The aryl hydrocarbon receptor mediates sex ratio distortion in the embryos sired by TCDD-exposed male mice

Abstract

Many reports describe an association between preconceptional paternal exposure to environmental chemicals, including the persistent organic pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with an increased number of female offspring. We chronically treated wild-type C57BL/6 male mice with TCDD to investigate a role for the aryl hydrocarbon receptor (AHR) transcription factor. These mice had a 14 % lower male:female sex ratio than control mice, which was not observed in TCDD-treated Ahr knock out mice. AHR target genes Cyp1a1 and Ahrr were upregulated in the liver and testis of WT mice and Ahr expression was higher in the epididymis (2-fold) and liver (18-fold) than in whole testis tissue. The AHR protein was localized to round spermatids, elongating spermatids, and Leydig cells in the testis of WT mice. These studies demonstrate AHR involvement in the sex ratio distortion of TCDD-exposed males and the need for evaluating the molecular and genetic mechanism of this process.

Keywords: AHR; Dioxin; Male reproduction; Paternal exposure; Sex ratio; TCDD.

Figure 1.. Mouse embryo sex ratio following TCDD exposure.

To identify genotypic male (XY) and female (XX) GD 15.5 embryos, genes specific to the X (Kdm5c) and Y (Kdm5d) chromosomes were detected by qPCR. For each adult male mouse, the overall sex ratio was determined by dividing the number of male offspring by the total number of all offspring across 2–9 litters. Data are presented as box and whisker plots. The line represents the median, while the dots represent individual male mice. The box represents 50% of the data and the whiskers are those data in the 25^th and 75^th quartiles (n=6–9 male mice). *Statistically significant differences (p<0.025) compared with WT CO.

Figure 2.. Determination of mouse sperm sex chromosome content by MP-FISH.

Sex chromosome content of caudal epididymal sperm was assessed using fluorescent probes specific for the X (green) and Y (red) chromosome. Sperm were counterstained with DAPI (blue). Images are representative from each treatment group (scale bar: 50 μm).

Figure 3.. Liver and testis gene expression analysis.

(A) Expression of hepatic mRNAs from the AHR signaling pathway (Ahrr, Cyp1a1) were assessed by qPCR. (B) Expression of testis mRNAs from the AHR signaling pathway (Ahrr, Cyp1a1) sex ratio determining genes (Slx, Sly), and sperm motility gene (Akap4) were assessed by qPCR. All expression values were normalized to vehicle treated WT mice and the Actb housekeeping gene. All data are presented as box and whisker plots. The line represents the median, while the dots represent individual male mice. The box represents 50% of the data and the whiskers are those data in the 25th and 75th quartiles (n=5–6). *Statistically significant differences (p<0.025) compared to WT CO. ^#Statistically significant differences (p<0.025) compared to KO CO.

Figure 4.. Localization of AHR in mouse testis.

Immunohistochemical detection of AHR in testis of male mice. Paraffin-embedded sections (5 μm) were stained using the AHR antibody (scale bar: 50 μm). AHR staining was localized to elongating spermatids (E), Leydig cells (L), and round spermatids (R). An IgG isotype control antibody was used as a control and showed no brown staining.

Figure 5.. Ahr mRNA expression.

Expression of Ahr mRNA in the testis, cauda epididymis, and liver was assessed by qPCR. All expression values were normalized to testis Ahr mRNA expression and the Actb housekeeping gene. All data are presented as box and whisker plots. The line represents the median, while the dots represent individual male mice. The box represents 50% of the data and the whiskers are those data in the 25th and 75th quartiles (n=3–5). *Statistically significant differences (p<0.05) compared to testis Ahr.