The influence of slow and ultra-rapid freezing on the organization of the meiotic spindle of the mouse oocyte.

Abstract

We have investigated the effect of ultra-rapid versus slow freezing on the meiotic spindle of the mouse oocyte. A slow freezing protocol [1.5 M dimethyl sulphoxide (DMSO)] and an ultra-rapid protocol (3.5 M DMSO/0.5 M sucrose) have been compared. Oocytes were fixed at different time points as follows: after prefreeze equilibration, immediately after thawing and after 60 or 180 min of post-thaw recuperation. The spindle was visualized with a monoclonal anti-alpha-tubulin antibody followed by an immunogold-silver staining technique. The chromosomes were stained with 4',6-diamidino-2-phenylindole (DAPI). Spindle morphology was classified as follows: normal barrel-shaped, abnormal shaped, partial/multipolar or absent. After slow freezing with 1.5 M DMSO, these spindle morphologies were found respectively in 67%, 17%, 11% and 6% of the oocytes after 60 min of post-thaw recuperation and in 72%, 11%, 11% and 6% after 180 min. After ultra-rapid freezing with 3.5 M DMSO/0.5 M sucrose, we observed the same categories of spindle morphology in 84%, 13%, 3% and 0% of oocytes after 60 min of post-thaw recuperation and in 86%, 7%, 7% and 0% after 180 min. The present study demonstrates that the majority of spindles exhibit a normal morphology after both slow and ultra-rapid freezing. The ultra-rapid freezing protocol preserved spindle integrity to the highest extent. Nevertheless, the occurrence of abnormal spindles and chromosome dislocation indicate that the genetic risk of oocyte freezing has to be evaluated in further detail.