Germ cell identification

Using specific markers for pre-meiotic and meiotic cells, we evaluated the germ cell progression in testicular tissue from prepubertal cats. Testicular cells were isolated by slicing with a scapel blade in Modified Ham`s F-10 Basal Medium–HEPES (Irvine Scientific) supplemented with 2mM L-glutamine, 1mM pyruvate, 100 IU/ml penicillin, 100 μg/ml streptomycin and 5% FBS. Cells suspensions were centrifuged at 300 × g for 8 minutes and resuspended in fresh Ham`s F10 medium (1:1). After, 20μl of suspension was smeared on a glass slide. The cells were fixed with 4% paraformaldehyde for 1 h at room temperature and permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 3 minutes. Cells were saturated in 5% BSA in PBS for 1 h at room temperature and incubated with the pre-meiotic marker anti-OCT4 (1:200, Abcam #ab137427) or the meiotic marker anti-BOULE (1:100, Abcam #ab28745) overnight at 4°C in a humidified chamber. Slides were washed in PBS twice and PBS-T once for 5 minutes each and then incubated with secondary antibodies (donkey anti-goat IgG-FITC, Santa Cruz Biotechnology #2024; 1:100 goat anti-rabbit IgG-TR, Santa Cruz Biotechnology #2780) for 1 h at 37°C in darkened container. The nucleus was stained with Hoechst 33342 (1:100, Sigma-Aldrich) in a humidified chamber for 10 minutes at room temperature and then, the slides were mounted with Vectashield medium (Vector laboratories) [36].

We evaluated 500 cells in duplicate per experimental group and calculated the proportion of cells with positive staining for both antibodies. Images were captured using an Olympus BX41 epifluorescence microscope (Olympus Corporation) with SPOT advanced software 5.0 (Diagnostic Instruments).