(360) Metabolic Regulation of Epigenetic Reprogramming at Fertilization

Carmen J. Williams¹, Paula Stein¹, Don A. Delker², Martín A. Estermann¹, Brian N. Papas², Zongli Xu³, Lenka Radonova¹, Virginia Savy¹

1. Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Durham, NC, USA

2. Integrative Bioinformatics Support Group, National Institute of Environmental Health Sciences, NIH, Durham, NC, USA

3. Biostatistics & Computational Biology Branch, National Institute of Environmental Health Sciences, NIH, Durham, NC, USA

 

Abstract Text:

In mice, abnormal calcium oscillations at fertilization negatively impact embryo development, implantation, and offspring health. It is unknown what mechanism links changes in calcium homeostasis at fertilization with these outcomes. This information is critical because calcium dynamics at fertilization are disrupted in patients with diabetes and inflammation or during assisted reproductive technology (ART) procedures, with unknown consequences. Upon fertilization, as the egg's calcium levels oscillate, there is a rapid rearrangement of epigenetic modifications, essential for successful maternal to zygotic transition (MZT). The epigenetic machinery uses metabolites, known as “epimetabolites”, which are generated by mitochondrial or nuclear enzymes. Among those, acetyl-CoA and lactyl-CoA are used for writing H3K27ac and H3K18la marks, critical for MZT. Interestingly, the activity of certain mitochondrial enzymes, such as pyruvate dehydrogenase (responsible for irreversible pyruvate to acetyl-CoA conversion), is regulated by intracellular calcium levels. We hypothesized that abnormal calcium exposure changes the availability of epimetabolites necessary for epigenetic reprogramming, priming the embryo for aberrant preimplantation development and long-term consequences on adult health. We developed a mouse model of dramatically increased calcium exposure at fertilization by targeting two plasma membrane calcium pumps (PMCA). PMCA-null eggs (dKO) had about 10 times more calcium than controls at fertilization (p< 0.0001). While fertilization rate was not affected, 40% of the embryos arrested their development between the 2-cell and 4-cell stage, suggesting that calcium impacts the MZT. Moreover, male offspring obtained from dKO females had higher fasting glucose than controls (p< 0.05) and their visceral fat had altered global DNA methylation and coordinate changes in expression of genes that regulate adipocyte function. To identify the mechanism, we focused on epigenetic changes occurring in the peri-fertilization window. dKO embryos had increased H3K27ac levels and decreased H3K18la levels at the 1-cell stage; these changes persisted until the 2-cell stage. Increased calcium exposure resulted in extremely low global transcription at the 1-cell (80% reduction, p< 0.0001) and 2-cell stages (p< 0.005) due to impairment of RNA polymerase I-mediated transcription. Similar results were obtained when ionomycin was used to raise intracellular calcium of wild-type fertilized eggs, a procedure used in ART clinics. Ultralow input CUT&Tag revealed significant differences in H3K27ac and H3K18la profiles between control and ionomycin-treated embryos at the 2C stage. We hypothesized that calcium stimulation of pyruvate dehydrogenase reduces the conversion of pyruvate to lactate, leading to decreased lactyl-CoA and H3K18la levels. Indeed, microinjection of exogenous lactyl-CoA before ionomycin treatment restored H3K18la and global RNA synthesis levels to that of controls. Together, these results demonstrate conclusively that excess calcium exposure at fertilization alters epigenetic reprogramming and does so in part by altering lactyl-CoA availability. These findings should be considered by ART clinics when selecting culture conditions and other treatments that impact calcium levels in human embryos.