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Advance Publication first posted on 23 February 2010 as Manuscript REP-09-0495

Activity of the Na,K-ATPase alpha4 isoform is important for membrane potential,


intracellular Ca2+ and pH, to maintain motility in rat spermatozoa.

Tamara Jimenez1, Gladis Snchez1, Eva Wertheimer2 and Gustavo Blanco1.


1. Department of Molecular and Integrative Physiology, University of Kansas Medical
Center, Kansas City, KS 66160, USA. 2. Department of Veterinary and Animal Sciences,
University of Massachusetts, Amherst, MA 01003, USA.

Short title: Na,K-ATPase 4 isoform mechanisms of action.

For correspondence: Gustavo Blanco,


Department of Molecular and Integrative Physiology,
3901 Rainbow Boulevard, Kansas City, Kansas 66160.
Phone: (913) 588-7405, Fax: (913) 588-7430,
email: gblanco@kumc.edu

Copyright 2010 by the Society for Reproduction and Fertility.

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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).

Interestingly, four structural variants, or isoforms of the Na,K-ATPase subunit,


named 1, 2, 3 and 4 are expressed in mammalian tissues. Each of these isoforms is
characterized by a cell-specific pattern of expression, and by particular enzymatic
properties (Blanco 2005a, Blanco 2005b). While 1 is found in nearly every tissue and it
is believed to maintain the basal ion gradients in the cell, the other polypeptides are
more restricted in their expression and appear to play tissue specific roles (Blanco &
Mercer 1998, Mobasheri et al. 2000). In particular, the 4 isoform is found in the testis,
where it is specifically expressed in the male germ cells after meiosis (Shamraj & Lingrel
1994, Woo et al. 1999, Blanco et al. 2000, Woo et al. 2000, Hlivko et al. 2006). The 4

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isoform is highly prevalent in spermatozoa and its activity corresponds to approximately


two thirds of the total Na+ and K+ active transport of the cells (Wagoner et al. 2005). The
only other Na,K-ATPase polypeptide present in sperm is the widely expressed 1
isoform (Wagoner et al. 2005). We have previously shown that the activity of 4 is
different from that of 1 and the other Na,K-ATPase isoforms. Thus, 4 exhibits
unique kinetic properties for its physiologic ligands, including a high affinity for Na+, a
low affinity for K+ and an intermediate affinity for ATP (Blanco et al. 1999, Sanchez et
al. 2006). Moreover, 4 has a distinctive very high sensitivity to ouabain, a characteristic
that most prominently distinguishes it from the 1 isoform (Blanco et al. 1999, Sanchez
et al. 2006). The presence of a Na,K-ATPase catalytic subunit with particular attributes,
such as those of 4, suggests that this polypeptide plays a specific role in the physiology
of sperm. In support of this, inhibition of 4 activity has been shown to diminish total
motility of spermatozoa (Woo et al. 2000, Sanchez et al. 2006).

Despite the progress made in understanding various characteristics of the expression


and activity of 4, its role in different aspects of sperm motility, and the mechanisms by
which this isoform supports sperm physiology remain unknown. Deciphering the
molecular basis of 4 action is an important step in comprehending how this isoform
contributes to male fertility. In the present work, we have studied the role of 4 in
different parameters of sperm movement and in a series of processes that are essential to
sperm motility. For this, we have taken advantage of the distinctive high sensitivity of 4
for ouabain, to specifically inhibit this isoform, and distinguish its activity from that of
the 1 isoform in rat spermatozoa. Our results show that 4, but not the 1 isoform, is

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important in maintaining sperm intracellular Na+ concentration ([Na+]i), membrane


potential, intracellular pH and cytoplasmic Ca2+ concentration ([Ca2+]i). The control of
these cell parameters is essential in sustaining sperm motility (Quill et al. 2006, Darszon
et al. 2007), suggesting that their regulation by 4 represent mechanisms through which
this Na,K-ATPase isoform supports motility of the male gamete.

