Starfish oocytes form intracellular ice at unusually high temperatures

Starfish oocytes, eggs, and embryos are popular models for studying meiotic maturation, fertilization, and embryonic development. Their large (170- to 200-μm) oocytes are obtainable in copious amounts and are amenable to manipulations that mammalian oocytes are not. The most formidable obstacle to working with marine oocytes is their seasonal availability, yet a successful means of preserving them for use during the nonreproductive season has not been reported. The aim of this study was to investigate the response of starfish oocytes to freezing with rapid and slow cooling rates under a variety of conditions to develop a cryopreservation protocol for these cells. Cryomicroscopic observation revealed that starfish oocytes in isotonic medium undergo intracellular ice formation (IIF) at very high subzero temperatures, such that the mean difference between the temperature of extracellular ice formation (T_EIF) and IIF (T_11F) was less than 3°C and the average T_11F was approximately between -4 and -6°C. Neither partial cellular dehydration nor addition of the cryopreservative dimethyl sulfoxide significantly depressed the T_11F. Under some conditions, we observed ice nucleation at multiple locations within the cytoplasm, suggesting that several factors contribute to the unusually high T_11F during controlled-rate freezing and thus vitrification may be a more suitable method for cryopreserving these cells.