48 Bisphenols modulate the anti-Müllerian hormone system in bovine granulosa cells cultured in vitro

Environmental agents with the ability to disrupt reproductive function, such as endocrine-disrupting compounds (EDCs), contribute to fertility decline in dairy cattle. There is evidence of an association between exposure of dairy cattle to EDC-contaminated drinking water/feed and reduced reproductive performance. Bisphenol A (BPA), a known EDC used in plastics, interferes with hormonal pathways and is implicated with poor fertility outcomes. As a result, analogues of BPA, such as bisphenol S (BPS), are now used as substitutes, despite limited research on potential reproductive adverse effects. Studies in our laboratory investigated nontraditional mechanisms of action of bisphenols that affect both farm animals and human fertility. Previously, we have shown that in vitro-cultured granulosa cells exposed to BPA, at the LOAEL (lowest-observed-adverse-effect level) dose (0.05 mg mL⁻¹), display a significant up-regulation of Smad1, Smad5, and Smad4 transcripts, principal mediators of the anti-Müllerian hormone (AMH) pathway. In addition, BPA down-regulates AMH in oocytes and embryos, while up-regulating its receptor, AMHRII, suggesting a direct interaction between BPA and the AMH system. AMH, an established marker of ovarian reserve, is associated with increased fertility. Thus, we hypothesise that bisphenols act through the AMH signalling pathway compromising oocyte competence. This study aimed to investigate the ability of bisphenols to alter AMHRII and Smad protein expression in bovine granulosa cells in vitro. Cumulus–oocyte complexes (COCs) were collected from ovaries obtained from a local slaughterhouse. Granulosa cells, isolated from the COCs, were cultured in vitro and treated at 1 and 12 h with physiologically relevant doses of BPA, BPS, and AMH alone (0.05 mg mL⁻¹, 0.05 mg mL⁻¹, 0.025 mg mL⁻¹, respectively) as well as BPA and BPS in combination with AMH. Protein levels of AMHRII and downstream Smad effectors (Smad1, Phospho-Smad1, and Smad4) were quantified by western blotting, using β-actin as a loading control. Densitometric analysis was performed using Image Laboratory and Chemidoc XRS + (BioRad) instrument. Following 12-h treatment with BPA in combination with AMH and with BPA alone, there was a significant decrease in the expression of Smad1 (P < 0.002, n = 3; P < 0.05, n = 3) and Phospho-Smad1 (P < 0.03, n = 5; P < 0.04, n = 5). There were no significant differences in AMHRII and Smad4 expression following 12 h of treatment. Moreover, there were no significant differences in any treatment group following 1 h of treatment. Results were statistically analysed by one-way ANOVA and Tukey’s multiple comparison test using GraphPad Prism 6 (GraphPad Inc.). These findings suggest that BPA, but not BPS, might modulate activation of the downstream effectors of the AMH system. To establish a direct link between BPA and the AMH receptor, additional receptor knock-down studies with Gapmers are being conducted to elucidate an alternative mechanism of bisphenols on the downstream mediators of the AMH pathway affecting fertility and reproductive outcome.

Funding for this research was from NSERC.