title: Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success.
abstract: Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H(2b)-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.
authors.affiliation: Max Planck Institute for Molecular Biomedicine, Münster, Germany.
authors.forename: Sebastian Thomas
authors.initials: ST
authors.lastname: Balbach
authors.forename: Telma Cristina
authors.initials: TC
authors.lastname: Esteves
authors.forename: Franchesca Dawn
authors.initials: FD
authors.lastname: Houghton
authors.forename: Marcin
authors.initials: M
authors.lastname: Siatkowski
authors.forename: Martin Johannes
authors.initials: MJ
authors.lastname: Pfeiffer
authors.forename: Chizuko
authors.initials: C
authors.lastname: Tsurumi
authors.forename: Benoit
authors.initials: B
authors.lastname: Kanzler
authors.forename: Georg
authors.initials: G
authors.lastname: Fuellen
authors.forename: Michele
authors.initials: M
authors.lastname: Boiani
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001755:Blastocyst / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D065150:Cellular Reprogramming / Q000502:physiology*; D016000:Cluster Analysis; D004249:DNA Damage; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D007700:Kinetics; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D053652:Nuclear Transfer Techniques; D059008:Time-Lapse Imaging; D019076:Transgenes
chemical_list: D000596:Amino Acids; D001120:Arginine