The effect of hyposmotic shock on motility and calcium signalling of human spermatozoa

Chloride (Cl\(^-\)) channels are thought to be implicated in volume regulation of human spermatozoa, a process vital for diverse cell functions. The Cl\(^-\) channel blockers DIDS, NPPB and niflumic acid were utilized to evaluate the influence of Cl\(^-\) channels on motility, morphology and [Ca\(^{2+}\)]\(_i\) signaling of human spermatozoa exposed to hyposmotic shock (295, 270 and 200 mOsm, equivalent to the osmotic conditions of cervical mucus, fallopian tubes, and non physiological respectively). At 295 and 270 mOsm, 50 and 100 μM DIDS inhibited motility whereas 400 μM abolished it. 400 μM DIDS augmented the percentage of sperm with coiled tails. Conversely, 100 μM NPPB and 200 μM niflumic acid did not affect motility or the number of coiled sperm. Immunodetection studies showed that DIDS inhibited serine/threonine phosphorylation of proteins in sperm cells, suggesting that DIDS (50, 100 and 400 μM) inhibits the production of cAMP. When sperm cells were exposed to hyposmotic shock there was a substantial increase in [Ca\(^{2+}\)]\(_i\) that exhibited a biphasic behaviour, with a transient peak of fluorescence immediately followed by a plateau with levels that remain more elevated than those observed during the control period, suggesting an important role for Ca\(^{2+}\) stores in volume regulation of human sperm. This increase in [Ca\(^{2+}\)]\(_i\) was independent of extracellular Ca\(^{2+}\). All the Cl\(^-\) channel blockers caused a significant rise in [Ca\(^{2+}\)]\(_i\) on their own, but only DIDS and niflumic acid attenuated the increase in [Ca\(^{2+}\)]\(_i\) caused by osmotic stress. Thus, though Cl- channel blockers have profound effects on motility and cause flagellar angulation in human sperm, their effects on [Ca\(^{2+}\)]\(_i\) signaling are such that conclusions on the role of Cl- channels in RVD of human sperm, based upon use of Cl\(^-\)-channel blockers, must be interpreted cautiously. In addition, DIDS may be affecting the HCO\(_3\)\(^-\) / Cl\(^-\) exchanger, preventing HCO\(_3\)\(^-\) entry into the cells, iii reducing activity of soluble adenylate cyclase and suppressing phosphorylation of proteins essential for sperm motility and volume regulation