Stimulation of β-Endorphin Secretion by Corticotropin-Releasing Factor in Primary Rat Leydig Cell Cultures*

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Abstract
CRF, a hypothalamic peptide, is a potent stimulator of POMC synthesis and secretion in the pituitary. POMC biosynthesis has been documented in the testis, specifically in Leydig cells, and recent studies suggest that CRF is synthesized locally in the testis. A reverse hemolytic plaque assay and immunocytochemistry with Leydig cell-specific antibodies were used to study the effect of CRF on secretion of the POMC peptide .beta.-endorphin (.beta.EP) from normal rat primary Leydig cell cultures. In enriched Leydig cell preparations incubated with .beta.EP antiserum (diluted 1:50) then with complement (diluted 1:25), approximately 15% of immunocytochemically identified Leydig cells formed plaques. Preabsorption of the antiserum with .beta.EP (2 .mu.g/.mu.l antiserum) overnight at 4 C abolished the formation of plaques. Increasing concentrations of CRF (from 10-1 to 10-7 M) resulted in an approximately 80% increase in both the percentage of plaque-forming cells and the mean plaque size. When the CRF antagonist CRF-(9-41) (10-6 M) was added in the presence of CRF, the increase in plaque number and average size did not occur. These results demonstrate that Leydig cells have functional CRF receptors and that .beta.EP secretion from these cells is stimulated by CRF.