Dnasa

Tubule cells isolation was performed by mechanical disaggregation in the presence of 0.1 mg/ml DNasa (Sigma, St Louis, MO) using a 21G needle from different segments of the seminiferous tubules isolation previously in PBS. Then, the solutions were filtered by through a mesh with a pore of 200 μm and then a pore of 70 μm of diameter. The filtered solutions were centrifuged for 3 min at 800 × g. Then the isolated cells were incubated in DMEM-F12 medium containing 3% BSA, for 30 min at 4°C. Primary antibody against ADAM17, or against ADAM10 coupled to phycoerytrin, was added diluted in blocking solution (1:250) and left to incubate overnight. The next day cells were washed three times with PBS and in the case of those incubated with the antibody against anti-ADAM17 they were dissolved in blocking solution with the corresponding secondary antibody conjugated with FITC (diluted 1:250) and incubated for one hour at 4°C. Then, cells were washed three times with PBS and the final pellet dissolved in PBS. The samples were analyzed by a flow cytometer (FAScanto) and 10,000 gated events were acquired in each sample. As controls, one autofluorescence sample, one sample with only primary antibody and one sample with only secondary antibody were analyzed. All date were analyzed with software FCS express V4.0 (De Novo Software, Los Angeles).

Sertoli cells were obtained from 17 day old male Sprague–Dawley rats’ testes killed by cervical dislocation. Testes were removed, decapsulated and placed in PBS containing 0.1 mg/ml collagenase (Sigma, St Louis, MO). Then, the tubules were washed three times in PBS. Tubule cells isolation was performed by mechanical disaggregation in the presence of 0.1 mg/ml DNasa (Sigma, St Louis, MO) using a 21G needle from different segments of the seminiferous tubules isolation previously in PBS. Then, the solutions were filtered by through a mesh with a pore of 200 μm and then a pore of 70 μm of diameter. Cells were resuspended in a solution containing PBS and distilled water (1:9) to produce a hypotonic shock, which destroys germ cells but not Sertoli cells. Then, the cells were filtered again, and the filtered solutions were centrifuged for 3 min at 800 × g and resuspended in DMEM-F12 medium without serum and containing only 10% antibiotic and antimycotic (Gibco®, Invitrogen, Carlsbad, CA). 1 × 10⁶ cells were cultured in DMEM-F12 medium supplemented with 10% antibiotic and antimycotic, pH 7.2 at 37°C and 5% CO₂. After 24 h the cells were washed and cultivated with fresh medium, in the same conditions as above for 4 days, after which, every germ cell is phagocyted by Sertoli cells. The culture obtained has at least 90% purity.