Gamete Biology

Journal of Assisted Reproduction and Genetics

, Volume 29, Issue 12, pp 1313-1325

First online:

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

  • Jeffrey P. McDermottAffiliated withDepartment of Molecular and Integrative Physiology, University of Kansas Medical Center
  • , Gladis SánchezAffiliated withDepartment of Molecular and Integrative Physiology, University of Kansas Medical Center
  • , Vargheese ChennathukuzhiAffiliated withDepartment of Molecular and Integrative Physiology, University of Kansas Medical Center
  • , Gustavo BlancoAffiliated withDepartment of Molecular and Integrative Physiology, University of Kansas Medical Center Email author 

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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.

Keywords

Testis Male germ cells Spermatogenesis Spermatozoa Sertoli cells