Purpose Expression of the Na,K-ATPase α4 isoform is required for sperm motility and fertility and is
controlled by the Atp1a4 promoter. Here, we have investigated the specific tissue, cell type and
developmental regulation of expression mediated by the Atp1a4 promoter.
Methods We have inserted the green fluorescent protein (GFP), downstream of the endogenous Atp1a4
promoter, in place of the Na,K-ATPase α4 gene, and used it as a marker for α4 expression in mice
Results Replacement of α4 by GFP completely disrupted α4 expression and activity, produced sperm
morphological and functional abnormalities, and caused infertility of Atp1a4null(GFP) male mice.
Immunoblot analysis of Atp1a4null(GFP) mouse tissues showed GFP expression only in testis and
epididymis. This particular expression pattern was found in adult, but not in mouse embryos or in 7, 18
day old mice. In agreement with expression of GFP, adult Atp1a4null(GFP) mouse testis and epididymis
displayed the typical fluorescence of GFP. Immunocytochemistry of testis and epididymis identified GFP
in more differentiated male germ cells, but not in spermatogonia, Leydig or Sertoli cells. Further analysis,
using immunoblot of fluorescently sorted testis cells with cell specific markers, detected GFP only in
spermatocytes, spermatids and spermatozoa.
Conclusions Our results show that the Atp1a4 promoter drives GFP expression exclusively in male germ
cells of the testis, where it restricts it to post-meiotic stages of spermatogenesis. These findings highlight
the exquisite spatial and temporal control of expression exerted by the Atp1a4 promoter on Na,K-ATPase
α4, which is particularly well suited to fulfill the special functions of spermatozoa.
Keywords: testis, male germ cells, spermatogenesis, spermatozoa, Sertoli cells.
The Na,K-ATPase is a plasma membrane enzyme that catalyzes the active transport of Na+ and K+
between the cell and its environment [1,2]. Function of the Na,K-ATPase is essential for maintaining key
parameters in the cell and this is why expression and activity of this ion transporter is highly regulated
. In mammalian cells, the Na,K-ATPase is expressed as various isozymes, which result from the
association of four different isoforms of the α subunit (α1, α2, α3 or α4) and three distinct isoforms of the
β polypeptide (β1, β2 or β3) [4,5]. Expression of these structurally and functionally different variants of
the Na,K-ATPase is not redundant but rather represents a mechanism that cells use to adapt Na+ and K+
transport to their specific physiologic needs [3,4,6,7].
The α4 polypeptide is the Na,K-ATPase isoform that is least abundantly expressed across the body. It
has been found in male germ cells of the testis, where it predominates in the sperm flagellum [8,9]. The
α4 isoform has particular affinities for Na+, K+ and ATP [10,11] and is important for maintaining
membrane potential, pH and the intracellular Na+ and Ca+2 levels of sperm . Moreover, the α4
polypeptide plays a primary role in sperm motility [8,13,14] and it is fundamental for sperm fertility since
knockout mice in which the α4 isoform has been deleted are completely sterile . While original
studies with specific antibodies identified α4 in testis male germ cells [8,10]; more recently, a work based
on RT-PCR analysis, reported the presence of Na,K-ATPase α4 mRNA in Sertoli cells, suggesting that α4
expression may not be solely confined to male germ cells, but could also extend to somatic cells .
This shows that other independent experimental approaches will be important to precisely understand the
tissue and cell type specific distribution of Na,K-ATPase α4. In addition, while studies of RNA, protein
and activity have helped follow the pattern of expression of the α4 isoform [8,9], a complete
characterization of the expression of α4 during different stages of development has not yet been
accomplished. Comprehending α4 expression pattern during development is an important step in
deciphering the biological relevance of this Na,K-ATPase isoform in male reproduction and fertility.
The Na,K-ATPase α4 isoform is encoded by the ATP1a4 gene, which is regulated by DNA sequences
located upstream the α4 coding sequences. These sequences, identified as the proximal promoter regions
of α4 (Atp1a4 promoter) have regulatory sites that drive the expression of the ATP1a4 gene [17,18]. In
the present work, we have taken advantage of the endogenous Atp1a4 promoter to drive the expression of
the Green Fluorescent Protein (GFP) in mice. This genetic approach allowed us to follow GFP expression
as a marker for the spatial and temporal regulation of protein expression exerted by the Atp1a4 promoter
in mice. We show that ATP1a4 promoter dependent expression of GFP is testis specific and exclusively
limited to the germ cells of the male gonad that have progressed to late stages of spermatogenesis. In
addition, our knock-in strategy has established a new mouse model that will be useful for future studies
related to the Na,K-ATPase α4 isoform.
Materials and Methods
Preparation of knockout animals
A mouse 129/SvEv BAC (BAC ID# bMQ-247N9) (Source BioScience) containing the Atp1a4
genomic locus was used to generate the Atp1a4 knockout gene-targeting vector. The targeting vector was
made by subcloning a GFP reporter gene with polyadenylation signal (Clontech) into the Atp1a4 start
site. Recombineering was employed to fuse the targeting vector to the BAC Atp1a4 locus .
Homologous recombination simultaneously inserted the vector and removed the Atp1a4 start methionine
in the first 62 nucleotides of exon 6, generating a frame shift mutation of the remaining DNA. The
targeting vector was linearized with PmeI and electroporated into EDJ22 (ATCC) ES cells (129SvEv cell
line) for homologous recombination. After neomyocin selection, isolated ES cell clones were screened by
PCR. Four clones were confirmed by Southern blotting with positive clones being further characterized
by karyotyping. Targeted ES clones were microinjected into C57BL/6 blastocysts and these were
implanted into pseudopregnant females. Resulting chimeras were mated to wild-type C57BL/6 mice to
generate F1 heterozygous offspring and breeding continued until a founder line was established.
