Different expression and activity of the α1 and α4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny

Abstract

Two catalytic isoforms of the Na,K-ATPase, a1 and a4, are present in testis. While a1 is ubiquitously expressed in tissues, a4 predominates in male germ cells. Each isoform has distinct enzymatic properties and appears to play specific roles. To gain insight into the relevance of the Na,K-ATPase a isoforms in male germ cell biology, we have studied the expression and activity of a1 and a4 during spermatogenesis and epididymal maturation. This was explored in rat testes at different ages, in isolated spermatogenic cells and in spermatozoa from the caput and caudal regions of the epididymis. Our results show that a1 and a4 undergo differential regulation during development. Whereas a1 exhibits only modest changes, a4 increases with gamete differentiation. The most drastic changes for a4 take place in spermatocytes at the mRNA level, and with the transition of round spermatids into spermatozoa for expression and activity of the protein. No further changes are detected during transit of spermatozoa through the epididymis. In addition, the cellular distribution of a4 is modified with development, being diffusely expressed at the plasma membrane and intracellular compartments of immature cells, finally to localize to the midregion of the spermatozoon flagellum. In contrast, the a1 isoform is evenly present along the plasma membrane of the developing and mature gametes. In conclusion, the Na,K-ATPase a1 and a4 isoforms are functional in diploid, meiotic and haploid male germ cells, a4 being significantly upregulated during spermatogenesis. These results support the importance of a4 in male gamete differentiation and function.

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

“…cDNA was generated by reverse transcription using the SuperScript First-Strand Synthesis System (Invitrogen) and oligo (dT) primers as described previously (33). The resulting first-strand cDNA was amplified using Na,K-ATPase isoform specific primers and under PCR conditions that ensure no cross-reactivity among the Na,K-ATPase isoforms.…”

“…Cell proteins (30 g) from homogenates of NHK and ADPKD cells that were grown to confluence on filter supports or plastic were analyzed by 8% SDS-PAGE for the ␣ and ␤ isoforms or 16% tricine gels for the ␥ subunit and blotted onto nitrocellulose membranes (Osmonics, Minnetonka, MN) as described (33,34). Primary antibodies that are specific for each Na,K-ATPase isoform were incubated overnight at 4°C.…”

“…Dose-response relations for the ouabain inhibition of Na,K-ATPase activity were fitted by Table 1. Characteristics of the primers that were used for RT-PCR analysis of Na,K-ATPase isoforms in normal and cystic human kidney epithelial cells equations assuming the presence of one or two enzyme populations with different affinity for ouabain as described previously (33). The validity of using a two versus a single component regression model for ouabain binding was statistically supported by applying the Snedecor F test (36).…”

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

“…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 [12].…”

“…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.…”