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Advance Publication first posted on 23 February 2010 as Manuscript REP-09-0495
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Abstract
While the function of the ubiquitous Na,K-ATPase 1 subunit has been well documented,
the role of the sperm specific 4 isoform of this ion transporter is less known. We have
explored the importance of 4 in rat sperm physiology, taking advantage of the high
sensitivity of this isoform for the inhibitor ouabain. Using concentrations that selectively
block 4 activity, we found ouabain to reduce, not only sperm total motility, but also
multiple parameters of sperm movement; including progressive motility, straight line,
curvilinear and average path velocities, lateral head displacement, beat cross frequency,
and linearity. According to a direct role of 4 on Na+ transport, ouabain inhibition of 4
increased [Na+]i in the male gamete. In addition, interference of 4 activity with ouabain
produced cell membrane depolarization, diminished pH and increased [Ca2+]i in
spermatozoa. Inhibition of 4 was sufficient to cause all these effects and additional
blockage of 1, the other Na,K-ATPase isoform expressed in sperm, with higher doses
of ouabain did not result in further changes in the cell parameters studied. These results
show that 4 is the Na,K-ATPase isoform primarily involved in controlling the
transmembrane Na+ gradient in sperm, and that 4 activity is necessary for maintaining
membrane potential, [Ca2+]i and [H+]i in the cells. The high dependence of sperm motility
on membrane excitability, [Ca2+]i and acid-base balance suggests that their regulation are
the mechanisms by which 4 maintains motility of the male gametes.
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Introduction
The Na,K-ATPase or Na pump is an enzyme of the plasma membrane of most cells
that transduces the energy from the hydrolysis of ATP to catalyze the exchange of
cytoplasmic Na+ for extracellular K+ (Kaplan 2002). In somatic cells, the ion gradients
generated by the Na,K-ATPase play a central role in maintaining cell volume and pH, in
keeping cell resting membrane potential, and in providing the chemical energy necessary
for the secondary transport of other ions, solutes, and water across the cell surface
(Hoffmann & Simonsen 1989, Gloor 1997, Feraille & Doucet 2001). The Na,K-ATPase
is an oligomer composed of two major polypeptides, the and subunits (Morth et al.
2007). The polypeptide constitutes the catalytic subunit of the Na,K-ATPase, directly
participating in the ion translocation and hydrolytic activity of the enzyme. It contains the
binding sites for Na+, K+, ATP and for the cardenolide, ouabain, which is a potent
inhibitor of the Na,K-ATPase (Kaplan 2002).
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Results
Inhibition of Na,K-ATPase 4 isoform inhibits multiple parameters of rat sperm
motility
We and others have previously shown that activity of the Na,K-ATPase 4 isoform is
important for motility of rat and human sperm (Woo et al. 2000, Sanchez et al. 2006).
However, previous experiments have only focused on general movement of the
spermatozoa and observations were based on bare visual determinations (Woo et al.
2000, Woo et al. 2002). To further study the relevance of 4, on not only total, but also
other parameters of sperm movement, we determined the effects of inhibition of this
isoform on sperm motility using computer assisted sperm analysis (CASA). We also
assessed the relative contribution of 4 to sperm motility and compared it with that of the
1 isoform. Extensive studies of the kinetic properties of the Na,K-ATPase shows that
each of the Na,K-ATPase isoforms expressed in sperm, 1 and 4, exhibit a marked
difference in affinity for the inhibitor ouabain (Blanco 2005a). In the rat, the ouabain
affinity of 4 is approximately 10,000 fold higher than that of 1 (Wagoner et al. 2005).
In this manner, differential sensitivity to ouabain has been used as a tool to specifically
inhibit 4 activity in a dose-selective manner, to study the particular function of this
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Na,K-ATPase, and in particular that of the 4 isoform, in sperm membrane potential. For
this, we treated rat spermatozoa in the absence and presence of 10-6 M and 10-3 M
ouabain and determined membrane potential of the cells, using the fluorescent indicator,
[DiSC3(5)] as described (Hernandez-Gonzalez et al. 2006). Figure 4A presents
representative traces for the fluorescence measured in untreated and ouabain treated cells.
