Osmotic tolerance and intracellular ion concentrations of bovine sperm are affected by cryopreservation

In this study, the effects of cryopreservation on osmoregulation and ion homeostasis in bovine sperm were studied. We determined: (1) the osmotic tolerance limits and cell volume response upon exposure to anisotonic conditions, (2) the intracellular pH and potassium concentration, and (3) expression and localization of proteins encoding for potassium and chloride ion channels. A flow cytometric approach was used for simultaneous assessment of cell volume and viability of propidium iodide stained sperm in anisotonic media. Osmotic tolerance was found to be decreased after cryopreservation, especially in the 120 to 60 mOsm/kg osmotic range. The critical osmolality at which half of the sperm population survived increased from 55 to 89 mOsm/kg. The osmotic cell volume response for viable sperm was similar before and after cryopreservation, with an osmotic inactive volume of about 70%. The intracellular pH, determined by recording changes in carboxyfluorescein fluorescence of sperm in media with different pH before and after addition of digitonin, decreased from 6.28 in diluted sperm to 6.16 after cryopreservation. The intracellular potassium concentration, determined using the potassium ionophore nigericin and incubation in media with various potassium concentrations, increased from 154 mM to 183 mM before and after cryopreservation, respectively. The levels of the chloride and potassium ion channel proteins chloride channel 3 protein (CLC-3) and two pore domain potassium channel 2 protein (TASK-2), as detected using Western blot analysis, were not affected by cryopreservation. Immunolocalization studies showed that CLC-3 is present in the acrosome and midpiece as well as in the upper and lower tail. In conclusion, cryopreserved sperm exhibit reduced tolerance to hypotonic stress, a decreased intracellular pH, and increased intracellular potassium level.