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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isoform in spermatozoa (Blanco 2005a). Thus, as we have previously reported, ouabain


in a concentration of 10-6 M is sufficient to completely inhibit 4 activity, without having
significant effects on the 1 polypeptide. A ouabain concentration of 10-3 M instead, will
cause inactivation of both the 1 and 4 isoforms (Wagoner et al. 2005). Based on this,
we treated rat spermatozoa in the absence and presence of 10-6 M and 10-3 M ouabain for
different times and measured the motility of the cells using CASA. As shown in Figure
1A, in medium without ouabain, rat sperm total motility diminished only slightly during
the entire incubation period of 2h. In contrast, ouabain inhibition of 4 was sufficient to
impair the percent of total motile spermatozoa in a time dependent manner. Further
inhibition of 1 with 10-3 M ouabain did not result in additional reduction of sperm
motility. Ouabain at 10-6 M, similar to 10-3 M, also diminished rat sperm progressive
motility (Figure 1B). In addition, ouabain selective blockage of 4 activity affected other
parameters of sperm motility, including straight line, curvilinear and average path
velocities, amplitude of lateral head displacement, beat cross frequency and linearity
(Figure 2A-F). Altogether, these results suggest that activity of the Na,K-ATPase is
important for sustaining multiple aspects of sperm flagellar movement, and that the 4
isoform, and not 1 is involved in this process.

Inhibition of 4 activity affects intracellular Na+ in rat spermatozoa


The low cytoplasmic concentration of Na+ that typically exists in most cells is a direct
consequence of the active ion transport function of the Na,K-ATPase (Kaplan 2002). This
Na+ gradient in turn is essential to many general and cell-specific processes (Feraille &
Doucet 2001, Kaplan 2002). We have previously demonstrated that the 4 isoform

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functions as an active Na,K-ATPase, catalyzing the ouabain-dependent hydrolysis of


ATP in rat sperm (Wagoner et al. 2005). However, the extent to which the activity of 4
as an ion transporter contributes to maintain sperm [Na+]i is unknown. To specifically
determine the role of 4 in [Na+]i maintenance, and to correlate its transport activity with
that of the 1 isoform, we treated rat spermatozoa without and with 10-6 M and 10-3 M
ouabain, and we measured Na+ in the cells using the fluorescent indicator, Sodium Green
Tetraacetate. Figure 3A shows a representative trace corresponding to the fluorescence
recorded in sperm without or with the indicated concentrations of ouabain. Figure 3B
summarizes the compiled values for the intensity of fluorescence in the cells from
different samples, expressed relative to the untreated controls. As expected, blockage of
the Na,K-ATPase with ouabain, even after a relatively short time of incubation with the
inhibitor (30 min), caused [Na+]i to increase in sperm by approximately 20%.
Interestingly, the major changes in [Na+]i were observed after inhibition of the 4 isoform
with 10-6 M ouabain, and no further significant increase in [Na+]i was detected after
inhibition of the Na,K-ATPase 1 polypeptide with 10-3 M ouabain. These results suggest
that the activity of the 4 isoform exerts a more prominent effect in maintaining the
[Na+]i in spermatozoa (Wagoner et al. 2005).

Function of 4 is important for sperm membrane potential


The transmembrane distribution of electrical charges, resulting from the uneven Na+
and K+ transport activity of the Na,K-ATPase contributes to maintain the resting
membrane potential that provides cells with the excitability required for their movement,
contraction, and transmission of impulses (Gloor 1997). We investigated the role of the

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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.

The 4 isoform contributes to pH balance in spermatozoa

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Regulation of intracellular pH is essential to the function of sperm (Darszon et al.


1999). An important mechanism that controls proton concentration in somatic cells
operates utilizing the transmembrane Na+ gradient generated by the Na,K-ATPase. In this
manner, the Na,K-ATPase indirectly controls pH in the cell (Bers et al. 2003, Despa et al.
2004). We explored whether a similar role for the Na,K-ATPase operates in spermatozoa.
Moreover, we determined whether 4, or the ubiquitous 1 isoform is involved in the
secondary regulation of H+ content in the male gamete. For this, rat spermatozoa in
modified Tyrodes medium were treated without and with 10-6 M and 10-3 M ouabain,
and changes in intracellular pH were followed using the fluorescent indicator, SNARF-1.
In order to study ouabain effects at a range of physiological pH values, experiments were
performed incubating the cells in media with pHs of 7.0, 7.2, 7.4 and 7.6. Under these
conditions, sperm viability, measured using the fluorescent marker, Dye Cycle Violet,
was 99 + 1%). Figure 5A shows a representative trace carried out at extracellular pH 7.4.
The initial segments in this trace correspond to samples treated without and with the
indicated ouabain concentrations, followed by recordings of samples clamped at preset
intracellular pH values, which served for calibration (Chow S. 2007). Figure 5B, depicts
the compiled data for controls and ouabain treated samples from a series of experiments
performed at the indicated extracellular pHs. As shown, the intracellular pH of sperm
raised after incubation in media with increasing pHs. Importantly, under the various pH
tested, ouabain caused acidification of the cells. In addition, in all cases, the primary
decrease in pH occurred after applying 10-6 M ouabain to the cells, with no significant
changes in proton levels after further treatment with 10-3 M ouabain. This suggests that
the Na,K-ATPase is a mechanism involved in acid-base balance in spermatozoa, and that

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the activity of the 4 isoform plays a fundamental role in maintaining proton


concentration low in the cells.

