Extra- and intracellular ice formation in mouse oocytes

Area of Science:

• Cryobiology
• Cell Biology
• Biophysics

Background:

• Cell survival during cryopreservation is critically dependent on preventing intracellular ice formation (IIF).
• The temperature at which supercooled cells nucleate ice is a key determinant of IIF.
• Mouse oocytes are a model system for studying factors influencing nucleation temperature.

Purpose of the Study:

• To investigate factors influencing intracellular ice nucleation temperature in mouse oocytes.
• To determine the effect of cryoprotective additive (CPA) concentration on nucleation temperature.
• To understand the role of the zona pellucida and cell deformation in IIF.

Main Methods:

• Controlled cooling experiments on mouse oocytes with varying concentrations of glycerol and ethylene glycol.
• Observation of intracellular ice formation (IIF) events, including visual cues like blackening or flashing.
• Calculation of unfrozen water fraction and solute concentrations at observed nucleation temperatures using phase diagram data.

Main Results:

• Increasing CPA concentration (glycerol or ethylene glycol) significantly decreased the nucleation temperature for IIF.
• Nucleation temperature dropped from -14°C in PBS to -41°C in 1M glycerol or 1.5M ethylene glycol.
• Cell deformation during initial freezing, particularly in the absence of CPA, often led to IIF.

Conclusions:

• Cryoprotective additives are crucial for lowering nucleation temperature and preventing IIF in mouse oocytes.
• The zona pellucida may impede but not block external ice growth, influencing IIF.
• Further research is needed to clarify the correlation between unfrozen water fraction, solute concentration, and IIF.