The effects of short-term culture and perifusion on LH-dependent steroidogenesis in isolated rat Leydig cells

Summary.Tumour Leydig cells and normal mature Leydig cells lost their steroidogenic response (pregnenolone and testosterone secretion) to LH after 24 h of culture. Immature cells showed a 2-fold increase in the basal pregnenolone secretion and no change in the LH-dependent pregnenolone secretion after 24 h of culture, whereas the LH-dependent steroidogenesis decreased after 48 h. None of the 3 cell preparations showed morphological signs of degeneration during a culture period of more than 7 days. Histochemical 3β-hydroxysteroid dehydrogenase activity in isolated immature Leydig cells disappeared during the first 2 days of the culture period and re-appeared after 5–7 days. Testosterone production by mature Leydig cells decreased during the first hours after exposure to LH, whereas pregnenolone secretion remained constant. From these results it was concluded that Leydig cells attached to plastic can be used for investigation of acute actions of LH on steroidogenesis. A perifusion technique for cells attached to plastic was developed and was applied to the kinetics of LH action on steroidogenesis in tumour and immature Leydig cells. The first stimulation of pregnenolone secretion occurred within 5 min, but a full stimulation was only obtained after 20–30 min. This was followed by a gradual decrease in the stimulated steroid secretion to ∼50% after 60 min.