Genotype analysis of tail biopsies was performed by PCR on isolated genomic DNA using the
REDExtract-N-Amp Tissue PCR Kit (Sigma).
All experimental protocols involving animals in this work were approved by the University of Kansas
Medical Center Institutional Animal Care and Use Committee. Spermatozoa from wild-type and
homozygous Atp1a4null(GFP) mice were obtained from the cauda of adult mice epididymides after swim-up
of the cells, as previously described . Sperm was resuspended in modified Tyrode’s medium,
containing: 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM Glucose, 0.8 mM
pyruvic acid, 4.8 mM lactic acid, 20 mM Hepes (pH 7.4), counted, and used for the different assays. For
some experiments, sperm was capacitated in modified Tyrode’s medium, supplemented with 1.7 mM
CaCl2, 25 mM sodium bicarbonate and 0.5% BSA.
Southern Blot Analysis
Southern blot was used to confirm the ES cell clones containing the α4 construct. DNA was first
digested with Bgl II and electrophoretically separated on a 0.8% agarose gel. After transfer to a nylon
membrane, DNA was hybridized with probes targeted to the 5’ external region of the Bgl II digested
DNA, or to the 3’ external region of the Bgl II digested DNA. After washing off the non-hybridized
probe, membranes were processed for autoradiography as described .
Polymerase chain reaction (PCR) analysis
PCR was used for analysis of genomic DNA. Reactions were performed using primers directed to
Na,K-ATPase α4 isoform sequences (sense 5′-TCAGACTTGCTCCCTTCACCTTTCCT-3′) and
antisense (5′-GTTTGCTTTGGCCATCCTCACCAT-3′) and to the GFP reporter sequence (5′-
TGTAGTTGCCGTCGTCCTTGAAGA-3′). Procedures were as described , using a first cycle of 120 s
at 94 °C, followed by 35 cycles at 94 °C for 15 s, 62 °C for 15 s, and 72 °C for 30 s.
Tissue samples were homogenized in RIPA buffer (1% NP-40, 0.25% NaDOC, 1mM Na3VO4, 1mM
NaF, 1mM EDTA, 150mM NaCl, 10 μM leupeptin, 10 μg/ml apotinin, 1mM PMSF and 50mM Tris (pH
7.4), centrifuged and the supernatant used for immunoblot analysis. Samples were subjected to
SDS/PAGE (7.5% gel) and immunoblotting, as previously described . The Na,K-ATPase α1 and α4
isoforms were identified using antisera generated against specific amino acids in the N-terminal regions
of the α isoforms in rabbit and chicken respectively as described . Other antibodies used to probe the
blots included mouse anti-PLZF (1:200 dilution; Calbiochem), rabbit anti-DDX4 (1 μg/ml; Abcam,
Cambridge, MA), rabbit anti-DMRT1 (1:4000 dilution; a kind gift from Leslie Heckert, University of
Kansas Medical Center), anti-KIF17b (1:1000 dilution), anti-GFP (1:2000 dilution; Abcam) and mouse
anti-beta tubulin (1:1000 dilution; Sigma-Aldrich). Horseradish peroxidase conjugated secondary
antibodies (Jackson ImmunoResearch Laboratories, Inc.) and chemiluminescence was used for detection.
Na,K-ATPase activity was assayed on sperm homogenates, through determination of the initial rate
of release of 32Pi from γ[32P]-ATP, as previously described . The ATPase activity of 10 μg of total
protein per sample was measured in a final volume of 0.25 mL in medium containing 120 mM NaCl, 30
mM KCl, 3 mM MgCl2, 0.2 mM EGTA, 30 mM Tris-HCl (pH 7.4), 3 mM ATP with 0.2 μCi γ[32P]-ATP
in the presence and absence of the indicated ouabain concentrations. Curve fitting of the experimental
data were performed using a Marquardt least-squares nonlinear regression computing program (Sigma
Plot; Jandel Scientific).
Sperm motility assay
Sperm motility was measured on spermatozoa from wild-type, and Atp1a4null(GFP) mice as previously
described . Samples were analyzed by computer-assisted sperm analysis (CASA), using the Minitube
Sperm-Vision Digital Semen Evaluation system (version 3.5, Penetrating Innovations). Total sperm
motility and different parameters of sperm movement were determined using the analytical setup
parameters defined before .
Histological and immunocytochemical analysis
Mouse testis and epididymis were dissected, fixed with Bouin’s fixative solution (Ricca Chemical
Company, Arlington TX), embedded in paraffin and sectioned to obtain 4 μm slices. After placing on
slides, samples were stained with hematoxylin/eosin, or they were processed for immunofluorescence as
described . Briefly, tissue was permeabilized with 0.3% Triton X100 in PBS. After blocking for 2 h at
room temperature with 0.1% BSA and 5% normal goat serum in PBS, the anti-GFP antibody was applied
overnight at 4 ˚C at a dilution of 1:2500. For immunochemical staining, biotinylated goat anti-rabbit
antiserum was used as the secondary antibody and detection was performed using the Vectastain ABC Kit
from Vector laboratories (Burlingame, CA) following the manufacturers’ instructions. For fluorescence
labeling, an Alexafluor-488 conjugated goat anti-rabbit antiserum (Molecular Probes, Eugene, OR) was
used as the secondary antibody. Digital images were obtained using a Nikon 80i microscope. Similar
protocols were used to explore expression of GFP in 16 day old whole mouse embryos.
