Common as well as unique methylation-sensitive DNA regulatory elements in three mammalian SLC9C1 genes.

The SLC9C1 gene (which encodes the NHE10 protein) is essential for male fertility in both mice and humans, however the epigenetic mechanisms regulating its testis/sperm-specific gene expression have yet to be studied. Here we identify and characterize DNA regulatory elements of the SLC9C1 gene across three mammalian species: mouse, rat, and human. First, in silico analysis of these mammalian SLC9C1 genes identified a CpG island located upstream of the transcription start site in the same relative position in all three genes. Further analysis reveals that this CpG island behaves differently, with respect to gene regulatory activity, in the mouse SLC9C1 gene than it does in the rat and human SLC9C1 gene. The mouse SLC9C1 CpG island displays strong promoter activity by itself and seems to have a stronger gene regulatory effect than either the rat or human SLC9C1 CpG islands. While the function of the upstream SLC9C1 CpG island may be divergent across the three studied species, it appears that the promoters of these three mammalian SLC9C1 genes share similar DNA methylation-sensitive regulatory mechanisms. All three SLC9C1 promoter regions are differentially methylated in lung and testis, being more hypermethylated in lung relative to the testis, and DNA sequence alignments provide strong evidence of primary sequence conservation. Luciferase assays reveal that in vitro methylation of constructs containing different elements of the three SLC9C1 genes largely exhibit methylation-sensitive promoter activity (reduced promoter activity when methylated) in both HEK 293 and GC-1spg cells. In total, our data suggest that the DNA methylation-sensitive elements of the mouse, rat, and human SLC9C1 promoters are largely conserved, while the upstream SLC9C1 CpG island common to all three species seems to perform a different function in mouse than it does in rat and human. This work provides evidence that while homologous genes can all be regulated by DNA methylation-dependent epigenetic mechanisms, the location of the specific cis-regulatory elements responsible for this regulation can differ across species.

Na+/H+ Exchangers (NHEs) in Mammalian Sperm: Essential Contributors to Male Fertility.

Na+/H+ exchangers (NHEs) are known to be important regulators of pH in multiple intracellular compartments of eukaryotic cells. Sperm function is especially dependent on changes in pH and thus it has been postulated that NHEs play important roles in regulating the intracellular pH of these cells. For example, in order to achieve fertilization, mature sperm must maintain a basal pH in the male reproductive tract and then alkalize in response to specific signals in the female reproductive tract during the capacitation process. Eight NHE isoforms are expressed in mammalian testis/sperm: NHE1, NHE3, NHE5, NHE8, NHA1, NHA2, NHE10, and NHE11. These NHE isoforms are expressed at varying times during spermatogenesis and localize to different subcellular structures in developing and mature sperm where they contribute to multiple aspects of sperm physiology and male fertility including proper sperm development/morphogenesis, motility, capacitation, and the acrosome reaction. Previous work has provided evidence for NHE3, NHE8, NHA1, NHA2, and NHE10 being critical for male fertility in mice and NHE10 has recently been shown to be essential for male fertility in humans. In this article we review what is known about each NHE isoform expressed in mammalian sperm and discuss the physiological significance of each NHE isoform with respect to male fertility.

Common as well as unique methylation-sensitive DNA regulatory elements in three mammalian SLC9C1 genes.

The SLC9C1 gene (which encodes the NHE10 protein) is essential for male fertility in both mice and humans, however the epigenetic mechanisms regulating its testis/sperm-specific gene expression have yet to be studied. Here we identify and characterize DNA regulatory elements of the SLC9C1 gene across three mammalian species: mouse, rat, and human. First, in silico analysis of these mammalian SLC9C1 genes identified a CpG island located upstream of the transcription start site in the same relative position in all three genes. Further analysis reveals that this CpG island behaves differently, with respect to gene regulatory activity, in the mouse SLC9C1 gene than it does in the rat and human SLC9C1 gene. The mouse SLC9C1 CpG island displays strong promoter activity by itself and seems to have a stronger gene regulatory effect than either the rat or human SLC9C1 CpG islands. While the function of the upstream SLC9C1 CpG island may be divergent across the three studied species, it appears that the promoters of these three mammalian SLC9C1 genes share similar DNA methylation-sensitive regulatory mechanisms. All three SLC9C1 promoter regions are differentially methylated in lung and testis, being more hypermethylated in lung relative to the testis, and DNA sequence alignments provide strong evidence of primary sequence conservation. Luciferase assays reveal that in vitro methylation of constructs containing different elements of the three SLC9C1 genes largely exhibit methylation-sensitive promoter activity (reduced promoter activity when methylated) in both HEK 293 and GC-1spg cells. In total, our data suggest that the DNA methylation-sensitive elements of the mouse, rat, and human SLC9C1 promoters are largely conserved, while the upstream SLC9C1 CpG island common to all three species seems to perform a different function in mouse than it does in rat and human. This work provides evidence that while homologous genes can all be regulated by DNA methylation-dependent epigenetic mechanisms, the location of the specific cis-regulatory elements responsible for this regulation can differ across species.

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm.

Na+/H+ exchangers (NHEs) are a family of ion transporters that regulate the pH of various cell compartments across an array of cell types. In eukaryotes, NHEs are encoded by the SLC9 gene family comprising 13 genes. SLC9C2, which encodes the NHE11 protein, is the only one of the SLC9 genes that is essentially uncharacterized. Here, we show that SLC9C2 exhibits testis/sperm-restricted expression in rats and humans, akin to its paralog SLC9C1 (NHE10). Similar to NHE10, NHE11 is predicted to contain an NHE domain, a voltage sensing domain, and finally an intracellular cyclic nucleotide binding domain. An immunofluorescence analysis of testis sections reveals that NHE11 localizes with developing acrosomal granules in spermiogenic cells in both rat and human testes. Most interestingly, NHE11 localizes to the sperm head, likely the plasma membrane overlaying the acrosome, in mature sperm from rats and humans. Therefore, NHE11 is the only known NHE to localize to the acrosomal region of the head in mature sperm cells. The physiological role of NHE11 has yet to be demonstrated but its predicted functional domains and unique localization suggests that it could modulate intracellular pH of the sperm head in response to changes in membrane potential and cyclic nucleotide concentrations that are a result of sperm capacitation events. If NHE11 is shown to be important for male fertility, it will be an attractive target for male contraceptive drugs due to its exclusive testis/sperm-specific expression.