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The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.

Assuntos

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Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed "OSKM" factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.

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Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.

RESUMO

In many cell types, epigenetic changes are partially regulated by the availability of metabolites involved in the activity of chromatin-modifying enzymes. Even so, the association between metabolism and the typical epigenetic reprogramming that occurs during preimplantation embryo development remains poorly understood. In this work, we explore the link between energy metabolism, more specifically the tricarboxylic acid cycle (TCA), and epigenetic regulation in bovine preimplantation embryos. Using a morphokinetics model of embryonic development (fast- and slow-developing embryos), we show that DNA methylation (5mC) and hydroxymethylation (5hmC) are dynamically regulated and altered by the speed of the first cleavages. More specifically, slow-developing embryos fail to perform the typical reprogramming that is necessary to ensure the generation of blastocysts with higher ability to establish specific cell lineages. Transcriptome analysis revealed that such differences were mainly associated with enzymes involved in the TCA cycle rather than specific writers/erasers of DNA methylation marks. This relationship was later confirmed by disturbing the embryonic metabolism through changes in α-ketoglutarate or succinate availability in culture media. This was sufficient to interfere with the DNA methylation dynamics despite the fact that blastocyst rates and total cell number were not quite affected. These results provide the first evidence of a relationship between epigenetic reprogramming and energy metabolism in bovine embryos. Likewise, levels of metabolites in culture media may be crucial for precise epigenetic reprogramming, with possible further consequences in the molecular control and differentiation of cells.

Assuntos

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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.

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The objective of Experiment 1 was to compare the effects of estradiol benzoate (EB) given 0 or 24h after the end of a progestagen treatment on ovulation and CL formation in anestrous cows. Twenty cows were treated with an intravaginal sponge containing 250 mg of medroxiprogesterone acetate (MPA). At sponge insertion, each cow received 3 mg EB and 10 mg MPA im. At device removal, cows received 0.7 mg EB either at that time (EB0) or 24h later (EB24). Ultrasound examinations and blood sampling to determine plasma progesterone concentrations were performed to detect ovulation and CL formation. Ovulation occurred in 77.8 and 81.8% cows in the EB0 and EB24 groups, respectively. Diameter of the ovulatory follicle (EB0 = 10.9 +/- 0.5mm; EB24 = 12.1 +/- 0.8 mm; P = 0.26) and the interval from sponge removal to ovulation (median = 3 days; P = 0.64) did not differ between treatments. Among the cows that ovulated (n = 16), short-lived CL were present in 2/7 and 2/9 cows in the EB0 and EB24 groups, respectively. Plasma progesterone concentrations and CL area did not differ between treatments (P > 0.05). In Experiment 2, cows were treated with the same protocol as in Experiment 1, but at sponge withdrawal all cows received 250 microg cloprostenol and timed artificial insemination (TAI) was performed 48 h after sponge removal. In Replicate 1 (n = 204 multiparous cows), pregnancy rates were 45.0 and 47.5% for EB0 and EB24, respectively (P > 0.05). In Replicate 2 (n = 69 primiparous cows) pregnancy rate did not differ between EB0 and EB24 (51.4% versus 52.9%). In conclusion, EB given 0 or 24h after the end of a progestagen treatment had the same effect on ovulation rate, time to ovulation, diameter of the ovulatory follicle, incidence of short-lived CL, luteal tissue area, and plasma progesterone concentrations of normal lifespan CL, and pregnancy rate after TAI in suckled beef cows.

Assuntos