Growing body of evidence identify nitric oxide (NO) as one of endogenous mediators elicited by stress exposure, but precise identification of downstream signaling pathway molecules are still missing. In this study, we examined the effects of acute (2h) and repeated (2 or 10 days, 2 hours daily) immobilization stress (IMO) on NO-cGMP signaling pathway in adult rat Leydig cells. Results showed that only repeated IMO was accompanied with a decrease in levels of eNOS and iNOS transcript and protein, followed by declined NO production. In parallel, TLDA analysis revealed that again only repeated IMO lowered expression of all cGMP specific and almost all dual specific PDEs in Leydig cells. Furthermore, PDE5 protein level was reduced by repeated IMO, suggesting that Leydig cells PDEs system is trying to preserve cGMP. In this scenario, increased level of PKG-I transcript and protein was observed. Moreover, repeated IMO increased level of StAR and PKG-I immunoprecipitated complex, further enhanced with ex vivo sGC stimulation, but reduced with sGC inhibition. Additionally, ex vivo inhibition of PKG-I abolished increased level of mature StAR protein in Leydig cells obtained from rats repeatedly exposed to IMO. The physiological significance of the presented results was suggested by the recovery of androgen production, reduced by acute IMO, but starts to recover after repeated IMO. Increased expressions of PKG-I and StAR complex after repeated IMO, accompanied with decreased PDE5 protein, may suggest the possible role of cGMP signaling in the adaptive response of Leydig cells to stress-impaired steroidogenesis. Taking together, presented results showed that repeated IMO affects cGMP signaling pathway localized in Leydig cells and that this signaling scenario exhibits significant impact on recovery of stress-impaired testicular steroidogenesis, mainly trough "overdrive" interaction between PKG-I and StAR protein.

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