Sorting of testis cells
Testis from adult (8 weeks) wild type and Atp1a4null(GFP) mice were dissected, decapsulated and placed
in DMEM medium supplemented with 2 mM glutamate, non-essential amino acids, 100 IU/ml penicillin,
100ug/ml streptomycin and 15mM HEPES (pH 7.4). Cells were dissociated following a modification of
the protocol previously described. Briefly, samples were first digested with 0.03 mg/ml
collagenase/dispase and 0.1 mg/ml DNase for 15 min with continuous agitation at 37oC. Dissociated
seminiferous tubules were centrifuged at 1,500 rpm for 5 minutes and further digested with 0.03
collagenase type IV-S for 20 min with continuous agitation at 37oC. After centrifugation at 2200 rpm for
10 minutes, cells were suspended to a final density of 5 x 106 cells/ml and were filtered through a cell
strainer (BD Biosciences, San Jose, CA). Dissociated cells were sorted according to their fluorescence at
488 nm using the Fluorescent-Activated Cell Sorter (FACS) Aria II cell sorter (BD Biosciences, San Jose,
CA). Separated GFP positive and negative cells were collected after centrifugation and samples used for
Atp1a4null(GFP) mice express GFP in place of the Na,K-ATPase α4 isoform
To determine the temporal and spatial pattern of expression of the α4 subunit, we have used a knock
in strategy to insert GFP in place of the Atp1a4 gene in mice (Atp1a4null(GFP) mice). The reporter GFP gene
with polyadenylation signal was introduced in the Atp1a4 start site in mouse embryonic stem (ES) cells.
This left GFP under the control of the Atp1a4 promoter and disrupted the Atp1a4 locus by removing the
remaining downstream sequence of exon 1 through exon 6 by homologous recombination (Fig 1A). ES
cell clones were screened by Southern blot analysis and positive clones were confirmed following the
strategy depicted in Figure 1B. One of the positive clones was chosen to make the chimeric mice. Once
this was generated, a founder line was established and the desired genotype of offspring was verified by
PCR (Fig. 2A). Further validation of the absence of the Na,K-ATPase α4 isoform and its substitution by
GFP was performed by immunoblotting of mouse sperm proteins. As shown in Figure 2B, genetically
modified mouse homozygous for the allele, completely lacked the α4 polypeptide and expressed GFP
instead. The null allele inheritance of mated heterozygous F1 mice (15 litters) followed Mendelian ratios
(27 wild-type, 52 heterozygous, and 29 homozygous mice). While Atp1a4null(GFP) mice lacked expression
of the α4 isoform, levels of α1, the other catalytic subunit of sperm Na,K-ATPase showed no change (Fig.
2B). Altogether, these results show that the Atp1a4 promoter is able to efficiently express GFP in place of
the α4 polypeptide and demonstrate that the effect of our genetic manipulation was Na,K-ATPase isoform
Atp1a4null(GFP) mice are devoid of Na,K-ATPase α4 isoform activity
As shown previously, the α4 isoform has a characteristically high affinity for the inhibitor ouabain
and is important for sperm morphology and motility [15,20]. To ascertain that insertion of GFP in the
Atp1a4null(GFP) mice produced the functional phenotype corresponding to disruption of the Atp1a4 gene, we
performed Na,K-ATPase activity and motility assays on sperm from the genetically engineered mice.
Determination of ouabain inhibition profiles of Na,K-ATPase activity in testis homogenates from wild
type mice showed a biphasic dose-response curve, with IC50 values in the nanomolar (2.04 + 0.31 x 10-9
M) and micromolar (2.34 + 0.63 x 10-5) range (Fig. 3A). This kinetic behavior corresponds to the
activities of the highly ouabain sensitive α4 and ouabain resistant α1 isoforms, respectively . In
contrast, inhibition of Na,K-ATPase by ouabain in Atp1a4null(GFP) mice testis showed a monophasic
response, lacking the enzymatic component characteristic of α4 and presenting a single IC50 value of 4.41
+ 1.10 x 10-5 M, which indicated the presence of only α1 in the sample (Fig. 3A). In agreement with the
absence of α4, total Na,K-ATPase activity was lower in Atp1a4null(GFP) than in wild type sperm (Fig. 3B).
These functional data further support the lack of expression of the α4 isoform in Atp1a4null(GFP) mice.
Atp1a4null(GFP) male mice are infertile
Atp1a4null(GFP) mice were overall phenotypically similar to wild-type mice. However, while
homozygous and heterozygous female mice and heterozygous male mice were fertile, producing normal
litter numbers and sizes, homozygous Atp1a4null(GFP) male mice were completely sterile. Controlled
matings of homozygous null males with wild type females yielded no pregnancies during a period of 3
months. This was not due to the inability of the mice to mate, as noted by the presence of vaginal plugs in
the females that have been mated with Atp1a4null(GFP) mice. Testis size, testis general morphology and
testis to body weight ratios were similar in the Atp1a4null(GFP) and wild type mice (data not shown). This
male specific abnormal reproductive phenotype resembles that previously found in α4 null mice  and
suggests first, that disruption of the Atp1a4 gene by GFP was efficient and second, it supports the
important role that the Na,K-ATPase α4 isoform has in male fertility.
