Solvent effects on cytoskeletal organization and in-vivo survival after freezing of rabbit oocytes

NBD-phallacidin revealed a polymerized actin distribution in the cortical region of the rabbit egg and along junctional feet. Staining with anti-alpha-tubulin antibody showed that the microtubule distribution was restricted to the barrel-shaped spindle. After cryoprotective treatment in the presence of propanediol, cortical polymerized actin was no longer visible within the egg and along junctional feet but filamentous actin was still present after treatment with dimethylsulphoxide. However, exposure to dimethylsulphoxide or propanediol led to the appearance of microtubules in the cytoplasm and to a disassembly of the spindle often associated with anomalies in chromosome position. Cytoplasmic microtubules formed by the action of propanediol were still present after freezing, thawing, and removal of the cryoprotectant, but after recovery of eggs in culture, they disappeared and barrel-shaped spindles were able to reform. When the effect of propanediol addition on in-vivo fertilization and development of frozen oocytes was examined, 39% (79/200) of frozen oocytes were fertilized and 9% (9/105) developed to normal fetuses, compared to 81% (38/47) and 32% (12/38) respectively for unfrozen control oocytes.