Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells.

Abstract

We have investigated the effects of insulin and somatomedin-C/insulinlike growth factor I(Sm-C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum-free medium containing transferrin, vitamin E, and insulin (5 micrograms/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm-C at nanomolar concentrations prevent the decline of both hCG receptors and hCG-induced cAMP production. This effect of both peptides was dose dependent with an ED50 of about 1 ng/ml and 5 ng/ml for SM-C and insulin, respectively. Insulin and Sm-C had no additive effect on these parameters. At nanomolar concentrations, Sm-C and insulin enhanced hCG-induced testosterone production but the effect of Sm-C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 micrograms/ml) was significantly higher than that of Sm-C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm-C stimulated [3H]-thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. In contrast, the mitogenic effects of insulin are mediated only through Sm-C receptors.