Pregnenolone

Pregnenolone, progesterone, 5α-progesterone, 3α-OH-5α-progesterone, 3β-OH-5α-progesterone, 5β-progesterone, 3α-OH-5β-progesterone, and 3β-OH-5β-progesterone were purchased from Steraloids Inc. (Newport, RI, USA). R1881 was purchased from Meilunbio Company (Dalian, China). DHT, BCA and K7174 were purchased from MCE (Shanghai Haoyuan Chemexpress, China). Doxycycline hyclate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tritium labelled androgens (R1881, Pregnenolone, progesterone, dehydroepiandrosterone, or androstenedione) were purchased from PerkinElmer (Waltham, MA, USA).

2-3 mg biopsy samples were minced and cultured with DMEM (Invitrogen, Waltham, MA), 10% FBS (ExCell Bio, China), and Penicillin-Streptomycin (100 x; Invitrogen, Waltham, MA, USA) in a 12-well plate at 37°C as previously described.³⁵ (link) Biopsy samples were treated with [³H] labelled pregnenolone (100, 000–200,000 cpm; final concentration was 48 nM) (PerkinElmer, Waltham, MA, USA). 250 μl medium was collected at 84 h for HPLC analysis. Then, samples were treated with β-glucuronidase (Novoprotein Scientific Inc., Shanghai, China) at 37°C for 2 h. Steroids were extracted with a mixture of ethyl acetate and isooctane (1:1), concentrated with a vacuum drier (Martin Christ Gefriertrocknungsanlagen, Osterode, Germany), and resuspended with a mixture of methanol and water (1:1). An Acquity Arc System (Waters, Milford, MA, USA) and a β-RAM model 5 in-line radioactivity detector (LabLogic Systems) were used to analyze metabolites in samples. A mixture of [³H] labelled androgens (AD, DHEA, Prog, Preg PerkinElmer, Waltham, MA, USA) was used as the standard to distinguish metabolites. The percentages of metabolites were calculated based on the area under curve (AUC) for each metabolite. For example, Preg % = (AUC of Preg)/ (AUC of Preg + AUC of all Preg metabolites) x 100 %.

Steroid metabolism and HPLC were performed as described previously.³⁵ (link) Briefly, cells or organoids were treated with [³H]-labeled steroids (pregnenolone, DHEA, AD; 100,000-200,000 cpm per well; PerkinElmer, Waltham, MA). Aliquots of the medium were treated with β-glucuronidase (Novoprotein Scientific Inc., Shanghai, China) and extracted using a mixture of ethyl acetate and isooctane (1:1). Steroids were analyzed using an Acquity Arc System (Waters, Milford, MA, USA) and a β-RAM model 3 in-line radioactivity detector (LABLOGIC, USA). The percentages of metabolites were calculated based on the area under curve (AUC) for each metabolite. For example, pregnenolone% = (AUC of pregnenolone) / (AUC of pregnenolone + AUC of all metabolites) x 100 %.