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
N. J. Stojkov,
Reproductive Endocrinology and Signaling Group, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia
Silvana A. Andric, PhD, Reproductive Endocrinology and Signaling Group, Department of Biology and Ecology, Faculty of Sciences at University of Novi Sad, Dositeja Obradovica Square 2, Novi Sad 21000, Serbia. E-mail: email@example.com
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 β-ADRs kinases in Leydig cells, thus giving new molecular insights into the role of cAMP/cGMP/adrenalin signalling in Leydig cells homeostasis.
Doxazosin (Doxa), an α1-selective adrenergic receptors (ADRs) blocker, is the most frequently prescribed medical therapy, alone or in combination, in the treatment of high blood pressure, symptoms related to metabolic syndrome, lower urinary tract symptom suggestive of benign prostatic hyperplasia (LUTS/BPH), problems with erectile dysfunction (ED) and in sexual health problems(reviewed in McConnell et al., 2003; Steers & Kirby, 2005; Kaplan et al., 2006; Wilt & MacDonald, 2006; Andersson & Gratzke, 2007; Lepor et al., 2012). Because LUTS/BHP, ED and metabolic syndrome have bidirectional connection with testosterone, it is important to investigate the impact of Doxa application on testosterone homeostasis. It has been shown recently that in vivo administration of Doxa (10 mg/kg BW for 15 days) highly decreases circulating levels of testosterone in rats (de la Chica-Rodríguez et al., 2008). On the contrary, combination Finansteride+Doxa induced a transient increase in plasma testosterone and a permanent reduction in dihydrotestosterone (Justulin et al., 2010). However, the consequences of Doxa application on elements that maintain testosterone homeostasis are not known.
Testosterone-producing Leydig cells, like all other steroid-producing cells, synthesize steroid hormones from a common precursor, cholesterol using the steroidogenic machinery comprising of cholesterol transporters, steroidogenic enzymes, and many regulatory molecules (Fig. 1, reviewed in Payne & Hales, 2004; Stocco et al., 2005). The steroidogenic function of Leydig cell is predominantly regulated by pituitary luteinizing hormone (LH) or its placental counterpart human chorionic gonadotropin (hCG). LH/hCG receptors activation (Zhang & Dufau, 2003) leads to stimulation of adenylyl cyclase (ADCY), accumulation in cAMP and activation of the cAMP-dependent kinase (PRKA). The phosphodiesterases (PDEs) terminate cAMP/cGMP signalling and have regulatory function in Leydig cells (Catt & Dufau, 1973; Dufau, 1998; Tsai & Beavo, 2011, 2012). Although Leydig cell steroidogenesis is mainly activated through LH/hCG receptors, regulation itself is a multi-compartmental process and includes neural (Selvage et al., 2006) and complex endocrine, paracrine and autocrine signalling pathways (reviewed in Saez, 1994; Gnessi et al., 1997; Payne & Hales, 2004) including cGMP (Andric et al., 2010a,b) and adrenergic signalling (Anakwe et al., 1985). In addition, many transcription factors are involved in regulation of the steroidogenic machinery expression (reviewed in Simard et al., 2005; Lavoie & King, 2009; Martinez-Arguelles & Papadopoulos, 2010; Midzak et al.,2011). All mentioned elements of steroidogenic machinery, might be involved in the regulation of testicular steroidogenesis, providing an adaptive mechanism by which testicular structures, including Leydig cells, recover from disturbed homeostasis.
Despite the wide clinical application of Doxa, the lack of fundamental knowledge about the ADRs milieu, cAMP/cGMP signalling and steroidogenic machinery in the steroid-producing cells such as Leydig cells hampers our ability to understand the short- and long-term consequences of Doxa application on steroid homeostasis. Accordingly, this study was designed to systematically evaluate the effect of Doxa po-application in clinically relevant dose (Kaye et al., 1986) on: the steroidogenic machinery homeostasis, cAMP/cGMP signalling and transcriptional profile of all ADRs in Leydig cells. The study followed the following: (i) The levels of circulating LH and androgens (T+DHT); (ii) The androgens levels in testicular interstitial fluid (TIF), extracted from testes and produced by testes in basal/hCG-stimulated environment; (iii) Ex vivo steroidogenic activity/capacity Leydig cells; (iv) The expression of transcripts for steroidogenic machinery elements (Fig. 1) including the enzymes/proteins (Lhr, Scarb1, StAR, Tspo, Cyp11a1, Hsd3b1/5, Cyp17a1, Hsd17b3/4, Cyp19a1) and transcriptional factors related to steroidogenesis (Sf1 – steroidogenic factor 1, Dax1-dosage-sensitive sex reversal adrenal hypoplasia critical region on chromosome X gene, Arr19 – androgen receptor corepressor 19kDa, Jak2 – Janus kinase 2, Stat5a – signal transducer and activator of transcription 5A, Ar, Esr1 – oestrogen receptor); (v) The transcriptional profile of cAMP signalling pathway elements including enzymes responsible for cAMP formation (ADCY), degradation (cAMP specific PDEs), and effects (PRKA); (vi) The transcriptional profile of cGMP signalling pathway elements including enzymes responsible for cGMP formation (GUCY1), degradation (cGMP-specific PDEs), and effects (protein kinase G, PRKG), as well as nitric oxide synthases (neural – Nos1, inducible – Nos2, endothelial – Nos3); (vii) The transcriptional profile of α/β-ADRs and β-ADRs kinases. Results showed that Doxa po-application disturbed testosterone homeostasis by changing the transcriptional ‘signature’ of steroidogenic machinery elements, cAMP/cGMP signalling and ADRs in Leydig cells.