As shown, the initial portion of the recordings represents the fluorescent emission
corresponding to the membrane potential of the cells, in the absence or presence of the
indicated ouabain concentrations. Each determination was followed by a calibration
curve, obtained in the same sample after the addition of the ionophore valinomycin and
increasing amounts of KCl. As reflected in the traces, valinomycin produced K+ leakage
from the cells and membrane hyperpolarization, while the subsequent addition of KCl,
caused a stepwise depolarization of the cells. This calibration method allowed calculation
of the cell membrane potential of the samples in the absence and presence of ouabain
(Hernandez-Gonzalez et al. 2006). Figure 4B, illustrates the values of membrane
potential for each experimental condition, averaged from different experiments. As
shown, 10-6 M ouabain produced depolarization of the cells, with a decrease in sperm
membrane potential of approximately 20 mV. A similar reduction in membrane potential
was obtained with 10-3 M ouabain. This suggests that the Na,K-ATPase importantly
contributes to maintaining membrane potential in rat spermatozoa, and that 4 is the
isoform primarily involved in this process.
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Discussion
The maintenance of differences in ion concentration between the environment and the
sperm cytoplasm has long been recognized as essential for normal motility of the male
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gamete (Darszon et al. 1999, Darszon et al. 2006). Of particular importance is the uneven
transmembrane distribution of Na+ and K+ generated by the Na,K-ATPase (Darszon et al.
1999). However, the specific contribution of different Na,K-ATPase isoforms in sperm
physiology is not precisely known. In this work, we have determined the role of the
Na,K-ATPase 4 isoform in various aspects of the motility of rat sperm, and have
explored the mechanisms by which this transporter functions in the male gamete.
Because animal models in which the 4 isoform has been deleted are not yet available,
our study has taken advantage of the unique high affinity of 4 to ouabain to specifically
inhibit this isoform (Woo et al. 2000, Wagoner et al. 2005). This method provides a
suitable pharmacological approach and is currently the best tool available to study the
function of the 4 isoform in the native spermatozoa. We show that inhibition of 4, with
relatively low amounts of ouabain, significantly affects multiple aspects of sperm
motility. The use of CASA has allowed us to establish in more detail the role of 4 on
different components of rat sperm movement, beyond the resolution given by previous
simple visual determinations. Thus, we found that activity of 4 is necessary for sperm
total and progressive motility, straight line, curvilinear and average path velocities, lateral
head displacement, beat cross frequency and linearity. The alterations in the parameters
of sperm motility we have studied have been used as important predictors of male fertility
in patients and for in vitro fertilization purposes (Moore & Akhondi 1996, Larsen et al.
2000, Shibahara et al. 2004). Based on these reports, the extent of the reduction in the
various parameters of sperm movement that we obtained after treatment with 10-6 M
ouabain is expected to interfere with normal male fertility (Moore & Akhondi 1996,
Larsen et al. 2000, Shibahara et al. 2004). Therefore, our results indicate that activity of
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the Na,K-ATPase 4 polypeptide widely influences sperm movement, and suggest that
this isoform is important to support motility and fertility of the male gamete. The broad
effect of inhibition of 4 activity on the various components of sperm motility suggests
that this isoform maintains sperm movement by acting through multiple different vital
parameters of sperm physiology.
We have found that the 4 isoform is primarily involved in maintaining the low
[Na+]i levels in rat spermatozoa. This agrees with our previous findings showing that 4
is responsible for most of the Na,K-ATPase catalytic activity of the cells (Wagoner et al.
2005). The importance of the 4 isoform as the main transport system for Na+ and K+ in
spermatozoa is also evident from our observation that 10-6 M ouabain caused maximal
depolarization of the sperm plasma membrane. The Na,K-ATPase itself is not the only
determinant of membrane potential, and the simultaneous leak of K+ via K+ channels is
the other necessary component for the maintenance of membrane electrical polarity in the
cell (Green 2004). Therefore, it is obvious that for the 4 isoform to influence plasma
membrane excitability of the male gamete, its action must be linked to that of K+
channels. Several K+ channels exist in spermatozoa (Darszon et al. 1999, Darszon et al.
2006) and future studies are necessary to determine if there is a specific functional
relationship between 4 and one of the particular K+ channel expressed in sperm. An
adequate membrane potential is essential for sperm motility and cell membrane
depolarization has been shown to be associated to infertility in asthenozoospermic
patients (Calzada & Tellez 1997). Our results therefore suggest that one of the
mechanisms by which the 4 isoform influences sperm motility is through its key role in
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maintaining the uneven transmembrane distribution of Na+ and K+, and the electrical
potential of the sperm plasma membrane.