Atp1a4null(GFP) mice show sperm morphological and functional abnormalities
When examined by CASA and under non capacitating media, sperm from Atp1a4null(GFP) mice showed
a drastic reduction in total and progressive sperm motility compared to wild-type spermatozoa (Fig. 4A
and B). Other parameters of sperm motility, such as straight line, curvilinear and average path velocities,
and amplitude of lateral head displacement were also decreased (data not shown). Moreover, when sperm
was incubated in capacitating medium, containing BSA and bicarbonate, the hyperactivation pattern of
motility that sperm normally achieves was drastically reduced in Atp1a4null(GFP) mice (Fig. 4C). In addition
to the defects in motility, sperm from Atp1a4null(GFP) mice also displayed abnormal morphology, consisting
in a bend between the mid- and the principal piece of the sperm flagellum (Fig. 4D). This corresponds to
the phenotype previously seen in α4-null sperm and is consistent with alterations in osmolarity of the cell
cytoplasm resulting from lack of the α4 isoform . Altogether, these data indicate that insertion of the
GFP in place of the Atp1a4 gene results in the phenotypical defects of sperm that are typical of the
absence of the α4 isoform .
GFP expression in Atp1a4null(GFP) mice is developmentally regulated and organ specific
Because GFP was placed under the Atp1a4 promoter, Atp1a4null(GFP) mice provided the opportunity to
use GFP as a substitute marker for the expression of the Na,K-ATPase α4 isoform. Therefore, we
examined expression of GFP at the protein level in testis and a series of other major organs of
Atp1a4null(GFP) mice at different stages of development. Three time points in the sexual maturation of mice
were chosen, since they represent milestones in the development of male germ cells in the testis: a) 7 days
post partum (7 dpp), when the testes contain germ cells at the stage of spermatogonia, b) 18 dpp, when
preleptotene, leptotene, and pachytene spermatocytes are present, but the gonad has no haploid
spermatids, and c) adult animals, in which cells at all stages of spermatogenesis are found and
spermatozoa are fully differentiated . Immunoblot analysis using an antibody against GFP identified
this protein exclusively in testis of adult mice (Fig. 5A, B and C). No GFP could be detected in any of the
tissues tested at 7 or 18 dpp. In addition, we explored expression of GFP in the mouse embryo, using
whole body sections of wild type and Atp1a4null(GFP) mice at embryonic day 16 day. As shown in Figure
5D, no GFP reactivity was found in either the wild type or Atp1a4null(GFP) embryos. These results show that
the Atp1a4 promoter drives expression of GFP in a tissue specific manner, being present in the testis and
only when mice have reached adulthood.
GFP fluorescence is present in testis and epididymis of adult Atp1a4null(GFP) mice
Another indication of Atp1a4 promoter driven expression of GFP in the male gonad during
development was obtained by directly following the fluorescence of whole testis samples at 488 nm.
Testis from 7 ddp, 18 ddp and adult Atp1a4null(GFP) mice were dissected and analyzed under regular and
fluorescent light. As shown in Figure 6A, no specific label corresponding to GFP was observed in wild
type and Atp1a4null(GFP) mouse testis at either 7 dpp or 18 dpp. Also, no GFP fluorescence was observed in
wild type adult testis. In contrast, significant label was detected in testis of adult Atp1a4null(GFP) mice
expressing GFP (Fig. 6A). In addition, we also studied direct fluorescence of wild type and Atp1a4null(GFP)
mouse epididymis. Similar to testis, GFP fluorescence was only apparent in epididymis of adult
Atp1a4null(GFP) mice (Fig. 6B). These observations further indicate that expression of GFP under the
ATP1a4 promoter takes place in both testis and epididymis of the adult Atp1a4null(GFP) mice.
GFP expression driven by the Atp1a4 promoter has a developmental and cell type specific pattern of
expression within Atp1a4null(GFP) mouse testis.
To further study expression of GFP within the testis, immunocytochemistry of testis sections was
performed, using an anti GFP antibody. As shown in Figure 7A, testis from wild type mice showed no
label for GFP either at 7 dpp, 18 dpp or adulthood. Similarly, GFP was not identified in Atp1a4null(GFP)
mouse testis at 7 and 18 dpp. In contrast, Atp1a4null(GFP) adult mouse testis presented GFP, which was
localized to the inner region of the seminiferous tubules (Fig 7A). When samples were viewed at higher
magnification, GFP staining was observed specifically at the later stages of male germ cell development
(Fig. 7B). No staining was observed in less differentiated male germ cells, such as spermatogonia and
early spermatocytes. In addition, the anti-GFP antibody did not label the Sertoli or the Leydig cells (Fig.
