Developmental exposure to real-life environmental chemical mixture programs a testicular dysgenesis syndrome-like phenotype in prepubertal lambs

Abstract

Current declines in male reproductive health may, in part, be driven by anthropogenic environmental chemical (EC) exposure. Using a biosolids treated pasture (BTP) sheep model, this study examined the effects of gestational exposure to a translationally relevant EC mixture. Testes of 8-week-old ram lambs from mothers exposed to BTP during pregnancy contained fewer germ cells and had a greater proportion of Sertoli-cell-only seminiferous tubules. This concurs with previous published data from fetuses and neonatal lambs from mothers exposed to BTP. Comparison between the testicular transcriptome of biosolids lambs and human testicular dysgenesis syndrome (TDS) patients indicated common changes in genes involved in apoptotic and mTOR signalling. Gene expression data and immunohistochemistry indicated increased HIF1α activation and nuclear localisation in Leydig cells of BTP exposed animals. As HIF1α is reported to disrupt testosterone synthesis, these results provide a potential mechanism for the pathogenesis of this testicular phenotype, and TDS in humans.

Keywords: Developmental toxicity; Environmental chemicals; Hypoxia inducible factor 1 alpha; Reproductive toxicity; Testicular dysgenesis syndrome.

Figure 1.

Histopathological findings in 8-week-old control and biosolids-exposed lamb testes (n = 11 per group). Representative images of haematoxylin / DDX4-DAB stained tissue sections from 8-week-old control and biosolids lambs, taken with a magnification of 100x (A and C respectively) and 400x (B and D respectively). Scale bars (bottom right of images) show 100 μm. Arrows indicate Sertoli-cell-only (SCO) seminiferous tubules. Relative proportions of germ cells to Sertoli cells (E) and percent of tubules which were SCO (F) (p = 0.014 and p = 0. 0082 respectively). Seminiferous tubule minimum Feret’s diameters (p = 0.013) (G). Boxes represent 25th to 75th percentile, horizontal bar indicates 50th percentile, whiskers indicate range excluding outliers, solid filled circles show outliers, open circles show individual data points, and red triangles show means.

Figure 2.

Heat map of DEGs plotted against z-score. Genes are ordered from top to bottom by increasing p-value.

Figure 3.

Log₂ fold change of testicular gene expression between biosolids-exposed and control animals (n = 11 per group). Boxes represent 25^th to 75^th percentile, horizontal bar indicates 50th percentile, whiskers indicate range excluding outliers, solid filled circles show outliers, open circles show individual data points, and red triangles show means.

Figure 4.

HIF1α in testes (n = 11 per group). Immunofluorescent staining of testes sections (A). Images to the right are of selections seen in images to the left, which were taken at 400x magnification. Blue staining is of DAPI and green of HIF1α. Scale bars (bottom left in overlay images) show 100 μm. Co-localisation analysis (B) performed on regions outside of seminiferous tubules shows significantly (p = 0.0032) more nuclear localisation of HIF1α in Leydig cells of biosolids-exposed animals than control. Manders’ Overlap Coefficients of the proportion of green signal overlapping with blue signal were determined by ImageJ. Western blots for HIF1α and α-Tubulin (C and D) show less (p = 0.0077) HIF1α in testes of biosolids-exposed animals than controls. Boxes represent 25^th to 75^th percentile, horizontal bar indicates 50th percentile, whiskers indicate range excluding outliers, solid filled circles show outliers, open circles show individual data points, and red triangles show means.