Ex vivo primary pituitary cell culture and GnRH stimulation.

Adult control or PitARKO mice without DHT insertion were sacrificed at diestrus by CO₂ asphyxiation and decapitated before their pituitaries were collected. The pooled pituitaries (3–5 pituitaries) were washed with 10 ml of 10% FBS DMEM (Cellgro, 10-013-CV) at 2500 g for 5 minutes. Afterward, the pituitaries were washed with 10 ml HBSS at 2500 g for 5 minutes, followed by sequential digestions with collagenase (Sigma # C0130, 2ml 1.5 mg/ml collagenase /10 pituitaries) for 2 hours at 37°C and in pancreatin solution (Sigma # P3292, 4.5 mg/ml pancreatin) for 15 minutes at 37°C. Finally, pellets were washed with 10% FBS DMEM followed by centrifugation at 2500 g for 5 minutes. The primary pituitary cells were counted and placed into Matrigel-coated 24-well (cell density is around 0.5 × 10⁶/well) or 384-well (1.5 × 10⁴/well) plates and incubated for 24 hours in DMEM-phenol red with 10% FBS, 100 U/ml penicillin, and 100μg/ml streptomycin (Gibco, ThermoFisher Scientific) (65–68). Cells were then cultured in 1 nM and 10 nM DHT or vehicle (no DHT) for 42 hours. We chose a 42-hour DHT incubation time because we observed that DHT inhibited GnRH-stimulated LH secretion in cultured primary pituitary cells after 36–48 hours of DHT treatment (data not shown). Cells were incubated with serum-free medium without phenol red for 3 hours before GnRH stimulation. GnRH was added into medium, and media were collected after 2-hour treatment. LH and FSH levels were measured by a Luminex assay or an ultrasensitive LH ELISA. Treatments were performed in triplicate or quadruplicates, and experiments were independently repeated at least 3 times.