Materials and methods
The antisera for StAR protein was generous gift from Professor Douglas Stocco (Clark et al., 1994), whereas the purified rabbit polyclonal antibody against HSD3B was generous gifts from Professor Ian Masson (Bain et al., 1991; Abbaszade et al., 1997). The rabbit polyclonal antibody recognizing type α/β isoforms of PRKG1 was obtained from Calbiochem (San Diego, CA, USA). Actin detection kit was purchased from Oncogene Research Product (San Diego, CA, USA), while the anti-mouse and anti-rabbit secondary antibodies linked to the horse-radish peroxidase were obtained from Kirkegaard and Pery Labs (Gaithersburg, MD, USA). The anti-progesterone-11-BSA serum No.337, anti-testosterone-11-BSA serum No.250 and anti-estradiol-11-BSA serum No.244 were kindly supplied by Gordon D. Niswender. The cAMP and cGMP EIA Kits were purchased from Cayman Chemicals (Ann Arbor, MI, USA), where the rat lutenizing hormone RIA kit was obtained from Alpco Diagnostics (Alpco Diagnostics, Windham, NH, USA). (1,2,6,73H(N)) labelled progesterone, testosterone and estradiol were obtained from Perkin–Elmer Life Sciences (Waltham, MA, USA). The RNeasy kit for total RNA isolation was purchased from Qiagen Co., GmbH, (Valencia, CA, USA), whereas Superscript III kit for cDNA preparation was obtained from Invitrogen (Carlsbad, CA, USA). TaqMan Low Density Rat Endogenous Control Panel, TaqMan Low Density Rat Phosphodiesterase Panel, TaqMan Low Density Rat GPCR Panel, TaqMan Universal PCR Master Mix and Power SYBR Green PCR Master Mix were purchased from Applied Biosystems (Carlsbad, CA, USA), whereas primers for real-time RQ-PCR were obtained from Integrated DNA Technologiest (Coralville, IA, USA). Medium 199 containing Earle's salt and l-glutamine (M199), DMEM/Nutrient Mixture F-12 Ham with l-glutamine and 15 mm HEPES (DMEM/F12), hCG (Chorionic gonadotropin, lyophilized powder, vial of 2500 IU), HEPES, penicillin, streptomycin, EDTA, Percoll, BSA fraction V, collagenase type IA, testosterone, isoproterenol (β-adrenergic agonist), trypan blue, β-glycerophosphate, tergitol (Niaproof 4, type 4), dithiothreitol, leupeptin and aprotinin were from Sigma (St. Louis, MO, USA). Doxa, α1 – selective adrenergic antagonist (in a form of Doxa mesylate), was obtained from Zdravlje AD (Leskovac, Srbija). All other reagents were of analytical grade.
All the experimental protocols were approved by the local Ethical Committee on Animal Care and Use at the University of Novi Sad operating under the rules of National Council for Animal Welfare and following statements of National Law for Animal Welfare (copyright March 2009). All experiments were performed and conducted in accordance with the National Research Council publication Guide for the Care and Use of Laboratory Animals (copyright 1996, National Academy of Sciences, Washington, DC) and NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80 23, revised 1996, 7th edition). All the experiments adhere to Guiding Principles in the Care and Use of Vertebrate Animals in Research and Training and were carried out in the Laboratory for Reproductive Endocrinology and Signaling, DBE, Faculty of Sciences at University of Novi Sad.
Animals and doxazosin application per os
Adult (3 months old, 250–270 g) male Wistar rats, bred and raised in the Animal Facility of the Department of Biology and Ecology (Faculty of Sciences, University of Novi Sad, Serbia), were used for the experiments. The animals were raised in controlled environmental conditions (22 ± 2 °C; 14/10 h light/dark cycle, lights on at 6 a.m.) with food and water ad libitum.
