Molecular interactions involving plasma membrane calcium ATPase 4 (PMCA4) in human semen and their role in fertility
Date
2014
Authors
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Publisher
University of Delaware
Abstract
Male factor infertility affects 1 in 20 men (Aitken et al. 2014) and one of its primary causes is reduced sperm motility (asthenozoospermia, AZ). In mice, deletion of Pmca4, which encodes the highly conserved Plasma Membrane Calcium ATPase 4 (PMCA4), the major Ca2+ efflux pump, leads to male infertility due to sperm motility defects (Schuh et al. 2004; Okunade et al. 2004). To date, the interacting partners of PMCA4 in sperm and the mechanism by which its absence results in loss of motility and ultimately infertility have not been elucidated. Further, PMCA4's presence and localization in human sperm has, up until now, only been speculated. We hypothesize that, as in endothelial and neuronal cells, in sperm PMCA4 interacts with eNOS (endothelial Nitric Oxide Synthase) and nNOS (neuronal Nitric Oxide Synthase), both of which are rapidly activated by Ca2+. Here we show for the first time that in human sperm PMCA4 is present on the posterior head, the neck, midpiece, and the proximal principal piece where it co-localizes with eNOS and nNOS. Fluorescence Resonance Energy Transfer (FRET) reveals that PMCA4 and the NOSs are within 10 nm apart. FRET efficiency was significantly higher (P <0.01) in capacitated (high cytosolic Ca2+ ([Ca2+]c) then uncapacitated (low cytosolic [Ca2+]c) sperm, indicating dynamic interactions. Co-immunoprecipitation (Co-IP) assays tested and validated the close association of PMCA4 and eNOS in capacitated, but not uncapacitated, sperm. Further, PMCA4 and the NOSs are present in the seminal plasma, specifically on prostasomes where co-IP showed them to be in a complex, similar to that in sperm. Finally, flow cytometric analysis demonstrated that following co-incubation of sperm and seminal plasma, eNOS can be delivered in vitro to sperm via prostasomes. Our findings allow us to support a novel complex in which PMCA4 interacts simultaneously with eNOS and nNOS in capacitated sperm to regulate their activity, and thus prevent elevated levels of nitric oxide (NO), which are known to induce AZ. Our studies suggest that mutations of PMCA4 could lead to AZ and infertility in humans.