Isolation method of Leydig cells from mature male Djungarian hamsters (Phodopus sungorus) and their steroidogenic activityin vitro

Abstract

For studies addressing the functions of Leydig cells, isolated cells are often better suited than intact animals. Here, the isolation procedure of Leydig cells from adult male Djungarian hamsters (Phodopus sungorus) is described. Cells were isolated using a procedure involving enzymatic dissociation and Percoll-gradient centrifugation. For each experiment, approximately 4.4 x 10(6) Leydig cells from six animals were obtained. The cells showed high steroidogenic responsiveness to physiological (ovine luteinizing hormone (oLH) and human chorionic gonadotropin (hCG)) and nonphysiological (forskolin) stimuli in vitro. Approximately 98% of cells were viable as assessed by trypan blue exclusion, and the purity varied from 80 to 95% as tested by 3 beta-hydroxysteroid dehydrogenase activity. Leydig cells were also identified by a bright yellow halo under phase-contrast microscopy. They contained numerous lipid droplets and showed round nuclei and prominent nucleoli. The cells responded to oLH, hCG and forskolin with an increased testosterone production in a dose-dependent manner. Dose-response curves in these studies suggest that Leydig cells of Djungarian hamsters undergo desensitization, probably due to down regulation of their LH/CG receptors.