The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (L-p), the permeability coefficients (P-s) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute L-p in the absence of permeant solutes and P-s and L-p in the presence of permeating solutes (CPAs) at 22degreesC. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.69+/-0.05 mum/min/atm (mean+/-SEM). Hydraulic conductivity (L-p) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 mum/min/atm in the presence of dimethylsulfoxide (Me2SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for P-s were estimated to be 1.72+/-0.36 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.3x10(-3) cm/min for Me2SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs. (C) 2004 Elsevier Inc. All rights reserved.
- entrapped fluorophore
- osmotic tolerance