Determination of the water permeability (Lp) of mouse oocytes at -25 degrees C and its activation energy at subzero temperatures.

Typically, subzero permeability measurements are experimentally difficult and infrequently reported. Here we report an approach we have applied to mouse oocytes. Interrupted cooling involves rapidly cooling oocytes (50 degrees C/min) to an intermediate temperature above the intracellular nucleation zone, holding them for up to 40 min while they dehydrate, and then rapidly cooling them to -70 degrees C or below. If the intermediate holding temperature and holding time are well chosen, high post thaw survival of the oocytes is possible because the freezable water is removed during the hold. The length of time required for the exit of the freezable water allows the water permeability at that temperature to be determined. These experiments used 1.5M ethylene glycol in PBS and included a transient hold of 2 min for equilibration at -10 degrees C, just below the extracellar ice formation temperature. We obtain an Lp=1.8 x 10(-3)microm min(-1)atm(-1) at -25 degrees C based on a hold time of 30 min yielding 80% survival and the premise that most of the freezable water is removed during the 30 min hold. If we assume that the water permeability is a continuous function of temperature and that its Ea changes at 0 degrees C, we obtain a subzero Ea of 21 kcal/mol; higher than the suprazero value of 14 kcal/mol. A number of assumptions are required for these water loss calculations and the resulting value of Lp can vary by up to a factor of 2, depending on the choices make.