Effect of cooling and exposure to ethylene glycol on in vitro maturation and embryo development of rhesus oocytes

The meiotic spindle of metaphase II-stage oocytes is damaged when mature oocytes are cooled to temperatures close to 0°C, as occurs during cryopreservation by equilibrium cooling. Since a spindle has not yet formed within a germinal vesicle-stage oocyte, it has been suggested that immature oocytes may be more resistant than metaphase II oocytes to cryopreservation by equilibrium cooling. To test this proposition, we examined the effects on rhesus macaque oocytes of chilling and exposure to ethylene glycol (EG) on their maturation and embryo development. A total of 202 cumulus-intact oocytes was collected from adult female rhesus monkeys that had been given follicle stimulating hormone for controlled ovarian hyperstimulation. Within two hours of their having been aspirated and prior to germinal vesicle breakdown, oocytes were either cooled to 0°C for 10 minutes or were exposed for 15 minutes at 35°C to 1.5 M EG to be tested as a possible cryoprotectant. After being exposed, oocytes were cultured in maturation medium, fertilized in vitro with rhesus spermatozoa, and cultured. The maturation rate and subsequent development into blastocysts of those oocytes that had been exposed to EG or cooled to 0°C did not differ significantly from untreated control oocytes. Additional germinal vesicle oocytes were exposed to 1.5 M EG at 35°C for 3 minutes and then supercooled to −7°C or frozen at −7°C or frozen at 0.5°C to −35°C. Rates of maturation and embryo development of oocytes cooled to or frozen at −7°C were significantly lower than rates for control oocytes; none of those frozen to −35°C even underwent maturation. These results suggest that germinal vesicle-stage oocytes may be less susceptible to injury resulting from chilling or exposure to ethylene glycol, but are still damaged by freezing.