Extra- and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes

Abstract: We are currently investigating factors that influence intracellular ice formation (IIF) in mouse oocytes and oocytes of the frog Xenopus. A major reason for choosing these two species is that while their eggs normally do not possess aquaporin channels in their plasma membranes, these channels can be made to express. We wish to see whether IIF is affected by the presence of these channels. The present Xenopus study deals with control eggs not expressing aquaporins. The main factor studied has been the effect of a cryoprotective agent [ethylene glycol (EG) or glycerol] and its concentration. The general procedure was to (a) cool the oocytes on a cryostage to slightly below the temperatures at which extracellular ice formation occurs, (b) warm them to just below the melting point, and (c) then re-cool them to −50°C at 10°C/min. In the majority of cases, IIF occurs well into step (c), but a sizeable minority undergo IIF in steps (a) or (b). The former group we refer to as low-temperature flashers; the latter as high-temperature flashers. IIF is manifested as abrupt blackening of the egg, which we refer to as “flashing.” Observations on the Linkam cryostage are restricted to Stage I and II oocytes, which have diameters of 200–300μm. In the absence of a cryoprotective agent, that is in frog Ringers, the mean flash temperature for the low-temperature freezers is −11.4°C, although a sizeable percentage flash at temperatures much closer to that of the EIF (−3.9°C). When EG is present, the flash temperature for the low-temperatures freezers drops significantly to ∼−20°C for EG concentrations ranging from 0.5 to 1.5M. The presence of 1.5M glycerol also substantially reduces the IIF temperature of the low-temperature freezers; namely, to −29°C, but 0.5 and 1M glycerol exert little or no effect. The IIF temperatures observed using the Linkam cryostage agree well with those estimated by calorimetry [F.W. Kleinhans, J.F. Guenther, D.M. Roberts, P. Mazur, Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry, Cryobiology 52 (2006) 128–138]. The IIF temperatures in Xenopus are substantially higher than those observed in mouse oocytes [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29–53]. Perhaps that is a reflection of their much larger size. [Copyright &y& Elsevier]