Bisphenols, plasticisers used in food containers, can transfer to food. Bisphenol A (BPA) has been described as an endocrine disruptor and consequently banned from the food industry in several countries. It was replaced by a structural analogue, Bisphenol S (BPS). BPA action on the steroidogenesis is one of the mechanisms underlying its adverse effects on the efficiency of female reproduction. This study aimed to determine whether BPS is a safe alternative to BPA regarding GC functions. Antral follicles (2–6 mm), of approximatively 1000 adult ewe ovaries, were aspired and GC purified. For 48 h, ovine GC were treated with BPA or BPS (from 1 nM to 200 µM) and the effects on cell viability, proliferation, steroid production, steroidogenic enzyme expression and signalling pathways were investigated. Dosages at and greater than 100 μM BPA and 10 µM BPS decreased progesterone secretion by 39% (P < 0.001) and 22% (P = 0.040), respectively. BPA and BPS 10 μM and previously mentioned concentrations increased oestradiol secretion two-fold (P < 0.001 and P = 0.082, respectively). Only 100 µM BPA induced a decrease (P < 0.001) in gene expression of the enzymes of steroidogenesis involved in the production of progesterone. BPA reduced MAPK3/1 phosphorylation and ESR1 and ESR2 gene expression, effects that were not observed with BPS. BPA and BPS altered steroidogenesis of ovine GC. Thus, BPS does not appear to be a safe alternative for BPA. Further investigations are required to elucidate BPA and BPS mechanisms of action.
Supplementary figure 1 : Cell viability and Progesterone secretion with ethanol control Cell viability and progesterone secretion was assessed in ovine granulosa cells (GC). Culture was performed during 48-h in complemented serum-free McCoy’s 5A media in presence or absence of ethanol (1/100,000,000 to 1/500) in concentrations corresponding to the different concentrations of bisphenols. The activity of adenylate kinase (A.) released into the culture supernatant by dead cells was assessed by bioluminescence (according to the manufacturer’s instructions). The progesterone concentration was determined in culture medium (B.). The results are expressed as the mean ± standard error of the mean (SEM) of six independent cultures, with each condition performed in duplicate, and normalised to the control condition of each culture experiment. Ethanol had not impact on cell viability and progesterone secretion in ovine GC.
Supplementary figure 2 : Gene expression of cumulus and granulosa cell markers The gene expression of two granulosa cell (GC) specific markers (anti-müllerian hormone [AMH] and AMH receptor [AMHR]) and two cumulus cell (CC) specific markers (Hyaluronic synthase 2 [HAS2] and pentraxin 3 [PTX3]) were assessed in ovine GC and CC (positive control). Specific oligonucleotide primers were used (A.). Expression of two GC specific markers was assessed in the ovine GC samples used in this study (B.). Presence of CC was investigated with two CC specific markers in ovine CC (positive control) and in the ovine GC samples used in this study (C.). Total messenger RNA (mRNA) was extracted from GC and reverse transcribed, and real-time polymerase chain reaction (qPCR) was performed. The geometric mean of two housekeeping genes (beta-actin [ACTB] and ribosomal protein L19 [RPL19]) was used to normalise gene expression. Gene expression of all samples are normalised to the mean of control condition. Markers of GC, AMH and AMHR, were present at similar levels between all GC samples. On the contrary, expression of markers of CC, HAS2 and PTX3, was highly reduced in GC samples used in this study.
Supplementary figure 3 : Progesterone secretion with medium containing phenol red or not Progesterone secretion was assessed in ovine granulosa cells (GC). The progesterone concentration was determined in culture medium after 48-h culture in complemented serum-free McCoy’s 5A media with or without phenol red and in presence of BPS 50 ìM or ethanol 1/2000 (control). The results are expressed as the mean ± standard error of the mean (SEM) of six independent cultures, with each condition performed in triplicate, and normalised to the control condition of each culture experiment. Bars with different letters indicate significant difference (p < 0.05). Red phenol had not impact on progesterone secretion in ovine GC and does not modulate BPS effect on progesterone secretion.