The steroidogenic activity of isolated Leydig cells from mature rats depends on the isolation procedure.

Abstract

Three different collagenase dispersion techniques for isolation of Leydig cells from testes of mature rats have been compared with respect to the yield and quality of the isolated cells. No Leydig cells could be isolated after perfusion of the testis with collagenase (10 mg/ml) followed by incubation at 37°C without shaking, whereas Leydig cells were obtained after incubation of decapsulated testis tissue with collagenase (1 mg/ml) and shaking (80 cycles/min or 1500 cycles/min). Of the total phenylesterase activity, which is localized in the endoplasmic reticulum of interstitial cells, 50 and 85% was released after shaking at 80 and 1500 cycles/, respectively. After sedimentation of the cells 14 and 56% of the microsomal esterase activity was present in the supernatant, whilst 6 and 10% of the esterase activity could be recovered in Ficoll‐purified cells. The high esterase activity in the supernatant and the low esterase activity in the purified cells indicated the presence of many broken cells. Testosterone production by cells prepared with the low shaking frequency could be stimulated 9‐fold by LH, whereas cells prepared with the high shaking frequency did not respond to LH. Cell preparations which could be stimulated with LH could also be stimulated (3‐fold) with NADPH, indicating the presence of damaged cells in addition to intact cells. The percentage of damaged cells in the preparations was estimated using an NADH‐dependent histochemical test for mitochondrial diaphorase activity, which shows activity when cell membranes are damaged. Using this criterion, it was found that about 30% of ficoll‐purified cells were damaged, whereas only 2% of cells which were pre‐incubated and attached to plastic appeared to be damaged. In***