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Differences prevailing in bovine in vitro produced blastocysts in the view of their post-transfer phenotypes three days ahead of implantation

ABSTRACT: Embryo transfer is largely used in cattle and classically performed at D7 (or D8) using unsorted D7 (or D8) blastocysts produced in vivo, or in vitro in defined media without serum or feeders. Outdated systems including serum and co-culture were however of interest for research purposes. We thus wondered whether embryos that would form a blastocoel at different times after fertilisation (D6 to D8) and stay in culture for up to 2 additional days (D6+1, D6+2, D7+1) would equally develop in vivo after temporary transfer to oestrus-synchronised recipients. Globally alike, those that survived up to D18 reached primitive streak stages and elongated to filamentous sizes similarly to in vivo (D18) or in vitro controls (classical D7-T7). Recovery rate differed between D6 and D8 embryos that were immediately transferred (58 vs 25% in D6-T6 vs D8-T8). With a reduced but intermediate survival (33%), the D6 embryos that stayed 2 more days in culture produced 7 times more IFN-tau at D18 than the immediately transferred D6 embryos. At the end of the culture, D6+2 embryos also displayed the higher number of differences with the D6 blastocysts. A “+1” phenotype emerged from the D6+1 and D7+1 embryos, that shared a larger gene set enrichment than the blastocysts they derived from, possible sign of a similar adaption to the in vitro environment. To the best of our knowledge, this is the first time that this culture system (B2, serum, co-culture) is used to study its impacts on the embryonic transcriptome prior to transfer. Initially reputed as beneficial to produce more expanding and hatching blastocysts, this culture system generated blastocysts that all dissembled in vivo developed ones (D7). Despite a loss of 40 to 60% in the two weeks after ET, no dying (vs surviving) signature was detectable in any of the transferred groups (D6, D6+2, D7+1, D8); was it rather a matter of developmental “pause”? Whether molecular differences prevailing to transfer partly reflected those induced by unfavourable conditions (or diapause) was therefore assessed.

