Glycerol permeability of human spermatozoa and its activation energy

Abstract

Glycerol has commonly been employed as a cryoprotectant in cryopreservation of human spermatozoa. However, the addition of glycerol into the sperm before freezing and the removal of glycerol from the sperm after freezing and thawing result in anisotonic environments to the cells, which can cause cell injury. To define optimal procedures for the addition/removal of glycerol and to minimize the cell injury, one needs to know the kinetics of glycerol permeation across the sperm plasma membrane at different temperatures. For this, one has to determine the permeability coefficient of glycerol ( P g) and its activation energy ( E a). Values of P g at different temperatures and at different glycerol concentrations were determined by measuring the time required for 50% spermolysis in hyperosmotic glycerol solutions which were hypotonic with respect to electrolytes. Value of the E a was determined assuming an Arrhenius type temperature dependence of P g. A dual fluorescent staining technique (propidium iodide and 6-carboxyfluoroscein diacetate) and flow cytometry were used to measure the spermolysis. The values of P g in 0.5, 1.0, 1.5, and 2.0 M glycerol at 22 °C are 1.62, 1.88, 1.68, and 1.54 × 10 −3 cm/min, respectively. The values of P g in 1 M glycerol at 0, 8, 22, and 30 °C are 0.33, 0.54, 1.88, and 2.60 × 10 −3 cm/min, respectively. The value of E a is 11.76 kcal/mol.