The extracellular vesicle tetraspanin CD63 journey from the testis through the epididymis to mature bull sperm

The important role of extracellular vesicles, which are considered key mediators of intercellular communication under physiological and pathological conditions, in various cellular processes, including those crucial for mammalian reproduction, has been increasingly studied. Tetraspanins, including CD63, are widely used as markers of extracellular vesicles, but they may also play a role in their biogenesis, cargo selection, cell targeting, and uptake. This study aimed to map the journey of the extracellular vesicle protein tetraspanin CD63 from the testis through the epididymis into mature bull sperm via an approach that included immunohistochemistry (immunofluorescence and immunoperoxidase staining), Western blot analysis, and immunoprecipitation analysis. We described the presence of CD63 in bull testicular and epididymal tissues, extracellular vesicles produced in these organs and spermatozoa during epididymal transit and after ejaculation. In addition, we revealed the nonuniform distribution of potential CD63 partners, such as CD9, integrin αV and syntenin-1, in the sperm head and tail and in extracellular vesicles. These findings contribute to understanding the complex mechanisms underlying sperm maturation and point to the possible involvement of tetraspanins and their associated partners, either as part of extracellular vesicles or sperm membranes, in these processes.