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.

Correlation of Abnormal Mitochondrial Distribution in Mouse Oocytes with Reduced Developmental Competence

“…This allowed assessment of the correlation between mitochondrial distribution and embryo developmental competence. Previous studies of vitrification in mouse oocytes have shown that incubation for 2 or 3 hrs resulted in a higher incidence of normal spindles than incubation for 1 hr, and that fertilization and blastocyst formation of vitrified mouse oocytes inseminated after 1 hr of incubation were significantly lower than for control specimens, but increased with insemination after incubation for 2 or 3 hrs (Aigner et al 1992;Chen et al 2001Chen et al , 2003. Therefore, activation of oocytes was observed after incubation for 30 min following vitrification and thawing.…”

High Survival Rate of Bovine Oocytes Matured In Vitro Following Vitrification

“…It has been reported in murine [42] and bovine [43] oocytes that the abnormal appearance of the spindle following the slow freezing and rapid thawing returned to be normal after a period of incubation. It has been reported that, in the context of slow freezing, a pre-incubation of 2 h was required for the oocytes before insemination to im prove the fertilizability and embryonic development of the freezing-thawed oocytes [41,44,45].…”

“…Incubation of frozen-thawed oocytes before insemination seems to allow recovery from the injury associated with the slow freezing procedure [42,43]. Pre-incubation of post-thawed bovine oocytes improves the fertilizability and embryonic developmental capacity [44,45].…”

Survival of Porcine Delipated Oocytes and Embryos after Cryopreservation by Freezing or Vitrification.

“…Damage of microtubles and microfilaments has been thought to be the major cause of loss or decrease of viability of oocytes after cryopreservation [35][36][37]. Cryo-injury to these elements is affected by the cryopreservation methods and cryoprotective agents used [35][36][37].…”