GONADOTROPIN RECEPTOR OCCUPANCY AND STIMULATION OF CAMP AND TESTOSTERONE PRODUCTION BY PURIFIED LEYDIG-CELLS - CRITICAL DEPENDENCE ON CELL CONCENTRATION

Rat testicular interstitial cells have been separated by discontinuous/continuous gradient of Percoll, yielding four cell fractions. The light cells in fraction I bound luteinizing hormone/human chorionic gonadotropin (LH/hCG) with high affinity but were not steroidogenic in response to hormone. Fraction II consisted mainly of germ cells. Although fraction III contained Leydig cells, this fraction was contaminated with germ cells and was less responsive to hormone as compared to the Leydig cells in fraction IV. The Leydig cells in fraction IV produced cAMP and testosterone in response to hormone action in a manner which was critically dependent upon cell concentration. The production of cyclic adenosine monophosphate (cAMP) in the presence of saturating concentrations of hCG (2.4 × 10t1̄0 M) was linear as a function of cell concentration up to 7.0 × 106 cells/1.25 ml and thereafter, a slight inhibition (26%) was seen at 10 × 106 cells/1.25 ml. The average value for cAMP production by hCG was 133.8 ± 8.5 pmol cAMP/2 × 106 cells. The production of testosterone was biphasic, increasing linearly up to 5 X 106 cells/1.25 ml and decreasing thereafter. Two million cells, in the presence of 2.4 × 10-10 M hCG, produced an average of 24.2 ± 1.7 ng of testosterone in reaction volumes ranging from 1 to 2 ml whereas the same number of cells only produced 5.1 ± 0.6 ng of testosterone in 250 μ1. The binding of 125I-labeled hCG to the same batch of cells increased with increasing cell concentrations as expected but under the conditions of maximal steroidogenesis at low cell concentrations (1.25 2.0, and 2.5 × 106 cells/1.25 ml), it was barely detectable. Thus, we conclude that there is an inverse relationship between the parameters of binding and biological response in purified Leydig cells. © 1990.