The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the transcriptome of aryl hydrocarbon receptor (AhR) knock-down porcine granulosa cells

Abstract

Background 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical, adversely affecting reproductive processes. The well-characterized canonical mechanism of TCDD action involves the activation of aryl hydrocarbon receptor (AhR) pathway, but AhR-independent mechanisms were also suggested. By applying RNA interference technology and Next Generation Sequencing (NGS) we aimed to identify genes involved in the mechanism of TCDD action in AhR knock-down porcine granulosa cells. Methods Porcine granulosa cells were transfected with small interfering RNAs targeting mRNA of AhR. After transfection, medium was exchanged and the AhR knock-down cells were treated with TCDD (100 nM) for 3, 12 or 24 h, total cellular RNA was isolated and designated for NGS. Following sequencing, differentially expressed genes (DEGs) were identified. To analyze functions and establish possible interactions of DEGs, the Gene Ontology (GO) database and the Search Tool for the Retrieval of Interacting Genes (STRING) database were used, respectively. Results The AhR gene expression level and protein abundance were significantly decreased after AhR-targeted siRNAs transfection of the cells. In TCDD-treated AhR knock-down cells we identified 360 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change [log2FC] ≥ 1.0). The functional enrichment analysis of DEGs revealed that TCDD influenced the expression of genes involved, among other, in the metabolism of vitamin A, follicular development and oocyte maturation, proliferation and differentiation as well as inflammation, stress response, apoptosis and oncogenesis. The three-time point study demonstrated that TCDD-induced changes in the transcriptome of AhR knock-down porcine granulosa cells were especially pronounced during the early stages of the treatment (3 h). Conclusions TCDD affected the transcriptome of AhR knock-down porcine granulosa cells. The molecules involved in the AhR-independent action of TCDD were indicated in the study. The obtained data contribute to better understanding of molecular processes induced by xenobiotics in the ovary.

Transgenerational Transmission of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Effects in Human Granulosa Cells: The Role of MicroRNAs

“…Here, by performing miRNA profiling in human granulosa cells after chronic TCDD exposure, we identified expression changes in miRNAs that target genes implicated in cancer-related signaling not only directly after acute or chronic exposure but also several days after the end of chronic exposure (i.e., in cells that were never exposed to TCDD). Several studies analyzed steroid hormone secretion, gene expression, the proteome, and the incidence of apoptosis in granulosa cells after TCDD exposure [5,7,8,20,23,25,[27][28][29][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53]. The present work was the first to assess the miRNome after acute and chronic TCDD exposure and in the following cell generations up to day 14 after chronic exposure.…”

“…Although the factors involved in TCDD-induced toxicity are still under investigation, several studies showed that the female endocrine system-disrupting effects of TCDD are, at least in part, caused by direct action on the ovaries [19,20]. Aromatic hydrocarbon receptors (AhR), the main mediator of TCDD intracellular action, are expressed in mammalian ovarian tissue, including human granulosa cells [19][20][21][22][23]. In porcine ovarian granulosa cells, TCDD alters the expression of genes involved in cell cycle regulation, proliferation, and follicular atresia [23][24][25][26][27][28] and it also modifies the expression of proteins involved in the rearrangement of the cytoskeleton and extracellular matrix and in the cellular response to stress [29].…”

“…Aromatic hydrocarbon receptors (AhR), the main mediator of TCDD intracellular action, are expressed in mammalian ovarian tissue, including human granulosa cells [19][20][21][22][23]. In porcine ovarian granulosa cells, TCDD alters the expression of genes involved in cell cycle regulation, proliferation, and follicular atresia [23][24][25][26][27][28] and it also modifies the expression of proteins involved in the rearrangement of the cytoskeleton and extracellular matrix and in the cellular response to stress [29]. In addition, many genes involved in cell proliferation and differentiation, inflammation, stress response, apoptosis, and oncogenesis are differentially expressed in AhR knock-down granulosa cells incubated with TCDD compared with non-exposed cells.…”

“…In addition, many genes involved in cell proliferation and differentiation, inflammation, stress response, apoptosis, and oncogenesis are differentially expressed in AhR knock-down granulosa cells incubated with TCDD compared with non-exposed cells. This suggests that TCDD may affect its target tissues also in an AhR-independent manner [23].…”