Characterization of extracellular vesicles from the boar reproductive tract: proteomic profiling and interaction with spermatozoa — ASN Events
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Characterization of extracellular vesicles from the boar reproductive tract: proteomic profiling and interaction with spermatozoa (#535)

Veronika Kraus 1 , Barbora Doleckova 1 2 , Ondrej Sanovec 1 3 , Michaela Frolikova 1 , Daniela Spevakova 1 , Zuzana Pilsova 4 , Aneta Pilsova 4 , Katerina Komrskova 1 5 , Ondrej Simonik 1 , Pavla Postlerova 1 4
  1. Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech Republic
  2. Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic
  3. Department of Physiology, Faculty of Science, Charles University, Prague, Czech Republic
  4. Department of Veterinary Sciences, Faculty of Agrobiology, Food, and Natural Resources, Czech University of Life Sciences Prague, Prague 6, Czech Republic
  5. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic

Background: Extracellular vesicles (EVs) are considered important mediators of intercellular communication within the male reproductive tract and contribute to sperm maturation. While EVs from boar seminal plasma (SP-EVs) have been partially characterized, the molecular composition and functional properties of epididymosomes from different regions of the boar epididymis remain poorly understood. This study aimed to isolate and characterize EVs from the epididymal fluids, as well as SP-EVs, and to investigate their interactions with boar spermatozoa.

Methods: EVs were isolated from epididymal fluids and SP using an optimised workflow combining differential centrifugation, filtration, and size-exclusion chromatography. EVs were characterized by electron microscopy, dynamic light scattering, nano-flow cytometry, and immunodetection of EV markers. EV protein cargo was analysed by LC–MS/MS proteomics, followed by Gene Ontology (GO) enrichment analysis. EV interaction with spermatozoa and cargo transfer were evaluated using lipophilic dye labelling and biotinylation assays.

Results: EVs of high purity were successfully isolated from all reproductive fluids while preserving their ability to interact with spermatozoa. Lipophilic dye and biotin labelling demonstrated EV binding and cargo transfer to spermatozoa. All EV populations were assessed for the presence of established EV markers as well as posttranslational protein modifications. Proteomic analysis identified distinct protein cargo across EV populations, and GO analysis indicated enrichment of proteins associated with signalling, immune modulation, and reproductive processes.

Conclusion: This study provides a comprehensive molecular characterization of EVs from the boar reproductive tract and demonstrates that isolated EVs retain the capacity to interact with spermatozoa and transfer molecular cargo. These findings expand current knowledge of boar reproductive EVs and provide a basis for future studies investigating EV-mediated mechanisms involved in sperm maturation and male fertility.

Acknowledgement: This study was supported by GA22-31156S, MEYS CR (LUAUS25072), IGA CULS (SV25-2-21230), Research programme Strategy AV21 Future of Assisted Reproduction (ART), and IBT CAS (RVO: 86652036).