Optimizing testosterone production by purified adult rat Leydig cells in vitro

We sought to establish conditions that increased the duration of testosterone production by fully differentiated adult rat Leydig cells in primary culture. A freshly isolated suspension of highly purified adult rat Leydig cells produced 83 ng testosterone/10(6) Leydig cells.h-1 when incubated in Medium 199 in a 1.5 ml microfuge tube with shaking for 3 h with a maximally stimulating concentration of ovine luteinizing hormone (LH). Unfortunately, adult rat Leydig cells that were allowed to attach only to a plastic culture dish flattened out, and testosterone production diminished rapidly. Leydig cells in Dulbecco's modified Eagles' medium-Ham's F12 (1:1; vol/vol) containing Cytodex 3 beads pre-equilibrated in culture medium containing fetal bovine serum attached to the beads and remained viable, but produced only 30 ng testosterone/10(6) Leydig cells.h-1 when incubated for 24 h with similar stimulation. Leydig cells similarly cultured and maximally stimulated with LH, responded to bovine lipoproteins (less than 1.222 g/ml) producing 105 ng of testosterone/10(6) Leydig cells.h-1 when incubated with 1 mg/ml bovine lipoprotein. Therefore, lipoproteins maintain the steroidogenic capacity of purified adult rat Leydig cells in primary culture for 24 h.