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Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos
Published on: August 18, 2022
Apoptotic processes and DNA cytosine methylation in mouse embryos arrested at the 2-cell stage
Dusan Fabian1, Alexandra Bukovská, Stefan Juhás
1Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovakia. fabian@saske.sk
Abstract:
The present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (alpha-amanitin, actinomycin D, TNF-alpha). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.
Insights
Early embryo development failures involve low apoptosis and significant DNA hypomethylation in 2-cell stage mouse embryos. Arrested embryos show widespread demethylation preceding apoptosis, impacting blastocyst development.
Area of Science:
- Developmental Biology
- Epigenetics
- Cell Biology
Background:
- Early embryonic development is crucial for reproductive success.
- Apoptosis and DNA methylation are key regulatory processes in development.
- Failures in these processes can lead to developmental arrest.
Purpose of the Study:
- To investigate the roles of apoptosis and DNA methylation reprogramming in early developmental failures.
- To analyze these processes in mouse embryos at the 2-cell stage.
- To correlate epigenetic changes with cell death markers.
Main Methods:
- In vitro culture of mouse 2-cell embryos.
- Chemical induction of developmental arrest and apoptosis (alpha-amanitin, actinomycin D, TNF-alpha).
- Cell-death assays (annexin V, TUNEL, caspase-3) and DNA methylation analysis (5-methylcytosine).
Main Results:
- Apoptosis in 2-cell stage embryos was minimal and delayed, appearing after 72 hours in arrested embryos.
- Nuclear condensation was the earliest apoptotic sign, preceding phosphatidylserine flip.
- Both normal and arrested embryos showed reduced DNA cytosine methylation; arrested embryos exhibited more pronounced, genome-wide demethylation.
- Demethylation onset overlapped with apoptotic events, with demethylated DNA more frequent in apoptotic cells at the blastocyst stage.
Conclusions:
- Apoptosis plays a limited role in early 2-cell stage arrest.
- Extensive DNA hypomethylation is a significant feature of developmental failure at this stage.
- Epigenetic reprogramming, specifically demethylation, is closely linked to cell death pathways in arrested embryos.
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