Abstract STUDY QUESTION Can 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 hamster test. PARTICIPANTS/MATERIALS, SETTING, METHODS Recombinant JUNO protein was secreted and purified from Drosophila melanogaster S2 cultures. Digital SPR was used to confirm JUNO–IZUMO1 binding kinetics and imaging flow cytometry was performed to assess the biological activity of recombinant JUNO. Protein–bead conjugation and activity were verified by immunochemistry and western blot. Human semen samples were obtained from donors aged 19–42 years, including both fresh ejaculates and cryopreserved samples. Sperm binding capacity, acrosome reaction and DNA fragmentation were analyzed using widefield fluorescence microscopy and flow cytometry, and the specificity of sperm–bead interaction were evaluated with anti-IZUMO1 monoclonal antibodies. MAIN RESULTS AND THE ROLE OF CHANCE Recombinant JUNO binds 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 (p ≤ 0.0001). Vitrified-based cryopreserved sperm displayed higher binding to JUNO-beads than conventionally cryopreserved samples (p ≤ 0.0001). Binding was significantly inhibited by an anti-IZUMO1 (2.5 µg/mL) antibody that blocks specifically the IZUMO1–JUNO interaction in vitrified samples (p ≤ 0.01), but not in conventionally cryopreserved sperm. Sperm bound to JUNO-beads were predominantly acrosome-reacted in both preservation methods; however, vitrified samples retained higher DNA integrity compared with conventionally cryopreserved samples. The assay proved robust across multiple donors and ejaculates, allowing classification into low- and high-binding capacity (LBC and HBC) groups, and data were validated using the hamster test. Pearson correlation analyses revealed only weak associations between total sperm motility and bead-binding parameters (|r| 0.27), indicating negligible or absent linear relationships. LARGE-SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was performed in vitro, and the number of semen donors was limited. As all participants were healthy donors, the population represents a selected fertile subpopulation. Further studies using samples from diverse patient populations are required to validate the assay’s potential as a predictor of male fertility. While sperm–egg binding is an essential prerequisite for fertilization, the JUNO-bead–based assay focuses on this initial interaction and does not capture downstream fertilization events. WIDER IMPLICATIONS OF THE FINDINGS This 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 INTEREST(S) This work is part of the project PID 2020-114109GB-I00 and PID2024-159920OB-I00 funded by MICIU/AEI/10.13039/501100011033 and by ERDF, EU 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.
Cots-Rodríguez et al. (Fri,) studied this question.