Testosterone propionate

Degarelix (as acetate), a third generation LHRH-Ant (Firmagon), was resuspended in sterile water for injection and administered s.c. to mice at a dose of 40 µg/g. Lupron (11.25-mg 3-mo depot), an LHRH-Ag, was prepared according to the manufacturer’s instructions and administrated intramuscularly to mice at a dose of 20 µg/g. Degarelix and Lupron were purchased from the MSKCC Pharmacy. Testosterone propionate (Sigma-Aldrich) was resuspended in peanut oil and injected daily s.c. (1 mg/mouse) in 100 µl. Surface antibodies against CD44 (IM7), EpCAM (G8.8), PDGFRa (APA5), PECAM-1 (390), CD45 (30-F11), and H-2Kk (AF3-12.1.3) were purchased from eBioscience; anti-Ly-51 (BP-1), CD34 (RAM34), CD62L (MEL-14), H-2Kb (AF6-88.5), IFN-γ (XMG1.2), IL-2 (JES6-5H4), c-Kit (2B8), CD3ε (145-2C11), CD25 (PC61), TER-119 (TER-110), and CD8α (53–6.7) were purchased from BD; anti-CD4 (RM4-5) and B220 (RA3-6B2) were purchased form Invitrogen; anti-CD44 (IM7), CD90.2 (30-H12), and IA/IE (M5/114.15.2) were purchased from BioLegend; and Ulex europaeus agglutinin 1 (UEA-1) was purchased from Vector Laboratories. For in vitro cultures of T cell differentiation (Fig. 2, A and B), cells were stained with antibodies to lineage (Lin)-specific markers: CD8, CD11c, NK1.1, CD3, CD4, B220, CD11b, and GR-1.

HaCaT and RAW264.7 cells were purchased from the American Type Cell Collection (ATCC, Manassas, VA, USA). Testosterone propionate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol extract of Chaenomeles sinensis was provided by Sunchang Institute of Health and Longevity (Sunchang, Korea). Dulbecco’s modified Eagle medium (DMEM), heat-inactivated FBS, PBS, and penicillin/streptomycin (Pen/Strep) were purchased from Welgene (Daegu, Korea). Polyclonal antibody against NF-E2-related factor 2 (NRF2) was purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal antibodies against 8-hydroxydeoxyguanosine (8-OHdG) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibody against 4-hydroxynonenal (4-HNE) was purchased from Abcam (Cambridge, MA, USA). 2,2’-diphenylpicrylhydrazyl (DPPH) was purchased from Cayman (Ann Arbor, MI, USA) and 2,2’-azinobis diammonium salt (ABTS) was purchased from Roche Diagnostics (Mannheim, Germany). Monoclonal antibodies against iNOS and COX-2 were purchased from BD Transduction Laboratories (Franklin Lakes, NJ, USA).

The 30 rats were divided into three groups for the following stage of treatment, which were as follows: i) Control group (n=10): Mice received a normal rodent diet and injection with olive oil at a volume equal to injections in the experimental groups; ii) PCOS-IR model group (n=10): 100 µg of testosterone propionate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was subcutaneously injected into animals, which was dissolved in 0.05 ml commercial corn oil. The rats were also fed a high-fat diet, consisting of 54.2% standard diet, 16.8% lard, 15% sucrose, 9% casein, 1% minerals, 1% vitamins and 3% malt dextrin. Following 8 consecutive weeks of treatment, the mice were sacrificed and the vaginal cytology was analyzed using the methylene blue staining method by LEICA DM1000 light microscope (Leica Microsystems GmbH, Wetzlar, Germany); iii) MitoQ₁₀ treatment group (n=10): Following establishment of the PCOS-IR animal model, 10 PCOS-IR rats were given clean drinking water which contained 500 µmol/l MitoQ₁₀ (Sigma-Aldrich; Merck KGaA) for 8 consecutive weeks. The solutions for MitoQ₁₀ were reformulated fresh every 3 days, and were stored at 4°C in a dark room.

Testosterone replacement was performed in ORX + T animals using testosterone propionate
(10 mg/kg, i.m.; Sigma, St Louis, MO, USA) that was administered every 5 days for 3 weeks.
Control and ORX animals were injected i.m. with vehicle (maize oil) instead. The
replacement was started 23 days after orchidectomy, when the atrophy of the
testosterone-dependent sexual accessory organs had already occurred (14 (link)).