Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity

The success rates for cryopreservation of immature oocytes from several spe cies including human remain low, in contrast to major improvements with mat ure oocytes. In this study, a new approach has been developed using a short exposure ultra-rapid freezing protocol, examining the effect of temperatur e and egg yolk (two factors which may be expected to influence membrane fle xibility) on the cryostability of immature mouse oocytes and cumulus comple xes. These two factors were tested in various patterns for their cryoprotec tive effect using ethylene glycol as the principal cryoprotectant. The resu lts showed that 37 degrees C pre- and post-freeze exposure significantly im proved both survival and normal spindle configuration after in-vitro matura tion. Egg yolk was found to produce further beneficial effects on both the oocyte and cumulus cell integrity, with the best effects being obtained at 37 degrees C with inclusion of egg yolk both before and after the freezing. This protocol produced >80% normal survival post-thaw with intact and atta ched cumulus complex, 84% maturation rate and 45% normal metaphase configur ation. In summary, a unique combination of high survival and meiotic normal ity together with good preservation of the attached cumulus cell mass has b een achieved using a simple new vitrification procedure.