REP-09-0495.full

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 Sánchez1, 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 α4 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: [email protected]

of 43 Reproduction Advance Publication first posted on 23 February 2010 as Manuscript REP-09-0495

Copyright © 2010 by the Society for Reproduction and Fertility.

mailto:[email protected]

2

Abstract

While the function of the ubiquitous Na,K-ATPase α1 subunit has been well documented,

the role of the sperm specific α4 isoform of this ion transporter is less known. We have

explored the importance of α4 in rat sperm physiology, taking advantage of the high

sensitivity of this isoform for the inhibitor ouabain. Using concentrations that selectively

block α4 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 α4 on Na+ transport, ouabain inhibition of α4

increased [Na+]i in the male gamete. In addition, interference of α4 activity with ouabain

produced cell membrane depolarization, diminished pH and increased [Ca2+]i in

spermatozoa. Inhibition of α4 was sufficient to cause all these effects and additional

blockage of α1, the other Na,K-ATPase α isoform expressed in sperm, with higher doses

of ouabain did not result in further changes in the cell parameters studied. These results

show that α4 is the Na,K-ATPase isoform primarily involved in controlling the

transmembrane Na+ gradient in sperm, and that α4 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 α4 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 α and β subunits (Morth et al.

2007). The α 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 α subunit,

named α1, α2, α3 and α4 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 α1 is found in nearly every tissue and it

is believed to maintain the basal ion gradients in the cell, the other α polypeptides are

more restricted in their expression and appear to play tissue specific roles (Blanco &

Mercer 1998, Mobasheri et al. 2000). In particular, the α4 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 α4

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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 α polypeptide present in sperm is the widely expressed α1

isoform (Wagoner et al. 2005). We have previously shown that the activity of α4 is

different from that of α1 and the other Na,K-ATPase α isoforms. Thus, α4 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, α4 has a distinctive very high sensitivity to ouabain, a characteristic

that most prominently distinguishes it from the α1 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 α4, suggests that this polypeptide plays a specific role in the physiology

of sperm. In support of this, inhibition of α4 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 α4, 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 α4 action is an important step in comprehending how this isoform

contributes to male fertility. In the present work, we have studied the role of α4 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 α4

for ouabain, to specifically inhibit this isoform, and distinguish its activity from that of

the α1 isoform in rat spermatozoa. Our results show that α4, but not the α1 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 α4 represent mechanisms through which

this Na,K-ATPase isoform supports motility of the male gamete.

Results

Inhibition of Na,K-ATPase α4 isoform inhibits multiple parameters of rat sperm

motility

We and others have previously shown that activity of the Na,K-ATPase α4 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 α4, 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 α4 to sperm motility and compared it with that of the

α1 isoform. Extensive studies of the kinetic properties of the Na,K-ATPase shows that

each of the Na,K-ATPase isoforms expressed in sperm, α1 and α4, exhibit a marked

difference in affinity for the inhibitor ouabain (Blanco 2005a). In the rat, the ouabain

affinity of α4 is approximately 10,000 fold higher than that of α1 (Wagoner et al. 2005).

In this manner, differential sensitivity to ouabain has been used as a tool to specifically

inhibit α4 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 α4 activity, without having

significant effects on the α1 polypeptide. A ouabain concentration of 10-3 M instead, will

cause inactivation of both the α1 and α4 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 α4 was sufficient to

impair the percent of total motile spermatozoa in a time dependent manner. Further

inhibition of α1 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 α4 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 α4

isoform, and not α1 is involved in this process.

Inhibition of α4 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 α4 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 α4

as an ion transporter contributes to maintain sperm [Na+]i is unknown. To specifically

determine the role of α4 in [Na+]i maintenance, and to correlate its transport activity with

that of the α1 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 α4 isoform

with 10-6 M ouabain, and no further significant increase in [Na+]i was detected after

inhibition of the Na,K-ATPase α1 polypeptide with 10-3 M ouabain. These results suggest

that the activity of the α4 isoform exerts a more prominent effect in maintaining the

[Na+]i in spermatozoa (Wagoner et al. 2005).

Function of α4 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 α4 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 α4 is the

isoform primarily involved in this process.

The α4 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 α4, or the ubiquitous α1 isoform is involved in the

secondary regulation of H+ content in the male gamete. For this, rat spermatozoa in

modified Tyrode’s 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 α4 isoform plays a fundamental role in maintaining proton

concentration low in the cells.

Activity of α4 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 α1 and α4 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 Tyrode’s 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 α4 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, K-

ATPase, and more specifically its α4 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 α4 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 α4 isoform has been deleted are not yet available,

our study has taken advantage of the unique high affinity of α4 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 α4 isoform in the native spermatozoa. We show that inhibition of α4, 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 α4 on

different components of rat sperm movement, beyond the resolution given by previous

simple visual determinations. Thus, we found that activity of α4 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 α4 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 α4 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 α4 isoform is primarily involved in maintaining the low

[Na+]i levels in rat spermatozoa. This agrees with our previous findings showing that α4

is responsible for most of the Na,K-ATPase catalytic activity of the cells (Wagoner et al.

