Work Description

Dataset Title: Supplementary Results Tables for "Developmental Programming: Differing impact of prenatal testosterone and prenatal bisphenol-A -treatment on hepatic methylome in female sheep"

Dataset contact: John Dou [email protected]

Dataset Creators:
Name: John F Dou
Email: [email protected]
Institution: University of Michigan Department of Epigemiology
ORCID: https://orcid.org/0000-0003-4577-8660

Name: Soundara Viveka Thangaraj
Email: [email protected]
Institution: University of Michigan Department of Pediatrics-Endocrinology
ORCID: https://orcid.org/0000-0003-4902-468X

Name: Yiran Zhou
Email: [email protected]
Institution: University of Michigan Department of Pediatrics-Endocrinology
ORCID: https://orcid.org/0000-0002-1965-8414

Name: Vasantha Padmanabhan
Email: [email protected]
Institution: University of Michigan Department of Pediatrics-Endocrinology
ORCID: https://orcid.org/0000-0002-8443-7212

Name: Kelly M Bakulski
Email: [email protected]
Institution: University of Michigan Department of Epigemiology
ORCID: https://orcid.org/0000-0002-9605-6337

Funding:
Research reported in this publication was supported by Eunice Kennedy Shriver National Institute of Child Health & Human Development, National Institutes of Health (NIH) under award R01HD099096 and P01 HD44232, and National Institute of Environmental Health Sciences R01 ES016541 and R01 ES 030374.

Key Points:
- We evaluated the impact of BPA and excess prenatal testosterone during pregnancy on offspring liver DNA methylation in Suffolk sheep
- Methylation impacts were associated with genes governing lipid and glucose metabolism, potentially underlying the observed metabolic disruptions such as insulin resistance and dyslipidemia
- Pathway analysis revealed that genes differentially methylated due to testosterone were involved in cellular organization, while those affected by BPA were enriched in signal regulation pathways
- The findings underscore how prenatal steroid excess and mimics influence epigenetic landscapes, contributing to metabolic disease programming, and offer insights into potential preventive strategies for metabolic dysfunctions in offspring

Research Overview:
Steroid hormones are integral to pregnancy and fetal development, regulating processes such as metabolism, inflammation, and immune responses. Excessive prenatal steroid exposure, through lifestyle choices or environmental chemicals, can lead to metabolic dysfunctions in offspring. The research focuses on how exposure to testosterone and BPA affects the liver’s DNA methylome, a key component of the epigenome influencing long-term health. Using Suffolk sheep, the study involved two cohorts: one exposed to prenatal testosterone and the other to BPA.

Methodology:
Suffolk sheep were maintained at the University of Michigan Sheep Research Facility (Ann Arbor, MI) where studies were conducted following the Institutional Animal Care and Use Committee of the University of Michigan approved protocol in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act.

In Cohort 1, pregnant sheep were randomly assigned to control and prenatal T treatment groups. The prenatal T treated group received biweekly intramuscular injections of T propionate (~1.2mg/kg; Millipore Sigma, St. Louis, MO) suspended in corn oil, from gestational day 30 to day 90. Liver tissue from five control and five prenatal T-treated sheep were used in this study.

In Cohort 2, pregnant sheep were randomly assigned to control and prenatal BPA treatment groups. Control and treatment groups were administered daily, subcutaneous injections of vehicle (corn oil) and 0.5mg/kg/day BPA (purity ≥ 99%, catalog number 239658; Aldrich Chemical Co, Milwaukee, Wisconsin) solubilized in corn oil, respectively. Liver tissue from four control and prenatal BPA treated sheep were used in this study.

