Male reproductive tract extracellular vesicles display region-specific heterogeneity in mice

As sperm transit the male reproductive tract, they undergo a series of dynamic changes, gaining motility, modifying lipid and protein content and refining their epigenetic composition. Extracellular vesicles are central to this post-testicular maturation and changes in their composition could directly impact male reproductive health, sperm quality and post-fertilisation development. This study aimed to characterise and compare extracellular vesicles isolated from distinct regions of the male reproductive tract. Extracellular vesicles were isolated from adult, male C57BL/6J cauda and caput epididymis (epididymosomes) and seminal vesicle fluid by precipitation and size exclusion chromatography. Isolated vesicles were characterised using nanoparticle tracking analysis, transmission electron microscopy, Western blotting and imaging flow cytometry. Epididymosomes and seminal fluid vesicles ranged from 110.26 to 121.26 nm in diameter, had a concentration of 109 to 1010 particles/cm3 and had a typical round, cup-shaped morphology. The size and concentration of extracellular vesicles from the caput were significantly larger than those from the cauda and seminal fluid. Imaging flow cytometry revealed that all isolated extracellular vesicles expressed CD81 and CD9 tetraspanins; however, CD63 was detected only in caput epididymosomes. Furthermore, there were significantly fewer CD9+ vesicles in seminal fluid EVs compared to epididymosomes. Using a range of bulk- and single-vesicle analytical approaches, we show that different regions of the male reproductive tract display distinct vesicle compositional phenotypes. However, additional studies are warranted to define the significance of this heterogeneity, their roles in regulating male reproductive health and the development of their offspring.