7B). These results support the idea that Atp1a4 promoter dependent expression of GFP takes place mainly
in male germ cells and not somatic cells of the testis and that it is limited to late stages of
Using also immunocytochemical analysis, we followed GFP expression in the epididymis from adult
Atp1a4null(GFP) mice. As shown in Figure 7C, specific label for GFP was identified in this organ and it was
only localized to the spermatozoa present in the lumen of the epididymal tubules. No specific GFP
reactivity was found in the epithelial cells lining the tubules. This indicates that GFP expression
controlled by the Atp1a4 promoter is confined to the differentiated male germ cells stored in the
GFP expression in adult Atp1a4null(GFP) mouse testis is limited to post meiotic male germ cells
To further determine the association of GFP expression with a particular cell type population in the
testis, we isolated testis cells from Atp1a4null(GFP) mice based on their fluorescence at 488 nm, by FACS
and subjected the isolated cell populations by immunoblot, using antibodies against cell type specific
markers. We used the Dsx- and mab-3-related transcription factor 1, DMRT1 to label Sertoli cells ,
the transcriptional repressor protein PLZF for spermatogonia , the DEAD family protein DDX4 as a
marker for spermatocytes and round spermatids  and the kinesin KIF17b as a postmeiotic marker of
male germ cells and spermatozoa . Also we included GFP as a control for cell sorting and tubulin as a
loading control. As shown in Figure 8A, before being subjected to cell sorting, dissociated cells from wild
type mice were positive for all cell markers, except for GFP. Dissociated cells from Atp1a4null(GFP) mice
showed the additional expression of GFP (Fig. 8A). In cells from Atp1a4null(GFP) mice, cell sorting
generated a GFP positive population that displayed the presence of GFP and were enriched in DDX4 and
KIF17b, indicating the presence of spermatocytes, round spermatids and spermatozoa, but had almost
undetectable levels of DMRT1 and PLZF, characteristic of Sertoli cells and spermatogonia (Fig. 8B). In
contrast, the cells that showed no fluorescence after sorting had no GFP, low levels of DDX4, no KIF17b
and high expression of DMRT1 and PLZF (Fig. 8B). Altogether, these results suggest that the Atp1a4
promoter drives expression of GFP in the testis seminiferous epithelium and specifically in the male germ
cells that have reached more differentiated stages of spermatogenesis.
In this work, we have used the expression of GFP, driven by the Atp1a4 promoter, as a reporter for
the expression of the Na,K-ATPase α4 isoform in mice. Different from previous studies, which detected
α4 using RT-PCR and immunoblotting techniques, this genetic approach provided us with an alternative
powerful means to define the tissue pattern of expression of α4 during development and its presence in
different cell types of the male gonad. In addition, knock in of GFP under the Atp1a4 promoter further
proved the function of α4 via disruption of the ATP1a4 gene. Introduction of GFP efficiently blocked
expression of the α4 isoform and eliminated the highly ouabain sensitive Na,K-ATPase activity typical of
α4. Moreover, replacement of α4 with GFP resulted in a phenotype that reproduced that of the α4 null
mice . This consisted in the Atp1a4null(GFP) male mice being completely infertile and exhibiting several
sperm alterations, including a bend in the sperm flagellum and a drastic reduction in sperm motility.
While expression of GFP in place of α4 affected sperm morphology and function, it did not alter the size
or macroscopic and histological morphology of the testis and epididymis. This suggests that expression of
α4 is not necessary in defining testis and epididymal structure or the development of either somatic or
male germ cells in those tissues. The similarity in phenotype between the α4 knock out mice  and the
present Atp1a4null(GFP) mice show that targeting of the Na,K-ATPase α4 isoform gene was appropriate and
suggests that the resulting defects in the mice are a consequence of the absence of the α4 isoform rather
than expression of GFP. Additional support for this are our previous data in which over-expression of
GFP as a fusion protein with the rat α4 isoform in mice increased sperm motility without affecting
fertility of the α4-GFP mice . The deficiency in α4 in Atp1a4null(GFP) mice was not compensated
through up-regulation of expression and activity of the other α isoform of the Na,K-ATPase of testis, the
α1 polypeptide. This lack of compensation agrees with our previous observations in α4 null mice  and
provides further evidence that Na,K-ATPase isoform diversity is a biologically relevant event, with the α4
isoform being specifically suited to fulfill the particular requirements of sperm function.
Concurrent with deletion of the α4 isoform, Atp1a4null(GFP) mice efficiently expressed GFP as detected
by the increase in intrinsic fluorescence and appearance of reactivity to the anti-GFP antibody in
immunoblots and histological sections of testis and epididymal samples. We found that GFP expression
was uniquely limited to the testis and epididymis. This protein localization coincides with previous
observations that found α4 RNA and protein in rat and human testis and in the mouse epididymis [14,27-
29]. The adequate expression of GFP under the Atp1a4 promoter further allowed us to follow the
developmental changes of GFP as an indicator of α4 temporal regulation of expression. GFP expression
in Atp1a4null(GFP) mice was only observed in testis and epididymis of adult mice, being undetectable in
whole body sections from Atp1a4null(GFP) mouse embryos, or in testis and epididymis from 7 and 18 day
old pre-pubertal mice. These results agree with previous observations, which showed that α4 expression
and hydrolysis of ATP dependent on α4 are significantly up-regulated with testis development in rats
[8,9]. Those studies however, showed differences regarding the precise onset of α4 expression and while
one of them detected low levels of α4 protein in testis at 7 and 18 days after birth , the other identified
α4 mRNA and protein in the male gonad at four and six weeks of age respectively . In agreement with
this last report, our present data indicate a late start for GFP expression by the Atp1a4 promoter, taking
place at post pubertal stages of testis development.
Our immunochemical and GFP based cell sorting experiments showed that the Atp1a4 promoter
directed expression of GFP specifically in male germ cells and not in Leydig or Sertoli cells of
Atp1a4null(GFP) mouse testis. These results differ from those of Konrad and coworkers, which reported
expression of the α4 isoform in Sertoli cells . In that study, however, the α4 isoform was identified at
the mRNA level using RT-PCR and not at the protein level. In addition, that work was performed in a rat-
derived Sertoli cell line and in Sertoli cells isolated from rat testis, in which cell homogeneity of the
preparation depended on the purification steps performed. Besides these differences, an alternative
explanation that may account for the finding of α4 message in Sertoli cells is the possibility that while
Sertoli cells can transcribe the α4 DNA into RNA, they are unable to translate the α4 message into
polypeptide molecules. Further experiments are needed to ascertain this last possibility. On the other
hand, in agreement with a specific male germ cell localization of the α4 isoform in testis are previous
observations which have used antibodies against the Na,K-ATPase α4 isoform [8,9].
