High oxygen levels during in vitro culture (IVC) can induce oxidative stress through accumulation of reactive oxygen species (ROS), negatively affecting embryo development. This study evaluated the effect of different O2 tensions during IVC on bovine blastocyst development and transcriptional status, considering transcription factors that play an essential role during early embryo development. For this purpose, embryos were produced in vitro by conventional protocols and cultured in two different oxygen tensions, physiological (5%) and atmospheric (20%). Expanded blastocysts were subjected to transcript quantitation analysis by RT-qPCR with Biomark™ HD System (Fluidigm, US), using 67 TaqMan assays specific for Bos taurus. Differences were observed in genes related to oxidation-reduction processes, DNA-dependent transcription factors, and factors related to important functional pathways for embryo development. Blastocyst rate was higher in the 5% O2 group and the number of cells was assessed, with the 5% O2 group having a higher number of cells. ROS concentration was evaluated, with a higher ROS presence in the 20% O2 group. Taken together, these results allow us to conclude that IVC of embryos at atmospheric O2 tension affects the expression of important transcription factors involved in multiple cell biology pathways that can affect embryo development, quality, and viability.

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Embryo morphokinetics suggests that the timing of the first embryonic cell divisions may predict the developmental potential of an embryo; however, correlations between embryonic morphokinetics and physiology are not clear. Here, we used RNA sequencing to determine the gene expression profile of in vitro-produced early- and late-dividing bovine embryos and their respective blastocysts, and compared these profiles to in vivo-produced blastocysts to identify differentially expressed genes (DEGs). Principal component analysis revealed that fast- and slow-dividing embryos possess similar transcript abundance over the first cleavages. By the blastocyst stage, however, more DEGs were observed between the fast- and slow-dividing embryo groups, whereas blastocysts from the slow-dividing group were more similar to in vivo-produced blastocysts. Gene ontology enrichment analysis showed that the slow-dividing and in vivo-produced blastocysts shared biological processes related to groups of up- or down-regulated genes when compared to the fast-dividing blastocysts. Based on these DEG results, we characterized the relationship between developmental kinetics and energy metabolism of in vitro-produced bovine embryos. Embryos from fast- and slow-dividing groups exhibited different pyruvate and lactate metabolism at 22 hr post-in vitro culture (hpc), glucose consumption at 96 hpc, and glutamate metabolism at 168 hpc. Glycogen storage was similar between cleavage-stage and morulae groups, but was higher in the blastocysts of the slow-dividing group. On the other hand, blastocysts of the fast-dividing group had a higher concentration of lipids. Taken together, these data identify transcriptomic and metabolic differences between embryos with different morphokinetics, suggesting that sorting embryos based on cleavage speed may select for different metabolic patterns. Mol. Reprod. Dev. 83: 324-336, 2016. © 2016 Wiley Periodicals, Inc.

Background The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this purpose, bovine embryos were in vitro produced using sexed semen (female), classified as fast (four or more cells) or slow (two cells) at 40 hpi and cultured until blastocyst stage, when they were analyzed.ResultsGenome-wide DNA methylation analysis identified 11,584 differently methylated regions (DMRs) (7976 hypermethylated regions in fast and 3608 hypermethylated regions in slow embryos). Fast embryos presented more regions classified as hypermethylated distributed throughout the genome, as in introns, exons, promoters, and repeat elements while in slow embryos, hypermethylated regions were more present in CpG islands. DMRs were clustered by means of biological processes, and the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Transcripts profiles from DM genes connected with these pathways were also assessed, and the most part disclosed changes in relative quantitation.ConclusionThe kinetics of the first cleavages influences the DNA methylation and expression profiles of genes related to metabolism and differentiation pathways and may affect embryo viability.Electronic supplementary materialThe online version of this article (10.1186/s13072-017-0171-z) contains supplementary material, which is available to authorized users.

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Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 +/- 0.65) when compared with non-starved cells (71.44 +/- 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.

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