Ovine fetal testis stage-specific sensitivity to environmental chemical mixtures

Abstract

Exposure of the fetal testis to numerous individual environmental chemicals is frequently associated with dysregulated development, leading to impaired adult reproductive competence. However, ‘real-life’ exposure involves complex mixtures of environmental chemicals (ECs). Here we test the consequences, for the male fetus, of exposing pregnant ewes to EC mixtures derived from pastures treated with biosolids fertiliser (processed human sewage). Fetal testes from continuously exposed ewes were either unaffected at Day 80 or exhibited a reduced area of testis immunostained for CYP17A1 protein at Day 140. Fetal testes from Day 140 pregnant ewes exposed transiently for 80 day periods during early (0-80 days), mid (30-110 days) or late (60-140 days) pregnancy, had fewer Sertoli cells and reduced testicular area stained for CYP17A1. Male fetuses from ewes exposed during late pregnancy also exhibited reduced fetal body, adrenal and testis mass, anogenital distance and lowered testosterone: collectively indicative of an anti-androgenic effect. Exposure limited to early gestation induced more testis transcriptome changes than observed for continuously exposed Day 140 fetuses. These data suggest that a short period of EC exposure does not allow sufficient time for the testis to adapt. Consequently, testicular transcriptomic changes induced during the first 80 days of gestation may equate with phenotypic effects observed at Day 140. In contrast, relatively fewer changes in the testis transcriptome in fetuses exposed continuously to ECs throughout gestation is associated with less severe consequences. Unless corrected by or during puberty, these differential effects would predictably have adverse outcomes for adult testicular function and fertility.

Developmental exposure to a real-life environmental chemical mixture alters testicular transcription factor expression in neonatal and pre-pubertal rams, with morphological changes persisting into adulthood

“…Despite this apparent phenotypic recovery, adult B rams exhibited an increased proportion of seminiferous tubules showing degeneration and depletion of elongating spermatids. This phenotype is regarded as the end stage lesion of low intratesticular testosterone (OECD, 2009), which agrees with previously published observations of lower plasma testosterone concentrations in the late gestation fetus and neonatal male offspring (Elcombe et al, 2022b;Lea et al, 2022), and may have an impact on sperm production, semen quality, fertility, and fecundity. When the adult phenotype is compared to younger male offspring (neonatal and prepubertal) with very similar gestational BTP exposure, a lessening of morphological changes with increasing time without exposure was mirrored in both the decrease in the numbers of differentially expressed genes, and the normalisation of transcription factor expression and activity.…”

“…Offspring of sheep grazed on BTP before and during pregnancy have shown a TDS-like phenotype (reduced germ cell numbers and greater rates of Sertoli-cell only seminiferous tubules) in neonatal rams (Elcombe et al, 2021), and in the pre-pubescent half-siblings of the adult rams presented here (Elcombe et al, 2022b). Following similar exposure, reductions in germ cell, Sertoli cell, and Leydig cell numbers has been seen in the mid-gestation (GD110) fetus (Paul et al, 2005), and in the late-gestation fetus (GD140) various exposure timings led to reductions in testes weight, total Sertoli cell numbers, and plasma testosterone concentrations (Lea et al, 2022). In the adult male offspring, there were no differences in terms of total germ cell populations and SCO tubules, which could suggest (partial) recovery from a TDS-like phenotype.…”

“…mTOR has previously been identified as affected by EC exposure in transcriptomic analyses of fetal, neonatal, and pre-pubertal biosolids ram testes (Elcombe et al, 2022b(Elcombe et al, , 2021Lea et al, 2022).…”

“…Of specific note was the identification of the mTOR signalling pathway as a site of disruption. mTOR has previously been identified as affected by EC exposure in transcriptomic analyses of fetal, neonatal, and pre-pubertal biosolids ram testes (Elcombe et al, 2022b, 2021; Lea et al, 2022). Alterations with mTOR signalling pathways due to low-level exposure should be of concern as mTOR is a crucial component of proper testicular development and spermatogenesis (Correia et al, 2020) and its disruption by chemical exposure (e.g., 4NP, DEHP, or BPA) has been shown to induce testicular autophagy in pubescent rodents (Duan et al, 2017; Fu et al, 2020; Quan et al, 2017).…”

“…A TDS-like phenotype (reduced germ cell numbers and greater rates of Sertoli-cell only (SCO) seminiferous tubules) has been previously reported in neonatal (1-day-old) and pre-pubertal (8-weeks-old) lambs whose mothers were grazed on BTP prior to and during pregnancy, with lower plasma testosterone concentrations also reported for the neonatal male offspring (Elcombe et al, 2022b, 2021). In the fetus (GD110 and GD140), across various exposure periods, fewer germ cells, Leydig cells, and Sertoli cells, and lower plasma testosterone concentrations have been reported (Lea et al, 2022; Paul et al, 2005). Results from adult rams would also suggest that BTP exposure (gestationally, and for 7 months during post-natal life, i.e., lactational / direct oral exposure) also results in a TDS-like phenotype in a subset of adult (19-months-old) rams (Bellingham et al, 2012).…”