title: Nuclear transfer of sand cat cells into enucleated domestic cat oocytes is affected by cryopreservation of donor cells.
abstract: In the present study, we used the sand cat (Felis margarita) as a somatic cell donor to evaluate whether cryopreservation of donor cells alters viability and epigenetic events in donor cells and affects in vitro and in vivo developmental competence of derived embryos. In Experiment 1, flow cytometry analysis revealed that the percentage of necrosis and apoptosis in cells analyzed immediately after freezing/thawing (61 vs. 8.1%, respectively) was higher than that observed in frozen/thawed cells cultured for 18 h (6.9 vs. 3.3%, respectively) or 5 days (38 vs. 2.6%; respectively). The relative acetylation level of H3K9 was lower in frozen/thawed cells (5.4%) compared to that found in cultured cells (60.1%). In Experiment 2, embryos reconstructed with frozen/thawed cells had a lower cleavage rate (85%; day 2) than did embryos reconstructed with cultured cells (95%), while development to the blastocyst stage (day 8) was not affected by cell treatment (17.0% with frozen/thawed cells vs. 16.5% with cultured cells). In Experiment 3, pregnancy rates were similar between both cell treatments (32% with frozen/thawed cells vs. 30% with cultured cells), but the number of embryos that were implanted, and the number of fetuses that developed to term was lower for embryos reconstructed with frozen/thawed cells (1.2 and 0.3%, respectively) than those reconstructed with cultured cells (2.6 and 1.8%, respectively), while the number of fetuses reabsorbed by day 30 was higher (75%) for embryos reconstructed with frozen/thawed cells than those reconstructed with cultured cells (31%). A total of 11 kittens from cultured cells and three kittens from frozen/thawed cells were born between days 60 to 64 of gestation. Most kittens died within a few days after birth, although one kitten did survive for 2 months. In Experiment 4, POU5F1 mRNA expression was detected in 25% of blastocysts derived from frozen/thawed cells, whereas 88 and 87% of blastocysts derived from cultured cells and by in vitro fertilization, respectively, expressed POU5F1. We have shown that cell cryopreservation increased the incidence of necrosis and apoptosis and altered epigenetic events in donor cells. Consequently, the number of embryos that cleaved, implanted, and developed to term-gestation and POU5F1 expression in derived blastocysts indirectly was affected.
authors.affiliation: Audubon Center for Research of Endangered Species, New Orleans, Louisiana 70131, USA. mgomez@auduboninstitute.org
authors.forename: Martha C
authors.initials: MC
authors.lastname: Gómez
authors.forename: C Earle
authors.initials: CE
authors.lastname: Pope
authors.forename: Robert H
authors.initials: RH
authors.lastname: Kutner
authors.forename: David M
authors.initials: DM
authors.lastname: Ricks
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Lyons
authors.forename: Mark
authors.initials: M
authors.lastname: Ruhe
authors.forename: Cherie
authors.initials: C
authors.lastname: Dumas
authors.forename: Justine
authors.initials: J
authors.lastname: Lyons
authors.forename: Mónica
authors.initials: M
authors.lastname: López
authors.forename: Betsy L
authors.initials: BL
authors.lastname: Dresser
authors.forename: Jakob
authors.initials: J
authors.lastname: Reiser
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015415:Biomarkers / Q000032:analysis; D001757:Blastomeres / Q000502:physiology; D002415:Cats / Q000502:physiology*; D015925:Cryopreservation; D004622:Embryo, Mammalian / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism / Q000502:physiology*
chemical_list: D015415:Biomarkers; D050814:Octamer Transcription Factor-3