Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells

ABSTRACT: We provide proof that the oocyte is able to reprogram two somatic nuclei at once and therefore is not limited in its reprogramming capacity to a single nucleus. Mouse ooplasts transplanted with 2 somatic cell nuclei simultaneously (double nuclear transfer) support preimplantation development and the derivation of tetraploid NT embryonic stem cells (tNT-ES cells). These cells are unique in that they embody the first case of cloned ES cells containing two reprogrammed somatic genomes at the same time and exhibit characteristics of true pluripotency. By assessing allelic expression of Oct4 in normal and double nucleus transplanted embryos throughout development we demonstrate that oocyte mediated reprogramming is embarked with a probabilistic component and that one reprogrammed genome can dominate the other during preimplantation development even if merged into the same nucleus. Animal handling and cell recovery: Oocytes from mice of the B6C3F1 strain were used for SCNT and ICSI. Cumulus cells (for SCNT) and sperm (for ICSI) were either from a B6C3F1 or OG2 donor the latter expressing an Oct4 promoter-driven GFP transgene. Additionally cells from pure C56Bl/6 or C3H mice were retrieved for SCNT. Female mice were sacrificed by cervical dislocation 14 h after hCG injection and oocytes retrieved from oviduct ampullae. Oocytes and cumulus cells were handled in Hepes-buffered CZB (HCZB) medium. Double nuclear transfer ICSI and embryo culture: Micromanipulations and embryo culture were performed at 30°C (room temperature). For SCNT oocytes were first enucleated (spindle-chromosome complex removed) before transfer of one two or three cell nuclei with a micropipette driven by a piezo actuator under Nomarski optics. The reconstructed oocytes were activated for 6 h in Calcium-free alpha-MEM supplemented with 10 mM SrCl2 and 5 micro-g mL-1 cytochalasin B. Intracytoplasmic sperm injection (ICSI) of one two or four into intact oocytes was used as the fertilization control condition using sperm from 8-week-old mice of B6C3F1 or OG2 strains. Embryos were placed in embryo culture medium and cultured at 37°C under 5% CO2. The activated oocytes were cultured in alpha-MEM at 37°C and under 5% CO2 until use. On day 5 after activation SCNT and ICSI blastocysts were processed for ESC derivation. Analysis of allelic expression of Oct4: RNA from single embryos was extracted using the ZR-RNA MicroPrep kit (Zymo Research). Complementary DNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Invitrogen) according to manufacturers instruction. A nested PCR was performed to amplify part of the Oct4 cDNA containing a BamH1 sensitive SNP. Primer sequences and PCR conditions are given in the supplementary methods. The PCR product was digested for 8h using 100 units of BamH1-HF (NEB) subsequently subjected to agarose gel electrophoreses and the results grouped into 3 categories (mainly C57Bl/6 balanced mainly C3H) according to the observed band patterns. Global transcriptome analysis by microarray: RNA samples to be analyzed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion) which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto a MouseRef-8 v2 expression BeadChip (Illumina) following the manufacturer instructions. After washing as recommended chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. Microarray data processing: The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was Background correction performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics was performed with in-house developed functions in Matlab. 8 samples were analyzed. tNT#1: tetraploid nuclear transfer, p17, double sedimentation; tNT#2: tetraploid nuclear transfer, p19, double sedimentation; tNT#3: tetraploid nuclear transfer, p25, double sedimentation; tNT#4: tetraploid nuclear transfer, p29, double sedimentation; tNT#5: tetraploid nuclear transfer, p18, double sedimentation; tNT#6: tetraploid nuclear transfer, p25, double sedimentation; NT#1: nuclear transfer, double sedimentation; NT#2: nuclear transfer, double sedimentation;

