Ab285350

GDF9 was measured in follicular fluid collected at the first oocyte retrieval from individual P1 and in follicular fluid from other six control women using an ELISA kit (CSB-E12925h, CUSABIO, China). All follicular fluid collected at the second oocyte retrieval from individual P1 had been used for western blot, so there was no remaining follicular fluid for ELISA. Wild-type, Gdf9^S415T/S415T, and Gdf9^Q308X/S415T female mice at the age of 10-12 weeks were executed at the stage of proestrus and their serum were collected. Serum levels of estradiol were measured using an ELISA kit (KGE014, R&D, USA), according to the manufacturer’s instructions, with sensitivity of 12.1 pg/mL. Serum levels of testosterone were measured using an ELISA kit (ab285350, abcam, UK), according to the manufacturer’s instructions, with sensitivity of 0.1 ng/mL. Three mice were included in each group.

1E6 RPM-Ascl1^WT or RPM-Ascl1^KO organoids were injected into the flanks (one tumor per mouse) of immunocompetent mice as described above. Mice were randomized into vehicle (5% D-Mannitol, Sigma M4125–500G) or Degarelix (15 mg/kg, Sigma SML2856–25MG) groups once tumors measured ≥ 150 mm³. Treatment was performed by subcutaneous delivery once every 14-days in a total of 100 uL. Tumor volumes were obtained and calculated as above. At sacrifice, blood was collected from mice and coagulated for 1hr on ice, followed by centrifugation at 1000 g for 30 min to isolate serum. Successful serum testosterone depletion was assessed by ELISA according to manufacturer recommendations (Abcam, ab285350).

The serum levels of testosterone (ab285350, Abcam, UK), estradiol (E2, ab285285, Abcam), luteinizing hormone (LH, CEA441Ra, USCN Life Science and Technology Company, Wuhan, China), follicle-stimulating hormone (FSH, CEA830Ra, USCN), and fasting insulin (FINS, ab273188, Abcam) were detected using commercial ELISA kits following the manufacturer’s protocols. Fasting plasma glucose (FPG) level was assessed with a blood glucose meter (Omron, USA). Homeostasis model assessment of insulin resistance (HOMA-IR) = FINS × FPG/22.5.