Fig 2: The exosome marker CD9 is highly expressed by cauda epididymal epithelial cells in intracellular structures.Immunolocalization of CD9 (red) in caput (A) and cauda (B) epididymal epithelial primary cells cultured in vitro. Nuclei were counterstained with DAPI (blue). Yellow arrow indicates intracytoplasmic localization. Scale bar: 10 µm. C: Western blot detection of CD9 (25 kDa) on Triton X-100 protein extracts of epididymal tubules dissected from the caput and cauda epididymal regions. Each lane contains the same amount of protein. Results are representative of at least three different experiments. D: Semi-quantitative analysis of the expression of CD9 using tubulin as a housekeeping gene. The highest expression was considered to be 100%, results are presented as average ± s.e.m. from three different experiments, * differs significantly p<0.05. Dissected and digested epididymal caput and cauda tubules and epithelial cells showed comparable amounts of total (tubulin) and epithelial cell cytokeratin 8 transcripts.

Fig 3: CD26 is associated with CD9 in microvesicles and has a synergistic role with CD9 in the molecular transfer to the corpus distal sperm. A: CD9-positive microvesicles were lysed in presence of BrijO10, CD9 (25 kDa) was immunoprecipitated and analyzed by western blot. CD26 (110 kDa) and CD224 (61 kDa) coimmunoprecipitated with CD9. Normal mouse IgG was used as the negative control. Semi-quantitative analysis of RNA expression of CD224 (B) and CD26 (C) in epididymal epithelial cells, using tubulin as the housekeeping gene and cytokeratin 8 as a marker of epithelial cells. The highest expression was considered to be 100%, results are presented as average ± s.e.m. from three different experiments, * differs significantly p<0.05. Primary cultures of epithelial cells from caput and cauda epididymal tubules show comparable amounts of total (tubulin) and epithelial cell (cytokeratin 8) transcripts. D: Relative fluorescence intensity on live corpus distal epididymal sperm after 60 min coincubation with DilC12-labeled CD9-positive microvesicles in the presence of normal IgG or anti-CD224, anti-CD26 or anti-CD9 and anti-CD26 antibody Fab fragments. The maximum fluorescence was considered to be 100%. Results are presented as average ± s.e.m. from three different experiments, * differs significantly p<0.05.

Fig 4: Epididymosomes have more affinity to transfer molecules to dead sperm.Relative DilC12 fluorescence intensity on live and dead corpus distal epididymal sperm after 60 min of coincubation with cauda epididymosomes or CD9-positive microvesicles (CD9-MV) labeled with DilC12. The maximum fluorescence of the control was considered to be 100%. Results are presented as average ± s.e.m. from three different experiments, * differs significantly p<0.05.

Fig 5: Membranous vesicle–sperm molecular transfer mechanism. A: CD9-positive microvesicles DilC12-labeled molecule localization on corpus distal epididymal sperm after coincubation for 1 hour at 37°C in sperm medium at pH 6.5. Similar results were obtained with sperm incubated with labeled epididymosomes (data not shown). B: Relative DilC12 fluorescence intensity on live corpus distal epididymal sperm after coincubation with cauda epididymosomes for 30, 60, and 90 min. C: Relative DilC12 fluorescence intensity on live corpus distal epididymal sperm after coincubation with cauda epididymosomes labeled with DilC12 (DilC12-labled Ep) or DilC12-labeled cauda epididymosomes diluted 1:1 with non-labeled epididymosomes (DilC12-labeled/non-labeled Ep) after 60 min of coincubation. D: Relative DilC12 fluorescence intensity on live caput epididymal sperm after coincubation with cauda epididymosomes and on corpus distal epididymal sperm after coincubation with CD9-positive microvesicles (CD9-MV) labeled with DilC12 for 60 min in the presence or absence of 1 mM ZnCl2 (Zn). In all cases, the maximum fluorescence was considered to be 100%. Results are presented as average ± s.e.m., * differs significantly p<0.05. Experiments were performed at least three times.