Methylomic and Transcriptomic Analysis of Genes in Stress Axis Tissues of Mature Brahman Cows Exposed to Prenatal Transportation Stress

Abstract

One’s health begins in utero and stress experienced prenatally is known to alter the postnatal methylome and transcriptome of offspring, resulting in various physiological disorders. Physiological, mental, and behavioral processes are modulated by circadian clock genes. Thus, the objectives of this project was to determine whether prenatal transportation stress (PNS) affected DNA methylation (DNAm) and gene expression, within key stress axis tissues of mature Brahman cows, as well as determine whether there were statistical differences in the expression of circadian clock genes within or among bovine hypothalamic paraventricular nucleus (PVN), anterior pituitary (PIT), amygdala (AMYG), adrenocortical (ADC), and adrenomedullary (ADM) tissues. The sample tissues were isolated and harvested humanely between 0800-1100 hours from 5-yr-old Brahman cows whose dams were either exposed to transportation during gestation (PNS, n = 6) or were non-transported (Control, n = 8). Total DNA and RNA were isolated for genome-wide DNAm and RNA-Seq analyses, respectively. Differential analysis was conducted in RStudio and the normalized gene counts of important circadian clock genes in Control animals were analyzed using the GLM and CORR procedures of SAS. DNAm analysis revealed 3, 64, 99, and 37 hypomethylated and 2, 93, 90, and 36 hypermethylated CpG sites (FDR < 0.15) within the PVN, PIT, ADC, and ADM, respectively. RNA-Seq analysis revealed 6, 25, and 5 differentially up-regulated genes (FDR < 0.15) in the PVN, PIT, and ADC, respectively, as well as 24 down-regulated genes in the PIT. Results revealed that the methylome and genome were altered, with several cellular processes potentially being modulated, including maintenance of the actin cytoskeleton, neurodevelopment, and synaptic function, as well as cell motility, survival, and homeostasis. Relative expression profiles of circadian clock genes differed (P < 0.01) within each tissue, with NPAS2 having greater expression in AMYG (P < 0.01) and PER1 having greater expression in all other tissues (P < 0.01). Expression among tissues also differed (P < 0.01) for individual circadian clock genes, with CLOCK expression being greatest within the adrenal tissues and NR1D1 expression being greatest within the other tissues (P < 0.01). The inability to repeatedly sample these tissues prevents longitudinal studies focusing on long-term impact of prenatal stress and the rhythmic expression of clock genes within and among stress tissues. Cell cultures and temporal relationships of these genes with tissue-specific endocrine products should be investigated to determine the dynamics of tissue methylomics and genomics over time, as well as define the roles of peripheral clock genes in regulation of metabolism and health.