Hepes buffered f12 dmem

Small seminiferous tubule segments were seeded, at a high density, in bicameral chambers and cultured in a serum-free culture medium as previously described (Geoffroy-Siraudin et al. 2010; Carette et al. 2015; Goldstein et al. 2016) . Incubations were carried out at 33°C in a water-saturated atmosphere of 95% air and 5% CO2. Each culture was carried out in four to seven replicates for 10 to 21 days, either in the absence or presence of the water sample to be tested. The complete culture medium was 15mM Hepes-buffered F12 / DMEM supplemented with 10µg / ml gentamycin, 10µU / ml nystatin, 10µg / ml insulin, 10µg / ml human transferrin, 10 -4 M vitamin C, 10µg / ml vitamin E, 10 -7 M testosterone, 3.3 10 -7 M retinoic acid, 3.3 10 -7 M retinol, 10 -3 M pyruvate (Sigma) and 1ng / ml FSH (NIH, Bethesda, MD).

For each culture, Sertoli cells were purified from testis of two 6-week-old chickens previously killed by electronarcosis as described previously (Guibert et al. 2011 (Guibert et al. , 2013)) . Sertoli cells were cultured in HEPES-buffered F12/ DMEM (Sigma) supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin (Sigma) and 5% FCS for 12 h at 37°C in a humidified atmosphere of 5% CO 2 . The medium was changed to 1% FCS every 48 h. Sertoli cells were seeded at a density of 5 × 10 5 cells/well and stimulated or not for 48 h with metformin (5 mM) followed by a short stimulation (15 min) with 10 ng/ mL ovine follicle-stimulating hormone (oFSH; Sigma) for immunoblotting or only with metformin to measure metabolite content (lactate, ATP). The metformin concentration was selected after kinetic and dose-response experiments performed on the ability to phosphorylate the AMPK, a molecular target of metformin, in chicken Sertoli cells. The 5-bromodeoxyuridine (BrdU) incorporation analysis was carried out on Sertoli cells seeded in chamber slides (2.5×10 4 cells/chamber). After stimulation, the media and cells were collected and stored at -80°C until analysis. At least three to eight different cultures were carried out as described in the figure legends.