Plasma membrane Ca2+ ATPase1 (PMCA1) and interacting partners: presence in cargos of exosomes from female fluids and its uptake by murine sperm
Date
2016
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Publisher
University of Delaware
Abstract
Plasma membrane Ca2+ ATPase4 (PMCA4) is the major Ca 2+ efflux pump in murine sperm. Its deletion leads to infertility in male mice, due to flagellar abnormalities and motility defects. PMCA4 is not only crucial in males but is expressed in females where oviductal sperm, prior to fertilization, acquire additional amounts to efflux increased Ca 2+ resulting from hyperactivation and the acrosome reaction. However, deletion of Pmca4 in female mice, has no effect on female fertility, despite Ca2+ efflux requirement which also occurs for oviductal ciliary action that is necessary for oocyte transport. As PMCA1 is the other ubiquitous PMCA isoform, it was predicted that it might act as a compensatory mechanism for PMCA4 loss in the female reproductive tract. Here I show that PMCA1 is expressed in tissues, luminal fluids and extracellular vesicles (EV) of the female tract, with increased levels seen in the oviduct during proestrus/estrus when mating occurs. Western analysis showed that PMCA1 is significantly upregulated in Pmca4 KO oviductal EVs (oviductosomes, OVS) compensating for Pmca4’s absence.
Transmission Electron Microscopy (TEM) of pellets obtained from ultracentrifugation of vaginal luminal fluid revealed the presence of EV, dubbed “Vaginosomes”. These were confirmed as exosomes/microvesicles based on size and biomarkers such as CD9 and HSC70, and were also visualized in situ using TEM of tissue sections. Vaginosomes were shown to contain PMCA1 via Western analysis. Western also showed the presence of nNOS (neuronal Nitric Oxide Synthase) and CASK (Ca2+/CaM-dependent Serine Kinase) in OVS, interacting partners of PMCA4 in sperm. Co-immunoprecipitation studies showed that these partners also interact with PMCA1 in oviductosomes.
For the first time, PMCA1 was localized on sperm and shown to co-localize with PMCA4 on the head, midpiece and proximal principal piece. Further, PMCA1 in OVS was shown to be delivered to the sperm surface in vitro following co-incubation of sperm in OLF. Further, the level of acquisition of PMCA1 by sperm from OLF was dependent on capacitation states, and is consistent with the presence of PMCA1 on the inner acrosomal membrane, where PMCA4 is also located. Our findings demonstrate that PMCA1 is the compensatory mechanism that accounts for the fertility of Pmca4 KO females and reveals for the first time the existence of EV in the vaginal luminal fluid.