Epigenetic effects of Bisphenol A on granulosa cells of mouse follicles during in vitro culture: An experimental study

BACKGROUND: Bisphenol A (BPA), a synthetic endocrine-disrupting chemical, is a reproductive toxicant. Granulosa cells have significant roles in follicle development, and KIT ligand (KITL) and Anti-Müllerian hormone (AMH) are essential biomolecules produced by them during folliculogenesis. OBJECTIVE: Due to the widespread use of BPA and its potential epigenetic effects, this study examined the impact of BPA on promoter methylation of amh and kitl genes in mouse granulosa cells. MATERIALS AND METHODS: Preantral follicles were isolated from ovaries of immature mice and cultured for eight days. Then, follicles were treated with 50 and 100 μM of BPA, and 0.01% (v/v) ethanol for 24 and 72 hr. Growth and degeneration of follicles and antrum formation were analyzed. The granulosa cells were isolated mechanically, and their extracted DNA was treated with sodium bisulfite. The promoter regions of the amh and kitl were analyzed with PCR and sequencing. RESULTS: BPA did not change follicle survival and antrum formation significantly (p = 0.41). However, the culture in the presence of 100 μM BPA had an inhibitory effect on growth. Before BPA treatment, the CpG of the kitl and amh promoters were unmethylated and partially methylated, respectively. While the percent of 5mC in the amh promoter reduced at 100 μM of BPA, it did not alter the kitl promoter methylation. CONCLUSION: BPA at higher concentrations has an inhibitory effect on follicle growth. Moreover, it seems that the epigenetic impact of BPA restricts to the demethylation of CpG sites.