CYTOSKELETON AND POLYPLOIDY AFTER MATURATION AND FERTILIZATION OF CRYOPRESERVED GERMINAL VESICLE STAGE MOUSE OOCYTES

Purpose: Our purpose was to assess the effect of cryopreservation on c ytoskeleton of germinal vesicle (GV) mouse oocytes and determine wheth er irreversible spindle damage and related digyny associated with cryo preservation of metaphase II (MII) oocytes can be avoided. Methods: Th e GV oocytes were cryopreserved using a slow-cooling (0.5 degrees C/mi n) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethylsulfox ide supplemented with 0.2 M sucrose and analyzed before and during fer tilization by multiple-label fluorescence and differential interferenc e contrast microscopy techniques, Results: When examined after in vitr o maturation, the vast majority (>95%) of cryopreserved and control oo cytes displayed nor mal microfilament and microtubule organization. Wi th respect to barrel-shaped spindle and nor mal chromosome alignment, no significant differences were observed between cryopreservation (78 and 86%, respectively) and control (85 and 95%, respectively) groups. In fertilization experiments, spindle rotation, formation of the secon d polar body, and pronuclear migration were displayed by similar perce ntages of cryopreserved (96, 94, and 37%, respectively) and control (9 8, 97 and 45%, respectively) oocytes, indicating normal functionality of the cytoskeleton during this period. However; pronuclear formation was significantly, inhibited by cryopreservation (81%) compared with c ontrols (100%). Regarding digyny and polyspermy, no significant increa se was observed after cryopreservation (3 and 10%, respectively) compa red with controls (3 and 6%, respectively). Conclusions: Cryopreservat ion of mouse oocytes at the GV stage is particularly advantageous to c ircumvent the spindle damage and increased digyny noted after cryopres ervation of MII oocytes.