Water transport and estimated transmembrane potential during freezing of mouse oocytes

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Abstract
The kinetics of water transport and the changes in transmembrane potential during freezing of mouse oocytes in isotonic phosphate buffered saline (PBS) were simulated using thermodynamic models. The permeability to water at 0°C,L pg , and the activation energy,E Lp , of metaphase II mouse oocytes from B6D2F1 mice were determined to be 0.044±0.008 μm/minatm and 13.3±2.5 kcal/mol during freezing at 2°C/min. The inactive cell volume was determined to be 0.214 with a correlation coefficient of 0.995, indicating that the oocytes closely follow the ideal Boyle-van't Hoff relation. The mean value of the oocyte diameter was 79.41±4.62 μm. These results were used to predict the behavior of mouse oocytes under various freezing conditions. The effect of the cooling rate on the cell volume and cytoplasm undercooling was investigated. The changes in transmembrane potential were also investigated during freezing of mouse oocytes. The computer simulations showed that at the beginning of the freezing process (−1°C), the fast growth of ice in the extracellular solution causes a sharp increase of the membrane potential. It is predicted that the change in membrane potential is substantial for almost all cooling rates. Estimations show that values as high as −90 mV may be reached during freezing. The hyperpolarization of the membrane may cause orientation of the dipoles within the membrane. For membrane proteins with 300 debye dipole moment, the theoretical prediction suggests that the percentage of dipoles aligned with the membrane potential increases from 16% at 0°C prior to freezing to 58% at −8°C after seeding of the external ice followed with a cooling at 120°C/min.