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

Abstract

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 (Atp1a4 null(GFP) mice). Results 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.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.

Identification of a VxP Targeting Signal in the Flagellar Na+/K+‐ATPase

“…To test if this region is involved in setting the differential localization, a set of chimeras with this region exchanged were analyzed. GFP‐α3 /α4 and GFP‐α4 /α3, were localized in patterns opposite to that of the parent constructs demonstrating that the targeting information lies within the intrinsically disordered region (Figure A,B). A subsequent pair of chimeras with only 24 amino acids substituted, GFP‐α3 /α4 and GFP‐α4 /α3, was generated and found to also have reversed localization patterns compared with the parent constructs (Figure C,D).…”

Methylation-dependent and independent regulatory regions in the Na,K-ATPase alpha4 (Atp1a4) gene may impact its testis-specific expression

“…As many promoters are positioned within the region between −700bp and +200bp relative to the TSS (Weber et al, 2007) and because this region is included in those that previous studies suggest 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 −700bp to +200bp 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).…”

Cardiotonic steroid ouabain stimulates expression of blood–testis barrier proteins claudin-1 and -11 and formation of tight junctions in Sertoli cells

“…This isoform has been exclusively detected in testes and was demonstrated specifically in spermatocytes and mature sperm (Blanco et al, 2000;Shamraj and Lingrel, 1994;Wagoner et al, 2005;Woo et al, 1999). Its function has been associated with fertility and sperm motility (Jimenez et al, 2011;Sanchez et al, 2006;Woo et al, 2000Woo et al, , 2002, and male α4 knockout mice produced sperm with morphological and functional abnormalities (and these animals are infertile) (McDermott et al, 2012). Interestingly, however, in a recent publication the α4 isoform of Na + ,K + -ATPase was shown to be involved in signalling events during capacitation of bovine sperm (Newton et al, 2010), so its physiological function in germ cells might go beyond mere involvement in cell motility.…”