70 Energy metabolites induce differential DNA methylation levels and transcription in trophectoderm and inner cell mass
Bibliographic record
Abstract
DNA methylation/demethylation is one of several epigenetic mechanisms by which metabolism regulates gene expression. More specifically, α-ketoglutarate (αKG) and succinate (Suc) are tricarboxylic acid cycle metabolites that may decrease and increase, respectively, the activity of DNA demethylases. Because pre-implantation embryos undergo reprogramming in both DNA methylation and metabolic pathways, it is possible that metabolic changes influence this epigenetic mark. To test that hypothesis, bovine embryos were in vitro produced by using standard protocols and, 8 h after fertilization, zygotes were transferred to synthetic oviductal fluid (SOF)-based culture medium (control, CO) or culture medium containing 4 mM dimethyl-αKG, or 4 mM dimethyl-Suc, where they remained until Day 4. Embryos were collected at Day 4 or remained in culture until Day 7, in control medium. Day 4 embryos were evaluated for DNA methylation levels by immunofluorescence detection of 5-methylcytosine (5mC) and cleavage rate. Day 7 embryos were also assessed for DNA methylation by immunofluorescence of 5mC, total cell number, blastocyst rates, and quantification of ACTB (housekeeping), DNMT1, DNMT3A, and DNMT3B transcript by RT-qPCR in trophectoderm (TE) and inner cell mass (ICM) separated by immunosurgery. The mRNA expression levels of were normalized to internal control ACTB and subsequently calculated using the 2−ΔΔCT method, using the control group for comparisons. All data were submitted to outlier detection using ROUT with Q = 1% followed by one-way analysis of variance (ANOVA) and Fisher’s least significant difference (l.s.d.) test in GraphPad Prism. αKG and Suc did not influence cleavage or blastocyst rates, total cell number, or cell allocation. αKG supplementation reduced 5mC fluorescence intensity in embryos assessed at Day 4 (CO: 12.8 ± 0.4 AU; αKG: 9.0 ± 0.2AU; P < 0.0001) and Day 7 (CO: 36.5 ± 0.7 AU; αKG: 23.5 ± 0.4 AU; P < 0.0001), whereas Suc incubation increased DNA methylation levels in embryos at Day 4 (CO: 12.8 ± 0.4 AU; Suc: 15.7 ± 0.3 AU; P < 0.0001) and Day 7 (CO: 36.5 ± 0.7 AU; Suc: 70.5 ± 0.5 AU; P < 0.0001). αKG increased expression of DNMT1 (P = 0.0438) in the ICM and led to lower levels of DNMT1 (P < 0.0001), DNMT3A (P = 0.0013), and DNMT3B (P = 0.0015) in TE cells. The culture with Suc increased DNMT1 (P = 0.0074), DNMT3A (P = 0.0186), and DNMT3B (P = 0.0286) in ICM. Regarding TE, Suc resulted in lower expression of DNMT1 (P < 0.0001), DNMT3A (P = 0.0017), and DNMT3B (P = 0.0052). In conclusion, both supplementations resulted in global DNA methylation changes without affecting embryo development rates or morphology. These changes were accompanied by alterations in transcript profiles between ICM and TE, with differences among treatments being more pronounced in transcripts from ICM. This is the first report of DNA demethylation–induced changes by analogues of TCA cycle metabolites during early reprogramming of the bovine embryo with prolonged effects in TE and ICM cells. This research was funded by FAPESP: 2017/18384-0; 2018/11668-6.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".