Histological Analysis of Testicular Tissue

For histology testes were fixed for 6 hours in Bouin’s solution and then embedded in Technovit 7100 resin. Sections (2.5μm) were cut and stained with H&E. Immunohistochemistry was carried out as described [11 (link),12 (link)]; testes were embedded in paraffin and sections (5μm) prepared and stained using anti-CYP11A1 (rabbit polyclonal, donated by Dr AH Payne, Stanford University) diluted 1/500 or anti-CYP17A1 (sc-46081 Santa Cruz, lot number F2014, Heidelberg, Germany) diluted 1/400 and using the appropriate secondary antibodies conjugated to biotin, diluted at 1/500 in the blocking serum. This step was followed by incubation with horseradish peroxidase labelled avidin-biotin complex (VectorLabs, Peterborough, UK). The revelation was undertaken with diaminobenzidine DAB (Immpact DAB;VectorLabs, Peterborough, UK). Slides were counterstained with haematoxylin, dehydrated and mounted with Pertex mounting medium (Cell Path, Hemel Hempstead, UK). Cell numbers were measured by the optical dissector as previously shown [8 (link), 9 (link)]. The cells were recognised by their position, round nucleus and relatively abundant cytoplasm. The total testis volume was estimated using the Cavalieri principle.