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

Abstract

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.

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.…”

“…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.…”

“…Hormones, cAMP/cGMP and NO measurement Progesterone (PROG), androgens (T+DHT), estradiol (E2) and LH levels were measured by radioimmunoassay (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. Progesterone measurements were assayed in duplicate, by RIA (the sensitivity: 6 pg per tube; the intra-assay coefficient of variation: 6.8%; the inter-assay coefficient of variation: 8.7%) using the anti-progesterone serum No.337 (Andric et al, 2007;Kostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011.…”