Bisphenol A and bisphenol S both disrupt ovine granulosa cell steroidogenesis but through different molecular pathways

Abstract

Background: Ovarian granulosa cells (GC) are essential for the development and maturation of a proper oocyte. GC are sensitive to endocrine disruptors, including bisphenol A (BPA) and its analogue bisphenol S (BPS), plasticisers present in everyday consumer products. BPA exhibits greater binding affinity for the membrane oestrogen receptor (GPER) than for the nuclear oestrogen receptors (ERα and ERβ). Here, we analysed the effects of BPA and BPS on the steroidogenesis of ovine GC in vitro, as well as their early mechanisms of action, the ovine being a relevant model to study human reproductive impairment. Disruption of GC steroidogenesis might alter oocyte quality and consequently fertility rate. In addition, we compared the effects of a specific GPER agonist (G-1) and antagonist (G-15) to those of BPA and BPS. Ewe GC were cultured with BPA or BPS (10 or 50 µM) or G-1 (1 µM) and/or G-15 (10 µM) for 48 h to study steroidogenesis.

Results: Both BPA and BPS (10 µM) altered the secretion of progesterone, however, only BPS (10 µM) affected oestradiol secretion. RNA-seq was performed on GC after 1 h of culture with BPA or BPS (50 µM) or G-1 (10 µM), followed by real-time PCR analyses of differentially expressed genes after 12, 24 and 48 h of culture. The absence of induced GPER target genes showed that BPA and BPS did not activate GPER in GC after 1 h of treatment. These molecules exhibited mainly independent early mechanisms of action. Gene ontology analysis showed that after 1 h of treatment, BPA mainly disrupted the expression of the genes involved in metabolism and transcription, while BPS had a smaller effect and impaired cellular communications. BPA had a transient effect on the expression of CHAC1 (NOTCH signalling and oxidative balance), JUN (linked to MAPK pathway), NR4A1 (oestradiol secretion inhibition), ARRDC4 (endocytose of GPCR) and KLF10 (cell growth, differentiation and apoptosis), while expression changes were maintained over time for the genes LSMEM1 (linked to MAPK pathway), TXNIP (oxidative stress) and LIF (cell cycle regulation) after 12 and 48 h, respectively.

Conclusion: In conclusion, although they exhibited similar effects, BPA and BPS impaired different molecular pathways in GC in vitro. New investigations will be necessary to follow the temporal changes of these genes over time, as well as the biological processes involved.

Keywords: Bisphenol; Endocrine disruptors; Ewe; Granulosa cells; Mechanisms of action; Steroidogenesis.

Fig. 1

The effects of the GPER-specific agonist (G-1) or antagonist (G-15) and/or bisphenol A (BPA) and bisphenol (BPS) on ovine granulosa cells (GC) steroidogenesis. The progesterone (A) and oestradiol (B) concentrations were determined in culture medium after 48 h of culture in complemented serum-free McCoy’s 5A media in the presence or absence (control) of BPA or BPS at 10 or 50 μM, and/or G-15 at 10 µM or G-1 at 1 μM. The results are expressed as the mean ± standard error of the mean of 10 independent cultures. Each condition was performed in duplicate and normalised to the control condition of each culture experiment. Bars with different superscripts indicate a significant difference (p ≤ 0.05). The actual control values were 37.07 ± 4.68 ng progesterone/mg protein (A) and 52.04 ± 2.21 pg oestradiol/mg protein B

Fig. 2

Differentially expressed genes (DEG) in ovine granulosa cells (GC) treated with bisphenol A (BPA), bisphenol S (BPS) or the GPER-specific agonist (G-1). After 1 h culture in complemented serum-free McCoy’s 5A media in the presence or absence (control) of BPA or BPS at 50 μM, or G-1 at 10 μM, six biological replicates were analysed with RNA-sequencing, and DEG were identified (padj ≤ 0.05). The Venn diagram (A) shows common and specific DEG from four comparisons: control versus BPA, control versus BPS, BPA versus BPS, and Control versus G-1. The heatmap (B) shows hierarchical clustering of DEG. The ordered list of heatmap genes have been annotated in Supplementary Table S3. Principle component analysis of gene expression in GC treated or not with BPA, BPS or G1 performed on expression values of 24 differential genes C

Fig. 3

Functional analysis of differentially expressed genes (DEG) from RNA-sequencing of ovine granulosa cells (GC). After 1 h of treatment in the presence or absence (control) of bisphenol A (BPA) or bisphenol S (BPS) at 50 μM, GC (six replicates per condition) were analysed with RNA-sequencing to obtain a list of DEG (p ≤ 0.05). The global clustering heatmap plot of functional sets of gene ontology (GO; p ≤ 0.01) terms was obtained by using ViSEAGO. From left to right are: the major processes, the cluster name, a heatmap of GO term counts from functional enrichment tests and a dendrogram based on Wang’s semantic similarity distance and Ward’s clustering criterion

Fig. 4

The effects of the GPER-specific antagonist (G-15) and/or bisphenol A (BPA) and bisphenol S (BPS) on gene expression of ovine granulosa cells (GC), according to changes over time. The expression of eight genes (CHAC1, JUN, LSMEM1, NR4A1, TXNIP, ARRDC4, KLF9 and LIF) were assessed in ovine GC. Gene expression was determined after 12, 24 and 48 h of culture in complemented serum-free McCoy’s 5A media in the presence or absence (control) of BPA or BPS at 50 μM. Total messenger RNA (mRNA) was extracted and reverse transcribed from ovine GC, then real-time polymerase chain reaction (qPCR) was performed. To normalise gene expression, the geometric mean of two housekeeping genes (β-actin [ACTB] and ribosomal protein L19 [RPL19]) was used. The results are expressed as the mean ± standard error of the mean of eight independent cultures and normalised to the mean of the control condition at 12 h. Statistical analysis was performed between the conditions of a same time (12, 24 or 48 h). Bars with different superscripts indicate a significant difference (p ≤ 0.05)