GONADOTROPIN STIMULATION OF CYCLIC ADENOSINE-MONOPHOSPHATE AND TESTOSTERONE PRODUCTION WITHOUT DETECTABLE HIGH-AFFINITY BINDING-SITES IN PURIFIED LEYDIG-CELLS FROM RAT TESTIS

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionicc gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, K(d), approximately 10(-10)M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable I-125-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250-mu-l) were used for in vitro hCG stimulation assays. Under these conditions, the I-125-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.