Adult rat Leydig cell cultures: Minimum requirements for maintenance of luteinizing hormone responsiveness and testosterone production

The aim of this study was to establish the minimum conditions required to maintain adult rat Leydig cell testosterone production and luteinizing hormone (LH) responsiveness in short term culture, at a level similar to that observed in vivo, which could be used to study factors which may have a delayed or chronic effect on Leydig cell function. Percoll gradient-purified adult rat Leydig cells (5.0 × 10⁴/250 μl) were cultured in Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) with 0.1% bovine serum albumin at 32°C for up to 3 days, with daily medium changes. A combination of submaximal rat LH (0.1 ng/ml) and a maximal concentration of rat serum lipoproteins (0.5 mg/ml) maintained testosterone production at between 5 and 15 ng/10⁶ cells/h; subsequent stimulation of the Leydig cells with a maximum dose of rat LH (8 ng/ml) over 24 h resulted in testosterone production of 75-240 ng/10⁶ cells/h on all 3 days of culture. However, the addition of 0.1% fetal calf serum instead of rat lipoproteins could not maintain LH-stimulated testosterone production in the same culture period. In cultures containing submaximal LH and lipoproteins, levels of testosterone production and responses to maximal LH stimulation were constant over the culture period when expressed as either testosterone production per 10⁶ cells plated, or testosterone production per μg DNA recovered at end of incubation. Reduction of the oxygen tension from 19% to 5%, or to 1% did not significantly alter testosterone production by Leydig cells under these established conditions. Although LH-stimulated immunoactive inhibin secretion by the cultured Leydig cells was enhanced by the presence of lipoprotein, maximal LH-stimulated inhibin levels were only partially maintained, and submaximal LH-stimulated inhibin secretion was non-detectable after the initial 24 h. This suggests that not all functions of the Leydig cells were maintained. Nevertheless, these culture conditions provide a simple, effective means of maintaining adult rat Leydig cell testosterone production and LH responsiveness for a period of at least 3 days in culture.