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Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success

Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success
Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success
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 H2b-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
1932-6203
e35322
Balbach, Sebastian Thomas
e3bff152-0246-4cb0-a9f5-6e6f25e0408b
Esteves, Telma Cristina
11040527-fb8c-4348-bb9d-e34f3cac6a23
Houghton, Franchesca Dawn
53946041-127e-45a8-9edb-bf4b3c23005f
Siatkowski, Marcin
dd890a0f-ff54-4665-ba27-40dbafb48c0b
Pfeiffer, Martin Johannes
9506ffba-9c3f-4dbd-9c78-29400220f27b
Tsurumi, Chizuko
0ad61aa2-5544-4c25-ba52-39411babee99
Kanzler, Benoit
bf9e7121-f3e7-41c0-a0c3-fbafa6421798
Fuellen, Georg
88e64995-0254-444d-ac28-64b3302c305b
Boiani, Michele
1979e7de-72f1-44fc-96c4-e1729afcbc6f
Balbach, Sebastian Thomas
e3bff152-0246-4cb0-a9f5-6e6f25e0408b
Esteves, Telma Cristina
11040527-fb8c-4348-bb9d-e34f3cac6a23
Houghton, Franchesca Dawn
53946041-127e-45a8-9edb-bf4b3c23005f
Siatkowski, Marcin
dd890a0f-ff54-4665-ba27-40dbafb48c0b
Pfeiffer, Martin Johannes
9506ffba-9c3f-4dbd-9c78-29400220f27b
Tsurumi, Chizuko
0ad61aa2-5544-4c25-ba52-39411babee99
Kanzler, Benoit
bf9e7121-f3e7-41c0-a0c3-fbafa6421798
Fuellen, Georg
88e64995-0254-444d-ac28-64b3302c305b
Boiani, Michele
1979e7de-72f1-44fc-96c4-e1729afcbc6f

Balbach, Sebastian Thomas, Esteves, Telma Cristina, Houghton, Franchesca Dawn, Siatkowski, Marcin, Pfeiffer, Martin Johannes, Tsurumi, Chizuko, Kanzler, Benoit, Fuellen, Georg and Boiani, Michele (2012) Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success. PLoS ONE, 7 (4), e35322. (doi:10.1371/journal.pone.0035322).

Record type: Article

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 H2b-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

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e-pub ahead of print date: April 2012
Organisations: Human Development & Health

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Local EPrints ID: 337452
URI: https://eprints.soton.ac.uk/id/eprint/337452
ISSN: 1932-6203
PURE UUID: 98b7a12a-0191-4e1f-9b06-8ab0d59f1731

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Date deposited: 26 Apr 2012 10:17
Last modified: 18 Jul 2017 06:03

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Contributors

Author: Sebastian Thomas Balbach
Author: Telma Cristina Esteves
Author: Marcin Siatkowski
Author: Martin Johannes Pfeiffer
Author: Chizuko Tsurumi
Author: Benoit Kanzler
Author: Georg Fuellen
Author: Michele Boiani

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