67 research outputs found

    Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos.

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    STUDY QUESTION Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? SUMMARY ANSWER There is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos. WHAT IS KNOWN ALREADY CIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown. STUDY DESIGN, SIZE, DURATION Because human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF). PARTICIPANTS/MATERIALS, SETTING, METHODS Single blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos. MAIN RESULTS AND THE ROLE OF CHANCE The genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P < 0.0001), and the frequency of whole chromosome or segmental aberrations was higher in embryos produced in vitro than in embryos derived in vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P < 0.01) and 84.6% of IVM-IVF embryos (P < 0.001). LARGE SCALE DATA Genotyping data obtained in this study has been submitted to NCBI Gene Expression Omnibus (GEO; accession number GSE95358) LIMITATIONS REASONS FOR CAUTION There were two main limitations of the study. First, animal models may not always reflect the nature of human embryogenesis, although the use of an animal model to investigate CIN was unavoidable in our study. Second, a limited number of embryos were obtained, therefore more studies are warranted to corroborate the findings. WIDER IMPLICATIONS OF THE FINDINGS Although CIN is also present in in vivo-developed embryos, in vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be applied with care. Additionally, our results encourage to refine and improve in vitro culture conditions and assisted reproduction technologies. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Agency for Innovation by Science and Technology (IWT) (TBM-090878 to J.R.V. and T.V.), the Research Foundation Flanders (FWO; G.A093.11 N to T.V. and J.R.V. and G.0392.14 N to A.V.S. and J.R.V.), the European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, EU324509 to J.R.V., T.V., O.T, A.D., A.S. and A.K.) and Horizon 2020 innovation programme (WIDENLIFE, 692065 to J.R.V., O.T., T.V., A.K. and A.S.). M.Z.E., J.R.V. and T.V. are co-inventors on a patent application ZL913096-PCT/EP2014/068315-WO/2015/028576 (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), licensed to Cartagenia (Agilent Technologies

    Maternal metabolic status and in-vitro culture conditions during embryonic genome activation deregulate the expression of energy-related genes in the bovine 16-cells embryo.

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    The molecular consequences of the metabolic stress caused by milk production of dairy cows in the early embryo are largely unknown. The objective was to determine the impact of dam metabolic status or in vitro culture during embryonic genome activation (EGA) on the transcriptomic profiles of bovine 16-cell stage embryos. Two days after synchronized oestrus, in vitro produced 2- to 4-cell stage embryos were endoscopically transferred in pools of 50 into the oviduct ipsilateral to the corpus luteum of lactating (LACT, n = 3) or nonlactating (i.e. dried off immediately at calving; DRY, n = 3) dairy cows. On Day 4, the oviducts were flushed to recover the embryos. Pools of five Day-2 embryos (n = 5) and Day-4 16-cell stage embryos obtained in vitro (n = 3) or from LACT or DRY cows were subjected to RNAseq. Temporally differentially expressed genes (DEG; FDR<0.05) between Day-2 and Day-4 embryos were determined considering the differences between the three conditions under which EGA occurred. Also, DEG between Day-4 embryos derived from the three conditions were identified. Functional analysis of the temporal DEG demonstrated that genes involved in ribosome, translation and oxidative phosphorylation in the mitochondria were strongly more expressed in Day-4 than Day-2 embryos. Comparison of Day-4 embryos that underwent EGA in vitro, or in LACT or DRY cows, identified DEG enriching for mitochondrial respiration and protein translation, including the mTOR pathway. In conclusion, exposure of the embryo to an unfavourable maternal metabolic status during EGA influences its transcriptome and potentially the competence for pregnancy establishment

    Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions

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    Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro-and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE's bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.Understanding gene expression patterns in response to altered environmental conditions atdifferent time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro (IVP) and in vivo produced blastocysts were used as controls. We compared gene-expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE’s bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates, however in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo vs. in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts

    CMV promoter is inadequate for expression of mutant human RyR2 in transgenic rabbits

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    Introduction Fundamental differences in Ca2+ homeostasis between mice and larger mammals require the validation of the mechanisms of arrhythmogenesis before translation into human pathophysiology. The purpose of this study was to create transgenic rabbits that express defective human cardiac ryanodine receptor (hRyR2) with a mutation (R4497C) causing a clinically relevant arrhythmogenic syndrome. Methods The construct pcDNA3-EGFP-hRyR2-R4497C with the CMV promoter was used to generate transgenic rabbits. The founder animals were created by microinjection and identified by PCR with specific primers for the EGFP sequence. The copy number of the transgene was quantified by real-time PCR using genomic DNA from blood cells. mRNA expression of EGFP-hRyR2-R4497C was quantified using RT-PCR with specific primers for the RyR2 and EGFP sequence. Protein expression of the transgene in heart and non-cardiac tissues was determined using immunoblots with antibodies directed against EGFP and RyR2. Results Real-time PCR in peripheral blood cells identified several rabbit lines with the construct integrated into their genome. Transcription levels of the transgene were low (Ct > 30). On the protein level, neither EGFP nor hRyR2 R4497C was detected in either cardiac or non-cardiac tissue. A truncated gene product (3′ end and central part of hRyR2 R4497C, but not EGFP) could be detected at the mRNA level in the heart. Discussion Lack of significant protein expression of the EGFP-RyR2 R4497C gene construct despite successful incorporation into the genomic DNA is due to combination of at least two factors: low mRNA expression, and truncation of the transgene on the mRNA level. Our results suggest that the CMV promoter may not be well suited for creating transgenic rabbits
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