Rupert P. Amann

Changes in ram sperm membranes during epididymal transit.

Abstract The sperm plasma membrane consists of three zones: a lipid bilayer that provides an impermeable barrier to uptake of extracellular, polar molecules; a charged lipid-water interface formed by the phospholipids and membrane-associated proteins; and a charged glycoprotein calyx with absorbed proteins. We quantitatively evaluated all three zones of membranes in testicular, cauda epididymal, and ejaculated ram sperm. Integrity of the lipid bilayer was assessed by measuring the aqueous isolated intracellular volume with water-soluble spin labels and electron spin resonance spectroscopy. Effects of rapid cooling to 0 °C (cold shock), of freezing on solid CO2, and of butylated hydroxytoluene (BHT) on membrane integrity were evaluated. Testicular, cauda epididymal, and ejaculated sperm all had an isolated intracellular volume of 20–24 μm3 per sperm. Isolated intracellular volume of testicular sperm was maintained (>90%) after cold shock, but reduced ~50% by freezing. BHT (1–3 m m ) did not protect testicular sperm against membrane damage. Cauda epididymal and ejaculated sperm retained 20–30% of their isolated intracellular volume after cold shock but not freezing. However, 4 m m BHT prevented freeze damage of ejaculated but not cauda epididymal sperm. The lipid-water interface of the membrane was studied using surface-directed spin labels; the negative charge density was found to increase during epididymal transit. Net charge of the glycoprotein calyx was detected using whole-cell isoelectric focusing. Testicular, cauda epididymal, and ejaculated sperm isofocused at pH 5.2, 4.7, and 4.8 after insertion at pH 6 into the preformed pH gradient. Mixtures of testicular plus cauda epididymal or testicular plus ejaculated sperm were separable. We conclude that each of the three zones of the plasma membrane of sperm changes during epididymal maturation.