Besides its direct role in Na+ and K+ transport, we also found that the 4 isoform
secondarily controls proton levels in spermatozoa, and ouabain inhibition of 4 caused a
pH decline of the sperm cytoplasm. This occurred after exposure of the cells to different
physiological pH values, suggesting that 4 is able to prevent sperm acidification over a
range of proton concentrations. The 4 isoform is not capable of directly transporting H+
(Blanco et al. 1999). Instead, and as has been shown for somatic cells (Bers et al. 2003,
Despa et al. 2004), the Na,K-ATPase can functionally couple to the Na+/H+ exchanger
(NHE) to regulate pH in sperm. In this manner, the 4 isoform provides spermatozoa
with an inwardly directed Na+ gradient that provides the electrochemical energy to drive
the secondary movement of protons out of the cell. This functional association between
the 4 isoform and NHE is supported by several other lines of evidence. First, tissue
unspecific forms (NHE1 and NHE5), and sperm specific types (sNHE and mtsNHE) of
the Na+/H+ exchanger is expressed in spermatozoa (Liu et al. , Darszon et al. 2001, Wang
et al. 2003, Wang et al. 2007). Second, these exchangers are expressed in the mid- and
principal segments of the sperm flagellum (Woo et al. 2002), thus, co-localizing with the
Na,K-ATPase 4 isoform (Woo et al. 2000, Wagoner et al. 2005, Sanchez et al. 2006),
which will favor their cooperative action. Finally, the ionophores nigericin and monensin,
which allow leakage of H+ out of the cells, are able to reestablish the motility that is lost
in spermatozoa after ouabain inhibition of the Na,K-ATPase (Woo et al. 2002). Our
results represent the first direct demonstration that activity of the Na,K-ATPase 4
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cells of vessels and in cardiac cells, and it is responsible for the cardiotonic effects of
digitalis in the heart (Blaustein et al. 1998, Blaustein et al. 2004). In those cells, the Na+dependent regulation of [Ca2+]i is mediated via a specific isoform of the Na,K-ATPase,
the 2 polypeptide (Golovina et al. 2003, Zhang et al. 2005). Both the 2 and NCX are in
close proximity in a common subcellular domain, which favors the functional interaction
between these ion transporters (Blaustein et al. 1998). The possibility of a mechanism for
Ca2+ regulation based on the coupled activity of the 4 isoform and NCX is supported by
the presence of a NCX transport system in spermatozoa (Wang et al. 2003). Interestingly,
NCX has been shown to be expressed at the middle piece of the sperm flagellum
(Krasznai et al. 2006, Bedu-Addo et al. 2008), where the Na,K-ATPase 4 isoform is
most abundant (Woo et al. 2000, Wagoner et al. 2005, Sanchez et al. 2006). Although
additional experiments will be necessary to further define the combined function of the
4 isoform and NCX, our results suggest that, similar to the 2 isoform, the 4
polypeptide is another Na,K-ATPase isoform involved in Ca2+ regulation, in this case,
specific to spermatozoa. Thus, 4 can be added to the list of ion transporters that control
[Ca2+]i in sperm (Bedu-Addo et al. 2008). Maintenance of sperm [Ca2+]i within a relative
limited range is critical to the motility of the male gamete (Bedu-Addo et al. 2008).
Studies performed in demembranated models, show that changes in free calcium
influence the curvature and symmetry of the sperm flagellum (Lindemann et al. 1992). In
this manner, the ability of the 4 isoform to control [Ca2+]i may represent another
mechanism by which this Na,K-ATPase polypeptide sustains sperm motility.
Previous work has studied the effects of ouabain in sperm of larger mammals.
Specifically, ouabain has been reported to elicit a series of responses in bull spermatozoa.
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In this species, ouabain does not modify total motility or amplitude of lateral head
displacement, but decreases progressive motility, curvilinear and average path velocities
(Thundathil et al. 2006). On the other hand, McGrady, found ouabain to cause a general
decrease in bull sperm flagellar wave (McGrady 1979). Our results on rat show that the
inhibitory effect of ouabain extends to a wide range of sperm motility parameters. These
disparities in results may depend on differences in the species studied, on the source of
sperm (ejaculated vs epididymal), or on the experimental conditions used.
Besides its effects on sperm motility, ouabain has also been shown to produce
capacitation in bull sperm through mechanisms that are independent of increases in
intracellular Ca2+ (Thundathil et al. 2006), but that involve activation of kinases and
protein phosphorylation events in the cells , 2009(Newton et al.), 2009). Our experiments
were designed to investigate rapid effects of ouabain and did not include long term
consequences on sperm capacitation. In contrast with the results in bull, we find that
ouabain inhibition of Na,K-ATPase in rat sperm induces a raise in intracellular Ca2+.
