Sex-specific effects of bisphenol A on the signaling pathway of ESRRG in the human placenta†

Abstract

Bisphenol A (BPA) exposure during pregnancy is associated with low fetal weight, particularly in male fetuses. The expression of estrogen-related receptor gamma (ESRRG), a receptor for BPA in the human placenta, is reduced in fetal growth restriction. This study sought to explore whether ESRRG signaling mediates BPA-induced placental dysfunction and determine whether changes in the ESRRG signaling pathway are sex-specific. Placental villous explants from 18 normal term pregnancies were cultured with a range of BPA concentrations (1 nM-1 μM). Baseline BPA concentrations in the placental tissue used for explant culture ranged from 0.04 to 5.1 nM (average 2.3 ±1.9 nM; n = 6). Expression of ESRRG signaling pathway constituents and cell turnover were quantified. BPA (1 μM) increased ESRRG mRNA expression after 24 h in both sexes. ESRRG mRNA and protein expression was increased in female placentas treated with 1 μM BPA for 24 h but was decreased in male placentas treated with 1 nM or 1 μM for 48 h. Levels of 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and placenta specific-1 (PLAC1), genes downstream of ESRRG, were also affected. HSD17B1 mRNA expression was increased in female placentas by 1 μM BPA; however, 1 nM BPA reduced HSD17B1 and PLAC1 expression in male placentas at 48 h. BPA treatment did not affect rates of proliferation, apoptosis, or syncytiotrophoblast differentiation in cultured villous explants. This study has demonstrated that BPA affects the ESRRG signaling pathway in a sex-specific manner in human placentas and a possible biological mechanism to explain the differential effects of BPA exposure on male and female fetuses observed in epidemiological studies.

Keywords: bisphenol A; estrogen-related receptor gamma; human placenta; placental dysfunction; sex specific manner.

Figure 1

Effects of BPA exposure on expression of ESRRG in term villous explants. (A) mRNA expression of ESRRG. (B) Quantification of ESRRG immunostaining. (C–J) Representative images of immunostained cultured villous explants. (C) Negative control. (D–G) 24 h of culture; (H–K) 48 h of culture. (D, H) Controls (0.05% (v/v) ethanol); (E, I) 1 pM BPA; (F, J) 1 nM BPA; (G, K) 1 μM BPA. One sample Wilcoxon test, median ± IQR. n = 18. Bar = 50 μm.

Figure 2

mRNA and protein expression of ESRRG in term villous explants from male and female fetuses after BPA exposure for 24 h. (A) mRNA expression of ESRRG. (B) Quantification of ESRRG immunostaining. (C) Negative control. Representative images of immunostained villous explants from male infants (D–G) and female fetuses (H–K). (D, H) Controls (0.05% (v/v) ethanol). (E, I) 1 pM BPA. (F, J) 1 nM BPA. (G K) 1 μM BPA. One sample Wilcoxon test, median ± IQR. Bar = 50 μm.

Figure 3

mRNA and protein expression of ESRRG in male or female villous explants treated with BPA for 48 h. (A) mRNA expression of ESRRG. (B) Quantification of ESRRG immunostaining. (C) Negative control. Representative images of immunostained placental explants from male fetuses (D–G) and female infants (H–K). (D H) Control (0.05% (v/v) ethanol). (E, I) 1 pM BPA. (F, J) 1 nM BPA. (G, K) 1 μM BPA. One sample Wilcoxon test, median ± IQR. Bar = 50 μm.

Figure 4

Effects of BPA on the mRNA levels of downstream genes of ESRRG. (A) HSD17B1; (B) CYP191.1; (C) HSD11B2; (D) PLAC1. One sample Wilcoxon test; n = 18. Median ± IQR. CYP191.1, cytochrome P-450; HSD17B1, 17β-hydroxysteroid dehydrogenase type 1; HSD11B2, 11β-hydroxysteroid dehydrogenase type 2; PLAC1, placenta specific-1.

Figure 5

The sex-specific effects of BPA on the mRNA levels of downstream genes of ESRRG’. (A) (HSD17B1, 24 h) and (B) (HSD17B1, 48 h); (C) (CYP191.1, 24 h) and (D) (CYP191.1, 48 h); (E) (HSD11B2, 24 h) and (F) (HSD11B2, 48 h). (G) (PLAC1, 24 h) and (H) (PLAC1, 48 h), mRNA levels. One sample Wilcoxon test, median ± IQR. CYP191.1, cytochrome P-450; HSD17B1, 17β-hydroxysteroid dehydrogenase type 1; HSD11B2, 11β-hydroxysteroid dehydrogenase type 2; PLAC1, placenta specific-1.

Figure 6

hCG and LDH levels in explant culture medium following BPA exposure. (A) hCG and (D) LDH levels in the culture medium from all villous explants after BPA treatment for 24 or 48 h (n = 6). (B) hCG levels in culture medium from explants from male and female for 24 h of culture, and (C) for 48 h of culture. (E) LDH levels in culture medium from explants from male and female for 24 h of culture, and (F) for 48 h of culture. n = 6. Median ± IQR, one sample Wilcoxon test.

Figure 7

Effects of BPA on the percentage of cells in cycle in villous explants cultured for 24 or 48 h. (A) Quantification of Ki67 staining. (B) Negative control. Representative images of ki67 staining in the placental explants cultured for 24 h (C–F) or 48 h (G–J). (C, G) Control group (0.05% ethanol); (D, H) 1 pM BPA; (E, I) 1 nM BPA; (F, J) 1 μM BPA. Black arrow, Ki67 positive cells. Kruskal-Wallis test, median ± IQR. n = 18. Bar = 50 μm.

Figure 8

Effects of BPA on the percentage of apoptotic cells in villous explants cultured for 24 or 48 h. (A) Quantification of M30 staining. (B) Negative control. Representative images of M30 staining after 24 (C–F) or 48 h (G–J) of BPA exposure. (C, G) Control (0.05% (v/v) ethanol); (D, H) 1 pM BPA; (E, I) 1 nM BPA; (F, J) 1 μM BPA. Black arrow, M30 positive cells. Kruskal-Wallis test, median ± IQR. n = 18. Bar = 50 μm.