Activity of 4 plays a role in sperm intracellular calcium maintenance


The Na+ gradient generated by the Na,K-ATPase is also important for the regulation
of Ca2+ levels in some cell types, such as muscle and cardiac cells. To explore if in
spermatozoa [Ca2+]i is regulated through the Na,K-ATPase, and to ascertain the relative
involvement of the 1 and 4 isoforms of the transporter in this event, we determined
Ca2+ concentration in rat sperm at the single cell level, using the fluorescent dye, Calcium
Green. This was performed after treatment of the cells in the absence and presence of 10-6
M and 10-3 M ouabain in Tyrodes modified medium. As shown in Figure 6A, ouabain
treatment resulted in significant increases in the Calcium Green signal in the cells,
indicating a raise in [Ca2+]i in spermatozoa. In addition, ouabain inhibition of 4 was
sufficient to cause the maximal ouabain-dependent augment of [Ca2+]i in the male
gamete. The use of 10-3 M ouabain, did not produce further changes in [Ca2+]i. Figure 6B
shows the quantification of the averaged data for [Ca2+]i for the control and ouabain
treated spermatozoa from various experiments. As shown, 10-6 and 10-3 M ouabain caused
an increase of approximately 60% of sperm [Ca2+]i. These data suggest that the Na, KATPase, and more specifically its 4 isoform, controls sperm [Ca2+]i.

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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isoform, is linked to the control of H+ levels in spermatozoa. This mechanism will be


important in preventing the raise of protons that takes place in the sperm cytoplasm as a
consequence of active movement of the cells. Further studies will determine which of the
several NHE systems are functionally coupled to the Na,K-ATPase 4 isoform. Changes
in pH have been shown to influence sperm motility, and in detergent extracted
preparations of rat sperm, variations in the proton concentration have been shown to
modulate sperm flagellar bending pattern and its response to cyclic adenosine 3', 5'monophosphate (cAMP) and Ca2+. (Lindemann et al. 1991). Therefore, regulation of the
proton concentration in the cells may represent one of the mechanism by which the Na,KATPase 4 isoform influences sperm motility.
Our results also show that the activity of the 4 isoform is functionally coupled to the
regulation of sperm [Ca2+]i. Our experiments were performed in the absence of
extracellular Ca2+. Therefore, the increase in [Ca2+]i secondary to ouabain inhibition of 4
is not due to Ca2+ internalization from the media through plasma membrane uptake
mechanisms, but rather depends on a decrease in the clearance of the cation from the cell
cytoplasm. The only transport mechanism that has been described for the elimination of
Ca2+ from the cytosol, and that is linked to the function of the Na,K-ATPase is the
Na+/Ca2+ exchanger, NCX (Lynch et al. 2008). In excitable cells, the function of the
NCX heavily relies on the inward flux of Na+ maintained by the Na,K-ATPase, to release
Ca2+ out of the cytoplasm (Zhang et al. 2005). The effect we observed resembles the
findings performed in excitable cells, and supports a functional coupling between the
Na,K-ATPase and the NCX to maintain sperm intracellular Ca2+ low (Bers et al. 2006).
This mechanism is particularly important in the regulation of [Ca2+]i in smooth muscle

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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.

Materials and Methods


Sperm Preparation
All experimental protocols used in this work were approved by the University of
Kansas Medical Center Institutional Animal Care and Use Committee. Spermatozoa were
obtained from the cauda of adult rat epididymides. For this, the epididymis was dissected,
its caudal portion was isolated and was cut at various points with a razor blade as
described (Hernandez-Gonzalez et al. 2006). The tissue was placed in modified Tyrodes
medium (Ecroyd et al. 2004), containing: 95mM NaCl, 4.7mM KCl, 1.2mM KH2PO4,
1.2mM MgSO4, 5.5mM Glucose, 0.27mM Pyruvic Acid, 0.25mM Lactic Acid, 40mM
Hepes and 20 mM Tris (pH 7.4), and was incubated for 10 min at 37 C. After this time,
spermatozoa were collected from the supernatant, centrifuged at 300 X g for 30 sec and
resuspended in modified Tyrodes medium. Cell were counted with the help of a
hemocytometer, and used for the different assays. All experiments were conducted in
modified Tyrodes medium.

