Phosphate buffered saline (pbs)

Briefly, tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval by boiling in citrate buffer (pH = 6, ZSGB-Bio, Beijing, China). The sections were then incubated overnight with anti-protein X antibody (Proteintech, Wuhan, China) at 4 °C. Following that, the sections were washed 3 times with PBS (ZSGB-Bio, Beijing, China) and incubated with secondary antibody (ZSGB-Bio, Beijing, China) for 1 h at ambient temperature. After three washes with PBS (ZSGB-Bio, Beijing, China), the sections were incubated with streptavidin-biotin-peroxidase complex (ZSGB-Bio, Beijing, China) for 1 h. Finally, the sections were visualized with domain antibodies and counterstained with hematoxylin (sigma inc., USA). Negative controls were prepared by omitting the primary antibody. Immunohistochemical results were scored by two independent pathologists using a semi-quantitative system based on staining intensity and percentage of positively stained cells.

The Leydig cell coverslips and testicular sections (4 μm) were fixed with 4% (v/v) paraformaldehyde for 30 min at 37 °C The slides were blocked using Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) before being incubated for 16–18 h at 4 °C with the primary antibodies anti-MT1/-MT2 (1:500 dilution) and anti-3β-HSD (1:500 dilution). The slides were incubated with secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 for 60 min at room temperature after being washed with PBS (1:400 dilution; ZSGB-BIO, Beijing, China). Finally, the nuclei were stained with DAPI (Thermo, 1:5000 dilution) for 5 min at room temperature. Then, the slides washed twice with PBS. The fluorescence images were captured using digital slide-scanning equipment (model: VS200, SLIDEVIEW, Olympus, Tokyo, Japan).

USCs were obtained from five healthy male adult donors (20–35 years old) using methods described previously [4 (link), 5 ]. Two hundred milliliters of fresh urine was collected from each donor. The samples were treated with 1% penicillin and streptomycin (Gibco, USA) and centrifuged for 10 min at 1500 rpm. The supernatant was discarded and the sediment was washed twice in phosphate-buffered saline (PBS, ZSGB-BIO, China). The cells from the pellet were seeded in 6-well plates and cultured in cell culture medium comprised of 50% Keratinocyte Serum-Free Medium (KSFM, Gibco, USA), 33.75% Dulbecco’s modified Eagle medium (DMEM, Gibco, USA), 11.25% Ham’s F-12 Nutrient Mixture (Gibco, USA), and 5% fetal bovine serum (FBS, Gibco, USA) supplemented by 5 ng/mL epidermal growth factor (EGF, Gibco, USA), 50 ng/mL bovine pituitary extract (Scienceu, USA), 0.4 μg/mL hydrocortisone (Sigma, USA), 5 μg/mL transferrin (Sigma, USA), 5 ng/mL bovine insulin (Sigma, USA), 0.18 mM adenine (Sigma, USA), 2 nM 3,3,5-triiodo-l-thyromine (Sigma, USA), 100 units/mL penicillin (Gibco, USA), and 100 μg/mL streptomycin (Gibco, USA). The medium was first changed 7 days after cell seeding and the non-adherent cells were removed. Culture medium was refreshed twice per week. When the cells reached 80% confluence, they were passaged at a ratio of 1:3. Cells at passage 3 were used in the following experiments.