The Heterogeneity of Isolated Adult Rat Leydig Cells Separated on Percoll Density Gradients: An Immunological, Cytochemical, and Functional Analysis

Intertubular cells, isolated from adult rat testes by collagenase dispersal under conditions designed to minimize cell damage, were fractionated on Percoll density gradients. In the gradient fractions, there was a close cellular correlation between the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), determined by cytochemistry, and other Leydig cell markers (nonspecific esterase, autofluorescence, and an antigen defined by monoclonal antibody LC-1C6). As the reagents for 3 beta HSD cytochemistry are excluded by intact membranes, Leydig cells with damaged plasma membranes were identified by 3 beta HSD reactivity in suspended cell preparations, and the total number of 3 beta HSD-positive (3 beta HSD+) cells in the same preparations was determined after lysis of the cell membrane. Whole cells were differentiated from cytoplasmic fragments by counterstaining with the nuclear dye propidium iodide, and the number of intact Leydig cells in each preparation was determined subsequently by subtracting the number of damaged nucleated 3 beta HSD+ cells from the total number of nucleated 3 beta HSD+ cells. The majority of intact isolated Leydig cells were found in gradient fractions of 1.054-1.096 g/ml density. Acute (3-H) basal and hCG-stimulated testosterone production per intact Leydig cell were dependent upon the concentration of Leydig cells per assay well, indicating that there is cooperativity among Leydig cells in vitro. There was no difference in steroidogenic function among intact Leydig cells from different fractions of the above density gradient range at assay concentrations greater than 10,000 Leydig cells/well. At lower cell concentrations, Leydig cells from gradient fractions of lower density (1.054-1.064 g/ml) produced slightly less testosterone in response to hCG stimulation than Leydig cells from more dense fractions (1.070-1.096 g/ml). Prolonging the exposure of isolated cells to the dispersal conditions caused declines in the apparent buoyant density and basal testosterone and hCG-stimulated cAMP and testosterone production of all Leydig cells, without detectable changes in cell integrity. The data indicate that both the absolute steroidogenic function and the functional heterogeneity of isolated intact Leydig cells are, at least in part, dependent upon the procedures used for their isolation.