Rats were handled daily during a 3-week period of acclimation before experiments. To mimic the most likely route of human exposure, rats were subjected to oral administration (po) of Doxa in clinically relevant dose for rat model (5 mg/kg BW) described before (Kaye et al., 1986). It is important to point out that the clinically relevant doses for humans stated in treatment guidelines are in the initial dosage of Doxa mesylate 1 mg once daily, and this is titrated to a maximum of 8 mg for BPH or 16 mg for hypertension. However, the mean plasma clearance values are 25 times higher in rats. The mean plasma clearance values for rats were 30 mL/min/kg BW, whereas those for human subjects were 1.2 mL/min/kg. The mean plasma half-life values were 1.2 h in rats, while the value of 9 h was reported for human volunteers (Kaye et al., 1986). Doxa oral bioavailability in the rat is approximately 50%, which is similar to the value of 63% reported for man at therapeutic doses. The long plasma half-life of Doxa provides the basis for once-daily dosing (Kaye et al., 1986). Groups of rats (six of rats in each) were left undisturbed (Control) or treated po with either water or Doxa once (1×Doxa), or for two (2×Doxa) or 10 (10×Doxa) consecutive days. Because Doxa tablets were dissolved in sterile distilled water, groups of control rats received water by oral gavage once, or for two or 10 consecutive days. Results were the same compared to undisturbed control, so they are presented as a one control application. In our experiments, treatment started every day at 6.30 a.m. and rats were quickly decapitated without anaesthesia 3.5 h after first, second or tenth Doxa application. The trunk blood was collected and individual serum samples were stored at −20 °C until assayed for LH and androgens (testosterone + dihydrotestosterone, T+DHT) levels.
Testicular interstitial fluid (TIF) collection
Collection of TIF was performed as described before (Janjic et al., 2012). Briefly, testes from control and Doxa-treated groups were quickly removed, decapsulated and placed on Falcon mesh No.100 (EMD Biosciences, San Diego, CA, USA) in a 50 mL plastic tube (one testes per tube), centrifuged at 100 g for 7 min at room temperature and TIF was collected. The volume of TIF was recorded and stored at −20 °C until assayed for androgens (T+DHT).
Primary testicular culture
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 −80 °C for steroid extraction and kept until extraction process (please see below). The remaining parts of each testes were divided into two halves and each half was placed individually in a vial with 1 mL Medium 199-0.1% BSA (Medium 199 containing Earl's salts, l-glutamine, sodium bicarbonate, 20 mm HEPES, antibiotics; pH 7.4, enriched with 0.1% BSA). The left half of testis was incubated only with medium, whereas the right half of testis was incubated with hCG (50 ng/mL) or isoproterenol (β-ADRs agonist). The contents of all incubation vials were gassed with 95% O2–5% CO2 and incubated for 60 min at 34 °C in a shaking water bath (120 oscillations/min). At the end of incubation, testicular preparations with medium were transferred to tubes, centrifuged at 1500 g for 5 min at 4 °C and individual samples of supernatants were stored at −20 °C until assaying for androgens (T+DHT) levels, while the tissue weight in pellet was measured and used as a source of RNA and proteins.
Extraction of steroids from testicular tissue
After TIF collection, pieces of testes from control and Doxa-treated groups were quickly cut, frozen, weighted and mechanically homogenized in phosphate-buffered saline (1xPBS, 1 : 5 w/v), following the extraction with three volumes (1 : 3 v/v) of diethyl ether three times. After evaporation of diethyl ether dry pellets were resuspended for determination of androgen content (Qamar et al., 2010; Janjic et al., 2012; Stojkov et al., 2012).
Preparation of purified Leydig cell and ex vivo cAMP/cGMP, NO and androgen (T+DHT) production
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, 2010a,b; Kostic et al., 2008, 2010, 2011). 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, 2010a,b; Kostic et al., 2008, 2010, 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 dissociated cells were diluted in 20 mL cold M199-0.5% BSA and placed on ice for 5 min to allow the seminiferous tubules to settle before filtering the supernatant through Mesh No.100 (Sigma Inc). The resulting cell suspension was centrifuged at 160g for 5 min at room temperature, and then the cell pellet was washed twice and resuspended in a corresponding amount (5 mL per testis) of DMEM/F12-0.1% BSA. 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, 2010a,b; Kostic et al., 2008, 2010, 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 10× 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 × 106 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. Fractions containing Leydig cells were collected from the 1.080/1.065 g/mL and 1.065/1.045 g/mL interfaces, washed in 50 mL M199-0.1% BSA and centrifuged at 200g for 5 min at room temperature. The cells were resuspended in a corresponding amount (2.5 mL per testis) of culture medium (DMEM/F12 supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin) and counted. 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, 2010a,b; Kostic et al., 2008, 2010, 2011). These data were not showed and served to us just as internal control. Purified Leydig cells were plated in 90 mm Petri dishes (5 × 106 cells in 5 mL culture medium per dish) and three to five replicates were cultured for secretion and expression analysis. After 2 h, cell-free media was collected and stored at −80 °C prior to the measurements of cAMP/cGMP and steroids levels in medium and cAMP/cGMP in cell content.
