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Chromosomal integrity of freeze-dried karyoplasts in mice
1Department of Biological Sciences, Asahikawa Medical University, 2-1-1-1 Midorigaoka-higashi, Asahikawa, 078-8510, Japan.
Abstract:
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 micromol/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.
Insights
Preserving mature oocytes without freezing is challenging. This study examined freeze-dried mouse oocyte karyoplasts, finding that extended pre-incubation improved chromosomal integrity for potential non-cryopreservation methods.
Area of Science:
- Reproductive Biology
- Cryobiology
- Cell Biology
Background:
- Mammalian oocyte preservation currently relies on cryostorage, with no established supra-zero temperature methods.
- Freeze-drying (FD) of Metaphase II (MII) oocytes has not been explored for chromosomal integrity due to rehydration failure.
- Developing non-cryopreservation techniques is crucial for advancing reproductive technologies.
Purpose of the Study:
- To investigate the chromosomal integrity of freeze-dried MII oocytes in mice.
- To assess the impact of pre-freeze-drying incubation on karyoplast stability.
- To explore the potential of freeze-drying as an alternative preservation method for oocytes.
Main Methods:
- Karyoplasts (spindle apparatus and cytoplasm) were isolated from MII mouse oocytes.
- Karyoplasts underwent pre-freeze-drying incubation at 4°C with γ-tocotrienol for up to 2 days.
- Freeze-dried karyoplasts were rehydrated, microinjected into enucleated oocytes, and parthenogenetically activated for chromosomal analysis.
Main Results:
- A significant portion of oocytes injected with karyoplasts freeze-dried after 8 hours to 2 days of pre-incubation showed normal chromosome constitution (27-29%).
- Insufficient pre-FD incubation (0-4 hours) resulted in severe chromosomal damage.
- While some reconstructed oocytes developed to the first cleavage metaphase (67-80%), chromosome stability remained low (3-29%) compared to fresh controls (51%).
Conclusions:
- Pre-incubation duration is critical for maintaining chromosomal integrity in freeze-dried mouse oocyte karyoplasts.
- Freeze-drying shows potential for oocyte preservation, but further optimization is needed to improve chromosomal stability.
- This study provides foundational data for developing non-cryopreservation methods for mammalian oocytes.
