Estradiol 3

Trunk blood was collected and allowed to clot at room temperature for 2 h in anticoagulant-free tubes, and then centrifuged at 3,500 g for15 min at 4°C. The clear supernatant was kept at −80°C until analysis. Serum samples were assessed for the determination of sex hormones levels (data presented in Supplementary Table S1): total testosterone (T), dihydrotestosterone (DHT), and estradiol (E2) levels. T and E2 levels were determined by Elecsys 2010 analyzer using the method of the electrochemiluminescence immunoassay (ECLIA). Standard commercial kits (Elecsys Testosterone II and Estradiol III, Roche Diagnostics, Mannheim, Germany) were used and the T and E2 levels were expressed in ng/ml and pg/ml, respectively. The sensitivities of the assays for T and E2 were 0.025 ng/ml and 5 pg/ml, respectively. Inter- and intra-assay coefficients of variance for T and E2 were 3.8% and 2.2%, and 5% and 3.9%, respectively. Serum DHT levels were measured using a sensitive kit (ALPCO Diagnostics, Salem, NH, USA) using the ELISA method and the values were expressed in pg/ml. The sensitivity of the assay for DHT was 6.0 pg/ml. Inter- and intra-assay coefficients of variance for DHT were 5.9% and 3.9%, respectively.

Serum samples were used to determine the levels of testosterone, progesterone, and estradiol. The levels of testosterone, estradiol, and progesterone were determined with an Elecsys 2010 analyzer using the electrochemical immunoassay (ECLIA) method. Standard commercial kits (Elecsys Testosterone II, Progesterone II, and Estradiol III Roche Diagnostics, Mannheim, Germany) were used. Testosterone and progesterone were expressed in ng/mL, while estradiol was expressed in pg/mL. The sensitivities for testosterone, progesterone, and estradiol were 0.025 ng/mL, 0.03 ng/mL, and 5 pg/mL, respectively. The inter- and intra-assay coefficients of variation for testosterone, progesterone, and estradiol were 3.8%, 3%, and 2.2%, 5%, and 3.9%.

Cultured cells were cultured in 12-well plates at a density of 1×10⁵ cells/ml in 1 ml culture medium as mentioned above. The culture medium was replaced with DMEM/F12 plus 10 nM of testosterone after 24 h when the cells reached 60–70% of cell confluency. The medium was then collected and stored at −80°C for hormonal analysis, and the remaining cells were lysed using SDS. The concentration of estradiol in the cell supernatants was measured using Estradiol III (Roche) according to the manufacturer’s instructions. The concentrations were then normalized according to the protein concentrations of the cell lysates measured using the bicinchoninic acid assay (Thermo Fisher).

Serum samples were assessed for the determination of sex hormone levels: total testosterone (T), progesterone (P4), and estradiol (E2) levels [20 (link)]. T, P4, and E2 levels were determined by using the Elecsys 2010 analyzer using the method of the electrochemiluminescence immunoassay (ECLIA). Standard commercial kits (Elecsys Testosterone II, Progesterone II, and Estradiol III Roche Diagnostics, Mannheim, Germany) were used, and the T and P4 levels were expressed in ng/ml and E2 was expressed in pg/ml. The sensitivities of the assays for T, P4, and E2 were 0.025 ng/ml, 0.03 ng/ml, and 5 pg/ml, respectively. Inter- and intra-assay coefficients of variance for T, P4, and E2 were 3.8%, 3%, and 2.2% and 5%, 5%, and 3.9%, respectively.