For extraction of cyclic nucleotide from content, Leydig cells were scraped with ethanol (Andric et al., 2010a,b; Kostic et al., 2011).
For the expression analysis of transcripts/proteins Leydig cell lysates were used as a source of RNA/protein.
Hormones, cAMP/cGMP and NO measurement
Progesterone (PROG), androgens (T+DHT), estradiol (E2) and LH levels were measured by radioimmunoassay (Andric et al., 2007, 2010a,b; Kostic et al., 2008, 2010, 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., 2008, 2010, 2011). Levels of androgens in serum or medium are referred to as testosterone+dihydrotestosterone (T+DHT), because the anti-testosterone serum No.250 showed 100% cross-reactivity with DHT (Andric et al., 2007, 2010a,b; Kostic et al., 2008, 2010, 2011). All samples were measured in duplicate in one assay (the sensitivity: 6 pg per tube; the intra-assay coefficient of variation: 5–8%; the inter-assay coefficient of variation: 7.5%). Estradiol levels in all samples were measured by RIA (Kostic et al., 2011), using the anti-estradiol serum No.244 already described (Korenman et al., 1974), also in duplicate in one assay (the sensitivity: 5 pg; the intra-assay coefficient of variation: 7.7%; the inter-assay coefficient of variation: 8.3%). For serum LH levels, all samples were measured in duplicate, in one assay (sensitivity less than 0.14 ng/mL; intra-assay coefficient of variation 4.2%; the inter-assay coefficient of variation: 6.8%), by RIA according to the manufacturer's protocol [ALPCO Diagnostic-LH (Rat) RIA] and the minimum detectable concentration has been assayed at 0.1 ng/mL (Andric et al., 2010a,b; Kostic et al., 2011). Level of cAMP in medium or in cell content of scraped purified Leydig cells was measured by the cAMP EIA Kit that permits cAMP measurement with a limit of quantification of 0.1 pmol/mL (at 80% B/B0) and IC50 of approximately 0.5 pmol/mL for acetylated cAMP samples (Kostic et al., 2008, 2011; Andric et al., 2010b). Level of cGMP in medium or in cell content of scraped purified Leydig cells was measured by the cGMP EIA Kit that permits cGMP measurement typically with the limit of quantification of 0.07 pmol/mL (at 80% B/B0) for acetylated cGMP (Andric et al., 2007, 2010a,b; Kostic et al., 2010). For measurement of nitrite (stabile metabolic product of NO) levels in the medium, sample aliquots were mixed with an equal volume of Griess reagent and the absorbance was measured at 546 nm (Green et al., 1982). Nitrite concentrations were determined relative to a standard curve derived from increasing concentrations of sodium nitrite (Andric et al., 2007, 2010a,b; Kostic et al., 2010).
RNA isolation and cDNA synthesis
Total RNA from purified rat Leydig cells and testes, were isolated using RNeasy kit reagent following a protocol recommended by the manufacturer (Quiagen, Valencia, CA, USA). Its concentrations and purity were determined spectrophotometrically (see Materials and methods in Table S5a & S5b). To eliminate residual genomic DNA, RNA samples (2 μg of total RNA) were treated with 2 IU DNase-I in a 20 μL reaction mixture. Following DNase-I treatment, the first strand cDNA was synthesized in duplicate reactions for each RNA sample and total RNA from each sample were reverse transcribed into cDNA in a 20 μL reaction mixture containing oligo (dT)18 primer and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). An aliquot of 5 μL of the RT reaction product (25 ng RNA calculating on starting RNA) was amplified with PCR reagent system. Negative controls consisting of non-reverse transcribed samples were included in each set of reactions. Quality of RNA and DNA integrity, were checked by using primers for RS16, GAPDH and β-actin, as described before by our group (Andric et al., 2007, 2010a,b; Kostic et al., 2008, 2010, 2011).
