INHIBITION OF STEROIDOGENESIS IN NEONATAL LEYDIG-CELLS BY UNKNOWN FACTOR(S) PRESENT IN SPENT MEDIA OF ANDROGEN-TREATED CULTURED TESTICULAR CELLS FROM ADULT-RATS

Treatment of cultured testicular cells from adult rats with Soc-dihydr otestosterone (DHT; 10(-6) M) or the synthetic androgen methyltrienolo ne (R1881; 10(-6) M) inhibited Leydig cell 3 beta-hydroxysteroid dehyd rogenase/Delta(5-4) isomerase (3 beta-HSD) enzyme activity, whereas no effect of both androgens on cultured cells derived from neonatal anim als could be observed. The inhibitory effect of DHT or R1881 on Leydig cell 3 beta-HSD enzyme activity, however, was abolished when adult ce lls were cultured in the presence of the antiandrogen cyproterone acet ate(CPA; 10(-5) M)or the protein synthesis inhibitor cycloheximide (CX ; 1 mu g/ml). Testicular cells from adult animals were also cultured i n the presence of the different treatments described above, and the sp ent media was collected and thereafter used as conditioned culture med ium (CCM) in subsequent experiments performed with neonatal cells. Dis persed testicular cells from neonatal rats were cultured for 12 days i n McCoy's 5a medium or in CCM derived from R1881-treated adult cells, and fresh culture medium or CCM was replaced every 2 days. The human c horionic gonadotropin (hCG)-stimulated testosterone production of neon atal cells was abolished in the presence of CCM derived from R1881-tre ated adult cells. Nevertheless, the steroidogenic response to hCG reco vered when neonatal cells were cultured for two additional days in McC oy's 5a medium. Treatment of neonatal cells with increasing concentrat ions of hCG (0.1-10 ng/ml) resulted in a dose-dependent augmentation i n Leydig cell 3 beta-HSD enzyme activity and testosterone production. A similar dose-dependent activation of steroidogenesis was observed in gonadotropin-stimulated neonatal cells cultured in the presence of R1 881 or CCM derived from untreated cultures of adult cells. In the same experiments the gonadotropin-stimulated steroidogenic activity of neo natal cells was almost completely abolished in the presence of CCM der ived from adult cells challenged with R1881 for 2 days. In contrast, n o inhibitory effect on hCG-stimulated steroidogenesis was observed whe n neonatal cells were cultured with CCM from cells treated with R1881 in combination with CPA or CX. The mechanism(s) whereby CCM from andro gen-treated adult cells inhibited neonatal Leydig cell steroidogenesis was also investigated. The full replication of hCG-stimulated steroid ogenesis elicited by the membrane-permeable cAMP analogue But(2)-cAMP (0.5 mM), the non-receptor activators of adenylate cyclase cholera-tox in (CT; 1 mu g/ml) and forskolin (FK; 50 mu M), or the phosphodiestera se inhibitor 1-methyl-3-isobutyl-xanthine (MIX; 0.1 mM) was abolished when fetal-neonatal Leydig cells were cultured in the presence of CCM derived from R1881-treated adult cells, suggesting that the inhibitory effect of CCM is exerted, at least in part, distal to the activation of the cAMP-protein kinase A pathway. These data show that CCM from an drogen-treated adult cells contains a newly synthesized factor(s) that has major inhibitory effects on neonatal cell steroidogenesis and sug gest that one or more of the cellular mechanism(s) involved in the ste roidogenic response to androgens differentiate spontaneously as pubert y approaches.