JUNO-coated beads as a functional assay to capture and characterize fertilization-competent human sperm.

study questionCan human fertilization-competent spermatozoa be captured through their ability to bind the oocyte receptor JUNO? SUMMARY ANSWER: JUNO-coated beads, which mimic the oocyte geometry, selectively bound acrosome-reacted spermatozoa with intact DNA, revealing that vitrification preserves functional sperm binding while slow cryopreservation increases non-specific interactions. WHAT IS KNOWN ALREADY: It is well established that sperm must undergo the acrosome reaction and expose the receptor IZUMO1 on the sperm head to bind specifically to JUNO on the oolemma. Studying the spermatozoa that reaches and engages with the oolemma, however, remains highly challenging due to the technical difficulty of recovering these sperm at the site of the molecular interaction. Bead-based models that contain oocyte receptors have therefore emerged as a powerful approach to functionally assess sperm-oocyte interactions, with promising applications for evaluating sperm quality. STUDY DESIGN SIZE DURATION: This was a cross-sectional experimental study including 21 semen donors of reproductive age recruited between January 2023 and June 2025. The JUNO-bead-based model was first validated using fresh human semen samples to establish the optimal sperm concentration and co-incubation time. Subsequently, two semen preservation methods, slow freezing and rapid freezing, were compared with respect to sperm-binding capacity to JUNO-coated beads, acrosomal status and DNA integrity. Finally, donors were classified according to sperm-binding capacity and validated by the hamster test. PARTICIPANTS/MATERIALS SETTING

methodsRecombinant JUNO protein was secreted and purified from MAIN RESULTS AND THE ROLE OF CHANCE: Recombinant JUNO bind human IZUMO1 ectodomain with a KD = 38 nM and specifically recognizes acrosome-reacted sperm and not acrosome-intact sperm. Human JUNO recombinant protein was successfully conjugated to oocyte-sized beads to generate a sperm-binding assay mimicking the geometry of the oocyte and experimental conditions of IVF. Human sperm bound specifically to JUNO-beads in a dose- and time-dependent manner, with highly significant differences compared to control beads ( LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This study was performed WIDER IMPLICATIONS OF THE

findingsThis study positions the JUNO-bead binding assay as a powerful functional model to investigate the biology of fertilization-competent sperm. By selectively capturing spermatozoa that have undergone the acrosome reaction and maintain DNA integrity, the model provides a unique experimental platform to study the molecular determinants of fertilization, to refine the selection of sperm for assisted reproduction, and to identify potential targets for novel contraceptive strategies. Beyond preservation protocols, these findings provide new functional evidence that sperm preservation method directly influences the molecular integrity required for fertilization, supporting vitrification as a superior approach over slow freezing. Moreover, the JUNO-bead assay emerges as a sensitive tool to reveal differences in sperm quality that are not captured by standard semen analysis, with potential applications in the optimization of assisted reproduction and fundamental research on the mechanisms that define the fertilizing spermatozoon. STUDY FUNDING/COMPETING INTERESTS: This work is part of the projects PID 2020-114109GB-I00 and PID2024-159920OB-I00 funded by MICIU/AEI/10.13039/501100011033 and by ERDF, EU and 23046/GERM/25 funded by FSRM/10.13039/100007801 to M.J.-M. This work was also supported, in part, by the Gates Foundation [INV-055841]. The conclusions and opinions expressed in this work are those of the author(s) alone and shall not be attributed to the Foundation. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. Protein production and characterization and biophysics infrastructure is supported by funding from a Canadian Institutes of Health Research Project Grant (PJT-203841) and Canada Foundation for Innovation John R Evans Leaders Fund (CFI-JELF) to J.E.L. The authors declare no conflicts of interest.