DNA replication is an integral part of the mouse oocyte's reprogramming machinery.
Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.
Max Planck Institute for Molecular Biomedicine, Münster, Germany.
Bingyuan
B
Wang
Max Planck Institute for Molecular Biomedicine, Münster, Germany.
Martin J
MJ
Pfeiffer
Max Planck Institute for Molecular Biomedicine, Münster, Germany.
Caroline
C
Schwarzer
Group of Computational Biology and Systems Biomedicine, Biodonostia Health Research Institute, San Sebastián, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.
Marcos J
MJ
Araúzo-Bravo
Max Planck Institute for Molecular Biomedicine, Münster, Germany.
Michele
M
Boiani
D000818:Animals; D065150:Cellular Reprogramming / Q000502:physiology*; D019976:Cloning, Organism / Q000379:methods*; D048749:Cytokinesis / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000502:physiology*; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D000071317:Nanog Homeobox Protein; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D009865:Oocytes / Q000502:physiology*; D016133:Polymerase Chain Reaction; D020554:Sperm Injections, Intracytoplasmic
D017931:DNA Primers; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse