Reproduction in Domestic Animals

Reprod Dom Anim 43 (Suppl. 2), 417–422 (2008); doi: 10.1111/j.1439-0531.2008.01193.xISSN 0936-6768Nuclear Transfer of Freeze-Dried Somatic Cells into Enucleated Sheep OocytesP Loi 1 , K Matzukawa 2 , G Ptak 1 , Y Natan 3 , J Fulka Jr 4 and A Arav 31 Department of Comparative Biomedical Sciences, Teramo University, Teramo, Italy; 2 National Institute of Livestock and Grassland Science,Tsukuba, Japan; 3 Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel; 4 Institute of AnimalProduction, Prague, Czech RepublicContentsLyophilization has been used since long time to preserveyeast and bacteria strains. Subsequently, a great deal ofefforts has been dedicated to the preservation in a dry state ofred blood cells and platelets. However, despite more than30 years passed by, no significant progress has been achieved.Recently, it has been reported that freeze-dried mice spermatozoawere able to generate normal offspring followinginjection into the mature mice oocytes. In this work, weprompted to apply the lyophilization protocol developed formice spermatozoa to sheep somatic cells (lymphocytes andgranulosa cells). More than 350 enucleated sheep oocyteswere injected with granulosa cells, and freeze dried using theprotocol developed for mice sperm cells. Transplanted nucleiorganized large pronuclei with fragmented DNA, but none ofthem entered the first mitosis. In the second part of theexperiments, trehalose and EGTA were found to reducesignificantly the extent of nuclear damage (65% and 55%intact nuclei in lymphocyte and granulosa cells, respectively)following freeze drying. Granulosa cells lyophilized withEGTA ⁄ trehalose and stored at room temperature for 3 yearswere used for nuclear transfer, and the injected oocytes werecultured in vitro for 7 days. Approximately 16% of the oocyteinjected with freeze-dried cells developed into blastocysts. Toconclude, we demonstrated for the first time that nucleatedcells maintain genomic integrity after prolonged storage in adry state, and we were able to achieve early embryonicdevelopment following injection of these cells into enucleatedsheep oocytes.IntroductionLyophilization has been used for the preservation offowl spermatozoa already in the 1950s (Polge et al.1949). The original protocol was applied later to otherspecies (Sherman 1954), but the results in terms ofoffspring production were contradictory (Saacke andMalquist 1961). The definitive proof that dry spermatozoaretain genetic integrity was established only whenmicrosurgical procedures were developed for bypassingthe lack of mobility of freeze-dried spermatozoa, andnormal mice were produced by intracytoplasmic sperminjection (ICSI) of freeze-dried sperm (Wakayama andYanagimachi 1998). A following paper from the samegroup demonstrated the preservation of genomic integrityin freeze-dried spermatozoa (Kusakabe et al. 2001),and more recently these results have been demonstratedin other species (Keskintepe et al. 2002; Liu et al. 2004).The possibility to store male gametes in a dry staterepresents a major breakthrough for storing and shippingmale gametes from mutants, transgenic and otherstrains of laboratory mouse. Lyophilization of spermcould also be an attractive way to store spermatozoa infarm animal species, and especially in human, whereICSI has become a routine procedure in assistedreproduction.The progressive reduction of large animals worldwide(Hilton-Taylor 2000; Margules and Pressey 2000) hassuggested to establish gene banks from species threatenedby extinction (Myers et al. 2000), with the aim touse the cells for somatic cell nuclear transfer (SCNT,Wilmut et al. 1997).Somatic cell nuclear transfer has indeed an obviouspotential for the multiplication of rare genotypes (Corley-Smithand Brandhorst 1999; Loi et al. 2001), but itswide application is prevented by the low efficiency interms of offspring outcomes. Furthermore, the majorityof the endangered mammals are practically unknownfrom a reproductive point of view. Therefore, thestorage of somatic cells for future use, once theprocedure of somatic cell cloning will be reliable, iscertainly a wise step to be undertaken. However, theestablishment of gene banks in the form of cell linesencounters several problems, represented by the costs ofliquid nitrogen. Recently, our group demonstrated thatsomatic cells rendered unviable by heat treatmentretained their potential to generate a normal lamb afternuclear transplantation (Loi et al. 2002). The mainscope of this work was to demonstrate the feasibility ofusing radical approaches for the nuclear reprogrammingof somatic cells, but, as in case of freeze-dried sperm, wealso established that lack of cell viability, as indicated bymassive membrane and cytoplasmic damage, is not anabsolute prerequisite for SCNT.In this work, we developed robust procedures for thepreservation of sheep somatic cells in a freeze-driedstate, and we tested their ability to direct early embryonicdevelopment of enucleated oocytes. The ensuingresults demonstrate for the first time the production ofnormal embryos from nuclear transfer of somatic cellsstored freeze dried for more than 3 years at roomtemperature.Materials and MethodsCell collection and freeze dryingIn the first experiment, granulosa cells dissociated fromin vitro-matured Cumulus Oocyte Complexes (COCs)from Sarda breed ewes were dispersed in 1 ml of HepesbufferedDMEM medium supplemented with 10% FCS;then 100 ll aliquots of the cell suspension were loadedinto 5 ml ampoules and plunged directly into liquidnitrogen. Frozen cells were then passed on pre-cooledstage of a freeze-drying apparatus (Freezone 4.5; LabconcoCorporation, Kansas City, MO, USA) andÓ 2008 Teramo University