Abstract
To test the effects of varying vitrification protocols on the cell cycle status and chromosomal integrity in cumulus-enclosed GV stage rat oocytes. Vitrified and thawed rat oocytes were labeled with fluorescent markers for chromatin, cell cycle activation, and f-actin and analyzed by conventional and laser scanning confocal microscopy. In all vitrification groups, significant alterations in cumulus cell connectivity, cell cycle status, and cytoplasmic actin integrity were observed following warming compared to fresh control oocytes. Based on the protein phosphorylation marker MPM-2, it is clear that warmed oocytes rapidly enter M-phase but are unable to maintain chromosome integrity as a result of multiple chromatin fusions. A prominent reduction in f-actin is evident in both the ooplasm and at the cortex of vitrified oocytes. Finally, an irreversible but irregular retraction of TZPs occurs on the majority of oocytes subjected to any of the vitrification protocols. These findings draw attention to undesirable consequences of immature oocyte vitrification that compromise cell cycle status and chromatin and cytoskeleton integrity that may not be evident until after fertilization.
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