Evaluation of Placentation and the Role of the Aryl Hydrocarbon Receptor Pathway in a Rat Model of Dioxin Exposure

Abstract

Background: Our environment is replete with chemicals that can affect embryonic and extraembryonic development. Dioxins, such as 2,3,7,8-tetrachlorodibenzo-$p$-dioxin (TCDD), are compounds affecting development through the aryl hydrocarbon receptor (AHR).

Objectives: The purpose of this investigation was to examine the effects of TCDD exposure on pregnancy and placentation and to evaluate roles for AHR and cytochrome P450 1A1 (CYP1A1) in TCDD action.

Methods: Actions of TCDD were examined in wild-type and genome-edited rat models. Placenta phenotyping was assessed using morphological, biochemical, and molecular analyses.

Results: TCDD exposures were shown to result in placental adaptations and at higher doses, pregnancy termination. Deep intrauterine endovascular trophoblast cell invasion was a prominent placentation site adaptation to TCDD. TCDD-mediated placental adaptations were dependent upon maternal AHR signaling but not upon placental or fetal AHR signaling nor the presence of a prominent AHR target, CYP1A1. At the placentation site, TCDD activated AHR signaling within endothelial cells but not trophoblast cells. Immune and trophoblast cell behaviors at the uterine-placental interface were guided by the actions of TCDD on endothelial cells.

Discussion: We identified an AHR regulatory pathway in rats activated by dioxin affecting uterine and trophoblast cell dynamics and the formation of the hemochorial placenta. https://doi.org/10.1289/EHP9256.

Figure 1.

Effect of TCDD exposure on pregnancy outcomes. (A) Schematic showing the treatment plan used in this study. Rats were treated on GD6.5 with corn oil (OIL) or TCDD and euthanized on GD13.5 or 18.5. (B) Fetal survival rate (%) after TCDD treatment as assessed on GD13.5 ($n=5$ pregnancies/group), *, $p<0.0001$. Fetal survival rate was calculated on a per pregnancy basis as the number of live fetuses/total fetuses times 100. (C) Detection of Cyp1a1 transcript levels in liver tissues of GD13.5 pregnant females exposed to OIL or different concentrations of TCDD using RT-qPCR ($n=5$ pregnancies/group), *, $p=0.006$. (D) Cyp1a1 expression in lung and spleen of OIL- or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW)-exposed pregnant rats at GD13.5 measured by RT-qPCR ($n=5$ pregnancies/group; lung, *, $p=0.0079$; spleen, $p=0.0079$). (E) Fetal and placental weights and fetal/placental weight ratio at GD18.5 following OIL or TCDD treatment ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) ($n=5$ pregnancies/group). (F) Representative images for CYP1A1 immunohistochemistry of embryonic day (ED) 18.5 fetus from pregnant rats treated with OIL or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW). Scale bar: 1 mm. (G) RT-qPCR measurement of Cyp1a1 transcripts in ED18.5 brain and liver tissues from OIL- or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW)-treated pregnant rats ($n=5$ pregnancies/group; fetal liver, *, $p=0.0079$; fetal brain, *, $p=0.0079$). Transcript expression level is relative to control. The delta-delta Ct method was used for relative quantification of gene expression for each sample normalized to 18S RNA. Data presented in (B) and (C) were analyzed using a one-way ANOVA followed by Tukey’s multiple comparison post hoc test and the Wilcoxon rank sum test (D, E, and G). The asterisks denote a statistically significant difference from the corresponding control. Bar graphs represent the $\text{mean}\pm \text{SEM}$. The mean and SEM values for data presented in this figure are shown in Tables S4–S8. Note: ANOVA, analysis of variance; BW, body weight; Cyp1a1, cytochrome P450, family 1, subfamily a, polypeptide 1; GD, gestation day; RT-qPCR, real-time quantitative polymerase chain reaction; SEM, standard error of the mean; TCCD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin.

Figure 2.

Measures of intrauterine endovascular trophoblast cell invasion in rats with gestational TCDD exposure. (A) Schematic showing the treatment plan used in this study. Rats were treated on GD6.5 with corn oil (OIL) or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) and euthanized on GD13.5. (B) Representative images for pan-cytokeratin (green) and CYP1A1 (red) immunostaining of GD13.5 placentation sites. The white arrow shows the depth of intrauterine endovascular trophoblast cell invasion. Scale bar: $500\mathrm{\mu }\mathrm{m}$. (C) Quantification of the depth of cytokeratin-positive cell penetration into the uterine mesometrial vasculature ($n=4–6/\text{group}$, *, $p=0.0001$). Depth of invasion was calculated as the ratio of the distance of trophoblast cell (cytokeratin positive) migration from the junctional zone into the uterus vs. the total distance between the junctional zone and the outer surface of the uterus. Assessments of the depth of invasion were performed at the center of each placentation site. (D) Schematic diagram depicting the metrial gland (uterine–placental interface). (E) RT-qPCR measurements of Prl5a1 and Prl7b1 transcripts in GD13.5 metrial gland tissues isolated from OIL- and TCDD-treated rats ($n=5/\text{group}$; Prl5a1, *, $p=0.0022$; Prl7b1, *, $p=0.0022$). Transcript expression level is relative to control. The delta-delta Ct method was used for relative quantification of gene expression for each sample normalized to 18S RNA. The level of significance was determined using the Wilcoxon rank sum test. Asterisks denote statistically significant differences from the corresponding controls. Bar graphs represent the $\text{mean}\pm \text{SEM}$. The mean and SEM values for data presented in this figure are shown in Tables S9 and S10. Note: BW, body weight; Cyp1a1, cytochrome P450, family 1, subfamily a, polypeptide 1; GD, gestation day; Prl5a1, prolactin family 5, subfamily a, member 1; Prl7b1, prolactin family 7, subfamily b, member 1; RT-qPCR, real-time quantitative polymerase chain reaction; SEM, standard error of the mean; TCCD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin.

Figure 3.

Generation of an AHR null rat model. (A) Schematic representation of the strategy for targeting Exon 2 of the Ahr gene. A red dashed line depicts the 342-bp deleted region. (B) Mendelian ratios of AHR heterozygous breeding and viability of AHR nulls. (C) Representative image of western blot analysis of AHR protein expression in WT and AHR null liver tissues. GAPDH was used as an internal control. (D) Transcript analysis of AHR target genes (Cyp1a1, Cyp1b1, and Ahrr) in WT and null (NULL) rat liver following corn oil (OIL) or TCDD ($25\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) treatment ($n=5/\text{group}$): Cyp1a1, Cyp1b1, and Ahrr, $p<0.0001$. The delta-delta Ct method was used for relative quantification of gene expression for each sample normalized to 18S RNA. (E) Number of ovulated oocytes recovered per female after gonadotropin stimulation of 4- to 5-wk-old WT or AHR null female rats ($n=6/\text{group}$). (F) Pregnancy success rates (sperm-positive females that became pregnant) of WT (+/+) and AHR null (–/–) rats in various breeding combinations ($n=6/\text{combination}$). (G) Average litter sizes from pregnancies generated by WT (+/+) and AHR null (–/–) rats in various breeding combinations ($n=6/\text{combination}$). Transcript expression level is relative to oil-treated respective genotype. The level of significance was determined using either the Wilcoxon rank sum test or a one‐way ANOVA followed by Tukey’s multiple comparison post hoc test where appropriate. Asterisks denote a statistically significant difference from the corresponding control. Bar graphs represent the $\text{mean}\pm \text{SEM}$. The mean and SEM values for data presented in this figure are shown in Tables S11–S14. Note: AHR, aryl hydrocarbon receptor; Ahrr, aryl-hydrocarbon receptor repressor; ANOVA, analysis of variance; BW, body weight; Cyp1a1, cytochrome P450, family 1, subfamily a, polypeptide 1; Cyp1b1, cytochrome P450, family 1, subfamily b, polypeptide 1; F, female; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Het, heterozygous; M, male; SEM, standard error of the mean; TCCD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; WT, wild type.

Figure 4.

Evaluation of TCDD-activated placental adaptations resulting from mating WT (WT), AHR heterozygous, and AHR null rats. (A) Effect of low and high TCDD dose ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW vs. $20\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ weight) on WT and AHR null (Null) fetal survival rate ($n=6/\text{group}$, *, $p<0.0001$). Fetal survival rate was calculated on a per pregnancy basis as the number of live fetuses/total fetuses times 100. (B) Schematic of the rat placentation site. (C) Representative images from placentation sites of WT females (WT F) mated with WT males (WT M) or AHR null females (Null F) mated with AHR null males (Null M). Pregnant females treated with corn oil (OIL) or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) at GD6.5 and euthanized on GD13.5. (D) Prl7b1 transcript abundance in GD13.5 metrial gland tissues isolated from WT F $\times$ WT M or Null F $\times$ Null M pregnancies treated with OIL ($n=5/\text{group}$) or TCDD ($n=6/\text{group}$; WT F $\times$ WT M, *, $p=0.0043$). (E) Survival rate of embryos generated by crossing Null F $\times$ WT M ($n=6/\text{group}$, *, $p<0.0001$). (F) Representative images of placentation sites from Null F mated to WT M or Ahr heterozygous females (Het F) mated to Null M. Pregnant females were treated with OIL or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) at GD6.5 and euthanized on GD13.5. Only placentation sites from null embryos (–/–) generated in the Het F $\times$ Null M pregnancies are presented. (G) Prl7b1 transcript abundance in GD13.5 metrial gland tissues isolated from Null F $\times$ WT M or from null placenta/fetal units (–/–) generated from Het F $\times$ Null M pregnancies treated with OIL ($n=5/\text{group}$) or TCDD ($n=6/\text{group}$); Het F $\times$ Null M, *, $p=0.0043$. Null placenta/fetal units from the Het F $\times$ Null M mating were identified by genotyping fetal tissues. In (C) and (F), placentation sites were interrogated by pan-cytokeratin (green) and CYP1A1 (red) immunofluorescence. Genotypes of maternal and placental tissues are indicated: $\text{WT},+/+$; $\text{Het},+/-$; Null, –/–. White arrows show the depth of intrauterine endovascular trophoblast cell invasion. Scale bar: $500\mathrm{\mu }\mathrm{m}$. In (D) and (G), the delta-delta Ct method was used for relative quantification of gene expression for each sample normalized to 18S RNA. Data presented in (A) and (E) were analyzed using a one‐way ANOVA followed by Tukey’s multiple comparison post hoc test, whereas data presented in (D) and (G) were analyzed using the Wilcoxon rank sum test. Asterisks denote a statistically significant difference from the corresponding control. Bar graphs represent the $\text{mean}\pm \text{SEM}$. The mean and SEM values for data presented in this figure are shown in Tables S15–S18. Note: AHR, aryl hydrocarbon receptor; ANOVA, analysis of variance; BW, body weight; cytokeratin, pan-cytokeratin; CYP1A1, cytochrome P450, family 1, subfamily a, polypeptide 1; F, female; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GD, gestation day; Het, heterozygous; M, male; Prl7b1, prolactin family 7, subfamily b, member 1; SEM, standard error of the mean; TCCD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; WT, wild type.

Figure 5.

Effects of TCDD on metrial gland (uterine–placental interface) transcript profiles. Transcript profiles were determined by RNA-sequencing (RNA-seq) of metrial gland tissue obtained from pregnant rats treated with corn oil (OIL) or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) at GD6.5 and harvested at GD13.5. The results are presented in a table (A) significantly upregulated and down-regulated transcripts in response to TCDD ($p<0.05$ and ${\log }_2$-fold change either direction $>1.5$). (B) Gene ontology analysis of top dysregulated genes. (C) A heatmap showing select differentially expressed genes in replicates of OIL- and TCDD-treated rats. (D) Distribution of NK cells within GD13.5 metrial glands of OIL- or TCDD-treated rats assessed by perforin immunohistochemistry ($n=3/\text{group}$). Scale bar: $500\mathrm{\mu }\mathrm{m}$. (E) NK cell numbers measured by CD161-flow cytometry ($n=3/\text{group}$). (F) Validation of RNA-seq results by RT-qPCR ($n=5/\text{group}$). The level of significance was determined using the Wilcoxon rank sum test (Klrd1, *, $p=0.0079$; Ly49s3c; Klra5, *, $p=0.0159$; Prf1, *, p = 0.0079; Gzmb, *, $p=0.0079$). Transcript expression level is relative to control. The delta-delta Ct method was used for relative quantification of gene expression for each sample normalized to 18S RNA. Asterisks denote a statistically significant difference from the corresponding controls. Bar graphs represent the $\text{mean}\pm \text{SEM}$. The mean and SEM values for data presented in this figure are shown in Tables S19 and S20. Note: BW, body weight; cytokeratin, pan-cytokeratin; down, down-regulation; GD, gestation day; Gzmb, granzyme B; Klra5, killer cell lectin-like receptor, subfamily a, member 5; Klrd1, killer cell lectin-like receptor subfamily D, member 1; Ly49s3c, Ly-49 stimulatory receptor 3; NK, natural killer; Prf1, perforin 1; RT-qPCR, real-time quantitative polymerase chain reaction; SEM, standard error of the mean; TCCD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; up, up-regulation.