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Sperm motility assays


Approximately 3 x 106 cells were resuspended in 300 l of the modified Tyrodes
medium described above. Because extracellular Ca2+ is fundamental for sperm motility,
1.7 mM CaCl2 was included in the medium. Cells in this solution were incubated without
and with 10-6 M or 10-3 M ouabain to selectively inhibit 4, or both the 1 and 4
isoforms (Wagoner et al. 2005). Incubation was performed at 37 C for different times
and for a total of 2h. Then, cells were labeled with 2 l of a 75 M stock of SITO 21, a
green fluorescent nucleic acid stain, which helps tracking cell movement. After 2 min
incubation with the dye, 7l aliquots from each sample were taken and placed into a glass
cell chamber 20 m in depth (Leja Products B.V., The Netherlands). The chambers were
viewed on an Olympus BX51microscope through a 20X phase objective and maintained
at 37 C on a heated platform. Viewing areas on each chamber were captured using a
CCD camera. Samples were analyzed by computer assisted sperm analysis (CASA),
using the Minitube SpermVision Digital Semen Evaluation system (version 3.5,
Penetrating Innovations, Verona, WI). Different sperm motility parameters were
analyzed, including total motility, progressive motility, curvilinear, average path and
straight line velocities, amplitude of lateral head displacement, beat cross frequency and
linearity. The analytical setup parameters used considered a cell identification, or cell size
area between 15 and 900 m2, a cutoff velocity corresponding to a minimum straight line
velocity of 5m/sec, a progressive motility threshold corresponding to a straight line
velocity of more than 20 m/sec, and a cutoff for low and medium average path
velocities of 10 m/sec and 120 m/sec respectively. Linearity was calculated from the

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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).

Intracellular Na+ measurements


Sperm intracellular Na+ determination was performed using a fluorimetric method as
described before (Hernandez-Gonzalez et al. 2006). Briefly, spermatozoa (20 X 106
cells/ml) were incubated in modified Tyrodes medium for 15 min at 37 C, in the
absence or presence of 10-6 M ouabain that inhibits only the 4 isoform, and 10-3 M
ouabain that blocks both the 1 and 4 isoforms (Wagoner et al. 2005). The cellpermeant nonfluorescent precursor of Sodium Green Tetraacetate at a final concentration
of 2.5 M was added and samples were further incubated for another 30 min at room
temperature. Cells were then washed twice by centrifugation at 300 X g for 5 min and
resuspended in 0.4 ml of fresh modified Tyrodes medium. Aliquots (50l each) were
placed in cuvettes with a total volume of 2.5 ml modified Tyrodes medium at room
temperature, and fluorescence of each sample was measured under continuous stirring at

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an excitation/emission wavelength of 507/532 nm. Changes in fluorescence were


expressed as percentage of the non treated controls.

Membrane potential assays in sperm


Membrane potential was determined using the fluorescent indicator [DiSC3(5)],
which has been successfully used to measure membrane potential in mammalian
spermatozoa (Plasek & Hrouda 1991, Espinosa & Darszon 1995, Zeng et al. 1995).
[DiSC3(5)] is charged and distributes across cellular membranes in response to
electrochemical gradients. As a result of binding to intracellular proteins, the dye exhibits
a slight shift in spectrum, providing reproducible estimates of plasma membrane
potential. Because [DiSC3(5)] also binds to mitochondria in their energized state, and this
can interfere with changes in the fluorescence signal, the mitochondrial membrane
potential was dissipated using m-chlorophenyldrazone (CCCP) as described (Demarco et
al. 2003). Following this method, sperm samples containing 2 X 106 cells/ml were treated
in modified Tyrodes medium, for 20 min at 37 C, with and without ouabain in
concentrations that inhibits only 4 (10-6 M), or both the 1 and 4 isoforms (10-3 M)
(Wagoner et al. 2005). Then, [DiSC3(5)], at a final concentration of 1 M, was added and
the samples were incubated at 37 C for an additional 8 min.

Sperm was further

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).

Intracellular pH measurements in sperm


Sperm intracellular pH was measured with the pH sensitive dye SNARF-1-AM as
described (Chow S. 2007). Spermatozoa (20 X 106 cells/ml) were incubated in modified
Tyrodes medium adjusted at different pHs (7.0, 7.2, 7.4 and 7.6) for 15 min at 37 C, in
the absence or presence of 10-6 M and 10-3 M ouabain. Then, the cell permeable nonfluorescent precursor SNARF-1-AM, at a concentration of 5 M was added and the cells
were incubated for another 30 min at 37 C. After centrifugation at 300 X g for 5 min,
cells were resuspended in 100l of fresh modified Tyrodes medium, and 15l aliquots
were transferred to cuvettes with a total volume of 2.5 ml modified Tyrodes medium.
For the calibrating controls, parallel samples were placed in cuvettes with 2.5 ml of the

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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).