Increases in Ca2+ have been reported as one of the biochemical changes that accompany
rodent sperm capacitation (Visconti et al. 1998). Therefore, it is conceivable that the 4
isoform may play a potential role in the regulation of sperm capacitation in response to
ouabain, also in rats. In sum, it is clear that while some of the responses to ouabain are
shared between bull and rat, others are not. Further studies are required to ascertain these
species specific differences in the response of sperm to ouabain.
Our data indicate that the role of the Na,K-ATPase in sperm motility, membrane
potential, intracellular pH and [Ca2+]i is isoform specific, and predominantly depends on
the activity of the 4, rather than the 1 polypeptide. We have previously shown that 4
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exhibits unique enzymatic properties and a particular response to the transported ions
(Blanco et al. 1999). In particular, when compared to the 1 isoform, 4 has a higher
apparent affinity for intracellular Na+, and small amounts of this cation selectively
stimulate 4 function. Therefore, the 4 isoform appears to be better suited to maintain
lower [Na+]i than 1. This predominant role of 4 in maintaining a steep Na+ gradient
across the sperm plasma membrane, places the isoform as the primary Na,K-ATPase
involved in sperm processes that depend on the Na+ gradient. Our results also suggest
that the 1 isoform may be playing a redundant role. Previous work showed that 1
contributes to approximately one third of the total Na,K-ATPase activity of rat
spermatozoa. In addition, 1 presents a broader distribution along the sperm flagellum,
compared to 4. Therefore, although activity of 1 is not significantly involved in the
sperm parameters studied in the present work, we cannot discard that it is performing a
role in the male gamete. In somatic cells, the widely expressed 1 isoform appears to
perform a housekeeping function, regulating the basal Na+ and K+ homeostasis in the
cells, while the other isoforms carry out cell specific tasks. In this manner, it is also
possible that in spermatozoa, the 1 isoform has some housekeeping role in
maintaining the basal [Na+]i gradient, while 4 is working to further control [Na+]i, and
secondarily regulate other ion levels, to sustain the high demands imposed by sperm
motility.
Based on our observations, we propose the following model for the mechanism of
action of the 4 isoform in the control of sperm motility. The ion transport activity of 4
is necessary to control the Na+ and K+ gradients that are important for maintenance of the
sperm membrane potential in concert with K+ channels. In addition, 4 generates the
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inward Na+ flux that is secondarily used by the NHE and NCX transport systems to drive
the movement of H+ and Ca2+ out of the cells respectively. The regulation of Na+, along
with the control of H+ and Ca2+ is crucial to sperm motility; therefore, the activity of 4 is
of high relevance to the physiology of the male gamete.
In conclusion, our work provides new evidence for the mechanisms by which the
Na,K-ATPase 4 isoform influences sperm motility. Future studies are underway to
further elucidate the molecular basis of 4 role on the physiology and fertility of the male
gamete.
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ratio between Straight Line Velocity and Curvilinear Velocity during the measurement
period. Amplitude of Lateral Head Displacement was obtained as the maximum distance
of the sperm head from the average trajectory of the sperm during the analysis period.
An average of 50 cells/field was captured, at a rate of 30 frames per field, and a total of
10 fields in each sample were analyzed. Therefore approximately 500 cells were analyzed
per condition in each experiment. Each field was taken randomly, scanning the slide
following a pre-established path to ensure consistency in the method. Experiments were
repeated 10 times; thus, allowing the observation of statistically significant numbers of
cells in the assays. The obtained data were expressed as percent of the motility of the
untreated control at the initial time (5 min).
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incubated for another 2 min with CCCP, at a final concentration of 1 M (HernandezGonzalez et al. 2006, Hernandez-Gonzalez et al. 2007). After this time period, 2.5 ml of
the suspension was transferred into a cuvette arranged with gentle stirring and maintained
at 37 C. Then, fluorescence at an excitation/emission wavelength of 620/670 nm was
recorded. After determining fluorescence of the different experimental conditions,
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calibration of the fluorescence changes into mV was performed in the same sample, by
adjusting the membrane potential of the cells as previously described (Espinosa &
Darszon 1995). Briefly, the K+ ionophore, valinomycin, was added at a final
concentration of 1M, to allow K+ to equilibrate across the plasma membrane. Then, the
K+ concentration was increased stepwise, to obtain final concentrations in the media of
9.9, 13.9, 21.9 and 37.9 mM KCl. After distribution of K+ in the cells, membrane
potential follows the Nernst equilibrium and can be calculated. The membrane potential
for the different KCl amounts added corresponded to -80, -67, -58, -45, and -30 mV,
respectively. The experimental sperm membrane potential in each case was linearly
interpolated using these data as plasma membrane potential versus arbitrary units of
fluorescence as previously described (Plasek & Hrouda 1991, Espinosa & Darszon 1995,
Zeng et al. 1995, Hernandez-Gonzalez et al. 2006).
