Direct Microscale Measurement of Mouse Oocyte Membrane Permeability to Water and Ethylene Glycol at Subzero Temperatures Using Cryomicroscopy

BACKGROUND: Investigation of cell osmotic behavior at subzero temperatures is of critical importance to the optimization of cooling procedures for cryopreservation. Based on established thermodynamic models, plasma membrane permeability coefficients for water and cryoprotectant agent (CPA) (L _p , P _CPA ) and their activation energies (E _a ^Lp , E _a ^PCPA ) are essential to predict the change of cell volume and composition of intracellular solutions corresponding to different cooling procedures. However, currently available methods to measure L _p at subzero temperatures suffer from technical difficulties due to ice formation and there are no generalized methods to measure P _CPA at subzero temperatures. OBJECTIVE: The present study aims to investigate cell osmotic behavior at subzero temperatures without ice formation. MATERIALS AND METHODS: In the study cells were directly injected into super-cooled CPA solutions mounted on a cryomicroscope, and the corresponding osmotic properties were measured. RESULTS: Using ethylene glycol (EG), the value of P _EG for mouse (CD-1) metaphase II oocytes at 0, -5, -10°C was determined to be 8.45 ± 1.20, 7.43 ± 0.91, 6.40 ± 1.10,x 10^-6 cm/min, respectively, and E _a ^PEG was calculated to be 3.9 kCal/mol. L _p in the presence of EG (L _p ^EG ) at 0, -5, -10 , -15°C was determined to be 7.0 ± 1.15, 4.90 ± 1.20, 2.44 ± 0.31, 1.20 ± 0.24, x10^-2 μm/min/atm, respectively, and E _a ^LP was calculated to be 15.5 kCal/mol. CONCLUSION: Comparing these values with those previously measured at superzero temperatures, we concluded that for mouse oocytes, the Arrhenius relationship for L _p ^EG is consistent at superzero and subzero temperatures, but the values of P _EG at subzero temperatures are much lower than the extrapolated values from the Arrhenius relationship at superzero temperatures, possibly caused by membrane phase transition at low temperatures.