Maternal Under- and Over-Nutrition during Gestation Alters Pancreatic DNA Methylation in Fetal Offspring

Poor maternal nutrition during gestation can increase insulin production and reduce β-cell proliferation in the pancreas tissue of the offspring with the effects persisting into postnatal life and across multiple generations. However, the mechanisms by which these changes occur are poorly understood. Changes to pancreas DNA methylation patterns during gestation may be mediating some of these effects. We hypothesized that maternal under- and over-nutrition during gestation would increase fetal pancreatic DNA methylation in the offspring. Twenty pregnant Western White-faced ewes were fed 100%, 60%, or 140% of NRC requirements for TDN for a ewe pregnant with twins beginning at d 30.2 ± 0.2 of gestation. Fetuses are referred to as CON, RES and OVER, respectively. Ewes (n = 6 to 7 per diet) were euthanized at d 135 of gestation and fetal pancreas tissue was collected. DNA was isolated, reduced representation bisulfate sequencing library preparation performed (3 to 4 fetuses per treatment per gender), and libraries were sequenced (Illumina MiSeq). Data were mapped to the Ovis aries reference annotation (Oar_V.3.1) using Bismark. Differentially methylated loci (DML; q ≤ 0.01; Table 1) and differentially methylated regions (DMR; P≤ 0.001; ∆ = 0.1; Table 1) were determined using the dispersion shrinkage for sequencing data analysis package in R.

Table 1: Total number of differentially methylated loci (DML) and regions (DMR) identified in the pancreas tissue of fetal offspring

Treatment Comparison	DML	DMR
CON vs. RES females	2,416	34
CON vs. RES males	1,514	64
CON vs. OVER females	1,874	48
CON vs. OVER males	608	25

Offspring from ewes fed 100% (CON), 60% (RES), or 140% (OVER) of NRC requirements for TDN from d 30.2 to d 135 of gestation.

In RES females, 62% of DMR are hypomethylated compared with CON females (P ≤ 0.001). In contrast, 93% of DMR are hypermethylated in RES males compared with CON males (P ≤ 0.001). In OVER, 66% and 80% of DMR are hypermethylated in females and males compared with CON females and males, respectively (P ≤ 0.001). These data demonstrate that maternal nutrition affects the DNA methylation patterns in the pancreas tissue of offspring in a diet-specific manner, and that the changes in pancreatic DNA methylation are gender-dependent.