ORGANISM(S): Bos taurus

PROVIDER: GSE96925 | GEO | 2017/03/23

SECONDARY ACCESSION(S): PRJNA380117

REPOSITORIES: GEO

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Project description:MAPK cascade in solution (no scaffold) Description This model describes a basic 3- stage Mitogen Activated Protein Kinase (MAPK) cascade in solution. This cascade is typically expressed as RAF= =>MEK==>MAPK (alternative forms are K3==>K2==> K1 and KKK==>KK==>K) . The input signal is RAFK (RAF Kinase) and the output signal is MAPKpp ( doubly phosphorylated form of MAPK) . RAFK phosphorylates RAF once to RAFp. RAFp, the phosphorylated form of RAF induces two phoshporylations of MEK, to MEKp and MEKpp. MEKpp, the doubly phosphorylated form of MEK, induces two phosphorylations of MAPK to MAPKp and MAPKpp. Rate constant Reaction a10 = 5. MAPKPH + MAPKpp -> MAPKppMAPKPH a1 = 1. RAF + RAFK -> RAFRAFK a2 = 0.5 RAFp + RAFPH -> RAFpRAFPH a3 = 3.3 MEK + RAFp -> MEKRAFp a4 = 10. MEKp + MEKPH -> MEKpMEKPH a5 = 3.3 MEKp + RAFp -> MEKpRAFp a6 = 10. MEKPH + MEKpp -> MEKppMEKPH a7 = 20. MAPK + MEKpp -> MAPKMEKpp a8 = 5. MAPKp + MAPKPH -> MAPKpMAPKPH a9 = 20. MAPKp + MEKpp -> MAPKpMEKpp d10 = 0.4 MAPKppMAPKPH -> MAPKPH + MAPKpp d1 = 0.4 RAFRAFK -> RAF + RAFK d2 = 0.5 RAFpRAFPH -> RAFp + RAFPH d3 = 0.42 MEKRAFp -> MEK + RAFp d4 = 0.8 MEKpMEKPH -> MEKp + MEKPH d5 = 0.4 MEKpRAFp -> MEKp + RAFp d6 = 0.8 MEKppMEKPH -> MEKPH + MEKpp d7 = 0.6 MAPKMEKpp -> MAPK + MEKpp d8 = 0.4 MAPKpMAPKPH -> MAPKp + MAPKPH d9 = 0.6 MAPKpMEKpp -> MAPKp + MEKpp k10 = 0.1 MAPKppMAPKPH -> MAPKp + MAPKPH k1 = 0.1 RAFRAFK -> RAFK + RAFp k2 = 0.1 RAFpRAFPH -> RAF + RAFPH k3 = 0.1 MEKRAFp -> MEKp + RAFp k4 = 0.1 MEKpMEKPH -> MEK + MEKPH k5 = 0.1 MEKpRAFp -> MEKpp + RAFp k6 = 0.1 MEKppMEKPH -> MEKp + MEKPH k7 = 0.1 MAPKMEKpp -> MAPKp + MEKpp k8 = 0.1 MAPKpMAPKPH -> MAPK + MAPKPH k9 = 0.1 MAPKpMEKpp -> MAPKpp + MEKpp Variable IC ODE MAPK 0.3 MAPK'[t] == d7*MAPKMEKpp[t] + k8*MAPKpMAPKPH[t] - a7*MAPK[t]*MEKpp[t] MAPKMEKpp 0 MAPKMEKpp'[t] == -(d7*MAPKMEKpp[t]) - k7*MAPKMEKpp[t] + a7*MAPK[t]*MEKpp[t] MAPKp 0 MAPKp'[t] == k7*MAPKMEKpp[t] - a8*MAPKp[t]*MAPKPH[t] + d8*MAPKpMAPKPH[t] + d9*MAPKpMEKpp[t] + k10* MAPKppMAPKPH[t] - a9*MAPKp[t]*MEKpp[t] MAPKPH 0.3 MAPKPH'[t] == -(a8*MAPKp[t]*MAPKPH[t]) + d8*MAPKpMAPKPH[ t] + k8*MAPKpMAPKPH[t] - a10*MAPKPH[t]*MAPKpp[t] + d10*MAPKppMAPKPH[t] + k10*MAPKppMAPKPH[t] MAPKpMAPKPH 0 MAPKpMAPKPH'[t] == a8*MAPKp[t]*MAPKPH[t] - d8* MAPKpMAPKPH[t] - k8*MAPKpMAPKPH[t] MAPKpMEKpp 0 MAPKpMEKpp'[t] == -(d9*MAPKpMEKpp[t]) - k9*MAPKpMEKpp[t] + a9*MAPKp[t]*MEKpp[t] MAPKpp 0 MAPKpp'[t] == k9*MAPKpMEKpp[t] - a10*MAPKPH[t]*MAPKpp[t] + d10*MAPKppMAPKPH[t] MAPKppMAPKPH 0 MAPKppMAPKPH'[t] == a10*MAPKPH[t]*MAPKpp[t] - d10* MAPKppMAPKPH[t] - k10*MAPKppMAPKPH[t] MEK 0.2 MEK'[t] == k4*MEKpMEKPH[t] + d3*MEKRAFp[t] - a3*MEK[t]*RAFp[t] MEKp 0 MEKp'[t] == -(a4*MEKp[t]*MEKPH[t]) + d4*MEKpMEKPH[t] + k6*MEKppMEKPH[t] + d5*MEKpRAFp[t] + k3*MEKRAFp[ t] - a5*MEKp[t]*RAFp[t] MEKPH 0.2 MEKPH'[t] == -(a4*MEKp[t]*MEKPH[t]) + d4*MEKpMEKPH[t] + k4*MEKpMEKPH[t] - a6*MEKPH[t]*MEKpp[t] + d6* MEKppMEKPH[t] + k6*MEKppMEKPH[t] MEKpMEKPH 0 MEKpMEKPH'[t] == a4*MEKp[t]*MEKPH[t] - d4*MEKpMEKPH[t] - k4*MEKpMEKPH[t] MEKpp 0 MEKpp'[t] == d7*MAPKMEKpp[t] + k7*MAPKMEKpp[t] + d9*MAPKpMEKpp[t] + k9*MAPKpMEKpp[t] - a7*MAPK[t]* MEKpp[t] - a9*MAPKp[t]*MEKpp[t] - a6*MEKPH[t]*MEKpp[t] + d6*MEKppMEKPH[t] + k5*MEKpRAFp[t] MEKppMEKPH 0 MEKppMEKPH'[t] == a6*MEKPH[t]*MEKpp[t] - d6*MEKppMEKPH[ t] - k6*MEKppMEKPH[t] MEKpRAFp 0 MEKpRAFp'[t] == -(d5*MEKpRAFp[t]) - k5*MEKpRAFp[t] + a5*MEKp[t]*RAFp[t] MEKRAFp 0 MEKRAFp'[t] == -(d3*MEKRAFp[t]) - k3*MEKRAFp[t] + a3*MEK[t]*RAFp[t] RAF 0.4 RAF'[t] == -(a1*RAF[t]*RAFK[t]) + k2*RAFpRAFPH[t] + d1*RAFRAFK[t] RAFK 0.1 RAFK'[t] == -(a1*RAF[t]*RAFK[t]) + d1*RAFRAFK[t] + k1*RAFRAFK[t] RAFp 0 RAFp'[t] == d5*MEKpRAFp[t] + k5*MEKpRAFp[t] + d3*MEKRAFp[t] + k3*MEKRAFp[t] - a3*MEK[t]*RAFp[t] - a5*MEKp[t]*RAFp[t] - a2*RAFp[t]*RAFPH[t] + d2* RAFpRAFPH[t] + k1*RAFRAFK[t] RAFPH 0.3 RAFPH'[t] == -(a2*RAFp[t]*RAFPH[t]) + d2*RAFpRAFPH[t] + k2*RAFpRAFPH[t] RAFpRAFPH 0 RAFpRAFPH'[t] == a2*RAFp[t]*RAFPH[t] - d2*RAFpRAFPH[t] - k2*RAFpRAFPH[t] RAFRAFK 0 RAFRAFK'[t] == a1*RAF[t]*RAFK[t] - d1*RAFRAFK[t] - k1*RAFRAFK[t] Generated by Cellerator Version 1.4.3 (6-March-2004) using Mathematica 5.0 for Mac OS X (November 19, 2003), March 6, 2004 12:18:07, using (PowerMac, PowerPC,Mac OS X,MacOSX,Darwin) author=B.E.Shapiro This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2010 The BioModels.net Team. For more information see the terms of use . To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

