title: Nuclear transfer of freeze-dried somatic cells into enucleated sheep oocytes.
abstract: Lyophilization has been used since long time to preserve yeast and bacteria strains. Subsequently, a great deal of efforts has been dedicated to the preservation in a dry state of red blood cells and platelets. However, despite more than 30 years passed by, no significant progress has been achieved. Recently, it has been reported that freeze-dried mice spermatozoa were able to generate normal offspring following injection into the mature mice oocytes. In this work, we prompted to apply the lyophilization protocol developed for mice spermatozoa to sheep somatic cells (lymphocytes and granulosa cells). More than 350 enucleated sheep oocytes were injected with granulosa cells, and freeze dried using the protocol developed for mice sperm cells. Transplanted nuclei organized large pronuclei with fragmented DNA, but none of them entered the first mitosis. In the second part of the experiments, trehalose and EGTA were found to reduce significantly the extent of nuclear damage (65% and 55% intact nuclei in lymphocyte and granulosa cells, respectively) following freeze drying. Granulosa cells lyophilized with EGTA/trehalose and stored at room temperature for 3 years were used for nuclear transfer, and the injected oocytes were cultured in vitro for 7 days. Approximately 16% of the oocyte injected with freeze-dried cells developed into blastocysts. To conclude, we demonstrated for the first time that nucleated cells maintain genomic integrity after prolonged storage in a dry state, and we were able to achieve early embryonic development following injection of these cells into enucleated sheep oocytes.
authors.affiliation: Department of Comparative Biomedical Sciences, Teramo University, Teramo, Italy. ploi@unite.it
authors.forename: P
authors.initials: P
authors.lastname: Loi
authors.forename: K
authors.initials: K
authors.lastname: Matzukawa
authors.forename: G
authors.initials: G
authors.lastname: Ptak
authors.forename: Y
authors.initials: Y
authors.lastname: Natan
authors.forename: J
authors.initials: J
authors.lastname: Fulka
authors.forename: A
authors.initials: A
authors.lastname: Arav
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D005260:Female; D005612:Freeze Drying / Q000379:methods / Q000662:veterinary; D008297:Male; D051379:Mice; D008855:Microscopy, Electron, Scanning / Q000662:veterinary; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009865:Oocytes / Q000166:cytology* / Q000502:physiology / Q000648:ultrastructure; D012756:Sheep / Q000502:physiology*; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure