Exogenous corticosterone administration during pregnancy in mice alters placental and fetal thyroid hormone availability in females

Abstract

Introduction: Maternal prenatal psychological stress is associated with adverse pregnancy outcomes and increased risk of adverse health outcomes in children. While the molecular mechanisms that govern these associations has not been fully teased apart, stress-induced changes in placental function can drive sex-specific phenotypes in offspring. We sought to identify and examine molecular pathways in the placenta that are altered in response to maternal prenatal stress.

Methods: We previously employed a mouse model of maternal prenatal stress where pregnant dams were treated with stress hormone (CORT) beginning in mid-gestation. Using this model, we conducted RNAseq analysis of whole placenta at E18.5. We used qRT-PCR to validate gene expression changes in the placenta and in a trophoblast cell line. ELISAs were used to measure the abundance of thyroid hormones in maternal and fetal serum and in the placenta.

Results: Dio2 was amongst the top differentially expressed genes in response to exogenous stress hormone. Dio2 expression was more downregulated in placenta of female fetuses from CORT-treated dams than both control placenta from females and placenta from male fetuses. Consistent with Dio2's role in production of bioactive thyroid hormone (T3), we found that there was a reduction of T3 in placenta and serum of female embryos from CORT-treated dams at E18.5. Both T3 and T4 were reduced in the fetal compartment of the placenta of female fetuses from CORT-treated dams at E16.5. Exogenous stress hormone induced reduction in thyroid hormone in females was independent of circulating levels of TH in the dams.

Discussion: The placental thyroid hormone synthesis pathway may be a target of elevated maternal stress hormone and modulate fetal programming of health and disease of offspring in a sex-specific fashion.

Keywords: Placenta; Prenatal stress; RNA-Seq; Sex-specific; Thyroid hormone.

Figure 1:. Differential gene expression in placenta from Vehicle and CORT-treated dams at E18.5.

A) Mouse mating treatment and collection strategy. B) Principal component analysis of placental transcriptomes. C) Heatmap of top 20 differentially expressed genes with FDR <0.05. N = 7–8 placenta from 4 dam/treatment – one male and one female placenta were selected from a single dam. D) Volcano plot showing 238 differentially expressed genes (red) at FDR <0.05. Dio2 is indicated. E) Box plot showing Dio2 expression as log₂(CPM) in placental samples stratified by sex and treatment. F-G) qPCR validation of Dio2 expression in female (F) and male (G) whole placenta. N = 4–5 placenta from independent dams. Gene expression was normalized to beta-actin and set relative to the Vehicle group. Data are presented as average fold change (2^-ddCt) ± SEM. Statistical analysis was performed on dCt values (see methods). **p < 0.01, unpaired t-test. (Figure 1A created with BioRender.com).

Figure 2:. Maternal CORT treatment reduces T3 levels in female placenta at E18.5.

A,B) Ratio of T3 (ng/mL) to total placental protein (μg/mL) in females (A) and males (B). C,D) Ratio of T4 (ng/mL) to total placental protein (μg/mL) in females (C) and males (D). n = 4 – 6 placenta/treatment from independent dams. *p = 0.03, Mann-Whitney Test.

Figure 3:. Circulating thyroid hormones in the fetus and dam at E18.5.

A) T3 concentration in serum from female embryos at E18.5. B) T3 concentration in serum from male embryos at E18.5. Serum was pooled across embryos of the same sex within a litter. N = 4–5 litters/treatment. *p = 0.02, Mann-Whitney Test. C) T3 concentration in maternal (dam) serum. N=5 dams/treatement. D) T4 concentration in maternal serum. N = 4 dams/treatment.

Figure 4:. Maternal CORT treatment does not alter thyroid hormone levels in whole placenta at E16.5.

A,B) DIO2 expression in female (A) and male (B) whole placenta. N = 3–4 placenta from independent dams. Gene expression was normalized to beta-actin and set relative to the Vehicle group. Data are presented as average fold change (2^-ddCt) +/− SEM. Statistical analysis was performed on dCt values (see methods). Ratio of T3 (ng/mL) to total placental protein (ug/mL) in females (B) and males (D). E,F) Ratio of T4 (ng/mL) to total placental protein (ug/mL) in females (E) and (F). n = 3 – 5 placenta/treatment from independent dams.

Figure 5:. Maternal CORT treatment alters thyroid hormone levels in trophoblasts from female embryos at E16.5.

Whole placenta were harvested at E16.5 from Vehicle and CORT-treated dams and the maternal decidua was dissected away. The remaining fetal placental tissue is termed “trophoblast.” A-B) DIO2 expression in female (A) and male (B) trophoblasts. N = 3–5 placenta from independent dams. Gene expression was normalized to beta-actin and set relative to the Vehicle group. Data are presented as average fold change (2^-ddCt) +/− SEM. Statistical analysis was performed on dCt values (see methods). C-D) Ratio of T3 (ng/mL) to total trophoblast protein (ug/mL) in females (C) and males (D). N = 4 trophoblast samples/treatment from independent dams. E-F) Ratio of T4 (ng/mL) to total lacental protein (ug/mL) in females (C) and (D). N = 4 trophoblast samples/treatment from independent dams. *p <0.03, Mann-Whitney Test). G-H) Circulating thyroid hormones in pregnant dams at E16.5. G) T3 concentration in serum from Vehicle and CORT treated dams. H) T4 concentration in serum from Vehicle and CORT treated dams. N = 4 dams/treatment.

Figure 6:. CORT treatment downregulates DIO2 in an extravillous trophoblast cell line.

Sw.71 cells were treated with Vehicle, 500ng/mL CORT, 500ng/mL CORT with 1uM RU486 or 1uM RU486 for A) 6 hours (N = 3 independent replicates) or B) 24 hours (N = 3–6 independent replicates). DIO2 expression was normalized to GAPDH and set relative to the Vehicle group. Data are presented as average fold change (2^-ddCt) +/− SEM. Statistical analysis was performed on dCt values (see methods), *p=0.01, **p<0.005, ***p<0.0001