Butylparaben

Mixed cultures of Sertoli and spermatogenic cells are prepared from three-week-old SD rats. Single cells are obtained by gravity sedimentation and use of 50% trypsin-EDTA. Cells are finally suspended at a concentration of 1 × 10⁸cell/ml and maintained for 24 h in Dulbecco’s modified Eagle medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovineserum. Thereafter, cells are maintained in serum-freemedium, which is changed every 24 h. At 72 h, spermatogenic cells are removed by treatment with hypotonic Tris buffer (20 mMTris–HCl, pH 7.4) for 5 min. The Sertoli cell-enriched cultures obtained are then maintained in serum-free medium for 24 h before being reseeded onto chamber slides. The samples are cultured for a further 5 days. Subsequently, they are exposed to 0, 1, 100 and 1000 nmol/ml butylparaben for 6 and 24 h. Some slides are stained with 0.5% toluidine blue for light microscopic observations, whereas the rest are used for vimentin immuno-histochemical analysis.