Our current data also show that the Atp1a4 promoter induces expression of GFP in male germ cells at
late stages of spermatogenesis. This is apparent from the immunocytochemical labeling of testis sections
with GFP and from the expression in GFP positive cells of DDX4 and KIF17b, markers for
spermatocytes, spermatids and spermatozoa respectively. However, while expression of GFP could be
barely detected in spermatocytes through the immunochemical analysis of testis sections (Fig. 7), it could
be identified in spermatocytes after cell sorting based on their GFP fluorescence. The finding of GFP
expression earlier in spermatogenesis (i.e. spermatocytes) using FACS as compared to
immunocytochemistry may depend on the higher sensitivity that the cell sorting approach has over the
immunocytochemical analysis. These results also suggest that expression of GFP in spermatocytes is low
and less than in spermatids and spermatozoa. Another indication that GFP expression in spermatocytes is
low comes from our immunoblot analysis, which could not detect GFP in testis of 18 dpp mice. Our
previous analysis of the α4 isoform expression in rat male germ cells separated via unit gravity
sedimentation showed that α4 is up-regulated during spermatogenesis and while α4 mRNA increases in
spermatocytes, α4 protein rises in spermatids . Therefore, it appears that the ATP1a4 promoter does
already drive low levels of GFP expression in spermatocytes. The ATP1a4 promoter driven expression of
GFP at late stages of spermatogenesis is consistent with our previous observations, which showed that the
proximal 5’ untranslated region of the human Na,K-ATPase ATP1a4 promoter contains CRE binding
elements that respond to the testis specific activator of transcription CREMτ , a transcription factor
that is a master controller of post-meiotically activated genes of male germ cells . Therefore, our
current data support the notion that the ATP1a4 promoter forms part of the transcription regulatory
machinery that is up-regulated after meiosis to serve important roles in the mature spermatozoa.
Our immunoblot and immunocytochemical data show that GFP expression is high in the
differentiated spermatozoa, which are clearly labeled by the anti-GFP antibody in the testis, epididymis
and after swim up of the cells. Lack of GFP labeling in prepubertal mouse testis, which is devoid of
spermatozoa also supports the notion that most of the protein expression driven by the Atp1a4 promoter is
aimed to the differentiated spermatozoa. Expression of GFP is primarily located to the mid-piece of the
sperm flagellum, a site that corresponds to that previously described for the α4 isoform [9,10,14].
However, some staining for GFP also appears in other segments of the sperm tail and little GFP appears
to be even present in the sperm head as seen in the immonocytochemical labeling of GFP in testis and
epididymis (Fig. 7). This wider localization of GFP compared to α4 is probably not surprising, since GFP
may not contain the structural determinants within its sequence to target this protein to the appropriate
domain on the sperm plasma membrane.
In conclusion, this new mouse model that we have generated shows that the Atp1a4 promoter directs
protein synthesis specifically in male germ cells of the testis at late stages of spermatogenesis. This
particular spatial and temporal transcriptional control of expression exerted by the Atp1a4 promoter is
consistent with the need of α4 for motility and fertility of the male gamete. In addition, this mouse line
represents a valuable tool for future studies of Na,K-ATPase α4 isoform expression and function.
We thank Renate Lewis and The Transgenic Vectors Core at Washington University in St. Louis for
generating the constructs used to engineer the mice used in this study, Leslie Heckert at University of
Kansas Medical Center (KUMC) for providing the Dmrt1 antibody and Stan Fernald (KUMC) for his
help with the microscopy images presented. Work was supported by NIH grants HD043044 and
1 Kaplan JH. Biochemistry of Na,K-ATPase. Annu Rev Biochem 2002;71:511-35.
2 Martin DW. Structure-function relationships in the NA+,K+-pump. Semin Nephrol 2005;25
3 Blanco G. Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation.
Semin Nephrol 2005;25 (5):292-303.
4 Blanco G, Mercer RW. Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in
function. Am J Physiol 1998;275 (5 Pt 2):F633-50.
5 Mobasheri A, Avila J, Cozar-Castellano I, Brownleader MD, Trevan M, Francis MJ, Lamb JF,
Martin-Vasallo P. Na+, K+-ATPase isozyme diversity; comparative biochemistry and
physiological implications of novel functional interactions. Biosci Rep 2000;20 (2):51-91.
6 Lingrel J, Moseley A, Dostanic I, Cougnon M, He S, James P, Woo A, O'Connor K, Neumann J.
Functional roles of the alpha isoforms of the Na,K-ATPase. Ann N Y Acad Sci 2003;986:354-9.
7 Jewell EA, Shamraj OI, Lingrel JB. Isoforms of the alpha subunit of Na,K-ATPase and their
significance. Acta Physiol Scand Suppl 1992;607:161-9.
8 Woo AL, James PF, Lingrel JB. Sperm motility is dependent on a unique isoform of the Na,K-
ATPase. J Biol Chem 2000;275 (27):20693-9.
9 Wagoner K, Sanchez G, Nguyen AN, Enders GC, Blanco G. Different expression and activity of
the alpha1 and alpha4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny.