At the conclusion of the second breeding season (~21 months of age), female offspring in the T cohort were subjected to ovariectomy to eliminate any confounding influences from varying steroid levels. Liver tissue samples were obtained during the artificially induced follicular phase, following established protocols. Early follicular phase estradiol levels were maintained using a subcutaneous estradiol implant, 1 cm in length. To synchronize estrous cycle, two controlled internal drug-release implants containing progesterone (CIDR-G; InterAg, Hamilton, New Zealand) were placed subcutaneously to mimic luteal phase levels of progesterone. The progesterone implants were removed after 14 days, and four 30-mm estradiol implants were inserted to produce late follicular phase levels of estradiol. Animals were euthanized 18 hours later during the late artificial follicular phase by a barbiturate overdose (Fatal Plus; Vortech Pharmaceuticals, Dearborn, MI). In the prenatal BPA-treated cohort, female offspring liver tissue samples were collected during the natural follicular phase after synchronization with two injections of Prostaglandin F2alpha, 11 days apart. Samples were taken 27 hours after the second Prostaglandin F2alpha injection. From all animals, liver tissue was collected from the tip of the left lobe, flash-frozen and stored at -80°C.

Whole genome bisulfite sequencing was employed to map DNA methylation across over 22 million CpG sites. DNA was extracted from 30 micrograms of frozen liver tissue using AllPrep DNA/RNA kit (Qiagen, Germantown, MD) using manufacturer protocol. A total of 200ng of DNA was used for each whole genome bisulphite sequencing. Library preparation and sequencing were carried out at the University of Michigan, Epigenomics Core. Libraries were quantified using the KAPA Library quantitation kit and pooled for sequencing with a S4 flowcell for 200 cycles (paired end, 100 base pairs in length) on a NovaSeq6000.

FastQC (v0.11.8) was used to assess quality of reads (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimming was done with TrimGalore (v0.4.5) (https://github.com/FelixKrueger/TrimGalore) with the parameters: –adapter AGATCGGAAGAGC -e 0.1 –stringency 6 –length 20 –nextseq 20. Reads were aligned to the ARS-UI-Ramb-v2.0 sheep genome build using Bowtie2 (v2.3.4) [46]. DNA methylation was called using MethylDackel (v0.4.0) (parameters -d 5 -D 2000 –mergeContext) (https://github.com/dpryan79/MethylDackel).

Data were analyzed in R statistical software (version 4.4.0). First, we filtered to DNA methylation sites (CpGs) with coverage of at least 10 reads in all samples. We conducted differentially methylated region analysis using the dmrseq package, with default parameters for determining candidate regions.

Files contained here:
This dataset contains full results tables for differentially methylated region analysis in the paper "Developmental Programming: Differing impact of prenatal testosterone and prenatal bisphenol-A -treatment on hepatic methylome in female sheep". Each table contains the following columns: chromosome (chr), start genomic position of region (start), end genomic position of region (end), width of region (width), number of CpGs in region (nCpG), test statistic (stat), p-value (pval), FDR corrected p-value (qval), gene located within or overlapping region (gene), mean difference between comparison groups (meanDiff).

-Supplemental_Table_1_prenatal_testosterone_differentially_methylated_regions.csv
Supplemental Table 1: Differentially methylated regions associated with prenatal-Testosterone treatment in sheep liver

-Supplemental_Table_3_BPA_differentially_methylated_regions.csv
Supplemental Table 3: Differentially methylated regions associated with prenatal-BPA treatment in sheep liver

-Supplemental_Table_6_Control_differentially_methylation_regions.csv
Supplemental Table 6: Differentially methylated regions in Controls between the prenatal-Testosterone and prenatal-BPA treated sheep.

Related Publication:
Dou J, Thangaraj, SV, Zhou Y , Padmanabhan V, Bakulski, KM. Developmental Programming: Differing impact of prenatal testosterone and prenatal bisphenol-A -treatment on hepatic methylome in female sheep. Forthcoming.

Use and Access:
This data set is made available under a Creative Commons Public Domain license (CC0 1.0).

To Cite Data:
Dou, J. F., Thangaraj, S. V., Zhou, Y., Padmanabhan, V., Bakulski, K. M. Supplementary Results Tables for "Developmental Programming: Differing impact of prenatal testosterone and prenatal bisphenol-A -treatment on hepatic methylome in female sheep" [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/m9nm-0375