Zonadhesin (ZAN) is a sperm protein currently known for its role in binding to the oocyte zona pellucida. Although its localization has been described in ejaculated and capacitated spermatozoa from mice, pigs and rabbits, its distribution following the acrosome reaction remains poorly understood. Here, we characterized ZAN localization in rabbit, boar, and bull spermatozoa under in vitro conditions mimicking early fertilization events. Ejaculated spermatozoa, swim-up-selected spermatozoa in capacitation medium, and acrosome reaction-induced spermatozoa were analyzed. Immunofluorescence was performed using an antibody against the MAM domain of ZAN to determine its localization and PNA-FITC to assess acrosomal status, while protein abundance was assessed by quantitative LC-MS/MS. Consistent with previous reports, ZAN was detected in the anterior region of intact spermatozoa. However, the MAM-specific antibody also revealed post-acrosomal localization. Species-specific patterns were observed in intact spermatozoa: rabbit and bull showed acrosomal staining with or without post-acrosomal labeling, whereas boar displayed predominant pre-equatorial and post-acrosomal localization. Although ZAN labeling decreased after acrosomal exocytosis, the protein was consistently retained in the post-acrosomal region. These findings suggest ZAN contributes not only to sperm–ZP binding but also to later fertilization events, including sperm–oolemma interaction and membrane fusion.

Zonadhesin (ZAN) is a sperm protein currently known to mediate binding to the oocyte zona pellucida. Although its localization has been described in ejaculated and capacitated spermatozoa from mice, pigs, and rabbits, its distribution following the acrosome reaction remains poorly understood. Here, we characterized the localization of ZAN in rabbit, boar, and bull spermatozoa under in vitro conditions designed to mimic early fertilization events. Ejaculated spermatozoa, spermatozoa selected by swim-up in capacitation medium and spermatozoa subjected to acrosome reaction induction were analyzed. Immunofluorescence was performed using an antibody targeting a domain of ZAN distinct from the previously studied domain, together with PNA-FITC to assess acrosomal status; protein abundance was assessed by quantitative LC-MS/MS. ZAN was detected mostly in the anterior region of intact spermatozoa, consistent with previous reports, but the distinct epitope also revealed post-acrosomal localization. Species-specific patterns were observed in intact spermatozoa, whereas following the acrosome reaction, ZAN was consistently detected in the post-acrosomal region. These findings identify a previously unrecognized post-acrosomal distribution of ZAN and suggest that this may contribute not only to sperm–ZP binding but also to later stages of fertilization, including sperm–oolemma interaction and membrane fusion.