Reproduction 2005;130 (5):627-41.
10 Woo AL, James PF, Lingrel JB. Characterization of the fourth alpha isoform of the Na,K-
ATPase. J Membr Biol 1999;169 (1):39-44.
11 Blanco G, Melton RJ, Sanchez G, Mercer RW. Functional characterization of a testes-specific
alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase. Biochemistry 1999;38
12 Jimenez T, Sanchez G, Wertheimer E, Blanco G. Activity of the Na,K-ATPase alpha4 isoform is
important for membrane potential, intracellular Ca2+, and pH to maintain motility in rat
spermatozoa. Reproduction;139 (5):835-45.
13 Woo AL, James PF, Lingrel JB. Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+
exchanger in sperm motility. Mol Reprod Dev 2002;62 (3):348-56.
14 Sanchez G, Nguyen AN, Timmerberg B, Tash JS, Blanco G. The Na,K-ATPase alpha4 isoform
from humans has distinct enzymatic properties and is important for sperm motility. Mol Hum
Reprod 2006;12 (9):565-76.
15 Jimenez T, McDermott JP, Sanchez G, Blanco G. Na,K-ATPase alpha4 isoform is essential for
sperm fertility. Proc Natl Acad Sci U S A;108 (2):644-9.
16 Konrad L, Dietze R, Kirch U, Kirch H, Eva A, Scheiner-Bobis G. Cardiotonic steroids trigger
non-classical testosterone signaling in Sertoli cells via the alpha4 isoform of the sodium pump.
Biochim Biophys Acta;1813 (12):2118-24.
17 Rodova M, Nguyen AN, Blanco G. The transcription factor CREMtau and cAMP regulate
promoter activity of the Na,K-ATPase alpha4 isoform. Mol Reprod Dev 2006;73 (11):1435-47.
18 Keryanov S, Gardner KL. Physical mapping and characterization of the human Na,K-ATPase
isoform, ATP1A4. Gene 2002;292 (1-2):151-66.
19 Lee EC, Yu D, Martinez de Velasco J, Tessarollo L, Swing DA, Court DL, Jenkins NA,
Copeland NG. A highly efficient Escherichia coli-based chromosome engineering system adapted
for recombinogenic targeting and subcloning of BAC DNA. Genomics 2001;73 (1):56-65.
20 Jimenez T, Sanchez G, McDermott JP, Nguyen AN, Kumar TR, Blanco G. Increased expression
of the Na,K-ATPase alpha4 isoform enhances sperm motility in transgenic mice. Biol Reprod;84
21 Blanco G, Sanchez G, Melton RJ, Tourtellotte WG, Mercer RW. The alpha4 isoform of the
Na,K-ATPase is expressed in the germ cells of the testes. J Histochem Cytochem 2000;48
22 Marty MS, Chapin RE, Parks LG, Thorsrud BA. Development and maturation of the male
reproductive system. Birth Defects Res B Dev Reprod Toxicol 2003;68 (2):125-36.
23 Matson CK, Zarkower D. Sex and the singular DM domain: insights into sexual regulation,
evolution and plasticity. Nat Rev Genet;13 (3):163-74.
24 Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun
RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 2004;36
25 Fujiwara Y, Komiya T, Kawabata H, Sato M, Fujimoto H, Furusawa M, Noce T. Isolation of a
DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific
expression in germ cell lineage. Proc Natl Acad Sci U S A 1994;91 (25):12258-62.
26 Chennathukuzhi V, Morales CR, El-Alfy M, Hecht NB. The kinesin KIF17b and RNA-binding
protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in
male germ cells. Proc Natl Acad Sci U S A 2003;100 (26):15566-71.
27 Shamraj OI, Lingrel JB. A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in
testis. Proc Natl Acad Sci U S A 1994;91 (26):12952-6.
28 Hlivko JT, Chakraborty S, Hlivko TJ, Sengupta A, James PF. The human Na,K-ATPase alpha 4
isoform is a ouabain-sensitive alpha isoform that is expressed in sperm. Mol Reprod Dev 2006;73
29 Underhill DA, Canfield VA, Dahl JP, Gros P, Levenson R. The Na,K-ATPase alpha4 gene
(Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene
(Atp1a2) on mouse chromosome 1. Biochemistry 1999;38 (45):14746-51.
30 Kosir R, Juvan P, Perse M, Budefeld T, Majdic G, Fink M, Sassone-Corsi P, Rozman D. Novel
insights into the downstream pathways and targets controlled by transcription factors CREM in
the testis. PLoS One;7 (2):e31798.
Figure 1. Knock in of GFP into the Na,K-Atpase Atp1a4 gene. A) Scheme showing the targeting strategy
used to introduce GFP into the Atp1a4 locus. B) Southern blot strategy employed to screen ES cell
clones. Identification of positive ES cell clones was performed using probes that specifically hybridized
to regions flanking the targeted α4 region. Wild-type (WT) and α4-knockin clones (Atp1a4null(GFP)
exhibited the predicted DNA bands of 17.0 kb and 7.9 kb for the 3’ end and 17.0 kb and 4.9 kb for the 5’
end. 1C2, 2G5, 2E12 adn 2G11 represent the different cell clones. As negative controls, genomic DNA
from the parental line, EDJ22, was used.
