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

Abstract

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.

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

“…The proportion of Leydig cells present in culture was determined by staining for HSD3B activity (Payne et al, 1980), and was found to be 95.3 ± 2.7%, while the viability was more than 90%. The steroidogenic capacity of Leydig cells (estimated by dose-dependent stimulation with hCG) and the activity of steroidogenic enzymes (estimated by incubating cells with increasing concentrations of steroid substrates), were in line with those previously published by others (Akinbami et al, 1994) as well as our group (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. These data were not showed and served to us just as internal control.…”

“…Primary culture of testes were prepared as described before by our group (Kostic et al, 2010). Briefly, after TIF collection, a small part of each testis, from control and Doxa-treated groups, was quickly cut, immediately frozen at À80°C for steroid extraction and kept until extraction process (please see below).…”

“…To follow ex vivo steroids production, and the expression of steroidogenic machinery elements (steroidogenic enzymes, proteins related to the steroidogenesis, transcription factors), cAMP signalling elements (ADCY, PRKA subunits, cAMP specific PDEs), cGMP signalling elements (GUCY, PRKG isoforms, cGMP-specific and dual PDEs) and ADRs, we used primary cultures of purified Leydig cells obtained from control and Doxa-treated rats prepared as described previously by our group (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. Primary cultures of purified Leydig cells were prepared from suspensions of interstitial cells.…”

“…Primary cultures of purified Leydig cells were prepared from suspensions of interstitial cells. Suspensions of interstitial cells were prepared according to Anakwe et al (Anakwe et al, 1985) with some modifications described previously by our group (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. Testes were quickly removed, decapsulated and placed in a 50-mL plastic tube (two testes per tube) containing 3 mL of collagenase solution (1.25 mg/mL collagenase Type I; 1.5% BSA; 20 mM HEPES in DMEM/F12) and incubated for 15 min at 34°C in a shaking water bath oscillating at 120 cycles/min.…”

“…The 0.2% trypan blue dye exclusion test (Sigma Inc) was used to determine total cell counts and to ensure that greater than 95% of the cells were viable. This suspensions of interstitial cells (Klinefelter et al, 1987) was used to prepare primary cultures of purified Leydig cells (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011 by centrifugation on a Percoll gradient consisting of four 2 mL layers of Percoll with densities of 1.090, 1.080, 1.065 and 1.045 g/mL (formed by mixing isotonic Percoll consisting of 109 concentrated DMEM/F12 enriched with 3% of BSA and the corresponding amount of Percoll and distilled water). A crude suspension of interstitial cells (approximately 35-40 9 10 6 cells), containing besides Leydig cells macrophages and endothelial cells (Klinefelter et al, 1987), was applied to each Percoll gradient and centrifuged at 500 g for 28 min at room temperature.…”