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 [15
]. 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 [15
] 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 [20
]. 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 [15
] 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
]. 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
]. 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 [9
], the other identified α4 mRNA and protein in the male gonad at four and six weeks of age respectively [8
]. 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 [16
]. 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
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. ), 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 [9
]. 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 [17
], a transcription factor that is a master controller of post-meiotically activated genes of male germ cells [30
]. 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
]. 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. ). 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.