FOLIC ACID SUPPLEMENTATION INDUCES GENE EXPRESSION CHANGES IN BOVINE OVIDUCT EPITHELIAL CELLS CULTURED IN VITRO

MANSILLA, MARIANO J.; GARCÍA, E. VANESA; BARRERA, ANTONIO DANIEL

Salta

Congreso; LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Folate is a well-known epi-nutrient that influences genomic methylation marks, which in turn, are crucial to orchestrate a tightly regulated pattern of gene expression through epigenetic mechanisms. In a previous study, we determined the presence and intra-oviductal concentration of folate, as well as, the existence of a fine-tuned regulation of the gene expression of its receptors and transporters in bovine oviduct epithelial cells (BOECs). Considering this, we hypothesized that folate could have a functional impact on BOECs. The present study aimed to assess the effect of different concentrations of folic acid (FA, synthetic form of folate) on cell health and gene expression levels in BOECs using a cell suspension culture system. BOEC explants from ampulla and isthmus regions were separately cultured in medium TCM-199 with FA in low (20 nM, usual FA content in culture medium, control group), physiological (1 uM, intra-oviductal concentration), elevated (10 uM) or supra-physiological (100 uM) concentration at 38.5 °C, 5% CO2 and 100% humidity during 24 h. Measurement of cytotoxicity was performed by evaluating LDH activity in the culture medium. Moreover, treated BOECs were processed for gene expression analysis using RT-qPCR. The genes examined were linked to important cellular processes including folate transport (FOLR1, FOLR2, FOLR3, SLC19A1, SLC46A1), DNA methylation (DNMT1, DNMT3A, DNMT3B), cell-cell interaction (CDH1), antioxidant activity (SOD2) and signaling pathways (TGFB1, MTOR). Cytotoxicity analysis showed a low LDH activity into the culture media of the experimental groups, indicating that FA did not affect epithelial cell integrity in the concentrations tested. Additionally, supplementation of culture medium with oviductal-like concentration of FA (1 uM) induced only the increase of mRNA levels for FOLR1 and FOLR3 in ampulla and isthmus explants, respectively, compared to the control group. In contrast, BOEC explants cultured in the presence of an elevated FA concentration (10 uM) showed increased mRNA expression levels of genes associated with folate transport (FOLR1, FOLR2, FOLR3, SLC19A1), DNA methylation (DNMT1, DNMT3A) and antioxidant activity (SOD2). Finally, it is worth noting that at supra-physiological concentration of FA (100 uM), transcriptional response in BOEC explants resulted in decreased mRNA levels for most analyzed genes with respect to control group in a region-dependent manner. These data allow us to suggest that fluctuations in extracellular folate levels can promote changes at molecular level in BOECs, providing new insights about the impact of maternal folate in the oviductal context.