Stable l glutamine

Dulbecco’s modified Eagle medium (DMEM) was obtained from GIBCO (Thermo Fisher Scientific, Waltham, MA USA). Fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT, USA). Stable L-glutamine, 1% non-essential amino acids, penicillin/streptomycin, and PBS were from Euroclone (Milan, Italy). Sitagliptin and Gly-Pro-amido-4-methylcoumarin hydrobromide (Gly-Pro-AMC) were from Sigma-Aldrich (St. Louis, MO, USA). Polycarbonate filters, 12 mm in diameter, 0.4 m in pore diameter, were from Transwell Corning Inc. (Lowell, MA, USA). Peptides Lup1 (LTFPGSAED) and Soy1 (IAVPTGVA) were synthetized by the company PRIMM (Milan, Italy) at >95% purity. Human serum was freshly collected from a healthy female volunteer.

Dulbecco’s modified Eagle’s medium (DMEM) was bought from GIBCO (Thermo Fisher Scientific, Waltham, MA, USA). Fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT, USA). Stable l-glutamine, 1% non-essential amino acids, penicillin/streptomycin, and chemiluminescent reagent were from Euroclone (Milan, Italy). Polycarbonate filters, 12 mm diameter, 0.4 µm pore diameter were from Transwell Corning Inc. (Lowell, MA, USA). Phenol red, PBS, bovine serum albumin (BSA), RIPA buffer, and the antibody against β-actin were from Sigma-Aldrich (St. Louis, MO, USA). The antibody against HMGCoAR was bought from Abcam (Cambridge, UK), that against PCSK9 from GeneTex (Irvine, CA, USA), that against phospho-HMGCoAR (Ser872) from Bioss Antibodies (Woburn, MA, USA), and that against LDLR from Pierce (Rockford, IL, USA). The antibodies against SREBP-2, rabbit Ig-HRP, mouse Ig-HRP, phenylmethanesulfonyl fluoride (PMSF), Na-orthovanadate inhibitors were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); the inhibitor cocktail Complete Midi from Roche (Basel, Switzerland). Mini protean TGX pre-cast gel 7.5% and Mini nitrocellulose Transfer Packs were purchased from BioRad (Hercules, CA, USA). The human proprotein convertase 9 immunoassay (Quantikine ELISA) was bought from R & D System (Minneapolis, MN, USA).

Testes were isolated and decapsulated in 0.1 M Phosphate Buffer. The seminiferous tubules were gently placed onto a small cube made of 1,5% agarose and soaked in culture medium for more than 24 h to replace water. The amount of medium was adjusted in order to cover half to four fifth of the height of agarose cubes. Tubules were maintained in incubator at 34 °C, 5% and controlled humidity overnight in the following culture medium: RPMI (Euroclone), 10% Fetal Bovine Serum (FBS) (Euroclone), 2 mM Stable L-glutamine (Euroclone), antibiotic antimycotic solution (A5955, Sigma-Aldrich). Seminiferous tubules were picked up from agarose cubes (Sigma) and fixed in 2% paraformaldehyde dissolved in PBS pH 7.2–7.4 for 10 min. A single fixed tubule was laid down onto a slide, covered with a coverslip and a gentle pressure was applied in order to allow cells to come out from the seminiferous tubule. Slides were then frozen in liquid nitrogen for further analyses.