Cryobiology of non-human primate oocytes

The responses to various stresses involved with cryopreserv-ation protocols were investigated using non-human primate oocytes. Fluorescence microscopy was used to assess the status of the F-actin microfilament system of rhesus monkey oocytes after exposure to different concentrations of glycerol. The F-actin organization around the cortex and hi the transzonal processes was modified by exposure to 1.0 or 2.0 M glycerol at ambient temperature. These effects were reduced significantly when exposure to glycerol was combined with cooling to 0°C. Cynomolgus monkey oocytes were also subjected to hyperosmotic stress and observed for morphological changes. An irregular shrinkage phenomenon was observed with germinal vesicle or metaphase I but not metaphase II (MIl) oocytes. The irregular shrinkage became uniform and spherical when the oocytes were pretreated with ethyleneglycol-bis-(β3-aminoethyl ether)N, N, N′, N′ tetraacetic acid (EGTA) before exposure to hypertonic solution. Also, in-vitro-matured MIl oocytes from cynomolgus monkeys were used to determine crucial biophysical parameters for freezing primate oocytes. The permeability of oocyte plasma membrane to water, Lpg, and its activation energy, ELP, were determined between 0 and -12°C in the absence of cryoprotective additives. The Lpg was found to be 3.8 ×10–14 m3N/s and the ELp was 141.5 kJ/mol. The pre-exponential kinetic and exponential thermodynamic parameters of intracellular ice formation were determined to be 8×108 m2/s and 2.2 ×109 K5 respectively. By combining models of water transport and intracellular ice formation, the cumulative fraction of oocytes with intracellular ice as a function of the cooling rate was also predicted, and it was shown to correlate reasonably with experimental observations.