233 EXPRESSION AND LOCALIZATION OF DNMT1 DURING EARLY BOVINE DEVELOPMENT
Bibliographic record
Abstract
DNA methylation of CG motifs is an important mechanism of transcriptional regulation. During embryonic development DNA methyltransferase 1 (DNMT1) has been implicated in the maintainance of gametic and embryonic epigenetic patterns. Here we report the characterization of DNMT1 expression patterns within in vitro-produced (IVP) bovine embryos. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries and either denuded (germinal vesicle, GV) or matured in vitro for 24 h (metaphase II, MII). Embryos at the 1-, 2-, 4-, 8-, 16-cell stage, and morula (Mo, Day 6 post-insemination, p.i.) and blastocyst (Days 7, 8, and 9 p.i.) stages were produced by in vitro fertilization and cultured in SOFm for appropriate times under a 5% O2, 5% CO2, and 90% N2 atmosphere. Oocytes/embryos were either snap frozen (-80°C) for DNMT1 mRNA and protein expression profiles analysis or wholemount fixed and permeabilized for DNMT1 immunostaining followed by laser confocal microscopy. DNMT1 RNA, determined by real-time PCR analysis, was present throughout bovine embryonic development (replications = 3, n = 10 embryos/pool and normalized by Histone H2A expression). Growing oocytes accumulated DNMT1 transcripts until the MII stage (20-fold increase), whereas after fertilization DNMT1 RNA levels decreased and remained constant until the 16-cell stage when DNMT1 RNA levels decreased further and then remains constant until the blastocyst (Day 8 p.i.) stage. Confocal analysis of DNMT1 immunostained oocytes/embryos (n = 20/stage) revealed that DNMT1 is localized in the cytoplasm of the oocyte and pre-implantation embryo with the exception of the 16-cell stage, when the enzyme is translocated to nucleus (confirmed by Hoechst co-localization). Moreover, a punctuate staining pattern was observed for DNMT1, which could be due to its association with the mitochondria or endoplasmic reticulum. Interestingly, in GV oocytes DNMT1 was present in localized areas of the nucleus, suggestive of nucleoli association. DNMT1 was observed in the majority of the nuclei in early blastocysts, while after expansion, DNMT1 accumulated in the cytoplasm of the trophectoderm and was localized in both the cytoplasm and the nucleus of the majority of cells within the inner cell mass. Western blot analysis revealed low levels of DNMT1 protein in oocytes and pre-implantation embryos with the exception of the 16-cell embryos and Mo stages during which a significant (P < 0.05) increase in the levels of DNMT1 protein was observed. Specificity of primers and conditions for the real-time PCR assay were confirmed by cDNA sequencing whereas specificity of the antibody used for immunofluorescence and western blot analysis was confirmed by amino acid sequencing. These results suggest the participation of DNMT1 in the bovine embryonic genome activation process, supporting a passive DNA demethylation process during the early cleavage stages in the bovine. The low expression profile of DNMT1 after morula stage indicates that other methylases are required for the maintenance of DNA methylation during blastocyst formation and expansion. This work was funded by CFIA, NSERC, CIHR, and OMAF.
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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".