Unknown,Transcriptomics,Genomics,Proteomics

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Smart-Seq of embryos injected with the mRNA of a recombinant OGA

ABSTRACT: 40-PE Smart-Seq of RNA isolated from single 2-cell embryos, single morulae, single blastocysts, single E7 embryos epiblast halves and single E7 trophectoderm halves. For all preimplantation stages, embryos were grown ex-vivo from three experimental groups of zygotes: zygotes injected with the mRNA of a recombinant OGA (named BtGH84) (Btgh_injected), zygotes injected with a catalytically dead version of BtGH84 (dBtgh_injected) (control nr.1) and non-injected zygotes (non_injected) (control nr.2). Postimplantation E7 embryos were grown from two experimental group of zygotes: Btgh-injected and dBtgh-injected (control) and transferred at the 2-cell stage to foster mothers. All samples from the same embryonic stage were processed for single embryo (or single-embryo single-tissue) Smart-Seq library preparation at the same time and then sequenced in the same NextSeq500 run. The different embryonic stages were processed on different days and sequenced in different NextSeq500 runs.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Charles Girardot

PROVIDER: E-MTAB-12981 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:An important facet of the oocyte to embryo transition in mammals is the rapid elimination of a subset of the maternal products that got accumulated during oogenesis. RNA studies support a view that the transition occurs quite rapidly. Whether this applies also for proteins remains to be determined beyond the very few cases known to date. We report that COPS3, the 3rd subunit of the COP9 signalosome complex, forms a large protein deposit in mouse oocytes (94th percentile of the riBAQ distribution). The one and only knock-out study had previously shown that Cops3 null (-/-) mouse embryos obtained from heterozygous intercrosses arrested after 5.5 dpc and were resorbed by 8.5 dpc mainly due to increased cell death in the epiblast (PMID: 12972600). However, more recent studies from our group ascribed Cops3 gene with a developmental role already at the 2-cell stage. Specifically, the sister blastomeres differ from each other in Cops3 mRNA levels and distribution, preceding the discordance of epiblast formation in derivative twin blastocysts, and implicating Cops3 in the molecular underpinnings of totipotency. This discrepancy between original knock-out result and recent observations can be resolved if we posit that the first requirement for Cops3 gene function was bridged by maternal protein that outlived the locus excision, consistent with the observation that Cops3 -/- blastocysts had the same immunostaining intensity as the +/- or +/+ counterparts in the original knock-out study. Thus, these contradicting observations support a hypothesis that 5.5 dpc may not have been the first time when the gene function was needed in development, but the second time. To test this hypothesis, pronuclear-stage mouse oocytes were subjected to an immunological method that directly targets the protein of interest by way of proteasomal degradation of the COPS3-antibody-TRIM21 complex. Briefly, pronuclear-stage mouse oocytes were microinjected with a mixture of the COPS3-specific purified monoclonal IgG antibody, mCherry-Trim21 mRNA and Oregon Green dextran beads (inert tracer). Injected oocytes were then allowed to develop and collected for lysis 24 hours later, when they reached the 2-cell stage but were not able to progress further. In addition to the COPS3, we targeted two additional proteins, OCT4 and TEAD4. The following seven experimental groups were examined by liquid chromatography-mass spectrometry (LC-MS/MS) using the pipeline described previously by Israel et al.: 1) non-manipulated 2-cell embryos; 2) 2-cell embryos from oocytes injected with Oregon Green dextran beads; 3) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA and Oregon Green; 4) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-COPS3 (Abcam 79698); 5) 2-cell embryos from oocytes injected with anti-COPS3 (Abcam 79698) and Oregon Green; 6) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-TEAD4 (Abcam 58310); 7) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-OCT4 (Abcam 181557).

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Smart-Seq of embryos injected with the mRNA of a recombinant OGA

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