Bisphenols effects on Granulosa Cell-Cell Communication

Abstract

Bisphenols are Endocrine Disrupting Compounds (EDCs) linked to negative fertility outcomes in humans. The most common one, bisphenol A (BPA) is a plasticizer that acts as an estrogen agonist. Alternatives such as BPS and BPF are becoming widespread and possess similar activities. The oocyte and nearby granulosa cells are particularly vulnerable to EDCs as they are dependent on hormone signaling. Granulosa-Granulosa cell communications are pivotal for proper development and are mediated by gap junction molecules, called connexins (Cxs). Cx37, Cx26, and Cx43 are crucial in follicular communication and are the focus of this study. Bisphenols can alter these genes which directly affects cell-cell communication and in turn proper development. This study uses an in vitro cell culture model of bovine granulosa cells as the bovine species is an excellent translational model for human reproductive toxicology. To determine optimal doses and exposure times, we performed a cytotoxic assay (CCK8) and treated cells with a low dose (0.5 μg/mL), a medium dose (5 μg/mL), the current reported LOAEL (50 μg/mL), and a high dose (0.5 mg/mL) of bisphenols for 1, 6, 12, 24, 48, and 72 hrs. The high dose was lethal at all time points for BPA/BPF, while BPS was toxic from 1 hrs (P=0.0016; n=4). The LOAEL dose was toxic for BPA at 6, 12, 24 hrs (P=0.018, 0.0021, 0.0042 respectively; n=7) and was lethal at 48/72 hrs. BPF at the LOAEL dose showed an increase in cell viability at 6 hrs which was significant at 12hrs (P=0.0119; n=5). This trend declined at 48/72 hrs but was not significant. BPS did not affect cell viability at any other dose and the medium dose did not affect viability for all bisphenols; however, the low dose of BPA/BPF showed a trend towards decreased viability at 48 hrs of treatment. Therefore, we looked at the effects of BPA/S/F at the low and LOAEL doses for 12/48 hrs. We used qPCR and western blotting to quantify transcripts and proteins of the three connexins. Our data show that Cx43 mRNA was significantly increased at the LOAEL dose for BPA/BPF at 12 hrs (p=0.02; n=6). Preliminary data on proteins indicate a decrease and increase in expression at 12hrs for BPA/BPF treatment, respectively, at the LOAEL dose. BPS shows no effect on Cx43. BPS low dose and BPA/BPF LOAEL doses at 12 and 48 hrs increase Cx37 mRNA. Preliminary data on the protein levels show a significant decrease of Cx37 protein at BPA low dose, but no changes after exposure to the other bisphenols. Lastly, Cx26 mRNA is significantly increased after BPA treatment at the LOAEL dose at 12 hrs (P<0.0001; n=5). This novel study investigates the effects of low/high doses and short-term/chronic exposure of bisphenols on cell-cell communication in granulosa cells and provides the basis for future studies on bisphenol toxicity in early development.