Morphological and transcriptomic alterations in neonatal lamb testes following developmental exposure to low-level environmental chemical mixture

Abstract

Exposure to anthropogenic environmental chemical mixtures could be contributing to the decline in male reproductive health. This study used the biosolid treated pasture (BTP) sheep model to assess the effects of exposure to low-dose chemical mixtures. Maternal BTP exposure was associated with lower plasma testosterone concentrations, a greater proportion of Sertoli cell-only seminiferous tubules, and fewer gonocytes in the testes of neonatal offspring. Transcriptome analysis highlighted changes in testicular mTOR signalling, including lower expression of two mTOR complex components. Transcriptomic hierarchical analysis relative to the phenotypic severity demonstrated distinct differential responses to maternal BTP exposure during pregnancy. Transcriptome analysis between phenotypically normal and abnormal BTP lambs demonstrated separate responses within the cAMP and PI3K signalling pathways towards CREB. Together, the results provide a potential mechanistic explanation for adverse effects. Exposure could lower gonocyte numbers through mTOR mediated autophagy, but CREB mediated survival factors may act to increase germ cell survival.

Keywords: Developmental toxicity; Environmental chemicals; Testicular dysgenesis syndrome.

Figure 1

Histopathological findings in neonatal control and biosolid lamb testes. Representative images of H&E stained tissue sections from 1-day old control (A) and biosolid (B) lambs taken at 40x magnification. Arrows indicate Sertoli cell-only (SCO) seminiferous tubules. Relative ratios of gonocytes to Sertoli cells (C) and proportions of SCO tubules (D) (p=0.0015 and p=0.0040 respectively). Plasma testosterone concentrations for control and biosolid lambs (E). Boxes represent 25^th to 75^th percentile, horizontal bar indicates 50^th percentile, whiskers indicate range excluding outliers, solid filled circles show outliers, open circles show individual data points, and red triangles show means.

Figure 2

Identification and determination of phenotypic subtypes. Hierarchical analysis of cDNA expression counts by iDEP (A) indicates varying response to exposure. Horizontal lines indicate individual genes, colours indicate difference in expression from the control average – red and blue indicate higher or lower gene expression, respectively. Hierarchical clustering trees were produced using genes with maximum expression levels in the top 75%, height of branching is proportional to average linkage correlations. Separation of biosolid lambs based on morphological data into two sub-groups (B): biosolid lambs which saw more of an effect (“susceptible”) and those which saw less of an effect (“resistant”), based on a threshold of 1x SD of control kernel density estimates and control value range.

Figure 3

Schematic representation of changes to testicular gene expression. Differential expression in biosolid susceptible (blue) and resistant (green) lambs (small arrows) compared to controls, and potential changes to cell signalling (large arrows) from normal developmental and spermatogenic signalling (grey arrows).