PSIII-3 Late-Breaking: Diisobutyl Phthalate (DIBP) at Low Doses Alters Gene Expression of Epigenetic Regulators in Bovine Granulosa Cells

Abstract

Abstract Phthalates are ubiquitous environmental contaminants and impact reproduction and epigenetic mechanisms. The use of diisobutyl phthalate (DIBP) has increased in the last decade. However, studies about DIBP's effects on female reproductive physiology are limited. Methylation of DNA and RNA are epigenetic processes that regulate gene expression, cell biology, and reproductive functions. How DIBP exposure regulates these epigenetic mechanisms in ovarian follicular cells remains to be unveiled. We hypothesize that DIBP at low doses alters the expression of RNA and DNA methylation regulators in bovine granulosa cells (GC). Bovine GC was isolated from small antral follicles (1-5 mm on surface diameter) from ovaries obtained at a slaughterhouse. Cells were cultured in vitro until reaching an average 70% confluence and treated with DIBP at levels detected in human blood (0 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL) combined with hormones that regulate ovarian folliculogenesis: FSH or FSH+IGF1. Cells were cultured for 24h for total RNA isolation (n = 5 pools) or for 48h for cell counting (n=5 pools). Relative mRNA abundance of RNA N6 methyladenine (m6A) methylation machinery (m6A writers: METTL3, METTL14; m6A erasers: ALKBH5, FTO), RNA 5-methylcytosine (m5C) methylation regulators (NSUN2, TRDMT1), and DNA methylation players (DNA methyltransferases: DNMT1, DNMT3A, DNMT3B; DNA demethylases: TET1, TET2, TET3) was determined via qPCR using 18S as housekeeping gene and expressed as 2-ΔΔCt. Cell proliferation was analyzed by cell counting using a MUSE cell analyzer. Data were analyzed via one-way ANOVA with GLM procedures of SAS for Windows. Normality was tested using Shapiro-Wilk test procedures and data considered not normal were transformed to log2 (Y + 0.6). Data sets that showed non-gaussian distribution after log-transformation was analyzed via the Kruskal-Wallis test using GraphPad Prism 9.2.0. Cell proliferation was not affected by DIBP treatments in combination with FSH or with FSH+IGF1. In GC treated with FSH in combination with DIBP, there was no significant effect on relative mRNA abundance of RNA or DNA methylation players. In combination with FSH+IGF1, GC treated with DIBP had lower (P < 0.05) METTL14 relative mRNA abundance at 1 ng/mL and 10 ng/mL than at 0 ng/mL and 100 ng/mL. TRDMT1 relative mRNA abundance was lower (P < 0.05) in the DIBP group at 10 ng/mL in comparison to groups 0 ng/mL and 1 ng/mL, but no 100 ng/mL. In addition, there was no significant effect on the relative mRNA abundance of DNA methylation regulators. In summary, GC treated with FSH+IGF1 and DIBP at low doses alters the gene expression of RNA methylation players in m6A and m5C RNA modifications in a non-monotonic dose-response, but does not alter mRNA abundance of DNA methylation regulators.