Age related changes of cAMP and MAPK signaling in Leydig cells of Wistar rats.

Here, we chronologically analyzed age-associated changes of cAMP- and MAPK-signaling in Leydig cells (LCs) in relation with decreased testosterone (T) production. In Wistar rats, decreased serum T observed in 12 to 24-month-old rats was not related to decreased serum LH concentration but to reduced luteinizing hormone receptor (Lhr/LHR) and time-coordinated reduction of steroidogenic gene expression (decreased Cyp11a1, Cyp17a1 in 12-month-old rats followed by decreased Star/StAR, Hsd3b/HSD3B, Hsd17b4, and increased Cyp19a1 later in life). The predecessors of age-related changes noted in LCs from 6 to 12-month-old rats were increased level of soluble adenylate cyclase (Adcy/AC) 10, increased JNK phosphorylation but suppressed P38 MAPK. At approximately the same time changed mRNA abundance for transcription factors important for steroidogenesis was detected (increased Nur77 and decreased Sf1, Dax1). Aging caused biphasic expression pattern of ERK1/2 and Nur77: increased in 12-month but decreased in LCs from 24-month-old rats. Further, decreased basal cAMP level observed from 12 to 24th month coincidence with increased expression of cAMP-specific phosphodiesterase (Pde)4a, Pde4b and regulatory subunit of protein kinase A (Prkar/PKAR). Exposing of senescent LCs to permeable cAMP-analog improved transcription of Sf1, Nur77, Star, Cyp11a1,Cyp17a1, but without effect on aging pattern of Dax1, Pde4a/b, Prkar2a, Lhr and MAPK genes. Collectively, results indicated that age-related LC dysfunction is accompanied with changes in MAPK and cAMP signaling and coordinated reduction in the expression of many of the genes that participate in T synthesis. The predecessors of aged-related changes are increased ratio of pJNK/JNK, AC10 and decreased P38 level in LCs from 6-month-old rats.

Orally applied doxazosin disturbed testosterone homeostasis and changed the transcriptional profile of steroidogenic machinery, cAMP/cGMP signalling and adrenergic receptors in Leydig cells of adult rats.

Doxazosin (Doxa) is an α1-selective adrenergic receptor (ADR) antagonist widely used, alone or in combination, to treat high blood pressure, benign prostatic hyperplasia symptoms, and recently has been suggested as a potential drug for prostate cancer prevention/treatment. This study was designed to evaluate the effect of in vivo Doxa po-application, in clinically relevant dose, on: (i) steroidogenic machinery homeostasis; (ii) cAMP/cGMP signalling; (iii) transcription profile of ADR in Leydig cells of adult rats. The results showed that po-application of Doxa for once (1×Doxa), or for two (2×Doxa) or 10 (10×Doxa) consecutive days significantly disturbed steroidogenic machinery homeostasis in Leydig cells. Doxa po-application significantly decreased circulating luteinizing hormone and androgens levels. The level of androgens in testicular interstitial fluid and that extracted from testes obtained from 1×Doxa/2×Doxa rats decreased, although it remained unchanged in 10×Doxa rats. Similarly, the ex vivo basal androgen production followed in testes isolated from 1×Doxa/2×Doxa rats decreased, while remained unchanged in 10×Doxa rats. Differently, ex vivo testosterone production and steroidogenic capacity of Leydig cells isolated from 1×Doxa/2×Doxa rats was stimulated, while 10×Doxa had opposite effect. In the same cells, cAMP content/release showed similar stimulatory effect, but back to control level in Leydig cells of 10×Doxa. 1×Doxa/2×Doxa decreased transcripts for cAMP specific phosphodiesterases Pde7b/Pde8b, whereas 10×Doxa increased Pde4d. All types of treatment reduced the expression of genes encoding protein kinase A (PRKA) regulatory subunit (Prkar2b), whereas only 10×Doxa stimulated catalytic subunit (Prkaca). Doxa application more affected cGMP signalling: stimulated transcription of constitutive nitric oxide synthases (Nos1, Nos3) in time-dependent manner, whereas reduced inducible Nos2. 10×Doxa increased guanylyl cyclase 1 transcript and PRKG1 protein in Leydig cells. Orally applied Doxa significantly disturbed the transcriptional 'signature' of steroidogenic machinery, cAMP/cGMP signalling and ADRs and ß-ADRs kinases in Leydig cells, thus giving new molecular insights into the role of cAMP/cGMP/adrenalin signalling in Leydig cells homeostasis.

Structural complexity of the testis and PKG I / StAR interaction regulate the Leydig cell adaptive response to repeated immobilization stress.

The role of the structural complexity of the testis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway was analysed in adult male rats exposed to acute and repeated immobilization stress (IMO). In whole testis preparations, exposure to acute and repeated IMO caused an increase in NO production. In contrast, NO production was inhibited in interstitial cell preparations after exposure to all types of stress. In purified Leydig cell preparations, NO production was inhibited only after exposure to prolonged IMO. These findings indicate that biologically active compounds released from various testicular compartments exert both stimulatory and inhibitory effects on NO production. TaqMan Low Density Array of rat phosphodiesterases revealed a decrease in the expression of cGMP-specific phosphodiesterase 5 (PDE5) in Leydig cells of animals exposed to repeated IMO. In contrast, the expression of cGMP-dependent protein kinase type I (PKG I), total and phosphorylated steroidogenic acute regulatory protein (StAR), and PKG I/StAR immunoprecipitated complex was increased during repeated exposure to IMO. The increase in both total and phosphorylated StAR formation was effectively blocked by inhibition of PKG I in vitro. Thus, increased expressions of PKG I and StAR complex, accompanied by decreased PDE5 activity, suggest that the NO-cGMP signalling pathway and consequent activation of the StAR protein regulate the adaptive response of Leydig cells to repeated IMO stress.

The adaptive response of adult rat Leydig cells to repeated immobilization stress: the role of protein kinase A and steroidogenic acute regulatory protein.

The ability of immobilization stress (IMO) to decrease Leydig cell steroidogenesis and serum androgen concentration 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 investigated whether the Leydig cells obtained from adult rats subjected to acute (15 min, 30 min or 2 h) and repeated (2 or 10 days, 2 h daily) IMO show adaptive mechanism(s) in response to stress-impaired steroidogenesis. The results showed that basal and human chorionic gonadotropin-stimulated cAMP production by Leydig cells isolated from rats 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 rats repeatedly exposed to IMO. Also, the phosphorylation and production of mature StAR protein was evident during exposure of rats to repeated IMO treatment. Treatment with cholesterol, the steroid substrate transported into mitochondria by StAR, significantly increased androgen and progesterone production by Leydig cells isolated from rats exposed to repeated IMO. In contrast, when other steroid substrates (22(R)-OH-cholesterol, pregnenolone, progesterone, Delta4-androstenedione) were present in the culture media, Leydig cell steroidogenesis was still reduced by IMO. Thus, PKA-mediated phosphorylation of StAR protein is an important mechanism in the adaptive response of Leydig cells to repeated IMO.