RF22 | PMON08 The effects of Bisphenol A on miR-21 expression and miR-21 mediated apoptosis in bovine granulosa cells.

Abstract

Abstract BPA, a widespread endocrine-disrupting compound (EDC), plays a significant role in the decline of female fertility. Granulosa cells are susceptible to EDCs and are important for oocyte competency. This research focuses on epigenetic mechanisms in granulosa cells that are disrupted by BPA, with a specific focus on microRNA-21(miR-21), which is crucial for oocyte maturation and primarily recognized as an antiapoptotic regulator. miR-21 targets the apoptotic genes PDCD4 and PTEN and limits their expression, resulting in an antiapoptotic effect. Previous studies in our laboratory demonstrate that BPA induces apoptosis and simultaneously increases miR-21 expression, leading to our hypothesis that BPA induces apoptosis in a miR-21 independent manner in bovine granulosa cells.To test this hypothesis, in vitro cultured granulosa cells were transfected with miR-21 inhibitor or scramble with or without BPA, followed by knockdown validation and apoptotic measurements. Using flow cytometry, transfection efficiencies were measured in time- and dose-dependent experiments, showing optimal knock-down greater than 95% at 0.5µM for 12 hrs. miR-21 expression measured by qPCR showed a significant decrease within inhibited groups (p=0.0008, n=3). Although PDCD4 and PTEN mRNA expression remained unaffected, PDCD4 protein expression is inhibited cells was significantly increased (p=0.0233, n=4), suggesting a miR-21 role in PDCD4 translation. PTEN protein was unaffected by miR-21 inhibition.In further experiments, transfected cells were exposed to BPA, and apoptosis was examined. Using Annexin V/PI staining, a significant decrease in healthy cells and an increase in early apoptotic cells within inhibited untreated groups was shown (p=0.0395 and 0.0193, n=3). This finding is likely due to antiapoptotic gene inhibition. BPA exposure resulted in significantly decreased healthy cells, increased late apoptotic cells (p=0.0047 and 0.0145, n=3), with no changes in necrotic cells. Furthermore, no differences were found between inhibited and non-inhibited groups, suggesting that BPA-induced apoptosis is miR-21 independent.Additionally, three proapoptotic (BAX, BAD, Casp-9) and two antiapoptotic (Bcl-2 and HSP70) genes were quantified by qPCR. Unexpectedly, BPA increased the expression of all genes at the mRNA level (p= 0.0021, 0.0283, 0.0143, 0.025, 0.0145, n=4). Quantification of protein levels will reveal whether this change is functional. Moreover, no differences were found between inhibited and non-inhibited groups, suggesting these changes are likely miR-21 independent. Lastly, initiator caspase 9 activity was measured after 6, 12, 24, 48, 72 hrs of BPA exposure, as caspases are primary effectors of apoptosis. 6 and 12hrs timepoints showed no changes; however, at 24 and 48 hrs significantly increased caspase activity was detected (p=0.0087 and 0.0014, n=4). At 72hrs, BPA exposure was lethal, resulting in insufficient cell numbers for measurements.Overall, this research focuses on effects of EDCs on apoptotic regulation within granulosa cells and aims to uncover epigenetic consequences of BPA's mechanism of action, ultimately affecting oocyte competency and fertility. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.