The Vitrification Of Metaphase II Mouse Oocyte Using Ethylene Glycol As Cryoprotectant



Introduction: The purpose of this study was to investigate the effect of vitrification procedure on the survival, fertilization, development and ultrastructure of metaphase (MII) mouse oocyte. Materials and Methods: Female NMRI mice 6-10 weeks old were superovulated using IP injection of 10 IU hMG and 10 IU hCG. Ovulated oocyte were collected from the ampullary portion of the oviducts at 12-13 hours after hCG injection. Cumulus cell mass were separated by 0.1% hyaluronidase. The oocyte were vitrified by solution of PBI contained 30 % (W/V) ficol 70, 0.5 M sucrose, 10.7% (W/V) acetamid and 10% (V/V) ethylene glycol and stored in liquid nitrogen. The frozen oocyte were thawed by sucrose and PBI solutions for 5 min, then the frozen and non-frozen oocytes were inseminated with epididymal sperm. For electron microscopy studies, the fertilized and unfertilized oocytes were fixed with 2.5 % glutaraldehyde and 1% osmium tetroxide. After dehydration with ethanol followed with propylene oxide, the oocytes were embeded in Araldite. Ultrathin sections were stained with uranyl acetate and leade citrate, then examined under TEM.
Results: The survival rate of vitrified oocytes were 80%. The fertilization and developmental rates of the vitrified group were not significantly different from the control. An extended subzonal space was noticed in vitrified group, morover, the cytoskeleton was disorganized and some mitochondrai were swell. In the fertilized vitrified oocytes, these changes were reversible and the ultrastructural feuture of these group was the same as fertilized control oocyte.
Conclusion: This vitrification method is a simple and usful procedure for cryopreservation of oocytes.