Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium—A revisit

Abstract

We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to −50°C or below occurs at temperatures where a critical fraction of the external water remains unfrozen (Cryobiology 51, 2005, 29-53; 54, 2007, 223-233). For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. (Cryobiology 38,1999, 403-407). Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic (Cryobiology 54, 2007, 212-222). Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. (Cryobiology 52, 2006, 401-416) on IIF in oocytes of the frog Xenopus. It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol. KeywordsIntracellular ice formation; oocytes; mouse; Xenopus; unfrozen fraction; phase diagrams Our laboratory has recently reported on several variables that influence the temperature at which intracellular ice forms in mouse oocytes and in oocytes of the frog Xenopus. [5,1] that are cooled rapidly enough to ensure that they undergo intracellular ice formation (IIF); namely 20°C/min and 10°C/min, respectively. One major variable affecting the temperature of IIFin mouse oocytes was the concentration of ethylene glycol (EG) or glycerol present in the *Corresponding author. Fax: +1 865 974 8027 e-mail address: pmazur@utk.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The bottom three lines of Table 1 and the bottom half of Table 2 show the computed values for the right-hand five columns based on the synthesized ternary phase diagrams [3]. The chief conclus...

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