Nuclear transfer of freeze-dried somatic cells into enucleated sheep oocytes

Area of Science:

• Reproductive biology and cryobiology
• Cellular and molecular biology

Background:

• Lyophilization is a long-standing method for preserving microorganisms like yeast and bacteria.
• Significant challenges persist in preserving mammalian cells, such as red blood cells and platelets, in a dry state.
• Recent success with freeze-dried mouse spermatozoa offers a potential avenue for preserving other cell types.

Purpose of the Study:

• To adapt and evaluate a lyophilization protocol, initially developed for mouse spermatozoa, for preserving sheep somatic cells (lymphocytes and granulosa cells).
• To assess the feasibility of using freeze-dried sheep somatic cells for nuclear transfer and subsequent embryonic development.

Main Methods:

• Sheep somatic cells (lymphocytes and granulosa cells) were subjected to lyophilization using a protocol optimized for mouse spermatozoa.
• Nuclear transfer was performed by injecting freeze-dried granulosa cells into enucleated sheep oocytes.
• Protective agents, trehalose and EGTA, were investigated to mitigate nuclear damage during freeze-drying.
• Oocytes injected with lyophilized cells were cultured in vitro to assess embryonic development.

Main Results:

• Initial attempts resulted in nuclear damage and failure of transplanted nuclei to enter mitosis.
• Trehalose and EGTA significantly reduced nuclear damage, preserving 65% and 55% intact nuclei, respectively.
• Sheep oocytes injected with freeze-dried granulosa cells (lyophilized with EGTA/trehalose) developed into blastocysts at a rate of approximately 16% after 7 days of in vitro culture.
• Freeze-dried cells stored at room temperature for 3 years were successfully used for nuclear transfer.

Conclusions:

• Nucleated mammalian cells can maintain genomic integrity after prolonged storage in a freeze-dried state.
• This study demonstrates the potential of lyophilization for long-term preservation of sheep somatic cells.
• Successful early embryonic development following nuclear transfer with freeze-dried cells opens new possibilities for assisted reproduction and cell banking.