The ability of immobilization stress (IMO) to decrease Leydig cell steroidogenesis and serum androgens level has been previously observed, but the possible mechanism(s) involved in the adaptation to prolonged or repeated stress have not been identified. In this study we investigate weather the Leydig cells obtained from adult rats subjected to acute (15 min, 30 min or 2h) or repeated (2 or 10 days, 2 hours daily) IMO causes mechanism(s) as adaptive response to stress-impaired steroidogenesis. Results showed that basal and hCG-stimulated cAMP production of Leydig cells isolated from animals exposed to both acute and repeated IMO was significantly reduced. Despite the reduced cAMP production, immunoblot analysis revealed increased immunoreactivity for both protein kinase A (PKA) and steroidogenic acute regulatory (StAR) protein in Leydig cells obtained from animals repeatedly exposed to IMO. Also, the PKA-dependent phosphorylation and production of mature StAR protein was evident during exposure of animals to repeated IMO treatment. The physiological significance of the presented results was suggested by the recovery of serum androgen level, also affected by 2 h or 2x2 h IMO, but which starts to recover after 10x2 h IMO, and significantly increased in vitro androgen and progesterone production in presence of cholesterol (the steroid substrate transported into mitochondria by StAR). Aditionaly, increase of stress-reduced androgen production after repeated IMO is not related with steroid gene expression, since real time RQ-PCR analysis reviled that both acute and repeated IMO decreased P450scc, 3betaHSD and P450c17 gene expression. In contrast, the level of StAR transcript was progressively increased in all IMO groups. Thus, PKA-mediated phosphorylation of StAR protein is an important cascade in the adaptive response of Leydig cells to repeated immobilization stress.

This work was supported by the Serbian Ministry of Science (Grant No. 143055) and Autonomic Province of Vojvodina (Grant No. 0667).