THE MELANOGENIC RESPONSE TO TESTOSTERONE IN SCROTAL EPIDERMIS: EFFECTS ON TYROSINASE ACTIVITY AND PROTEIN SYNTHESIS

Abstract

ABSTRACT Pigmentation of the scrotum of the black-pelted rat, as expressed through melanocyte melanogenic activity, is controlled by androgens. Castration decreased in vitro incorporation of [14C] tyrosine into melanin. Testosterone pre-treatment for 4 days increased melanin radioactivity over castrate controls; the increment in vitro was prevented by an inhibitor of protein synthesis (cycloheximide) added to the incubation. However, cycloheximide only partially blocked melanin synthesis when added to tissue from animals hormone treated for 6 days in vivo, and was ineffective in tissue from intacts. Bulk protein synthesis in vitro (incorporation of [14C] tyrosine or -leucine) was not affected by castration or testosterone treatment but was uniformly inhibited by cycloheximide. The data suggest that new synthesis of specific protein in vitro was necessary for initial hormone-stimulation of melanogenesis, but with longer exposure to hormone sufficient protein was pre-synthetized in vivo to permit melanogenesis during incubation with the inhibitor. Radioautographs of epidermis incubated with [14C] tyrosine showed grains concentrated over macromolecular aggregates in melanocytes, a pattern not altered by cycloheximide. Though available for incorporation into general tissue protein. [14C] tyrosine was apparently incorporated preferentially into melanin by melanocytes. DOPA (3,4-dihydroxyphenylalanine) added to incubations in cofactor amounts did not affect decreased melanin synthesis after castration and appears, therefore, not to be rate limiting in that decrease. Tissue uptake of free [14C] tyrosine or — leucine during incubation was lower than normal in castrate epidermis; uptake was elevated by testosterone treatment. Concentrations appeared sufficient in all preparations to suggest that availability is not rate limiting for synthesis of melanin or protein; however, a possible influence on amino acid permeability for melanocytes remains undetermined. Tyrosinase activity was present in both particulate and cytosol fractions of epidermis but decreased significantly after castration only in the cytosol. Testosterone increased particulate activity after 4 days and soluble activity after 9 days of treatment. These and findings above are consistent with a model that tyrosinase is synthesized and incorporated into melanosome structure within 4 days testosterone treatment; with longer treatment synthesis may then exceed that required for melanosome assembly and tyrosinase appears in the soluble milieu.