Reproduction in Domestic Animals

Dry Cells for Nuclear Transfer 421Table 2. In vitro development of enucleated oocytes injected withfreeze-dried and fresh, control granulosa cellsSource of cells Cultured 2–8 Cells Morula BlastocystGranulosa control 129 43 31 27 (20.9%) aGranulosa freeze-dried 160 52 28 25 (15.6%) bThe cultures were maintained for 7–8 days in the medium SOF plus amino-acidsand BSA, with FCS added on day 4. Reconstructed embryos were checked every24 h for development.a–b p = 0.56.nerable to the combined osmotic ⁄ dehydration stressimposed the freeze-drying process.These preliminary observations, and the followingreports published on the topics (Kaneko et al. 2003),prompted us to formulate alternative test medium forfreeze-drying somatic cells. Our first target was to avoidthe massive DNA damage evident in rehydrated cellsdried with the sperm protocol.Trehalose produced by some plants, yeasts, sporesand a range of unicellular organism confers them theability to survive conditions of almost absence of water(Crowe et al. 1992). The mechanism by which trehaloseconfers desiccation tolerance is not clear. Trehalose perse is thought to replace the shell of water on the surfaceof macromolecules, proteins in particular (Prestrelskiet al. 1993) while on lipids it seems that its protectiveeffect is due to the inhibition of phase transitiontemperature of membranes (Leslie et al. 1994). Remarkably,the induced threalose expression in fibroblastsconferred desiccation tolerance for 5 days (Guo et al.2000). Based on these indications, trehalose wasincluded in the formulation of the freeze-dryingmedium.As reported in results, freeze-dried medium containing0.1 M trehalose was highly effective in protecting thenuclear compartment, for about half of the cells hadintact DNA after rehydration. The best results observedin lymphocytes, comparing to granulosa cells (65% vs55%, respectively), p < 0.05) was probably due to thesmaller size of the former cell.Enucleated oocytes reconstructed with freeze-driedgranulosa cells developed a pronucleus-like structureand started to cleave an equal rates compared withcontrol ones (Table 2), and 16% of them reached theblastocyst stage (Fig. 2). This result was quite surprising,because if we consider that about half of thegranulosa cells displayed damaged DNA followingrehydration, we would have expected a much lowerproportion of oocytes injected with dried cells developingto blastocyst stage. Exonuclease and recombinationactivity both increase during oogenesis, and fully growneggs have found to catalyse homologous recombination,ligation and illegitimate recombination of exogenousDNA (Lehman et al. 1993), suggesting that multiplepathways are available in the oocyte’s cytoplasm for therepair of double strand break. Therefore, it is possiblethat DNA damage induced by the dehydration arerepaired by the oocyte and do not affect the development,at least to blastocyst stage.Our results showed for the first time the developmentalpotential of freeze-dried cells after the nucleartransplantation. However, while in the original reportsperm cells were kept lyophilized for 4 months prior touse for ICSI, the cells used in our study were storedfreeze dried for more than 3 years at room temperature.Therefore, we believe that our method is a majorcontribution towards alternative ways or the establishmentand maintaining cell lines for nuclear transfer.Cloning by nuclear transfer is a very dynamic field,where joint efforts of embryologist and molecularbiologist are producing knowledge of nuclear reprogrammingmechanism at very rapid pace (Gurdon et al.2003; Meissner and Jaenisch 2006), letting foreseeing asuccessful application of cloning procedures in themedium term. Unfortunately, the list on endangeredspecies is not limited to mammals, but a wide range ofminor vertebrates are also disappearing quickly. However,cloning procedures have been recently adapted tovery unconventional animal models with two papersreporting about successful cloning of zebra fish (Ju et al.2004) and Drosophila (Haigh et al. 2005) these accomplishmentsbroad the range of species that can potentiallybenefit from cloning technology.To conclude, we demonstrated for the first time thatsomatic cells stored in a freeze-dried state are able todirect embryonic development of enucleated oocytes tillblastocyst stage. These results represent a substantialbreakthrough innovative for the establishment ofgenetic banks from endangered animals.ReferencesCorley-Smith GE, Brandhorst BP, 1999: Preservation ofendangered species and populations: a role for genomebanking, somatic cell cloning, and androgenesis? MolReprod Dev 53, 363–367.Crowe JH, Hoekstra FA, Crowe LM, 1992: Anhydrobiosis.Annu Rev Physiol 54, 579–599.Guo N, Puhlev I, Brown DR, Mansbridge J, Levine F, 2000:Trehalose expression confers desiccation tolerance on humancells. Nat Biotechnol 18, 168–171.Gurdon JB, Byrne JA, Simonsson S, 2003: Nuclear reprogrammingand stem cell creation. Proc Natl Acad Sci U S A30, 11819–11822.Haigh AJ, Macdonald WA, Lloyd VK, 2005: The generationof cloned Drosophila melanogaster. Genetics 169, 1165–1167.Hilton-Taylor C, 2000: (compiler): IUCN Red List ofThreatened Species. IUCN ⁄ SSC, Gland, Switzerland andCambridge, UK.Johnson RT, Rao PN, 1970: Mammalian cell fusion: inductionof premature chromosome condensation in interphasenuclei. Nature 226, 717–722.Ju B, Huang H, Lee KY, Lin S, 2004: Cloning zebrafish bynuclear transfer. Methods Cell Biol 77, 403–411.Kalogeropoulos N, Christoforou C, Green AJ, Gill S,Ashcroft NR, 2004: chk-1 is an essential gene and isrequired for an S-M checkpoint during early embryogenesis.Cell Cycle 3, 1196–1200.Kaneko T, Whittingham DG, Yanagimachi R, 2003: Effect ofpH value of freeze-drying solution on the chromosomeintegrity and developmental ability of mouse spermatozoa.Biol Reprod 68, 136–139.Keskintepe L, Pacholczyk G, Machnicka A, Norris K, CurukMA, Khan I, Brackett BG, 2002: Bovine blastocyst developmentfrom oocytes injected with freeze-dried spermatozoa.Biol Reprod 67, 409–415.Ó 2008 Teramo University