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above media at preset pHs (6.0, 7.0, 7.2, 7.4, 7.6 and 8.0). These samples were further
treated with 2 g/ml of the ionophore nigericin for 20 min, to permeabilize the cells and
clamp the intracellular pH to the preset values in the media (Chow S. 2007). After this
incubation time, sequential fluorescence of the experimental samples (untreated and
ouabain treated cells), followed by samples for pH calibration, were measured at an
excitation of 488 nm and an emission ratio of 640/580 nm. The pH dependent spectral
shift of SNARF-1 allowed determination of the cell pH based on the calibration curves
obtained in media of preset pH as described (Chow S. 2007).
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Statistical Analysis
Experiments were repeated at least three times using a minimum of triplicate
determinations. Statistical significance of differences between controls and ouabain
treated samples was determined by the students t-test, using Sigma Plot software (Jandel
Scientific, San Rafael, CA). Statistical significance was defined as P< 0.05.
Declaration of interest
There is no conflict of interest that could be perceived as prejudicing the impartiality of
the research reported.
Funding
This work was supported by National Institutes of Health grants HD043044, HD055763
and HD38082.
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Figure Legends
Figure 1. The Na,K-ATPase 4 isoform is important for rat sperm motility. Cells were
isolated from the cauda of rat epididymides and were treated without and with 10-6 M and
10-3 M ouabain in modified Tyrodes medium. (A) total, and (B) progressive motility.
Sperm movement was determined using CASA for the indicated times. Cells from 10
different fields per time point were analyzed. Symbols represent untreated cells (), or
cells treated with 10-6 M () and 10-3 M ( ) ouabain. Each value is the mean and bars
represent the standard errors of the mean of ten experiments. Values were expressed as
percent of motility of the untreated control at the earliest time point, taken as 100%.
Maximal values for total and progressive motility were 76
2 % and 46
7 %
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represent untreated cells (open bars), or cells treated with 10-6 M ouabain (stripped bars)
and 10-3 M ouabain (black bars). Values significantly different from the control are
indicated with an asterisk, with P< 0.001. No statistical differences were found between
samples treated with 10-6 M and 10-3 M ouabain.
Figure 3. The Na,K-ATPase 4 isoform is important in maintaining the low [Na+]i in rat
sperm. Rat caudal epididymal spermatozoa were collected and incubated in modified
Tyrodes medium without and with 10-6 M and 10-3 M ouabain for 30 min at 37 C. The
fluorescent indicator Sodium Green Tetraacetate was used to follow changes in [Na+]i.
(A) Representative trace recorded at a excitation/emission wavelength of 507/532. (B)
Compiled data from three different experiments performed in triplicate. Values were
expressed relative to the untreated controls. Bars represent the mean + standard errors of
the mean. Values significantly different from the control are indicated with an asterisk,
with P= 0.008 for 10-6 M ouabain vs untreated control and P= 0.01 for 10-3 M ouabain vs
untreated control. No statistical differences were found between samples treated with 10-6
M and 10-3 M ouabain (P= 0.67).
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620/670. Calibration was performed with the addition of valinomycin (indicated in the
graph with an arrow (Val), followed by the sequential addition of increasing amounts of
KCl. The initial medium contained 5.9 mM KCl and the subsequent KCL additions
(indicated with the letters a to d), resulted in the following final KCL concentrations in
mM: (a) 9.9, (b) 13.9, (c) 21.9 and (d) 37.9, which corresponded to the indicated
membrane potentials (Em) in mV respectively. (B) Compiled data from three different
experiments each performed in triplicate. Bars represent the mean + standard errors of the
mean. Values significantly different from the untreated control are indicated with an
asterisk, with P= 0.03 for 10-6 M ouabain vs untreated control and P= 0.04 for 10-3 M
ouabain vs untreated control. No statistical differences were found between samples
containing 10-6 M and 10-3 M ouabain (P= 0.9).
are indicated with an asterisk, with P< 0.001 for both 10-6 M and 10-3 M ouabain vs
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untreated control at all extracellular pHs. No statistical differences were found between
samples containing 10-6 M and 10-3 M ouabain for all pH samples (P > 0.4).
30
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