F9397

Flutamide (F9397; Sigma‐Aldrich, St. Louis, MO) or vehicle (sesame oil; S3547; Sigma‐Aldrich) pretreatment was given subcutaneously 24 hr before experimentation to allow the full effect of Flutamide in the perinatal rat (Lieberburg et al., 1980). Male and female pups were randomized to standard dose Flutamide (10 mg/kg), high dose Flutamide (50 mg/kg) or vehicle control (10 μl/g body weight sesame oil).

Human endometrial tissue (proliferative phase, n = 20) was obtained from women undergoing surgery for non-malignant gynaecological conditions. None of the women were receiving hormonal therapy or suffering from endometriosis. Cycle phase was determined as previously reported37 (link). Written informed consent was obtained from all subjects prior to surgery, and ethical approval was granted by the Lothian Research Ethics Committee (LREC 10/S1402/59). Methods were carried out in accordance with NHS Lothian Tissue Governance guidelines. Primary endometrial stromal cells (ESC) were isolated from proliferative phase endometrium as described previously37 (link). Decidualization was induced by addition of decidualization media (DM: RPMI 1640, 2% charcoal-stripped FCS, 0.1 mg/ml 8-Br-cAMP (Sigma B5386), 1 μM progesterone (Tocris, Cat no. 2835). Some cells were incubated with the antiandrogen flutamide for the duration of the culture period (10 μM; Sigma F9397). Control cultures were incubated with RPMI 1640, 2% charcoal-stripped FCS and equivalent volume of vehicle control (DMSO). To assess the time-dependent accumulation of secreted products treatments were maintained for the duration of each time point. ESC were treated for 1, 2, 4 and 8 days as indicated.

Prednisone (P6254, Sigma-Aldrich) was resuspended in DMSO (D4540, Sigma-Aldrich) at 5 mg/mL each week. Daily treatment was 1 mg/kg body weight prednisone in 50 μL PBS given every day via i.p. injection. Vehicle treatment was the equivalent amount of DMSO proportional to body weight in 50 μL PBS. Weekly treatment was 1 day of prednisone treatment (1 mg/kg prednisone in 50 μL PBS) and 6 subsequent days of vehicle treatment (DMSO in 50 μL PBS). Unanesthetized mice were injected daily at 7 am with sterile BD Micro-Fine IV Insulin Syringes (14-829-1A, Thermo Fisher Scientific). Mice were weighed weekly and body weight was used for dosing calculations. Short-term (4-week) treatments consisted of 5 weekly prednisone injections total, with the final injection given 2 days prior to sacrifice. Long-term (38-week) treatments consisted of 39 weekly prednisone injections total, with the final injection given 2 days prior to sacrifice. AR activity was suppressed via the competitive antagonist flutamide (F9397, Sigma-Aldrich), which was administered at 15 mg/kg in 90% corn oil/10% ethanol. ER activity was suppressed via the competitive antagonist fulvestrant (I4409, Sigma-Aldrich), which was administered at 5 mg/kg in 95% corn oil/5% DMSO. Sex steroid receptor antagonists were administered daily as 100-μL i.p. injections.