3β hsd

Serial sections of musk gland were incubated with primary polyclonal antibody (200 μg/ml, 1:200 dilution) against AR (Abcam) for 12 h at 4 °C. Serial sections of testis were incubated with primary polyclonal antibody (200 μg/ml, 1:200 dilution) against StAR (Santa Cruz Biotechnology), P450scc (Abcam) or 3β-HSD (Abcam) for 12 h at 4 °C. The sections were then incubated with a second antibody, goat anti-rabbit IgG conjugated to biotin and to peroxidase with avidin, using a rabbit ExtrAvidin staining kit (ZSGB-BIO), followed by visualizing with 0.5 mg 3,3-diaminobenzidine (Solarbio) solution in 1 ml of 0.05 M Tris–HCl buffer, pH 7.6, plus 0.4 μl H₂O₂.

Melatonin was obtained from Selleckchem (USA). D-Glucose was obtained from Sigma-Aldrich (St. Louis, MO, USA). NAC (N-acetyl-L-cysteine) was purchased from Beyotime Biotechnology (Shanghai, China). The primary antibodies against STAR, P450, AMPK, P-AMPK, COXIV, Cytc, NRF1, Beclin-1, ATG7, ATG12, ATG5 and LC3 were obtained from Cell Signaling Technology (Beverly, MA, USA); 3β-HSD, SIRT1, mtTFA, ATPB, PGC1-α and acetyl-Lysine were purchased from Abcam Biotechnology (Cambridge, MA, USA); BNIP3L, SOD2 and Tubulin 1-α were obtained from Beyotime Biotechnology (Shanghai, China); and LXR, GPX4, GPX5 and Actin were purchased from Proteintech Group (Wuhan, China). The goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Zhongshan Gold Bridge Biotechnology Co. Ltd. (Beijing, China). Alexa Fluor 488 donkey anti-mouse Immunoglobulin G (IgG) (H + L), MitoTracker™ Green FM and MitoSOX™ Red Mitochondrial Superoxide Indicator were obtained from Invitrogen (Carlsbad, CA, USA). The goat anti-mouse IgG/Alexa Fluor/594 were purchased from Zhongshan Gold Bridge Biotechnology Co. Ltd. (Beijing, China). Membrane and Cytosol Protein Extraction Kit, Cell Mitochondria Isolation Kit, Reactive Oxygen Species Assay Kit and Mitochondrial membrane potential assay kit with JC-1 were obtained from Beyotime Biotechnology (Shanghai, China).

Insulin-like 3 (INSL3) is an important hormone secreted by fetal Leydig cells to induce the testis descent [18 (link)]. 3β-HSD is the important androgen-biosynthesizing enzyme in the fetal Leydig cells [19 (link)]. Immunohistochemical staining of INSL3 or 3β-HSD was performed as was stated above. INSL3 or 3β-HSD staining was performed using rabbit monoclonal antibodies against INSL3 (AB6790, Abcam) and 3β-HSD (Abcam), respectively. Leydig cell cytoplasmic density and background area density were measured using image analysis software (Image-Pro Plus; Media Cybernetics) according to the manufacturer’s instruction. More than 50 fetal Leydig cells were evaluated in each testis and the density each samples was averaged.