Intracellular Ca2+ determination


Changes in [Ca2+]i were determined using the cell permeable fluorescent dye Calcium
Green-1-AM (June CH 2007). Cells were treated in modified Tyrodes medium without
Ca2+, and in the absence and presence of 10-6 M or 10-3 M ouabain to either inhibit the 4
or also the 1 isoform (Wagoner et al. 2005). During this incubation time, spermatozoa
were loaded with Calcium Green-1-AM, used at a final concentration of 5M. After
washing the cells twice in modified Tyrodes medium, by centrifuging at 300 X g for 5
min, cells were resuspended in fresh medium and placed on slides. To favor the adhesion
to the slides, cells were inmobilized on Cell-Tak coated slides (BD Biosciences, Bedford,
MA) as previously described (O'Toole et al. 2000). Cells were then subjected to confocal
microscopy. The fluorescence images of individual cells were collected using a Nikon
scope and a 20 X objective at an excitation/emission of 488/515-530 nm. The samples
were maintained at 37C employing a heated device regulated by the system acquisition
control. After images were obtained, analysis of the collected data was performed using

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Metamorph software (Molecular Devices, Downingtown, PA). A total of 60 cells per


experimental condition were analyzed in each experiment, and results were expressed
relative to the control, in which ouabain was ommited.

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 %

respectively. Differences were found to be statistically significant only between the


samples treated with either 10-6 M or 10-3 M ouabain and the untreated controls, with P<
0.001.

Figure 2. Activity of the Na,K-ATPase 4 isoform is important for several parameters of


rat sperm motility. Rat spermatozoa from the cauda of epididymides were isolated and
treated in modified Tyrodes medium in the absence and presence of 10-6 M and 10-3 M
ouabain. Sperm motility was determined using CASA and different parameters were
analyzed after 1h treatment without and with the indicated amounts of ouabain, using the
SpermVision system. (A) straight line velocity, VSL; (B) average path velocity, VAP;
(C) curvilinear velocity, VCL; (D) amplitude of lateral head displacement, ALH; (E) beat
cross frequency, BCF; and (F) linearity. Cells from 10 different fields per condition were
analyzed. Bars represent the mean + standard errors of the mean of ten experiments. Bars

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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).

Figure 4. Na,K-ATPase 4 isoform activity contributes to maintain rat sperm membrane


potential. Rat spermatozoa from the cauda epididymides were isolated and incubated for
30 min in modified Tyrodes medium in the absence and presence of 10-6 M and 10-3 M
ouabain. Then, membrane potential of the cells was determined using the fluorescent
indicator [DiSC3(5)] as explained in Materials and Methods. (A) Representative traces
for the control and ouabain treated cells, obtained at an excitation/emission wavelength of

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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).

Figure 5. The Na,K-ATPase 4 isoform is important for acid-base balance in rat


spermatozoa. Rat spermatozoa from the cauda epididymides were isolated and incubated
for 30 min in modified Tyrodes medium in the absence and presence of 10-6 M and 10-3
M ouabain. Intracellular pH of rat spermatozoa was measured using the fluorescent
indicator SNARF-1. (A) Representative trace obtained at an excitation/emission ratio of
640/580 nm. Calibration was performed after incubation in media of preset pHs and 2
M of the ionophore nigericin. (B) Effect of ouabain on sperm intracellular pH,
determined at various extracellular pH values (7.0, 7.2, 7.4 and 7.6) within the
physiological range. Compiled data from four different experiments performed in
triplicate. Bars represent the mean

standard errors of the mean. Statistical differences

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).

Figure 6. The Na,K-ATPase 4 isoform functions as a regulator of rat sperm [Ca2+]i.


Rat spermatozoa from the cauda epididymides were isolated and incubated for 30 min in
modified Tyrodes medium in the absence and presence of 10-6 M and 10-3 M ouabain.
Calcium Green-AM was used to determine [Ca2+]i. (A) Images from a representative cell
from untreated controls or samples incubated with ouabain. Pictures were taken using a
Nikon inverted confocal microscope and analyzed using Metamorph software. Scale bar
at the bottom right corresponds to 10m. (B) Compiled data from three different
experiments. Bars represent the mean + standard errors of the mean. Values significantly
different from the untreated control are indicated with an asterisk, with P< 0.001 for both
10-6 M and 10-3 M ouabain vs untreated control. No statistical differences were found
between samples containing 10-6 M and 10-3 M ouabain (P=0.1).

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