Abstract
While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism.
Highlights
Oocyte-induced reprogramming after somatic cell nuclear transfer (SCNT) affords unparalleled insight into the molecular mechanisms that enable nuclei to go from the differentiated to the pluripotent state
Localization of enhanced green fluorescent protein (EGFP) to mitochondria – a key precondition to use the Cox8-EGFP construct to study nucleus-cytoplasmic interaction during reprogramming – was shown by cryo-immuno electron microscopy, where EGFP was detected by anti-GFP antibody labeled by protein A gold
Mitochondria-specific EGFP labeling was confirmed in embryonic stem cells (ESCs)
Summary
Oocyte-induced reprogramming after somatic cell nuclear transfer (SCNT) affords unparalleled insight into the molecular mechanisms that enable nuclei to go from the differentiated to the pluripotent state. Within hours of SCNT, the ooplasm (i.e. the oocyte once deprived of its chromatin) confers the nucleus transplant epigenetic changes that can lead to an embryo-like gene expression pattern. To this end, gene expression must change both qualitatively and quantitatively (reprogramming). In SCNT embryos, mRNA levels are lower compared to fertilized controls during early cleavages [2,4], while protein levels are poorly characterized In this context, it is reasonable to hypothesize that after SCNT, the ooplasmic environment signals the nucleus, so that mRNA and protein levels can be adjusted
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