ORGANISM(S): Mus musculus

SUBMITTER: Marcos Araúzo-Bravo

PROVIDER: E-GEOD-36468 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We provide proof that the oocyte is able to reprogram two somatic nuclei at once and therefore is not limited in its reprogramming capacity to a single nucleus. Mouse ooplasts transplanted with 2 somatic cell nuclei simultaneously (double nuclear transfer) support preimplantation development and the derivation of tetraploid NT embryonic stem cells (tNT-ES cells). These cells are unique in that they embody the first case of cloned ES cells containing two reprogrammed somatic genomes at the same time and exhibit characteristics of true pluripotency. By assessing allelic expression of Oct4 in normal and double nucleus transplanted embryos throughout development we demonstrate that oocyte mediated reprogramming is embarked with a probabilistic component and that one reprogrammed genome can dominate the other during preimplantation development even if merged into the same nucleus. Animal handling and cell recovery: Oocytes from mice of the B6C3F1 strain were used for SCNT and ICSI. Cumulus cells (for SCNT) and sperm (for ICSI) were either from a B6C3F1 or OG2 donor the latter expressing an Oct4 promoter-driven GFP transgene. Additionally cells from pure C56Bl/6 or C3H mice were retrieved for SCNT. Female mice were sacrificed by cervical dislocation 14 h after hCG injection and oocytes retrieved from oviduct ampullae. Oocytes and cumulus cells were handled in Hepes-buffered CZB (HCZB) medium. Double nuclear transfer ICSI and embryo culture: Micromanipulations and embryo culture were performed at 30°C (room temperature). For SCNT oocytes were first enucleated (spindle-chromosome complex removed) before transfer of one two or three cell nuclei with a micropipette driven by a piezo actuator under Nomarski optics. The reconstructed oocytes were activated for 6 h in Calcium-free alpha-MEM supplemented with 10 mM SrCl2 and 5 micro-g mL-1 cytochalasin B. Intracytoplasmic sperm injection (ICSI) of one two or four into intact oocytes was used as the fertilization control condition using sperm from 8-week-old mice of B6C3F1 or OG2 strains. Embryos were placed in embryo culture medium and cultured at 37°C under 5% CO2. The activated oocytes were cultured in alpha-MEM at 37°C and under 5% CO2 until use. On day 5 after activation SCNT and ICSI blastocysts were processed for ESC derivation. Analysis of allelic expression of Oct4: RNA from single embryos was extracted using the ZR-RNA MicroPrep kit (Zymo Research). Complementary DNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Invitrogen) according to manufacturers instruction. A nested PCR was performed to amplify part of the Oct4 cDNA containing a BamH1 sensitive SNP. Primer sequences and PCR conditions are given in the supplementary methods. The PCR product was digested for 8h using 100 units of BamH1-HF (NEB) subsequently subjected to agarose gel electrophoreses and the results grouped into 3 categories (mainly C57Bl/6 balanced mainly C3H) according to the observed band patterns. Global transcriptome analysis by microarray: RNA samples to be analyzed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion) which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto a MouseRef-8 v2 expression BeadChip (Illumina) following the manufacturer instructions. After washing as recommended chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. Microarray data processing: The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was Background correction performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics was performed with in-house developed functions in Matlab.

Project description:Following intracytoplasmic sperm injection (ICSI), in vitro culture (IVC) in ART media causes imbalance in the blastocyst's inner cell mass (ICM)with reduction of the primitive endoderm compartment compared to non-IVC blastocystsFollowing embryo transfer (ET), these imbalances do not influence birth rates, and the postnatal effects are inconspicuous. ICSI alters gene expression in mouse embryos. Choice of embryo culture media has been correlated with birth and body growth rates in human studies. No analysis has yet been made combining ICSI and IVC in the mouse. We produced mouse concepti by ICSI followed by IVC and ET so as to mimic,at least in part, the way human embryos are produced and handled in the clinical practice. Fertilized oocytes were cultured in two sets of sequential ART media, compared with an optimized mouse medium and with the mouse oviduct. Embryos were analyzed or implanted to produce newborns. In total, 3706 fertilized mouse oocytes, 1672 blastocysts and 78 newborns were examined during the course of the study. Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and embryo recipients, respectively. B6C3F1 oocytes were fertilized by intracytoplasmic C57Bl/6 sperm injection and were allowed to cleave in HTF/MultiBlast and in ISM1/ISM2 sequential culture protocols as well as in optimized mouse medium (KSOM(aa)) and in vivo in the CD1 mouse oviduct. Cleavage and blastocyst rates were analyzed by Wilcoxon / Kruskal-Wallis test and the blastocysts were split in two subgroups:one subgroup was analyzed for cell lineage allocation and for transcriptome, the other subgroup was transferred to genital tract, allowed to develop to term and characterized for birth rate, litter size, neonatal bodyweight and behavior using ANOVA F-test and t-test. The effects of ART media on blastocyst rates exceed those of ICSI. Protein and mRNA analysis reveal that IVC has a marked effect on the primitive endoderm lineage of ICSI blastocysts. In contrast to preimplantation, birth rates after ET are indistinguishable not only among ICSI but also in comparison to non-injected controls. At birth, ICSI-IVC-ET concepti are outwardly normal and weigh the same as non-ICSI controls. Confounding effects of subfertility, lifestyle and genetic heterogeneity are reduced to a minimum in the hybrid inbred mouse model compared to human patients. Confounding effects of polyspermy and parthenogenetic activation are prevented by ICSI. There is more to ART than ICSI, IVC and ET, and only a small number of ART media were examined. Preimplantation effects of ART culture media can effectively be modeled in the mouse because the in vitro environment is suboptimal for both mouse and human. However, in the optimal uterine environment of their own species, mouse and human ART blastocysts may be supported and selected for differently, depending on species-specific features such as the kinetics of implantation and the endometrium. 14 samples were analyzed. HTF/MultiBlast: Mouse multiblastocysts in HTF medium, 3 biological rep ISM1/ISM2: Mouse blastocysts in ISM1 and ISM2 medium, 3 biological rep KSOM(aa): Mouse blastocysts in KSOM(aa) medium, 2 biological rep oviduct: Mouse oviduct, 3 biological rep micromanipulation control: Mouse micromanipulation control, 3 biological rep

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Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells

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