Green fluorescence protein driven by the Na,K-ATPase α4 isoform promoter is expressed only in male germ cells of mouse testis

Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA.
Journal of Assisted Reproduction and Genetics (Impact Factor: 1.72). 11/2012; 29(12). DOI: 10.1007/s10815-012-9876-x
Source: PubMed


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.

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 (Atp1a4 ( null(GFP) ) mice).

Replacement of α4 by GFP completely disrupted α4 expression and activity, produced sperm morphological and functional abnormalities, and caused infertility of Atp1a4 ( null(GFP) ) male mice. Immunoblot analysis of Atp1a4 ( null(GFP) ) mouse tissues showed GFP expression in testis. 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 Atp1a4 ( null(GFP) ) mouse testis displayed the typical fluorescence of GFP. Immunocytochemistry of testis 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. While epididymis showed GFP expression, this was confined to the spermatozoa within the epididymal tubules.

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.

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    • "For each sample (kidney and sperm) a total of twenty clones, from two biological replicates, were analyzed by bisulfite sequencing. 4 D.L. Kumar et al. / Gene xxx (2015) xxx–xxx Please cite this article as: Kumar, D.L., et al., Methylation-dependent and independent regulatory regions in the Na,K-ATPase alpha4 (Atp1a4) gene may impact its testis-specific expression, Gene (2015), may be involved in the tissue-specific expression of this gene in human and mouse (Blanco et al., 2006; McDermott et al., 2012), we choose the −700 bp to +200 bp region of Atp1a4 (corresponding to the genome coordinates Chr1:174,188,355–174,189,255 of the mm9 mouse genome assembly) for analysis as a promoter (Mα4-Promoter). Although our analysis of Atp1a4 did not uncover a cluster of CpG dinucleotides characteristic of a CpG island in the Mα4-Promoter, this region does contain individual CpG dinucleotides that could potentially play an important role in control of Atp1a4 expression. "
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    ABSTRACT: The α4 Na,K-ATPase is a sperm-specific protein essential for sperm motility and fertility yet little is known about the mechanisms that regulate its expression in germ cells. Here, the potential involvement of DNA methylation in regulating the expression of this sperm-specific protein is explored. A single, intragenic CpG island (Mα4-CGI) was identified in the gene encoding the mouse α4 Na,K-ATPase (Atp1a4), which displayed reduced methylation in mouse sperm (cells that contain α4) compared to mouse kidney (tissue that lacks α4 expression). Unlike the intragenic CGI, the putative promoter (the -700 to +200 region relative to the transcriptional start site) of Atp1a4 did not show differential methylation between kidney and sperm nevertheless it did drive methylation-dependent reporter gene expression in the male germ cell line GC-1spg. Furthermore, treatment of GC-1spg cells with 5-Aza2-Deoxycytidine led to upregulation of the α4 transcript and decreased methylation of both the Atp1a4 promoter and the Mα4-CGI. In addition, Atp1a4 expression in mouse embryonic stem cells deficient in DNA methytransferases suggests that both maintenance and de novo methylation are involved in regulating its expression. In an attempt to define the regulatory function of the Mα4-CGI, possible roles of the Mα4-CGI in regulating Atp1a4 expression via methylation-dependent transcriptional elongation inhibition in somatic cells and via its ability to repress promoter activity in germ cells were uncovered. In all, our data suggests that both the promoter and the intragenic CGI could combine to provide multiple modes of regulation for optimizing the Atp1a4 expression level in a cell type-specific manner.
    Gene 09/2015; DOI:10.1016/j.gene.2015.09.003 · 2.14 Impact Factor
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    ABSTRACT: Modeling neurological disorders using zebrafish increases rapidly as this model system allows easy access to all developmental stages and imaging of pathological processes. A surprising degree of functional conservation has been demonstrated between human genes implicated in neurodegenerative diseases and their zebrafish orthologues. Zebrafish offers rapid high throughput screening of therapeutic compounds and live imaging of pathogenic mechanisms in vivo. Several recent zebrafish studies functionally assessed the role of the sodium-potassium pump (Na(+)/K(+)-ATPase). The Na(+)/K(+)-ATPase maintains the electrochemical gradients across the plasma membrane, essential for e.g. signaling, secondary active transport, glutamate re-uptake and neuron excitability in animal cells. Na(+)/K(+)-ATPase mutations are associated with neurological disorders, where mutations in the Na(+)/K(+)-ATPase α2 and α3 isoforms cause Familial hemiplegic migraine type 2 (FHM2) and Rapid-onset dystonia-parkinsonism (RDP)/Alternating hemiplegic childhood (AHC), respectively. In zebrafish, knock-down of Na(+)/K(+)-ATPase isoforms included skeletal and heart muscle defects, impaired embryonic motility, depolarized Rohon-beard neurons and abrupt brain ventricle development. In this review, we discuss zebrafish as a model to assess Na(+)/K(+)-ATPase isoform functions. Furthermore, studies investigating proteomic changes in both α2- and α3- isoform deficient embryos and their potential connections to the Na(+)/K(+)-ATPase functions will be discussed.
    Neuroscience & Biobehavioral Reviews 09/2013; 37(10). DOI:10.1016/j.neubiorev.2013.09.013 · 8.80 Impact Factor
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    ABSTRACT: Most of our knowledge on the biological role of the testis-specific Na,K-ATPase alpha 4 isoform derives from studies performed in non-human species. Here, we studied the function of human Na,K-ATPase alpha 4 after its expression in transgenic mice. Using a bacterial artificial chromosome (BAC) construct containing the human ATP1A4 gene locus, we obtained expression of the human α4 transgene specifically in mouse sperm testis and, in the sperm flagellum. The expressed human alpha 4 was active, and compared to wild-type sperm, those from transgenic mice displayed higher Na,K-ATPase alpha 4 activity and greater binding of fluorescently labeled ouabain, which is typical of the alpha 4 isoform. The expression and activity of endogenous alpha 4 and the other Na,K-ATPase alpha isoform present in sperm, alpha 1, remained unchanged. Male mice expressing the human ATP1A4 transgene exhibited similar testis size and morphology, normal sperm number and shape, and no changes in overall fertility compared to wild-type mice. Sperm carrying the human transgene exhibited enhanced total motility and an increase in multiple parameters of sperm movement, including higher sperm hyperactive motility. In contrast, no statistically significant changes in sperm membrane potential, protein tyrosine phosphorylation, or spontaneous acrosome reaction were found between wild-type and transgenic mice. Altogether, these results provide new genetic evidence for an important role of human Na,K-ATPase alpha 4 in sperm motility and hyperactivation, and establishes a new animal model for future studies of this isoform. Mol. Reprod. Dev. 2015. © 2015 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 01/2015; 82(3). DOI:10.1002/mrd.22454 · 2.53 Impact Factor
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