Etiocholanolone

Interstitial cells were isolated from adult WT and Tspo gKO mice ranging in age from 56 to 97 days. Mice were asphyxiated with CO₂ and testes were removed. The tunica of the testis was removed, and seminiferous tubules were separated from interstitial cells by digestion (10 minutes, 34°C) in 0.25 mg/mL collagenase (Aldrich-Sigma)/13 mg/mL DNase (Aldrich-Sigma) in M-199 (Thermo Fisher Scientific) followed by unit-gravity sedimentation in M-199 supplemented with 1% bovine serum albumin [23 (link)]. Histochemical staining with nitro blue tetrazolium for 3βHSD activity with 0.4 mM etiocholanolone (Steraloids) as the steroid substrate [24 (link)] indicated the presence of an enriched Leydig cell population, containing 20% to 25% 3βHSD-positive cells. Cells were incubated at a concentration of 2.33 × 10⁵ in a final volume of 1 mL for 2 hours in a shaking water bath at 34°C. Cells were incubated in buffered Dulbecco MEM:Ham F12 culture medium (Aldrich-Sigma) alone, or with a stimulating dose of 50 ng/mL human chorionic gonadotropin (hCG) (National Hormone Pituitary Program). Testosterone production was assayed by ELISA as described above.