IMPACT OF NUTRIENT IMBALANCE ON THE EPIGENETIC SIGNATURE AND GENE EXPRESSION OF BOVINE EARLY EMBRYOS

The Developmental Origins of Health and Disease (DOHaD) theory emphasizes the long-term health implications of maternal nutritional imbalances during pregnancy. This study focuses on how variations in energetic substrates during preimplantation development affect gene expression, differential transcript usage (DTU), and early developmental outcomes in bovine embryos. Using in vitro production techniques, bovine blastocysts were produced in standard and modified serum-free culture medium with either restricted (0.5-fold) or excess (1.5-fold) energy levels compared to the standard. RNA was extracted from pools of five expanded blastocysts per treatment, with quality assessments ensuring RNA integrity numbers (RIN) of 8.6-10. The sequencing was performed on a NovaSeq 6000 platform, producing 150 bp paired end reads, which were mapped and quantified using Salmon or HISAT2/HTSEQ. Differential gene expression analysis, conducted with DESeq2 in R, revealed only a few genes with significant changes (adjusted p<0.1), and Principal Component Analysis (PCA) showed distinct clustering just in embryos exposed to increased energy substrates. In agreement with these findings, the blastocyst rate and morphology, and the expression of lineage markers (SOX2, CDX2, and NANOG) remained largely consistent across treatments, suggesting that early embryonic development is resilient to mild nutritional fluctuations. Additionally, differential transcript usage (DTU) - which has been implicated in disease onset through changes in transcript isoforms, driven by mechanisms like alternative splicing or promoter activity shifts - has been explored. By analyzing RNA-Seq data with specific pipelines (DRIMSeq and DEXSeq), the study identified nearly 6000 genes with multiple isoforms, of which approximately 90 exhibited differential expression in response to energy level variations, resulting in 250-280 isoforms affected according to the culture conditions. Enrichment analysis highlighted molecular functions linked to 'palmitoyl-CoA hydrolase activity' and 'acyl CoA hydrolase activity,' consistent with the onset of metabolic adjustments. Furthermore, 'histone deacetylase binding' was affected in the increased energy treatment, while 'transcription factor binding' was more prominent under energy restriction, pointing to interference with epigenetic and nuclear reprogramming mechanisms. Immunofluorescence and confocal microscopy confirmed an involvement of histone acetylation, showing a 25-50% increase in the signal intensity of in embryos exposed to higher energy substrates, a change that could have long-lasting implications for gene regulation and developmental outcomes. Conversely, changes in H4 acetylation under low-energy substrate conditions were not observed. These results align with the DOHaD framework, suggesting that while early embryos may compensate for energetic imbalances, such adjustments could predispose the offspring to health issues later in life through heritable epigenetic modifications. Combining our observations, we propose a model whereby embryonic plasticity is achieved through the switching of transcript isoforms mediated by histone modifications. Overall, these findings contribute to a deeper understanding of how early energetic imbalances impact embryonic development at the molecular level, revealing a role of DTU in the onset of epigenetic modifications that may lead to DOHaD.