Aims: Embryos produced in vitro are used in human assisted reproductive technology and animal industries. However, the quality and epigenetic landscape of these embryos differ from those of embryos collected from the uterus. Follicular fluid (FF) influences the oviduct and regulates its environment. In the present study, we examined the effects of short-term exposure of early-stage embryos to low concentration of FF
Methods: Experiment-1. In vitro-fertilized embryos were cultured with 1% FF from 18 to 48 h post-insemination (pi), and DNA methylation (5mC) and blastulation rates were examined. Experiment-2. Embryos treated with FF (48 h pi) were subjected to RNA-seq to predict upstream regulators. Experiment-3. 5mC and histone modifications were examined in TGFB1-treated embryos, which were then subjected to RNA-seq. Experiment-4. FF collected from individual cows was rated based on the concentration of TGFB1, and the effects of TGFB1-rich or -poor FF on 5mC and embryonic development were examined. Experiment-5. Granulosa cells corresponding to rich or poor FF were subjected to RNA-seq.
Results: Experiment-1. FF improved embryonic development and reduced the levels of 5mC in the 8-cell and blastocyst stages. Experiment-2. Differentially expressed genes (DEGs) revealed that TGFB1 was a significant upstream regulator. Experiment-3. TGFB1 reduced the levels of 5mC in the 8-cell and blastocyst stages, increased the levels of TET3 and H3K4me3, and decreased the levels of H3K9me3 in the 8-cell stage. Pathways enriched by DEGs included focal adhesion pathways. Experiment-4. TGFB1-rich FF improved embryonic development and reduced 5mC compared to poor FF. Experiment-5. DEGs of granulosa cells revealed that the top upstream regulator of rich FF granulosa cells was TGFB1.
Conclusion: TGFB1 plays a major role in follicle formation, induces DNA demethylation, and improvement of development.