CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation

Abstract

Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+) did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

Figure 1. Protocols used to isolate epididymosomes or small microvesicles by differential centrifugation of epididymal fluids.

Figure 2. A population of small CD9-positive membranous vesicles with distinct protein content is present in the cauda epididymal fluid.

Epididymosomes and small membranous vesicles were purified from caput (A) and cauda (B) epididymal fluid. Vesicle size was evaluated by Zetasizer NanoZS. The experiment was performed three times in duplicate. C: Cauda small membranous vesicle size, structure, and shape were evaluated by electron microscopy. Scale bar: 100 nm. D: Protein content of epididymosomes and small membranous vesicles purified from caput and cauda epididymal fluid was evaluated by the Bradford technique, and the proportion was calculated by considering epididymosomes as 100%. Measurements were performed in epididymal fluid from three different individuals. E: Western blot detection of CD9 (25 kDa) on Triton X-100 protein extracts from small membranous vesicles recovered from caput, corpus distal, and cauda epididymal fluid. Each lane contains the same amount of protein. Results are representative of three different experiments. F: Western blot detection of CD9 (25, 27, and 33 kDa), P25b, GliPr1L1, ELSPBP1, MIF, and AKR1B1 on Triton X-100 protein extracts from cauda epididymosomes (Epidid.) and CD9-positive microvesicles (CD9-MV). Each lane contains the same amount of protein. Molecular standards are indicated on the left side of the figure. Results are representative of three different experiments.

Figure 3. 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.

Figure 4. Localization of CD9 in the epididymal duct.

Immunohistological localization of CD9 in different sections of the bovine epididymis: caput (Ca), corpus (Co), and cauda (Cd). Inserts represent negative controls. Immune complexes are observed in red and nuclei are counterstained in blue.

Figure 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.

Figure 6. 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.

Figure 7. 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.