The Effect Of C-myb On HCG-induced Testosterone Secretion In Isolated Rat Leydig Cells

Objective: To investigate the effect of c-myb antisense oligodeoxynucleotides(c-myb ASODNs) on hCG-induced testosterone secretion in isolated rat Leydig cells, and the effects of cAMP, verapamil, cycloheximide, dexamathasone on the expression of c-Myb oncoprotein were also observed. Methods: (1)Testosterone secretion of isolated rat Leydig cells was measured by radioimmunoassay. (2) C-Myb protein of isolated rat Leydig cells was detected by immunohistochemistry. Results: (1) HCG (12×10-3IU/ml)significantly increased testosterone secretion and the percentage of positive stained Leydig cells for c-Myb protein (P＜0.01).(2) C-myb ASODNs inhibited hCG-induced testosterone secretion of isolated rat leydig cells in a dose-dependent manner. At the same time the percentage of positive stained for c-Myb protein was also remarkably decreased. But nonsense tat ODNs had no similar effect on Leydig cells. (3) Further experiments showed that dbcAMP(10-4mol/L) obviously reversed the inhibitory effect of c-myb ASODNs on hCG-induced testosterone secretion and increased the percentage of positive stained for c-Myb protein. (4) Verapamil(10μmol/L), a Ca2+ channel blocker, decreased the percentage of positive stained Leydig cells for c-Myb protein, and also lowered hCG-induced testosterone secretion in isolated rat Leydig cells. (5) Cycloheximide(50μg/ml), a Protein synthesisinhibitor, decreased the percentage of positived stained Leydig cells and hCG-induced testosterone secretion in isolated rat Leydig cells. (6) Dexamethasone (10-8mol/L)decreased the percentage of positive stained leydig cells and hCG-induced testosterone secretion in isolated rat Leydig cells. These effects could be blocked by RU486(10-8mol/L), a dexamethasone antagonist. Conclusion: (1) The results indicated that c-myb is closely in correlation with hCG-induced testosterone secretion.And the mechanism might be mediated by cAMP -protein kinase A pathway and IP3-DG-calmodulin protein kinase pathway, which in turn activates the expression of c-myb proto-oncogene in isolated rat Leydig cells. (2) cycloheximide can decrease hCG-induced testosterone secretion,which might be through inhibiting c-Myb synthesis of Leydig cells. (3) Dexamethasone can decrease hCG-induced testosterone secretion, which might be through regulating c-myb expression of Leydig cells.