Project description:Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain M-bM-^@M-^\unclonableM-bM-^@M-^] for unknown reasons. Here we examined whether neurons from adult mice could be cloned, using a combination of two epigenetic approaches. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark - dimethylated histone H3 lysine 9 (H3K9me2) - and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (per embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrated for the first time that adult neurons could be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos. Comparative gene expression analyses using blastocysts of cloned embryos were performed by microarray. Cloned embryos were produced with three different types of donor cells (neonatal Sertoli cells, CA1 pyramidal cells and Purkinje cells) and all cloned embryos were treated with Trichostatin A (TSA). Each embryos were cultured for 96 h and blastocysts derived from each donor cell types were subjected to gene expression microarray. For comparison of gene expression, the data sets of control sex- and genotype-matched embryos produced by in vitro fertilization and SCNT-derived blastocysts from cumulus cells treated with TSA from our previous paper (Inoue K. et al. Science 2010) were also used.

Project description:Background: Since the development of in vitro embryo production in cattle, different supplements have been added to embryo culture media to support development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birth weights and low viability in calves (Large Offspring Syndrome). Global gene expression analysis of bovine embryos through RNA sequencing produced in different conditions (in vivo, in vitro with serum added and in vitro without addition of serum) can provide valuable information to optimize culture media for in vitro embryo production, which is necessary to obtain healthy offspring. Results: Here, we used RNA sequencing to examine the effect of in vitro embryo production, either in serum or in serum-free conditions, on the global gene expression pattern of individual bovine blastocysts. For this purpose, we compared in vitro produced with in vivo derived embryos. The embryos produced in serum had ten times more genes differentially expressed than the embryos produced in serum-free conditions (1,106 vs. 110). Importantly, in vitro production appeared to have a different impact on the embryos according to their sex, with male embryos showing more deviations in their gene expression pattern than their female counterparts. In particular, embryos produced in the presence of serum showed the largest difference, with male embryos having eight times more genes differentially expressed than the females (1,561 vs. 200). This difference was reduced when the embryos were produced in serum-free conditions, where the male embryos had only about double the number of genes differentially expressed than their female counterparts (64 vs. 35). The pathways affected by in vitro production differed depending on the supplementation. While embryos produced in serum conditions had altered genes involved in RNA processing and mitosis, embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism. Conclusions: Serum supplementation had a major impact on the gene expression pattern of the embryos. On the contrary, the transcriptome of embryos produced in serum-free conditions resembled more in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by in vitro production, especially after serum supplementation.

Project description:In present, interspecies cloning and interspecies-pregnancy were studied for endangered species rescue. However, the low implantation and survival ratio, spontaneous abortion, and unknown reason embryos absorption are the common and difficult problems of interspecies-pregnancy. In order to discover the mechanism of interspecies-pregnant failure and find ways to overcome the xeon-pregnant obstacles, we chosen the rat embryos pregnant in mouse uterus as a interspecies-pregnancy model. Three groups were set, mouse embryos to mouse recipients (MM) as control group, rat embryos to mouse recipients (RM), and rat and mouse embryos to mouse recipients together (RMM) as experiment groups. The former studies showed that rat embryos live no longer than day 7 of mouse pregnancy (D7). Our results showed that rat embryos survived to D7, and still existed to day 9 of mouse pregnancy (D9) in RM group. Surprisingly, the rat embryos survived to day 13 of the mouse gestation (D13) in RMM group. Microarray analysis was used to detect the global-gene expression profile changes of the whole implantation sites among the three groups at D7 and D9. By this way, we screened out the genes promoting the implanted rat embryos development in a mouse uterus which helped the rat embryos survive to D13 in RMM group compared with RM group, and the genes hindering the rat embryos development in a mouse uterus which prevented rat embryos living longer than D7 in RM group and D13 in RMM group compared with MM group. These findings provide insights into the mechanism of interspecies pregnant failure and new idea for interspecies pregnant studies. Experiment Overall Design: microarray was use to screen the genes among the day 7 and day 9 implantation sites of rat embryos implantation sites in a mouse uterus between rat embryos transfer to mouse recipients and rat embryos transfer to mouse recipients with mouse embryos. The mouse day7 and day 9 embryos implantation sites were use as control. Experiment Overall Design: totally 6 samples were analyzed, each samples two replications (one of them had three replications).

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Differences prevailing in bovine in vitro produced blastocysts in the view of their post-transfer phenotypes three days ahead of implantation

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