94 A POLARIZED CELL-CULTURE SYSTEM TO STUDY THE EFFECTS OF ELEVATED SERUM NONESTERIFIED FATTY ACID CONCENTRATIONS ON THE BOVINE OVIDUCTAL MICROENVIRONMENT IN VITRO

Abstract

The detrimental consequences of elevated serum nonesterified fatty acid concentrations (NEFA) typical in negative energy balance, obesity, or type II diabetes have previously been demonstrated in ovarian follicles. However, regardless its key role in reproduction, the consequences of elevated NEFA on oviductal physiology are unclear. Therefore, the aim of this study was to 1) determine whether serum NEFAs can be found in the oviductal microenvironment using a polarized oviductal epithelial cell culture and more specifically to 2) study the effect of the BSA gradient and the direction of the NEFA gradient. Bovine oviductal epithelial cells from slaughterhouse oviducts in Day 3 to 5 of the luteal phase were mechanically isolated and cultured for 7 days in a flask. Cells were transferred in a polarized oviductal epithelial cell culture-system (Snapwell^® polyester membrane, pore size 0.4 µm, 4 × 10⁶ pores cm^–2) with an apical (A) and basal (B) medium supporting cell growth until the transepithelial electric resistance reached 700 Ωcm² (Day 6). Then, cells were exposed to a transmembranary BSA (0.75 or 3% BSA) or NEFA gradient (0 or 360 µM NEFA: 115 µM oleic acid, 140 µM stearic acid, 105 µM palmitic acid) for 48 h in 4 experimental setups: 1) A and B containing 0.75% BSA and B supplemented with NEFA, 2) same as in 1 but NEFAs were added in A, 3) A containing 0.75% BSA and B supplemented with 3% BSA and NEFA, 4) same as in 3 but with 3% BSA + NEFA in A and B contained 0.75% BSA. Samples were analyzed for total NEFA and specific fatty-acid concentrations by photometric and gas chromatographic assays. In total, 72 wells in 4 replicates were cultured and analyzed. Data were processed by paired sample t-tests and student t-tests. Exposure to NEFA did not alter the transepithelial electric resistance. In Exp. 1, the total NEFA concentration in A increased with 0.013 µM (21.11%) over 48 h of NEFA exposure in the basal compartment explained by a significant rise of stearic (8.52 µM, 20.68%) and oleic acid (12.86 µM, 45.17%) accompanied with only a 19.45% decrease of total NEFA in B (P < 0.05). When the transport direction was reversed in Exp. 2, the total NEFA concentration decreased 53.4% in A, with no obvious NEFA rise in B. Implementing a BSA gradient (Exp. 3 and 4) was associated with a slight decrease (13 and 15.7%, respectively; P < 0.05) of NEFA concentration in the supplemented compartment, but no obvious NEFA increase in the opposite compartment could be detected. Overall, these results suggest that NEFA transport in this culture system is tightly regulated and can only be influenced by the transport direction of the NEFA. Further research should focus on transport and metabolisation of the fatty acids added.