Abstract
Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca(2+) efflux pump, plasma membrane Ca(2+)ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4-64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes.
Highlights
The mechanism(s) for delivery of transmembrane proteins to sperm via exosomes/microvesicles is unknown
Our results provide support for the mechanism and reveal that fusion can be blocked by exogenous ligands, fibronectin, and vitronectin, for ␣51and ␣v3 integrins, their Arg-Gly-Asp recognition motif, and anti-␣v antibodies
Effect of Adhesion Peptide, Arg-Gly-Asp (RGD), for Integrins on OVS-Sperm Fusion—Having demonstrated the presence of ␣vintegrin subunit on mouse sperm and OVS, we investigated its involvement in their fusion
Summary
The mechanism(s) for delivery of transmembrane proteins to sperm via exosomes/microvesicles is unknown. Results: Using SR/SIM and a lipophilic dye, fusion detected for delivery of PMCA4 wasinhibited by blocking integrin/ligand interactions. Conclusion: Integrins on sperm and microvesicles play a role in microvesicle fusion for cargo delivery. Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca2؉ efflux pump, plasma membrane Ca2؉ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Immunofluorescence colocalized OVS with the ␣v integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. Our results provide evidence that receptor/ligand interactions, involving ␣v3 and ␣51integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be involved in cargo delivery of prostasomes, epididymosomes, and uterosomes
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