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 chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, 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 125I-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 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the 125I-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.
Browne
E S
ES
Department of Physiology and Endocrinology, Medical College of Georgia, Augusta 30912.
Bhalla
V K
VK
D000818:Animals; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D006063:Chorionic Gonadotropin / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D000242:Cyclic AMP / Q000096:biosynthesis*; D007457:Iodine Radioisotopes; D007985:Leydig Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051381:Rats; D013739:Testosterone / Q000096:biosynthesis*
D006063:Chorionic Gonadotropin; C034923:HCG, deglycosylated; D007457:Iodine Radioisotopes; D013739:Testosterone; D000242:Cyclic AMP