Cordycepin

Chemicals and reagents used in this study were of analytical grade or higher. Ammonia solution, glacial acetic acid, and HPLC grade acetonitrile were purchased from Merck, Darmstadt, Germany. Sodium dihydrogen orthophosphate and sodium hydroxide were obtained from Ajax Finechem Pty Ltd., Auckland, New Zealand. Cyclodextrins, cordycepin, 2′-deoxyguanosine, uridine, and 2′-deoxyadenosine were commercially available from Wako Pure Chemical Industry, Ltd., Osaka Japan. Sodium pyrophosphate decahydrate, adenosine 5′-triphosphate disodium salt (ATP), adenosine 3′-monophosphate (AMP), adenosine 5′-diphosphate disodium (ADP), adenosine, and cucurbit[n]urils were obtained from Sigma-Aldrich Co. St. Louis, MO, USA. Cytidine, 2′-deoxyuridine, inosine, thymidine, and 2′-deoxyinosine were purchased from FUJIFILM Wako Pure Chemical Corporation, Japan. Guanosine was obtained from Alfa Aesar, Thermo Fisher Scientific, Heysham, UK. Acridine orange (AO) and berberine (BE) was purchased from Invitrogen Company, Eugene, Oregon, OR USA. Deionized water was prepared using a Millipore Milli Q-Plus system (Millipore, Bedford, MA, USA). Deuterium oxide (D₂O) was purchased from Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA.

Primary rat-testicular cells (5 × 10⁴ cells/well) were cultured on collagen type IA-coated dishes in DMEM/F12 medium (Fujifilm Wako Pure Chemical) supplemented with 15% horse serum, 2.5% fetal bovine serum (FBS), antibiotics, and antimycotics at 37 °C under 5 % CO₂ in humidified air. The human prostate cell lines LNCaP and PC3 (Japanese Collection of Research Bioresouces Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan) were cultured in RPMI 1640 medium (Nakalai Tesque, Kyoto, Japan) supplemented with 10% FBS, antibiotics, and antimycotics at 37 °C under 5% CO₂ in humidified air. The human endometrial glandular epithelial cell line EM1 was also cultured in DMEM/F12 (Fujifilm Wako Pure Chemical) supplemented with 10% FBS, antibiotics, and antimycotics.
Rat-testicular cells were pretreated with CM (100 µg/mL) or cordycepin (0.5 mM, Fujifilm Wako Pure Chemical) for 1 h, and then treated with ovine LH (NIDDK-oLH-26; 10 or 100 ng/mL, provided by Dr. A. F. Parlow, National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA, USA) or dibutyryl-cyclic AMP (Db; 0.1 or 0.5 mM, Tokyo Chemical Industry Co., Tokyo, Japan) for 1.5, 4, or 24 h to test the effects on steroidogenesis.