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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.
Highlights
Somatic cell nuclear transfer (NT) into mouse oocytes efficiently yields pluripotent stem cells [1], which are functionally and transcriptionally indistinguishable from those derived from fertilized embryos [2,3,4]
While direct cell reprogramming induced by transcription factors tolerates different cell division rates [8], an NT embryo that fails to adapt to the embryonic cleavage regime may be selected against
We used an interference bandpass filter (580/10 nm) for bright field to exclude harmful wavelengths, and generated a mouse line ubiquitously and constitutively expressing a histone H2b-GFP transgene. With these tools we determined cell cycle lengths of the first four cell cycles of mouse embryos cloned from cumulus cells and control embryos fertilized by intra-cytoplasmic sperm injection (ICSI), during culture in a-MEM (Figure 1A; movies S2, S3)
Summary
Somatic cell nuclear transfer (NT) into mouse oocytes efficiently yields pluripotent stem cells [1], which are functionally and transcriptionally indistinguishable from those derived from fertilized embryos [2,3,4]. Pregnancies from NT mouse embryos are rare, and cloned offspring are exceptional [5]. To explain the discrepancy between stem cell and pregnancy rates, it has been proposed that paternal alleles of genes involved in trophectodermal differentiation and placenta formation may be refractory to reprogramming [6]. Critical embryonic genes must be activated in less than 24 hours (embryonic genome activation) in order for the somatic nucleus to support cleavage when maternal transcripts disappear [7]. Since embryonic development proceeds as precisely timed sequence of events [9,10,11] and is characterized by rapid successions of DNA replication and mitotic divisions lacking notable G1 or G2 phases, the long cell cycle of differentiated cells [12] must be reprogrammed after NT to comply with the embryonic program
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