Bisphenol S Impaired Human Granulosa Cell Steroidogenesis in Vitro

Abstract

Bisphenol S (BPS) is a structural analog of the endocrine disruptor bisphenol A (BPA); it is the main BPA replacement in the plastics industry. Previous studies have shown that BPA and BPS exhibit similar effects on reproduction in fish and rodent species. BPS reportedly alters steroidogenesis in bovine granulosa cells. Luteinised granulosa cells collected from 59 women who were undergoing an in vitro fertilization procedure were cultured for 48 h in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). BPS exposure was investigated by assessing follicular fluids from these 59 women for their BPS content. Culture medium, cells, total messenger RNA (mRNA) and total protein extracted from the luteinised granulosa cells were examined for oestradiol and progesterone secretion, cellular proliferation, viability, gene expression, steroidogenic enzyme expression and cell signaling. BPS was measured in follicular fluids using mass spectrometry. Exposure of granulosa cells to 10 or 50 µM BPS for 48 h induced a 16% (p = 0.0059) and 64% (p < 0.0001) decrease, respectively, in progesterone secretion; 50 µM BPS decreased oestradiol secretion by 46% (p < 0.0001). Ten µM BPS also tended to reduce CYP11A1 protein expression by 37% (p = 0.0947) without affecting HSD3B1 and CYP19A1 expression. Fifty µM BPS increased ERRγ expression. Environmental levels of BPS (nanomolar range) did not induce changes in steroidogenesis in human granulosa cells. The effects of BPS were observed after only 48 h of BPS exposure. These acute effects might be similar to chronic effects of physiological BPS levels.

Keywords: Bisphenol S; endocrine disruptors; granulosa cells; proliferation; steroidogenesis; women.

Figure 1

Effect of Bisphenol S (BPS) on human granulosa cell (HGC) viability. HGC underwent 48-h culture in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). (A.) HGC viability was assessed using the Live/Dead Viability/Cytotoxicity Kit for mammalian cells. The results of three independent experiments per condition are expressed as the percentage of living cells in each condition. (B.) HGC viability was assessed on spent 48-h culture media using the Bioluminescence Cytotoxicity Assay Kit. Data are expressed as the mean relative light units ± standard error of the mean (SEM) of six independent cultures. Bars with different superscripts indicate a significant difference (p ≤ 0.05).

Figure 2

Effect of Bisphenol S (BPS) on human granulosa cell (HGC) cellular proliferation. HGC underwent 48-h culture with 10 µM bromodeoxyuridine/5-bromo-2’-deoxyuridine (BrdU), in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). The cell proliferation was normalised to the control condition of each culture. The results are expressed as the mean ± standard error of the mean (SEM) of five independent experiments with four replicates per condition. Bars with different superscripts indicate a significant difference (p ≤ 0.05).

Figure 3

Effect of Bisphenol S (BPS) on human granulosa cell (HGC) progesterone secretion. HGC underwent 48-h culture in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). The progesterone concentration was measured in culture media, and its value was normalised by the protein concentration in each well. Data are expressed as ng progesterone per µg protein and normalised to the control condition. The results of seven independent experiments are presented as the mean ± standard error of the mean (SEM). Bars with different superscripts indicate a significant difference (p ≤ 0.05).

Figure 4

Effect of Bisphenol S (BPS) on human granulosa cell (HGC) oestradiol secretion. HGC underwent 48-h culture in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). The oestradiol concentration was measured in culture media, and its value was normalised by the protein concentration in each well. Data are expressed as pg oestradiol per µg protein and normalised to the control condition. The results of seven independent experiments are presented as mean ± standard error of the mean (SEM). Bars with different superscripts indicate a significant difference (p ≤ 0.05).

Figure 5

Effect of Bisphenol S (BPS) on steroidogenic enzyme expression. HGC underwent 48-h culture in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). Proteins were then extracted and separated using electrophoresis on 4–12% (w/v) sodium dodecyl sulphate polyacrylamide gels. After electrotransfer to nitrocellulose membranes, the proteins were probed with HSD3B (A), CYP11A1 (B), CYP19A1 (C) and VCL antibodies. Bands on the blots were quantified, and the data are expressed in arbitrary units as the ratio of HSD3B (n = 5), CYP11A1 (n = 3) or CYP19A1 (n = 5) to VCL. The results are expressed relative to the control as the mean ± standard error of the mean (SEM). # indicates a tendency (p < 0.10).

Figure 6

Effect of BPS on HGC gene expression. The expression of six hormonal receptors (oestrogen receptors (A): oestrogen receptor 1 [ESR1], oestrogen receptor 2 [ESR2], Oestrogen-related receptor γ [ESRRG] and G protein-coupled oestrogen receptor [GPER] and (B) androgen receptor [AR], progesterone receptor [PR]), and two genes involved in steroidogenesis (B: CYP17A1 and StAR), was determined in HGC after 48-h culture in complemented serum-free McCoy’s 5A media in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). Total messenger RNA (mRNA) was extracted from HGC and reverse transcribed, and real-time polymerase chain reaction (qPCR) was performed. The geometric mean of two housekeeping genes (Glyceraldehyde-3-phosphate dehydrogenase [GAPDH] and ribosomal protein L19 [RPL19]) was used to normalise gene expression. The results are expressed as mean ± standard error of the mean (SEM) of six independent cultures and normalised to the mean of control condition. No letter in common indicates a significant difference (p < 0.05).

Figure 7

Western blot analysis of MAPK3/1 signalling pathway activation in human granulosa cell (HGC) after Bisphenol S (BPS) exposure. HGC underwent a time response (0 to 60 min culture) in the presence or absence of BPS 10 µM. Proteins were then extracted and separated using electrophoresis in 4–12% (w/v) sodium dodecyl sulphate polyacrylamide gels. After electrotransfer to nitrocellulose membranes, the proteins were probed with phospho-MAPK3/1 and total MAPK3/1 antibodies. Bands on the blots were quantified, and the data are expressed in arbitrary units as the ratio of phospho- to total MAPK3/1 as the mean ± standard error of the mean (SEM) of six independent experiments. Different letters indicate significant differences (p ≤ 0.05). abc letters correspond to differences in MAPK3/1 phosphorylation between 10 µM BPS timepoints, and xyz letters correspond to differences in MAPK3/1 phosphorylation between control timepoints.

Figure 8

Female follicular fluid exposure to Bisphenol S (BPS). Follicular fluids from 59 women (in abscissa) were collected. Assays that measured BPS glucuronide (BPSG) were performed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC MS/MS). The limit of quantification (LOQ) value, presented with a horizontal black line, was 1.17 nM (0.5 ng/mL) for BPSG in female follicular fluid. The BPSG level is represented for each of the 59 women in nM.