Comparison of ultrastructure and morphology of mouse ovarian follicles after conventional and direct cover vitrification using different concentrations of ethylene glycol

Background: Many attempts have done to improve cryopreservation of mammalian ovaries using simple, economical and efficient technique “vitrification”.
Objective: The aim of the present study was to compare the mouse ovaries cryopreservation by direct cover vitrification (DCV) using different concentrations of ethylene glycol (EG) with conventional vitrification methods (CV).
Materials and Methods: Ninety NMRI mice were sacrificed by cervical dislocation; their ovaries were divided into three main experimental groups: control or non-vitrified group, CV group and DCV groups with 4, 6 and 8M EG as cryoprotectant. After vitrification-warming, the viability of mechanically isolated follicles and the morphology of ovarian follicles by light and electron microscopes were studied.
Results: The normality of primary and preantral follicles in non-vitrified and CV groups were higher than those achieved by DCV groups (p<0.001). The survival rates of isolated follicles in non-vitrified, CV and DCV groups with 4M, 6M and 8M ethylene glycol were 98.32, 96.26, 84.10, 85.46 and 84.56 %, respectively and in DCV groups it was lower than other groups (p<0.001). The ultrastructure of ovarian follicles was well preserved in CV technique. The follicles in DCV groups appeared to have vacuolated oocyte with nuclear shrinkage and irregular distribution of cytoplasmic organelles. Their mitochondria were located mainly in the sub cortical part of the oocyte and the granulosa cells demonstrated some signs of degeneration.
Conclusion: DCV of mouse ovarian tissue using only EG has induced some alteration on the fine structure of follicles. The integrity of mouse ovarian tissue was affected by DCV technique more than CV.

Keywords
Cryopreservation, Direct cover vitrification, Ethylene glycol, Preantral follicle, Ultrastructure