Mapping Gene Expression Changes in the Fetal Rat Testis Following Acute Dibutyl Phthalate Exposure Defines a Complex Temporal Cascade of Responding Cell Types1

Abstract

Phthalates are chemical plasticizers used in a variety of consumer products; in rodents, they alter testicular development, leading to decreased testosterone synthesis and maldevelopment of the reproductive tract. Here, our goals were to discover a set of biomarker genes that respond early after relatively low-dose-level dibutyl phthalate (DBP) exposure and map the responding testicular cell types. To identify testicular phthalate biomarker genes, 34 candidate genes were examined by quantitative PCR at 1, 2, 3, or 6 h after exposure of Gestational Day 19 rats to DBP dose levels ranging from 0.1 to 500 mg/kg body weight. Twelve genes (Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Fos, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a) were identified with increased expression by 1-3 h at 100 or 500 mg/kg DBP, and 7 of these 12 genes had increased expression by 6 h at 10 mg/kg DBP. Using in situ hybridization of fetal testis cryosections from DBP-exposed rats, the temporal cellular expression of 10 biomarker genes was determined. Genes with a robust response at 1 h (Dusp6, Egr1, Fos, and Thbs1) were induced in peritubular myoid cells. For Egr1 and Fos, the interstitial compartment also showed increased expression at 1 h. Cxcl10 and Nr4a1 were induced by 1-3 h in both sparsely located interstitial cells and peritubular myoid cells. By 3 h, Stc1 was induced in Leydig cells, and Edn1, Ier3, and Tnfrsf12a were increased in Sertoli cells. These data reveal a complex early cascade of phthalate-induced cellular responses in the fetal testis, and for the first time suggest that peritubular myoid cells are an important proximal phthalate target cell.

Mono-(2-ethylhexyl) Phthalate Directly Alters the Expression of Leydig Cell Genes and CYP17 Lyase Activity in Cultured Rat Fetal Testis

“…In vivo/in utero , phthalates have been shown to have broadly inhibitory effects on the expression of genes encoding steroidogenic enzymes. Thus, in addition to Cyp17a1 , the genes encoding cytochrome P450scc and 3beta-HSD have also been shown to be inhibited by phthalates [16], [17], [34], [35], [36]. The doses of phthalates used in these experiments may also induce changes through systemic effects on the pregnant mother or the fetus itself.…”

The presence of macrophages and inflammatory responses in an in vitro testicular co-culture model of male reproductive development enhance relevance to in vivo conditions

“…Consistent with the current study, short term DEHP exposure (3 hours) has been shown to upregulate gene expression of KC in human macrophage-like THP-1 cells in vitro (Nishioka et al 2012). Similarly, 3 hours after DBP exposure (500mg/kg), Johnson, et al observed increased KC gene expression in rat testis in vivo (Johnson et al 2007). In the 3D-TCS, increased levels of KC protein were observed at early timepoints (significant impacts detected at 8 hours) suggesting that KC mediates early responses to phthalate exposure in the 3D-TCS in manner similar to what is observed in vivo .…”

Cellular and Molecular Effect of MEHP Involving LXRα in Human Fetal Testis and Ovary

“…Therefore, the observed decrease in PPARγ mRNA probably reflects the loss of gonocytes induced by MEHP exposure in the cultured human testis [16]. Concerning the decrease in NGFIB mRNA expression, transcriptomic analyses performed in rodent fetal testes have revealed that in vivo exposure to different phthalates increases the mRNA expression of NGFIB and NOR1 , [21], [32], [33]. On the contrary we observed here a decrease in the human fetal testis and in fetal germ cells, exhibiting here a new specificity of the human species.…”

“…Indeed, for PPARγ , both mRNA and protein expression are down-regulated [30], [31]. Transcriptomic analyses also reported that NGFIB (NR4A1) and NOR1 (NR4A3), two orphan members of the NR superfamily, are up-regulated following phthalate exposure [21], [32], [33]. Furthermore, species-differences have been described.…”