L2376

Six different lipid fractions were studied, including FFA (linolenic; Sigma‐Aldrich, L2376, and linoleic acid; Sigma‐Aldrich, 62240), TG (glyceryl trilinolenate ≥97% and glyceryl trilinoleate ≥98%; Sigma‐Aldrich, T7140), CE (cholesteryl linoleate ≥98%; Sigma‐Aldrich, C0289), and PL (Lecithin Granulat, biosym.dk, 16637). The PL contained both LA and LNA.
Due to a very low FA content (9.9 g/kg), straw was chosen as carrier for the lipid fractions used for the BH experiment. The lipid fractions were solubilized in 100 ml diethyl ether and mixed with finely ground wheat straw (particle size: <1 mm). The mixture was prepared for all samples at once, and each incubated sample contained either 5 mg FFA, 10 mg TG or CE, or 15 mg of PL. After mixing, these mixtures were placed in a fume cupboard, and the added ether was allowed to evaporate, while the mixture was frequently stirred to ensure homogeneous samples. Mixtures containing lipid fractions with LNA (FFA and TG) were placed under a stream of N₂ to prevent oxidation of the LNA.

All cell culture-related media were purchased from GE Healthcare Life Sciences (Thermo Fisher Scientific, Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Corning^® (Corning, NY, USA). A penicillin–streptomycin solution was purchased from TOKU-E (Bellingham, WA, USA). The n-3 PUFAs we used were α-linolenic acid (ALA, cis,cis,cis-9,12,15-octadecatrienoic acid, L2376, Sigma-Aldrich, St. Louis, MO, USA), EPA (cis-5,8,11,14,17-eicosapentaenoic acid, E2011, Sigma-Aldrich), and DHA (cis-4,7,10,13,16,19-docosahexaenoic acid, D2534, Sigma-Aldrich), and the n-6 PUFAs we used were linoleic acid (LA, 9-cis,12-cis-linoleic acid, L1376, Sigma-Aldrich) and arachidonic acid (AA, cis,cis,cis,cis-5,8,11,14-eicosatetraenoic acid, A3611, Sigma-Aldrich). Polyethyleneimine (PEI) and polybrene were purchased from Millipore (Darmstadt, Germany). A protease inhibitor cocktail was purchased from Roche (Basel, Switzerland).

Primary GC culture and bovine serum albumin (BSA)-FA conjugate preparations were done as described earlier (21 (link)). For testing the effects of actual follicular concentration of ALA (Sigma-Aldrich,L2376) and cis-9, trans-11CLA (Sigma-Aldrich,16413) the media were replaced every 48 h with supplemented α-MEM media containing different concentrations of ALA (20, 40, and 80 μM) or cis-9, trans-11 CLA (15, 30, and 60 μM) as BSA conjugates or non-conjugated BSA as vehicle control (Sigma-Aldrich A7030). All control wells received the same volume of BSA as the test groups with highest fatty acid concentration. The conditioned media collected on the 8th day of culture were stored at −20°C for steroid hormone estimation, while the remaining cells were lysed for RNA isolation.