Identification of exosomes/microvesicles in the oviductal luminal fluid: characterization of molecular interactions in cargo delivery to sperm using nanoscopy, TEM, and PMCA4 as a model
| Author(s) | Aldossary, Amal Ali K. | |
| Date Accessioned | 2015-06-15T12:21:07Z | |
| Date Available | 2015-06-15T12:21:07Z | |
| Publication Date | 2014 | |
| Abstract | Plasma Membrane Calcium ATPase 4, PMCA4, is the major Ca2+ efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. Here I show that Pmca4 mRNA, 4a and 4b variants, and PMCA4a protein are highly expressed in the female reproductive luminal fluids (LF) during estrus. Transmission electron microscopy (TEM) of the pellet recovered from ultracentrifugation of the oviductal LF revealed membrane vesicles with exosomal/microvesicle orientation, size, and shape, and with the characteristic CD9 exosomal biomarker. Thus, I dubbed these exosomes/microvesicles "oviductosomes" (OVS), to which PMCA4 was immunolocalized. Co-incubation of sperm with OVS resulted in up to a ~3-fold increase of sperm PMCA4a. To determine the mechanism of delivery of PMCA4 via OVS, I defined an experimental approach for the assessment of OVS-sperm interaction at a nanoscale level, using a lipophilic dye and 3D SR-SIM. Co-incubation assays detected fusion of pre-labeled OVS with sperm, primarily over the head and midpiece. Fusion was confirmed by TEM, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Both OVS and sperm carry αv-subunit of αvβ3 integrin. In capacitated and acrosome-reacted sperm, fusion was significantly inhibited by blocking integrin-ligand interactions via exogenous ligands, vitronectin and fibronectin, and their Arg-Gly-Asp recognition motif. To examine the potential impact of transfer of OVS-derived PMCA4 on sperm, Ca 2+ -ATPase activity, Ca2+ loading, and motility assays were performed. Following in vitro co-incubation of Pmca4 KO sperm with OVS from WT females, the Ca2+ -ATPase activity was significantly increased, intracellular Ca2+ levels significantly decreased, and progressive motility rates significantly increased. Thus, the results are consistent with the acquisition of oviductal PMCA4 in vivo to meet the demand of an increased requirement for Ca 2+ efflux in sperm during their storage, capacitation, and the acrosome-reaction, and to maintain sperm viability. Knowledge of OVS, identified for the first time, and their transfer of oviductal PMCA4 (and likely other fertility-modulating proteins) to sperm just prior to fertilization, provides new insights into molecular interactions at the fertilization site. This has important implications for increasing the fertilizing competence of sperm in the in vitro fertilization clinic. | en_US |
| Advisor | Martin-DeLeon, Patricia A. | |
| Degree | Ph.D. | |
| Department | University of Delaware, Department of Biological Sciences | |
| Unique Identifier | 911034570 | |
| URL | http://udspace.udel.edu/handle/19716/16794 | |
| Publisher | University of Delaware | en_US |
| URI | http://search.proquest.com/docview/1661456433?accountid=10457 | |
| dc.subject.lcsh | Mice. | |
| dc.subject.lcsh | Adenosine triphosphatase. | |
| dc.subject.lcsh | Seminal vesicles. | |
| dc.subject.lcsh | Sperm-ovum interactions | |
| dc.subject.lcsh | Cell membranes. | |
| dc.subject.lcsh | Oviduct. | |
| dc.subject.lcsh | Spermatozoa. | |
| dc.subject.lcsh | Calcium. | |
| Title | Identification of exosomes/microvesicles in the oviductal luminal fluid: characterization of molecular interactions in cargo delivery to sperm using nanoscopy, TEM, and PMCA4 as a model | en_US |
| Type | Thesis | en_US |