title: Oviductosome-Sperm Membrane Interaction in Cargo Delivery: DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM).
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.
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware 19716.
authors.forename: Amal A
authors.initials: AA
authors.lastname: Al-Dossary
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware 19716.
authors.forename: Pradeepthi
authors.initials: P
authors.lastname: Bathala
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware 19716; Delaware Biotechnology Institute, Newark, Delaware 19711.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Caplan
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware 19716. Electronic address: pdeleon@udel.edu.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Martin-DeLeon
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D055354:Exosomes / Q000378:metabolism*; D005187:Fallopian Tubes / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D005306:Fertilization; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000032:analysis; D016023:Integrins / Q000032:analysis / Q000378:metabolism*; D008297:Male; D008561:Membrane Fusion; D051379:Mice; D046529:Microscopy, Electron, Transmission; D016253:Microscopy, Immunoelectron; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000032:analysis / Q000378:metabolism*; D021381:Protein Transport; D013094:Spermatozoa / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D005456:Fluorescent Dyes; D016023:Integrins; D053499:Plasma Membrane Calcium-Transporting ATPases
keywords: calcium ATPase; exosome; fertilization; integrin; sperm