Figure 6.

Evaluation of measures of AHR pathway activation in placentation sites of rats treated with oil or TCDD. (A) A representative image of pan-cytokeratin (green) and CYP1A1 (red) immunostaining of a GD13.5 placentation site from a pregnant female rat treated with TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) at GD6.5. Scale bar: $500\mathrm{\mu }\mathrm{m}$. Expression of Cyp1a1, Cyp1b1, and Ahrr in the GD13.5 (B) metrial gland or (C) labyrinth zone tissues from pregnant rats treated with corn oil (OIL) or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) at GD6.5 ($n=6/\text{group}$). Metrial gland: Cyp1a1, *, $p=0.0079$; Cyp1b1, *, $p=0.0079$; and Ahrr, *, $p=0.0079$; labyrinth zone: Cyp1a1, *, $p=0.0022$ and Cyp1b1, *, $p=0.0022$; Ahrr, $p=0.0022$. Asterisks denote a statistically significant difference from the corresponding control. Representative immunofluorescence images for (D) CD31 and CYP1A1 colocalization and (E) RECA1 and CYP1A1 colocalization in GD13.5 metrial gland and labyrinth zone tissues from pregnant rats treated with corn oil (OIL) or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) at GD6.5. Scale bar: $50\mathrm{\mu }\mathrm{m}$. Cyp1a1 expression in (F) rat trophoblast stem cells and (G) rat arterial endothelial cells following vehicle or TCDD treatment for 24 h ($n=6$). Cyp1a1 transcript levels were measured by RT-qPCR (*, $p<0.0001$). Transcript expression level is relative to control. Data were analyzed using the Wilcoxon rank sum test (B and C) and an ANOVA followed by Tukey’s multiple comparison post hoc test (F and G). Bar graphs represent the $\text{mean}\pm \text{SEM}$. The mean and SEM values for data presented in this figure are shown in Tables S21–S24. Note: Ahrr, aryl-hydrocarbon receptor repressor; ANOVA, analysis of variance; BW, body weight; CD31, cluster of differentiation 31; Cyp1a1, cytochrome P450, family 1, subfamily a, polypeptide 1; Cyp1b1, cytochrome P450, family 1, subfamily b, polypeptide 1; GD, gestation day; JZ, junctional zone; LZ, labyrinth zone; MG, metrial gland; RECA1, Rat Endothelial Cell Antigen-1; RT-qPCR, real-time quantitative polymerase chain reaction; SEM, standard error of the mean; TCCD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin.

Figure 7.

Mapping cell dynamics within the metrial gland (uterine–placental interface) of pregnancies exposed to oil or TCDD using scRNA-seq. Metrial glands were harvested on GD13.5 from pregnant rats treated with corn oil (OIL) or TCDD ($2\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ BW) at GD6.5 and analyzed by scRNA-seq. (A) A flow diagram illustrating the study design and analysis strategy. (B) Expression of canonical markers identifying cell clusters. (C) Number of differentially expressed genes (DEGs) following TCDD treatment in various cell clusters. Volcano plots showing DEGs between OIL and TCDD samples in (D) natural killer (NK) cells, (E), macrophages (Mϕ), and (F) endothelial cells (EC). Red dots represent up-regulated genes, whereas blue dots represent down-regulated genes. (G) Expression of Ahr, Cyp1a1, and Cyp1b1 in stromal (ST) cells, NK cells, mesenchymal (MES) cells, Mϕ, EC, and smooth muscle cells (SMC). Data were analyzed by a two-sided Wilcoxon-rank-sum test, $\text{FDR}<0.05$, $\text{logFC}>0.15$. Additional lists of relevant differentially regulated transcripts and pathway analysis are provided in Excel Tables S2–S8. Note: Ahr, aryl hydrocarbon receptor; BW, body weight; Cyp1a1, cytochrome P450, family 1, subfamily a, polypeptide 1; Cyp1b1, cytochrome P450, family 1, subfamily b, polypeptide 1; down, down-regulation; FDR, false discovery rate; GD, gestation day; logFC, log fold change; scRNA-seq, single cell RNA sequencing; TCCD, 2,3,7,8-tetrachlorodibenzo-$p$-dioxin; up, up-regulation.