Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry

Abstract

Intracellular ice formation (IIF) plays a central role in cell damage during cryopreservation. We are investigating the factors which trigger IIF in Xenopus oocytes, with and without aquaporin water channels. Here, we report differential scanning calorimeter studies of Xenopus control oocytes which do not express aquaporins. Stage I to VI oocytes (which increase progressively in size) were investigated with emphasis on stage I and II because they are translucent and can also be studied under the cryomicroscope. Measurements were made in 1, 1.5, and 2 M ethylene glycol (EG) in frog Ringers plus SnoMax. A multistep freezing protocol was used in which the samples were cooled until extracellular ice formation (EIF) occurred, partially remelted, slowly recooled through the EIF temperature, and then rapidly (10 °C/min) cooled. EIF in the 1, 1.5, and 2 M EG occurred at −6.4, −7.8, and −8.9 °C, respectively. Freezing exotherms of individual stage I–VI oocytes were readily visible. A general trend was observed in which the IIF temperature of the early stage oocytes (I–III) was well below T EIF while the later stages (IV–VI) froze at temperatures much closer to T EIF. Thus, in 1.5 M EG, T IIF was −21.1, −25, and −26.6 °C in stages I–III, but was −17 and −8.5 °C for stage IV and V–VI. Concurrently, the percentage of oocytes in which IIF was observed fell dramatically from a high of 40 to 72% in early stages (I–III) to a low of only 7% in stage V–VI because, particularly in the later stages, IIF was hidden in the EIF exotherm. We conclude that early stage oocytes are a good model system in which to investigate modulators of IIF, but that late stage oocytes are damaged during EIF and infrequently supercool.