Fig 1.
Experimental design, timeline, and tag-seq analysis pipeline.
(A) Timeline for prenatal bisphenol exposure, maternal care observations, and offspring growth, neurodevelopmental and behavioral outcomes. Offspring brains were collected at the bolded ages. (B) Tag-seq analysis pipeline for PND 10 medial prefrontal cortex and amygdala. Fastq files were filtered and trimmed using cutadapt, aligned to the rat genome using STAR, and sorted using samtools. Gene counts were compiled using bedtools and normalized using DEseq2. Differential gene expression analysis was done using DEseq2. Normalized, log-transformed counts were used for weighted gene co-expression network analysis (WGCNA) and the clustered eigengene modules were analyzed. Gene sets from eigengene module with significant main effects or interactions were extracted and analyzed for enriched GO biological processes, Transcription Factor (TF) Protein-Protein Interactions, and ENCODE and ChEA Consensus TFs from ChIP-X.
Fig 2.
Prenatal bisphenol exposure was associated with reduced eye-opening prevalence at PND 15 after accounting for postnatal licking/grooming from PND 1–5, particularly in male pups.
(A) No main effects of prenatal treatment were found for eye-opening prevalence at PND 15. Marginal interactions between prenatal treatment and postnatal licking/grooming were found for (B) total pups within a litter and (C) male pups only, but not (D) female pups only. After accounting for postnatal licking/grooming, all prenatal bisphenol exposure groups showed reduced eye-opening prevalence compared to the Corn Oil group for total pups within a litter and male pups only. Bar plots are displayed with mean +/- SEM with individual datapoints. Scatterplots are displayed with linear regression lines for each prenatal treatment group. * p < 0.05 main effect of prenatal treatment; ✤ p < 0.10 interaction between prenatal treatment and postnatal maternal care.
Table 1.
Summary of conditional post-hoc tests between the corn oil group and prenatal bisphenol treatment groups at the 16th, 50th, and 84th values of postnatal maternal care.
Fig 3.
Prenatal bisphenol exposure was associated with reduced attentional set shifting performance after accounting for postnatal nest attendance from PND 1–5 for male and female offspring.
No main effects of prenatal treatment were found for (A) trials to reach criterion or (B) percent correct trials in the attentional set shifting response task. A marginal interaction between prenatal treatment and postnatal nest attendance was found for (C) percent correct trials for male offspring. A significant interaction between prenatal treatment and postnatal nest attendance was found for (D) trials to reach criterion for female offspring. After accounting for postnatal nest attendance, the 50 μg/kg BPA (male and female offspring) and 150 μg/kg Mixed BP (female offspring only) exposure groups showed reduced attentional set shifting performance compared to the Corn Oil group. Bar plots are displayed with mean +/- SEM with individual datapoints. Scatterplots are displayed with linear regression lines for each prenatal treatment group. * p < 0.05 main effect of prenatal treatment; ★ p < 0.05 interaction between prenatal treatment and postnatal maternal care; ✤ p < 0.10 interaction between prenatal treatment and postnatal maternal care.
Fig 4.
Prenatal bisphenol exposure was associated with reduced anxiety-like behavior in the open field after accounting for postnatal nest attendance from PND 1–5 for male offspring.
No main effects of prenatal treatment were found for (A) time spent in open arms or (B) time spent in the closed arms of the elevated plus maze (EPM), although there was a significant interaction of prenatal treatment and offspring sex on time spent in the closed arms of the EPM. Significant main effects of offspring sex were found for (C) time spent in the center and (D) time spent in the corners of the open field, where female offspring were more anxious than male offspring. (E) A significant main effect of postnatal nest attendance was found for time spent in the center in the open field for male offspring. A significant interaction between prenatal treatment and postnatal nest attendance was found for time spent in the center in the open field for (E) male offspring but not (F) female offspring. After accounting for postnatal nest attendance, the 50 μg/kg BPA and 50 μg/kg Mixed BP exposure groups showed reduced anxiety-like behavior in the open field compared to the Corn Oil group for male offspring only. Bar plots are displayed with mean +/- SEM with individual datapoints. Scatterplots are displayed with linear regression lines for each prenatal treatment group. * p < 0.05 main effect of prenatal treatment; ❡ p < 0.05 interaction between prenatal treatment and offspring sex; ❖ p < 0.05 main effect of offspring sex; ● p < 0.05 main effect of postnatal maternal care; ★ p < 0.05 interaction between prenatal treatment and postnatal maternal care.
Table 2.
Summary of differentially expressed genes compared to the corn oil group.
Fig 5.
The interactive effects of prenatal bisphenol exposure and postnatal maternal care on transcriptomic profiles in the male medial prefrontal cortex.
There were significant main effects or interactions with prenatal treatment and postnatal maternal care for the (A) black module, (B) brown module, (C) lightcyan module, (D) tan module, and (E) yellow module. Scatterplots are displayed with linear regression lines for each prenatal treatment group. ● p < 0.05 main effect of postnatal maternal care; + p < 0.10 main effect of postnatal maternal care; ★ p < 0.05 interaction between prenatal treatment and postnatal maternal care; ✤ p < 0.10 interaction between prenatal treatment and postnatal maternal care.
Fig 6.
The interactive effects of prenatal bisphenol exposure and postnatal maternal care on transcriptomic profiles in the female medial prefrontal cortex.
There were significant main effects or interactions with prenatal treatment and postnatal maternal care for the (A) brown module and (B) turquoise module. Scatterplots are displayed with linear regression lines for each prenatal treatment group. ● p < 0.05 main effect of postnatal maternal care; ★ p < 0.05 interaction between prenatal treatment and postnatal maternal care.
Fig 7.
The interactive effects of prenatal bisphenol exposure and postnatal maternal care on transcriptomic profiles in the male amygdala.
There were significant main effects or interactions with prenatal treatment and postnatal maternal care for the (A) blue module and (B) green module. Scatterplots are displayed with linear regression lines for each prenatal treatment group. * p < 0.05 main effect of prenatal treatment; ★ p < 0.05 interaction between prenatal treatment and postnatal maternal care; ✤ p < 0.10 interaction between prenatal treatment and postnatal maternal care.
Fig 8.
The interactive effects of prenatal bisphenol exposure and postnatal maternal care on transcriptomic profiles in the female amygdala.
There were significant main effects or interactions with prenatal treatment and postnatal maternal care for the (A) black module and (B) yellow module. Scatterplots are displayed with linear regression lines for each prenatal treatment group. ^ p < 0.10 main effect of prenatal treatment; * p < 0.05 main effect of prenatal treatment; ★ p < 0.05 interaction between prenatal treatment and postnatal maternal care.