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 mu m d iameter) from 14 clay old mice were cryopreserved by a slow freezing p rotocol with dimethyl sulphoxide and then matured in vitro for 12 days after rapid thawing. Minor freeze damage observed after 1 day of in-v itro culture included ablation of the theca cell layer and granulosa c ell dehydration, resulting in disruption of intercellular contacts wit h the oocyte and between granulosa cells. Of the follicles, 24% were i rreversibly 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 grow n oocytes ovulated in vitro was not different from that of unfrozen co ntrols. Cryopreserved early preantral follicles matured in vitro respo nded to stimulus with human chorionic gonadotrophin in a similar way t o 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 fe rtilize and develop to hatched blastocysts.