Chromosomal integrity of freeze-dried karyoplasts in mice

In mammalian species, there is currently no way to preserve mature oocytes at supra-zero temperatures without cryostorage. Metaphase II (MII) oocytes freeze-dried in any medium or solution cannot be revived after rehydration. Therefore, the injurious effects to chromosomes of freeze-drying MII oocytes have not been reported. The aim of this study was to examine the chromosomal integrity of freeze-dried MII oocytes in mice. Spindle apparatuses with small amounts of cytoplasm, “karyoplasts” so-called, were removed from MII mouse oocytes. Before freeze-drying (FD), the karyoplasts were incubated at 4 °C for up to 2 days (pre-FD incubation) in EGTA/Tris-HCl buffered solution supplemented with 20 μmol/l γ-tocotrienol. After freeze-drying, the freeze-dried karyoplasts were rehydrated and microinjected into enucleated MII oocytes. Parthenogenetic activation of the reconstructed oocytes was performed to analyze the chromosomes at the first cleavage metaphase. A portion of normally activated oocytes that had been injected with fresh karyoplasts (51 %) and karyoplasts freeze-dried after pre-FD incubation for 8 h to 2 d (27 %–29 %) exhibited normal chromosome constitution. Insufficient pre-FD incubation (0–4 h) caused severe chromosomal damage. By contrast, almost all parthenogenetic embryos (98 %) reconstructed via fusion of fresh karyoplasts and enucleated oocytes maintained normal chromosome constitution. Some MII oocytes reconstructed and activated parthenogenetically using freeze-dried karyoplasts could develop the first cleavage metaphase (67–80 %) while retaining chromosome stability (3–29 %). Further improvements in FD procedures should enhance the chromosomal integrity of freeze-dried karyoplasts.