Figure 2. Characterization of founder lines of the Atp1a4null(GFP) mice. A) PCR genotyping of mice was
performed on genomic DNA extracted from tail clips of the mice. Distinct products of 405 and 531 bp
correspond to wild-type and Atp1a4null(GFP) mice respectively. Samples containing wild-type DNA spiked
with the targeting vector was used as a positive control, whereas samples with no DNA were used as a
negative control. B) Immunoblot analysis of GFP expression in sperm lysates from wild type and
homozygous Atp1a4null(GFP) mice. Anti-α4 antiserum generated in chicken and anti-α1 antiserum generated
in rabbit, followed by horseradish peroxidase conjugated secondary antibodies and chemiluminescence
were used for detection. Tubulin was used as a loading control.
Figure 3. Sperm from Atp1a4null(GFP) mice lack Na,K-ATPase α4 activity. A) Dose-response curves for the
inhibition of Na,K-ATPase activity by ouabain. Assays were performed on testes homogenates, using 10
μg total protein, at saturating concentrations of all physiological ligands of the Na,K-ATPase, in the
presence of the indicated concentrations of ouabain. The curves represent the best fit of the experimental
data to highly ouabain-sensitive and ouabain-resistant α4 and α1 isoforms of the Na,K-ATPase. Each
value is the mean ± SEM of three experiments. B) Maximal Na,K-ATPase activity for α4 and α1 isoforms
in wild type and Atp1a4null(GFP) mice. Activity of the α4 and α1 isoforms was distinguished based on the
difference in the ouabain affinity that characterizes these isoforms. Total Na,K-ATPase activity
corresponded to that inhibitable by 10−3 M ouabain. Hydrolysis of ATP by α4 was determined as that
sensitive to 10−6 M ouabain. Activity of α1 corresponded to the difference in ATP hydrolysis obtained at
10−6 and 10−3 M ouabain.
Figure 4. Sperm from Atp1a4null(GFP) mice show morphological and functional abnormalities. A-C) Sperm
from Atp1a4null(GFP) mice are asthenoospermic. Sperm in modified Tyrode’s medium was subjected to
CASA analysis to determine sperm total (A), progressive (B) and hyperactive motility. Bars represent the
mean ± SEM of three experiments. Values significantly different from the controls are indicated with an
asterisk, with P values ranging between 0.05 and 0.001. D) Sperm from Atp1a4null(GFP) mice present
flagellar bending. Sperm was fixed in buffered formalin phosphate, permeabilized with Triton X-100 and
labeled with anti-GFP antibody and Alexa 488 conjugated goat anti-rabbit IgG. Images were obtained
using a Nikon Eclipse 80i scope (Nikon Instruments, Melville, NY) under a 40X objective.
Figure 5. GFP is expressed in testis of Atp1a4null(GFP) mice. (A-C) Immunoblot analysis of GFP in different
tissues from adult, 18 dpp and 7 dpp Atp1a4null(GFP) mice. A total of 50 μg of protein per sample was
loaded onto a SDS-PAGE gel, transferred to a nitrocellulose membrane and analyzed with anti-GFP
antibody. Tubulin was used as a loading control. Representative gels are shown. (D) Immunochemical
analysis of GFP in embryos. Whole body sections from 16 day old wild type and Atp1a4null(GFP) mouse
embryos were fixed, embedded in paraffin and sectioned. GFP was detected by immunocytochemistry,
using anti-GFP antibody, followed by biotinylated goat anti-rabbit antiserum the Vectastain ABC Kit.
Hematoxylin was used as a counterstain.
Figure 6. Testis and epididymis of Atp1a4null(GFP) mouse show GFP fluorescence. Tissues were dissected
and whole mount light and fluorescent microscopy for testis (A) and epididymis (B) from 7 dpp, 18 dpp
and adult mice was determined. Samples were examined using a Leica MZFLIII stereomicroscope (Leica
Microsystems, Buffalo Grove, IL) and images were captured with an Olympus DP72 digital camera.
Figure 7. GFP is expressed in male germ cells of the testis and in spermatozoa in the epididymis of
Atp1a4null(GFP) mice. (A, B) Testis from 7 dpp, 18 dpp and adult mice were fixed overnight in Bouin’s
fixative and embedded in paraffin. Tissue section was performed using a Leica CM3050S cryostat.
Samples were hydrated, permeabilized with 0.3% Triton X-100, and quenched with 3% H2O2. Samples
were labeled with anti-GFP overnight followed by incubating with a biotinylated goat anti-rabbit IgG.
Staining and detection was performed following manufactures instruction (Vectastain ABC Kit, Vector
laboratories, Burlingame, CA). Samples were counter stained with hematoxylin, dehydrated, and mounted
in Permount Mounting Media (Sigma Chemical Co, St. Louis, MO). Images were captured using a Nikon
Eclipse 80i scope (Nikon Instruments, Melville, NY), using a 40 X (A) or 100 X (B) objective. (C)
Epididymis from wild type and Atp1a4null(GFP) mice were dissected and processed for
immunocytochemistry, using an anti-GFP antibody and fluorescently labeled secondary antibodies. DAPI
was included to stain the cell nuclei. Clear field images were obtained using regular light microscopy.
Figure 8. GFP is expressed in male germ cells of the testis late in spermatogenesis. Cells were isolated
from adult testis of wild type and Atp1a4null(GFP) mice and subjected to immunoblot analysis, before (A) or
after sorting (B), based on their GFP fluorescence at 488 nm using FACS. Blots were probed using
antibodies against GFP, Dmrt1, PLZF, DDX4 and KIF17b. Tubulin was also determined in the samples
as a control for gel loading.