2005). The importance of the α4 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 α4 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 α4 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 α4 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 α4 isoform

secondarily controls proton levels in spermatozoa, and ouabain inhibition of α4 caused a

pH decline of the sperm cytoplasm. This occurred after exposure of the cells to different

physiological pH values, suggesting that α4 is able to prevent sperm acidification over a

range of proton concentrations. The α4 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 α4 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 α4 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 α4 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 α4

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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 α4 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,K-

ATPase α4 isoform influences sperm motility.

Our results also show that the activity of the α4 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 α4

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 α2 polypeptide (Golovina et al. 2003, Zhang et al. 2005). Both the α2 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 α4 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 α4 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

α4 isoform and NCX, our results suggest that, similar to the α2 isoform, the α4

polypeptide is another Na,K-ATPase isoform involved in Ca2+ regulation, in this case,

specific to spermatozoa. Thus, α4 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 α4 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 α4

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 α4, rather than the α1 polypeptide. We have previously shown that α4

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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 α1 isoform, α4 has a higher

apparent affinity for intracellular Na+, and small amounts of this cation selectively

stimulate α4 function. Therefore, the α4 isoform appears to be better suited to maintain

lower [Na+]i than α1. This predominant role of α4 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 α1 isoform may be playing a redundant role. Previous work showed that α1

contributes to approximately one third of the total Na,K-ATPase activity of rat

spermatozoa. In addition, α1 presents a broader distribution along the sperm flagellum,

compared to α4. Therefore, although activity of α1 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 α1 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 α1 isoform has some “housekeeping” role in

maintaining the basal [Na+]i gradient, while α4 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 α4 isoform in the control of sperm motility. The ion transport activity of α4

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, α4 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 α4 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 α4 isoform influences sperm motility. Future studies are underway to

further elucidate the molecular basis of α4 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 Tyrode’s

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 Tyrode’s medium. Cell were counted with the help of a

hemocytometer, and used for the different assays. All experiments were conducted in

modified Tyrode’s medium.

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Sperm motility assays

Approximately 3 x 106 cells were resuspended in 300 µl of the modified Tyrode’s

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 α4, or both the α1 and α4

isoforms (Wagoner et al. 2005). Incubation was performed at 37 ºC for different times

and for a total of 2h. Then, cells were labeled with 2 µl of a 75 µM stock of SITO 21, a

green fluorescent nucleic acid stain, which helps tracking cell movement. After 2 min

incubation with the dye, 7µl aliquots from each sample were taken and placed into a glass

cell chamber 20 µm 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 ºC 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 µm2, a cutoff velocity corresponding to a minimum straight line

velocity of 5µm/sec, a progressive motility threshold corresponding to a straight line

velocity of more than 20 µm/sec, and a cutoff for low and medium average path

velocities of 10 µm/sec and 120 µm/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 Tyrode’s medium for 15 min at 37 ˚C, in the

absence or presence of 10-6 M ouabain that inhibits only the α4 isoform, and 10-3 M

ouabain that blocks both the α1 and α4 isoforms (Wagoner et al. 2005). The cell-

permeant nonfluorescent precursor of Sodium Green Tetraacetate at a final concentration

of 2.5 µM 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 Tyrode’s medium. Aliquots (50µl each) were

placed in cuvettes with a total volume of 2.5 ml modified Tyrode’s 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 Tyrode’s medium, for 20 min at 37 ˚C, with and without ouabain in

concentrations that inhibits only α4 (10-6 M), or both the α1 and α4 isoforms (10-3 M)

(Wagoner et al. 2005). Then, [DiSC3(5)], at a final concentration of 1 µM, 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 µM (Hernandez-

Gonzalez 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 1µM, 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

Tyrode’s 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 non-

fluorescent precursor SNARF-1-AM, at a concentration of 5 µM 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 100µl of fresh modified Tyrode’s medium, and 15µl aliquots

were transferred to cuvettes with a total volume of 2.5 ml modified Tyrode’s 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 µg/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 Tyrode’s medium without

Ca2+, and in the absence and presence of 10-6 M or 10-3 M ouabain to either inhibit the α4

or also the α1 isoform (Wagoner et al. 2005). During this incubation time, spermatozoa

were loaded with Calcium Green-1-AM, used at a final concentration of 5µM. After

washing the cells twice in modified Tyrode’s 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 37°C 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 student’s 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 α4 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 Tyrode’s 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 α4 isoform is important for several parameters of

rat sperm motility. Rat spermatozoa from the cauda of epididymides were isolated and

treated in modified Tyrode’s 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 α4 isoform is important in maintaining the low [Na+]i in rat

sperm. Rat caudal epididymal spermatozoa were collected and incubated in modified

Tyrode’s 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 α4 isoform activity contributes to maintain rat sperm membrane

potential. Rat spermatozoa from the cauda epididymides were isolated and incubated for

30 min in modified Tyrode’s 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 α4 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 Tyrode’s 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

µM 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 α4 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 Tyrode’s 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 10µm. (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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