SLOW AND ULTRARAPID FREEZING OF FULLY GROWN GERMINAL VESICLE-STAGE MOUSE OOCYTES - OPTIMIZATION OF SURVIVAL RATE OUTWEIGHTED BY DEFECTIVE BLASTOCYST FORMATION

Purpose: The cryopreservation of mature metaphase II-stage mouse oocyt es is associated with decreased fertilizability, spindle damage, and i ncreased polyploidy. Therefore, we investigated the outcome of cryopre servation of immature germinal vesicle-stage mouse oocytes. Methods: O ocytes were punctured from Graafian follicles in primed F1 hybrid mice and were then released into maturation medium containing the meiotic inhibitor dibutyryl cyclic AMP. Both slow and ultrarapid freezing prot ocols with dimethyl sulfoxide, 1,2-proponediol, or a mixture of both a gents were tested. We recorded morphological survival rates, in vitro maturation rates, and two-cell and blastocyst formation rates. Each gr oup of frozen oocytes was compared with both unfrozen germinal vesicle -stage oocytes and metaphase II-stage oocytes.Results: An optimal cryo survival rate of 78% was reached after ultrarapid freezing with 3 M di methyl sulfoxide followed by one-step dilution, but a decreased rate o f two-cell formation was observed. Freezing with a combination of dime thyl sulfoxide and 1,2-propanediol did not improve this fertilization- decreasing effect. Very low cryosurvival rates after freezing with 1,2 -propanediol indicated its inappropriateness for ultrarapid freezing o f immature oocytes. The rates of in vitro maturation were equivalent f or frozen-thawed and freshly collected germinal vesicle-stage oocytes, independent of the freezing protocol used. We report, nevertheless, a s a general characteristic for both slow and ultrarapid freezing of fu lly grown germinal vesicle-stage oocytes, that the in vitro developmen t up to the blastocyst stage is inhibited despite full nuclear maturat ion. Conclusion: We report that cryopreservation of immature germinal vesicle-stage oocytes is invariably associated with a low developmenta l capacity after fertilization. The rate of in vitro nuclear maturatio n did not equate with developmental competence. This in turn suggests the importance of cytoplasmic maturation for embryonic development.