JUNO-Coated Beads as a Functional Assay to Capture and Characterize Fertilization-Competent Human Sperm

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 molecular interaction. Bead-based models that content 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 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. Participants/materials, setting, methods Recombinant JUNO protein was expressed and purified in Drosophila melanogaster S2 cells, and protein-bead conjugation was verified by immunochemistry. 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 assessed using widefield fluorescence microscopy and flow cytometry. Specificity of sperm-bead binding was evaluated with anti-IZUMO1 monoclonal antibodies. Main results and the role of chance 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 the in vitro fertilization. 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 ug/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. 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 potential of the assay as a predictor of male fertility. 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 funded by MCIN/AEI/10.13039/501100011033 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 biophysics infrastructure is supported by funding from Canada Foundation for Innovation John R Evans Leaders Fund (CFI-JELF) to J.E.L. The authors declare no conflict of interest. Lay summary Identifying and studying the sperm cell that is truly capable of fertilizing an egg is a major challenge, because this process occurs precisely when the sperm binds to the egg and penetrates it. In this study, we developed a model based on microscopic beads that mimic the shape of the egg and are coated with JUNO, a receptor essential for sperm–egg recognition. These JUNO-beads capture only those sperm cells with the right molecular and cellular properties to bind to the egg membrane and enable fertilization. Using this system, we compared sperm samples preserved through slow freezing (cryopreservation) or rapid freezing (vitrification). We observed that vitrified samples retained a higher proportion of sperm with fertilizing characteristics, sperm that had undergone the acrosome reaction but maintained intact DNA. Furthermore, by applying this model to samples from different donors, we were able to classify them according to their high or low binding capacity to JUNO-beads. Overall, this approach provides a new way to “capture” and evaluate fertilizing sperm, offering potential applications for improving sperm quality assessment in assisted reproduction, and a valuable tool for studying the defining features of the fertilizing sperm cell.

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