A morphological and functional study of the effect of slow freezing followed by complete in-vitro maturation of primary mouse ovarian follicles.

Mechanically isolated intact early preantral follicles (100-130 microns diameter) from 14 day old mice were cryopreserved by a slow freezing protocol with dimethyl sulphoxide and then matured in vitro for 12 days after rapid thawing. Minor freeze damage observed after 1 day of in-vitro culture included ablation of the theca cell layer and granulosa cell dehydration, resulting in disruption of intercellular contacts with the oocyte and between granulosa cells. Of the follicles, 24% were irreversibly damaged and had a collapsed oocyte. The remaining majority of the follicles had an intact oocyte as evaluated by ultrastructural analysis. Follicles with an intact oocyte were cultured in vitro and, after an initial retarded development, the final number of fully grown oocytes ovulated in vitro was not different from that of unfrozen controls. Cryopreserved early preantral follicles matured in vitro responded to stimulus with human chorionic gonadotrophin in a similar way to controls, with mucification of the oocyte-cumulus complex, germinal vesicle breakdown and extrusion of the first polar body of the oocyte. These cryopreserved, in-vitro matured oocytes had the potential to fertilize and develop to hatched blastocysts.