Testosterone

Levels of testosterone (R&D System) from serum and levels of IL-1α, IL-23 and IL-1β (Invitrogen) from the testis were measured by ELISA, according to the manufacturer’s instructions. Blood was removed from the heart, settled for 30 min at room temperature and centrifuged at 15,000 × g for 10 min at RT. Serum supernatant was collected and stored at −20 °C until used. Total protein content was quantified using the BioRad DC Protein Assay kit.

Granulosa cells were collected from ovaries of immature (3-week-old) mice at 6 h following injections of eCG^{19 (link)}. Cells were seeded onto serum-recoated 24- or 96-well plate. Granulosa cells were treated with 100 ng/ml FSH (NIDDK, Torrance, CA)/DMEM/F12 medium, testosterone (10 ng/ml, Sigma)/ethanol, and/or RA (1 μM, Sigma)/dimethyl sulfoxide (DMSO) in the presence or absence of 1% (v/v) fetal bovine serum. Some granulosa cells were treated with 0.05, 0.5, or 5 μM aphidicolin (Sigma)/DMSO or 100 ng/ml GDF9 (mouse recombinant GDF9, R&D Systems, Inc., Minneapolis, MN) in the presence of FSH, testosterone, and 1% (v/v) fetal bovine serum.

Testosterone, estradiol, progesterone, corticosterone, IgG, IgM, TNF-α, and IL-6 levels in mice sera were measured at the age of 14 weeks. Testosterone (R&D Systems, USA), estradiol (Cayman Chemical, USA), progesterone (Cayman Chemical, USA), corticosterone (ALPCO, USA), IgG (ICL, USA), IgM (ICL, USA), TNF-α (Anogen, Canada), IL-6 (Anogen, Canada) in sera were detected, respectively, by using the immunoassay kits according to the manufacturer's protocol.

The KGN human granular carcinoma cell line was cultured in DMEM/F12 (Gibco, NY, United States) containing 10% charcoal-stripped fetal bovine serum (Thermo Fisher Scientific, MA, United States) and 1% antibiotics (mixture of penicillin, streptomycin, and neomycin) (Gibco) in an incubator at 37°C and 5% CO₂ (Thermo Fisher Scientific). The cells were passaged every 2–3 days.
For FSH stimulation, the cell culture medium was replaced with a fresh medium containing 10 ng/ml recombinant FSH (rFSH, MyBioSource Inc.) as previously described (Foldesi et al., 1998 (link)). To investigate the activity of the androgen receptor (AR) and melatonin receptor 1A (MTNR1A), 10 nmol/L testosterone (R&D Systems) or 10 nmol/L melatonin (Sigma-Aldrich, MO, United States) was supplemented in a fresh medium as the substrates. In addition, cAMP (Sigma-Aldrich) of different concentrations was added to the fresh medium to promote aromatase function.

The mouse sex hormone levels were measured at 5 months. The exact levels of testosterone (R&D Systems), luteinizing hormone (LH) (Shanghai Xinle Biotechnology), and follicle stimulating hormone (FSH) (Elabscience) in serum were detected using immunoassay kits according the manufacturer's protocols.