO1‐JUNO and IZUMO4‐JUNO protein interactions. This is the first study that reports human IZUMO1, IZUMO2, and IZUMO4 expression/localization and involvement in gamete interaction. Findings presented enhance our understanding of the molecular interactions leading to fertilization and may contribute to male infertility diagnosis and treatment.