RF22 | PMON09 BPA and BPF, but not BPS, alter pro- and anti-apoptotic transcript levels in granulosa cells, affecting cell viability.

Endocrine-disrupting compounds (EDCs) elicit adverse responses in the body, including the disruption of female reproductive functions. A common EDC is Bisphenol A (BPA), which is a frequently used plasticizer. Due to the negative effects of BPA on human health, production of BPA-free products using analogs, such as BPS and BPF, has increased; yet their effects are still largely unknown. Adequate oocyte maturation requires proper functioning of the surrounding environment, consisting of granulosa cells that impact oocyte competency. Granulosa cells are also susceptible to BPA disruption, affecting oocyte maturation and, ultimately, fertility. In our lab, we showed that BPA and BPS significantly increased apoptosis in bovine embryos through increased DNA fragmentation (Saleh et al., 2021). However, the apoptotic pathway used is not fully characterized. Thus, the aim of this study is to investigate how bisphenols affect granulosa cell viability and through which apoptotic pathway. In vitro cultured granulosa cells were exposed for 12 or 48 hrs to six treatment groups: control, vehicle, estradiol, BPA, BPS and BPF at a low dose (0.005 mg/mL) and at the BPA Lowest Observed Adverse Effect Level (LOAEL-0.05 mg/mL). Live cells were counted using trypan exclusion. A significant decrease in the number of live cells after BPA treatment at the LOAEL dose for 12 hrs was observed (P=0.02, n=9). mRNA expression of three proapoptotic (BAX, BAD, Casp9) and two antiapoptotic (HSP70 and BCl-2) genes was quantified by qPCR. A significant increase in BAX transcript levels after 12 hrs of exposure to BPA and BPF at the LOAEL dose (P=0.046, P= 0.033, n=6) as well as in BAD (P= 0.049, n=3) and Casp9 (P=0.049, n=4) levels after BPA exposure at the LOAEL dose was detected. Interestingly, at the 48 hr timepoint, a significant increase in BAD (P= 0.018, n= 9), BAX (P=0.002, n=10), and Casp9 (P=0.015, n=5) transcript levels was observed only following BPF treatment at the LOAEL dose. Despite expecting a decrease, both anti-apoptotic transcripts, at 12 and 48 hrs, showed increased levels following exposure to BPA and/or BPF at the LOAEL dose. This suggests an early first line of defense or shock response. Future protein analysis will help in further understanding these unexpected findings. To quantify apoptosis levels, flow cytometry together with Annexin V/PI staining was used. Results indicate that after 48 hrs of treatment, BPA at the LOAEL dose significantly increases late apoptosis (P=0.004, n=5) and necrosis (P=0.0014, n=5), while showing a significant decrease in the number of healthy cells (P=0.0007, n=5). Preliminary data also show a similar pattern after BPA treatment for 12 hrs. This research aims to further our knowledge of the mechanisms by which bisphenols affect oocyte competency and suggests that BPF might have equivalent potency and effects to BPA. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.