Ultrastructure Of Primary Follicle After Vitrification Of Mouse Ovary

Introduction: This study was performed to investigate the effect of vitrification procedure using ethylene glycol as cryoprotectant on the ultrastructure and morphology of primary follicles of mouse ovarian tissue.
Materials and Methods: Ovaries from 8-10 weeks old NMRI mice were vitrified using a solution of T6 medium containing 30% (W/V) Ficol 70, 0.5 M Sucrose, 10.7% (V/V) Acetamide and 40% (V/V) Ethylene glycol (EGFS 40) and stored in liquid nitrogen. After thawing the vitrified ovarian tissues were washed by 1 M sucrose solution and equilibrated by T6 medium. For histology, the frozen-thewed and fresh ovarian tissues were fixed using Bouin"s solution. The fixed tissues were embedded in paraffin wax, serially sectioned at 6 micrometer and stained with Hamatoxylin and Eosin. For electron microscopic studies, the frozen-thawed ovarian and control tissues were fixed with 2.5% Glutaraldehyde and 1% Osmiume tetroxide. After dehydration with ethanol the tissue were embedded in Epon 812 resin. Ultrathin sections were stained with Uranyl acetate and Lead citrate, then examined under TEM.
Results: The proportion of morphologically normal follicles was observered frozen and fresh ovaries. At the light microscopic level almost all of the primary follicles appeared normal in frozen and non-frozen ovaries. In electron microscopic study the integrity of cell organelles, nuleus of oocyte and follicular cells were well preserved, but some mitochondria were swell, their cristae disappeared in some parts, perivitilline space was electron lucent and there was few cortical granule in cortex of oocyte.
Conclusion: The results of this study showed that the vitrification method, using ethylene glycol as cryoprotectant did not cause any harmful damage to follicular cells and oocytes of primary follicles, thus this procedure could be an efficient method for storage of immature oocytes within ovary especially before cancer therapy.