Plasma membrane calcium ATPase 4 co-ordinates calcium and nitric oxide signaling in regulating murine sperm motility and fertility
Date
2014
Authors
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Publisher
University of Delaware
Abstract
Reduced sperm motility or asthenozoospermia is the primary cause of male infertility. In mice, asthenozoospermia leading to infertility results from a deletion of Pmca4 , which encodes the highly conserved Plasma Membrane Calcium ATPase 4 (PMCA4), the major calcium efflux pump in murine sperm. However, to date the mechanism underlying the loss of PMCA4, which results in the motility defects, is unknown. In somatic cells PMCA4, in addition to its role in calcium efflux, is known to modulate nitric oxide (NO) signaling by negatively regulating the production of NO via nitric oxide synthases (NOS) in endothelial (eNOS) and neuronal (nNOS) cells. Both eNOS and nNOS are activated by calcium. In the absence of PMCA4 there are elevated levels of intracellular calcium and consequently increased levels of NO, leading to increased amounts of the highly reactive peroxynitrite, which reacts with polyunsaturated fatty acids. Since the sperm membrane is enriched in polyunsaturated fatty acids, it is highly susceptible to attack from peroxynitrite with resulting lipid peroxidation and ultimately a decrease in sperm motility. Therefore, in exploring the potential mechanism for the loss of fertility in Pmca4 null mice, the goal of this study was to determine if eNOS and nNOS are interacting partners in sperm, and if the degree of interaction is calcium-dependent. Using co-localization, co-immunoprecipitation, and Fluorescence Resonance Energy Transfer (FRET) assays, we investigated the association of PMCA4 and the NOSs in uncapacitated and capacitated (where levels of calcium are elevated) sperm. PMCA4 was shown to co-localize with eNOS and nNOS on the cell membrane of sperm. Co-immunoprecipitation assays detected an association between PMCA4 and eNOS and nNOS, with the interaction being stronger in capacitated versus uncapacitated sperm. FRET analysis showed an energy transfer efficiency of 35% in capacitated sperm versus 23% in uncapacitated sperm, revealing that PMCA4 and eNOS are less than 10 nm apart in both capacitated and uncapacitated sperm. The analysis showed an efficiency rate of 24% in capacitated and only 6% in uncapacitated sperm for PMCA4 and nNOS, revealing an intimate interaction between PMCA4 and nNOS only in capacitated sperm. These results identify eNOS and nNOS as interacting partners with PMCA4 in sperm. The data support the hypothesis that PMCA4 is closely associated with the NOSs under high concentrations of intracellular calcium. During its efflux activity, PMCA4's association with eNOS and nNOS would expose them to a local environment of decreased calcium, therefore negatively regulating them. Our findings show that co-ordination and integration of calcium and nitric oxide signaling are involved in maintaining normal sperm motility and fertility. Analysis of Pmca4 null sperm motility assay showed that L-NAME, a known inhibitor of NOSs, decreased motility in capacitated Pmca4 null sperm from 1.15% in the untreated sample to 0.07% when treated with low levels of L-NAME and 0.15% at high levels of L-NAME. In uncapacitated sperm there is a dose dependent decrease in motility when treated with L-NAME (14.8%, 5.31%, and 3.20%). In testes of Pmca4 nulls TUNEL assays revealed that the rate of apoptosis is significantly (p < 0.001) higher than that in wild-type, consistent with earlier findings of increased levels of nitric oxide synthase activity in these mutants. Importantly, while apoptotic activity did not differ significantly (p > 0.05) between WT and the nulls in the interstitial spaces, it did for each type of germ cells.