Foetal and post-natal exposure of sheep to sewage sludge chemicals disrupts sperm production in adulthood in a subset of animals

Abstract

Exposure to ubiquitous, environmental chemicals (ECs) has been hypothesized as a cause for declining male reproductive health. Understanding the long-term effects of EC exposure on reproductive health in humans requires animal models and exposure to 'real life', environmentally relevant, mixtures during development, a life stage of particular sensitivity to ECs. The aim of this study was to evaluate the effects of in utero and post-natal exposure to environmentally relevant levels of ECs, via sewage sludge application to pasture, on the adult male sheep testis. Hormones, liver concentrations of candidate ECs and Sertoli and germ cell numbers in testes of adult rams that were exposed to ECs in sewage sludge in utero, and until weaning via maternal exposure, and post-weaning via grazing pastures fertilized with sewage sludge, were quantified. Evaluated as a single group, exposure to sludge ECs was without significant effect on most parameters. However, a more detailed study revealed that 5 of 12 sludge-exposed rams exhibited major spermatogenic abnormalities. These consisted of major reductions in germ cell numbers per testis or per Sertoli cell and more Sertoli cell-only tubules, when compared with controls, which did not show any such changes. The sludge-related spermatogenic changes in the five affected animals were significantly different from controls (p < 0.001); Sertoli cell number was unaffected. Hormone profiles and liver candidate EC concentrations were not measurably affected by exposure. We conclude that developmental exposure of male sheep to real-world mixtures of ECs can result in major reduction in germ cell numbers, indicative of impaired sperm production, in a proportion of exposed males. The individual-specific effects are presumed to reflect EC effects on a heterogeneous population in which some individuals may be more susceptible to adverse EC effects. Such effects of EC exposure in humans could have adverse consequences for sperm counts and fertility in some exposed males.

Figure 1

Sertoli cell-only (SCO) tubules in sewage sludge-exposed animals. SCO tubules were defined as those that contained either no VASA positive germ cells (A) or only a few spermatogonia (Sg, arrows) scattered around the basement membrane (B). In contrast, tubules scored as non-SCO (C) either had many Sg scattered around the basement membrane or Sg plus other germ cell types were present (*). Scale bar = 50 μm.

Figure 2

Mean average testis weight in control (C) and sewage sludge-exposed (S) rams. Three rams in the S group (blue circles) had testis weights below the lowest control value and markedly lower than the rest of the S group (s small, p < 0.05). Bars represent mean ± SEM.

Figure 3

Mean serum concentrations of (A) Testosterone, (B) LH, (C) FSH and (D) INHA in control and sewage sludge-exposed rams. Blue circles = sludge-exposed animals with a low testis weight (as in Fig. 2). Bars represent mean ± SEM.

Figure 4

VASA immunostaining of germ cells (GC) in control and sludge-exposed rams. VASA staining shows the phenotype of control animals (A) and two distinct groups of sludge-exposed animals: S1, which had normal GC counts compared with controls (B) and S2, which had statistically significant lower GC counts than controls (C). Note that although S1 animals had normal GC numbers, some also had occasional Sertoli cell-only (SCO) tubules (*), whereas S2 animals all exhibited varying degrees of GC loss and more frequent SCO tubules were evident. Scale bar = 100 μm.

Figure 5

Sertoli cell number in control and sewage sludge-exposed rams. WT-1 immunoexpression, which was unaffected by sludge exposure or germ cell loss, was used to identify Sertoli cells for counting. (A) Control, (B) S1 sludge-exposed, (C) S2 sludge-exposed. Scale bar = 50 μm. (D) Mean Sertoli cell number was not significantly different between control and sludge-exposed animals (S1 + S2). Blue circles = sludge-exposed animals with a low testis weight (as in Fig. 2). Bars represent mean ± SEM.

Figure 6

Germ cell numbers/volume in control and sewage sludge-exposed animals. Mean ± SEM data are shown as total germ cell (GC) number per testis (A), GC number per Sertoli cell (SC) (B), the absolute nuclear volume of GC (GCAV) per testis (C), the nuclear volume of GC per Sertoli cell (GCAV : SC) (D) or the number of spermatocytes (E) or round spermatids (F) per testis. Overall, values were not significantly different between control and sludge-exposed rams. However, there were two distinct groups within the sludge-exposed group. S1 were not different to C animals whereas five animals (S2) had GC counts significantly lower than the lowest control (*p < 0.001). The dotted black line represents the lowest control value for each parameter. Red lines show the statistical differences between control and S2 and between S1 and S2. Blue circles = sludge-exposed animals with a low testis weight (as in Fig. 2).