Aquaporins in the human testis and spermatozoa - identification, involvement in sperm volume regulation and clinical relevance.

Despite the high water-permeability of human spermatozoa, little is known about the identity and the role of aquaporins (AQP) in them or germ cells. Using ejaculates from donors, sperm AQPs were identified by western blotting followed by the analysis of mRNA with RT-PCR. Protein expression in the testis and spermatozoa was localized by immunocytochemistry. Inhibitors were used to investigate the involvement of aquaporins in water transport when ejaculated spermatozoa were swollen in medium mimicking uterine hypo-osmolality by quinine that blocks volume regulation. Sperm AQP7 and AQP8 in 39 infertile patients and 11 healthy donors were quantified by flow cytometry. AQP1 was absent from spermatozoa. Sperm and testicular AQP7-9 had nucleotide sequences identical to those of somatic cells but AQP8 mRNA also showed shorter variants. AQP7 was expressed abundantly by round and elongated spermatids and ejaculated spermatozoa, AQP8 by all germ cells and spermatozoa, and AQP9 rarely by spermatocytes or Sertoli cells. Protein bands showed specificity by western blotting for AQP7 and AQP8 but not AQP9. The absence of sperm AQP9 was further suggested by the ineffectiveness of its inhibitor phloretin in blocking quinine-induced swelling, but HgCl(2,) which inhibits AQP8, was effective. Sperm AQP7 expression was correlated with progressive motility and was lower in patients than in donors. Sperm AQP8 expression was inversely correlated with the extent of sperm coiling, which is a swelling phenomenon, but showed no difference between patients and donors. In conclusion, AQP7 and AQP8 were identified in human spermatozoa and could play a role in glycerol metabolism and water transport respectively.

Potassium softens vascular endothelium and increases nitric oxide release.

In the presence of aldosterone, plasma sodium in the high physiological range stiffens endothelial cells and reduces the release of nitric oxide. We now demonstrate effects of extracellular potassium on stiffness of individual cultured bovine aortic endothelial cells by using the tip of an atomic force microscope as a mechanical nanosensor. An acute increase of potassium in the physiological range swells and softens the endothelial cell and increases the release of nitric oxide. A high physiological sodium concentration, in the presence of aldosterone, prevents these changes. We propose that the potassium effects are caused by submembranous cortical fluidization because cortical actin depolymerization induced by cytochalasin D mimics the effect of high potassium. In contrast, a low dose of trypsin, known to activate sodium influx through epithelial sodium channels, stiffens the submembranous cell cortex. Obviously, the cortical actin cytoskeleton switches from gelation to solation depending on the ambient sodium and potassium concentrations, whereas the center of the cell is not involved. Such a mechanism would control endothelial deformability and nitric oxide release, and thus influence systemic blood pressure.

Channels for water efflux and influx involved in volume regulation of murine spermatozoa.

The nature of the membrane channels mediating water transport in murine spermatozoa adjusting to anisotonic conditions was investigated. The volume of spermatozoa subjected to physiologically relevant hypotonic conditions either simultaneously, or after isotonic pre-incubation, with putative water transport inhibitors was monitored. Experiments in which quinine prevented osmolyte efflux, and thus regulatory volume decrease (RVD), revealed whether water influx or efflux was being inhibited. There was no evidence that sodium-dependent solute transporters or facilitative glucose transporters were involved in water transport during RVD of murine spermatozoa since phloretin, cytochalasin B and phloridzin had no effect on volume regulation. However, there was evidence that Hg(2+)- and Ag(+)-sensitive channels were involved in water transport and the possibility that they include aquaporin 8 is discussed. Toxic effects of these heavy metals were ruled out by evidence that mitochondrial poisons had no such effect on volume regulation.

Mutation analysis of the X-chromosome linked, testis-specific TAF7L gene in spermatogenic failure.

The precise temporal and spatial expressions of specific transcription regulation factors (TRF) have long been considered essential for spermatogenesis. Recently, it has been speculated that mammals have evolved more specialised TRF genes. In the human, the TAF7L gene may be essential for maintenance of spermatogenesis. In this study, we investigated the possible role of the TAF7L gene located on the X chromosome in testicular function and spermatogenic failure. In a case-controlled retrospective study, we recruited 16 infertile males with consistent, nonobstructive azoospermia and with normal serum follicle-stimulating hormone (FSH) levels. Twenty age-matched men with normal spermatogenesis with the same ethnic background (Caucasian) were recruited as controls. Their genomic DNA was screened for sequence changes in the coding regions and part of the flanking introns of the TAF7L gene by direct sequencing. Amino acid sequence was compared with the NCBI standard sequence (BC043391). Semen analysis and hormone evaluation were performed. We observed six sequence variations in four patients, consisting of two point mutations, one each in exon 9 and 13 and one six-basepair deletion in exon 13 with concomitant changes in amino acid. One additional nucleotide exchange was observed in intron 8. Most of these changes were also found in eight controls with the exception of changes in exon 13. A meta-analysis including the present study and literature data suggests a possible association of the point mutation in exon 13 with infertility. There was no association or relationship with reproductive hormones. In conclusion, the sequence variants in the cDNA sequence observed are common polymorphisms. The changes in intron 8 appear novel. We report for the first time that most of the alterations are not associated with gonadal dysfunction, while the sequence variant in exon 13 may represent a risk factor for spermatogenic failure.