Real time polymerase chain reaction and relative quantification
The relative expression of the genes (Bustin, 2002) was quantitated by real time polymerase chain reaction (PCR) and two types of chemistries used to detect PCR products: SYBR®Green-based and TLDA® (TaqMan Low Density Array)-based detection.
SYBR Green: The relative expression of the genes by using SYBR Green for amplicon detection and ROX as an internal reference dye was performed as previously described (Andric et al., 2010a,b; Kostic et al., 2010, 2011). Standard PCR settings were used in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) in presence of an aliquot of 5 μl of the RT reaction product (25 ng RNA calculating on starting RNA) as well as specific forward (F) and reverse (R) primers. The primers were designed by using software Primer Express 3.0 (Applied Biosystems) and full genes sequences from National Center for Biotechnology Information Entrez Nucleotide database (www.ncbi.nlm.nih.gov/sites/entrez). The primers sequences used for real time PCR analysis including GenBank accession codes for full genes sequences are given in Supplemental Materials and methods (Tables S1–S5). Gapdh were also measured in the same samples and used to correct variations in RNA content among samples. A melt curve analysis was performed to ensure a single product was generated. The relative quantification of gene expression was calculated using ABI Prism 7900HT sequence detection system software and Relative Quantification Manager (Applied Biosystems). Relative quantification of each gene was performed in duplicate, three times for each gene and twice for each of three independent in vivo experiments (Andric et al., 2010a,b; Kostic et al., 2010, 2011).
TaqMan Low Density Array Rat Phosphodiesterase Panel Assays: The expression of the genes for PDEs in Leydig cells obtained individually from all rats was analysed in relative quantification real time PCR by using the TLDA Rat Phosphodiesterase Panel Assay and ABI Prism 7900HT Sequence Detection System and Relative Quantification Manager Software as described previously by our group (Andric et al., 2010a,b; Kostic et al., 2010, 2011). The Gapdh/Actb genes were used as endogenous controls and quantitated in the same real time PCR as a part of TaqMan Low Density Rat Panels, and then used to correct for variations in RNA content among samples. Each sample was run in duplicate or triplicate, three times for each gene, for each of three independent in vivo experiments.
Protein extraction and Western blot analysis
After incubation, Leydig cells (5 × 106 per well) were washed twice with ice-cold PBS and lysed in a 1 mL buffer containing 20 mm HEPES, 10 mm EDTA, 40 mm β-glycerophosphate, 1% tergitol, 2.5 mm MgCl2, 1 mm dithiothreitol, 0.5 mm 4-(aminoethyl)-benzenesulfonyl fluoride hydrochloride, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and a cocktail of phosphatase inhibitors (0.05 mm (-)-P-bromotetramisol oxalate, 10 μm cantharidin, and 10 nm microcystinLR; pH 7.5). Testes were washed with ice-cold PBS and lysed in the same lysis buffer (Andric et al., 2007, 2010a,b; Kostic et al., 2008, 2010, 2011). Protein concentrations were estimated by the Bradford method using BSA as a standard (Bradford, 1976).
For Western blot analysis, equal amounts of proteins were mixed (1 : 1 v/v) with the SDS-PAGE loading buffer, denatured for 5 min at 95 °C, and loaded on SDS-PAGE 16% gels. All gels were analysed by one-dimensional SDS-PAGE, using a discontinuous buffer system, and proteins were transferred to a PVDF membrane, Immobilon-P (EMD Biosciences, San Diego, CA, USA), using a wet transfer, according to the manufacturer's recommendation. The immunodetection of the StAR protein was performed by using antisera against StAR protein generously supplied by Professor Douglas Stocco (dilution 1 : 1000; Clark et al., 1994), HSD3B was detected with antibody kindly provided by Professor Ian Masson (dilution 1 : 1000; Bain et al., 1991; Abbaszade et al., 1997). The PRKG1 was detected by using rabbit polyclonal antibody recognizing type α/β isoforms of PRKG1 (Calbiochem; dilution 1 : 1000), whereas actin was detected by using actin detection kit (Oncogene Research Product; dilution 1 : 5000). The reactive bands were always determined with a luminol-based kit (Pierce, Rockford, IL, USA), and the reaction was detected by an enhanced chemiluminescence system, using X-ray film. The immunoreactive bands were analysed as two-dimensional images using the Image J (version 1.32; http://rsbweb.nih.gov/ij/download.html). The OD of images is expressed as volume (OD x area) adjusted for the background, which gives arbitrary units of adjusted volume (Andric et al., 2007, 2010a,b; Kostic et al., 2008, 2010, 2011).
For in vivo studies the results represents group means ± SEM values of individual variation (six rats per group per experiment). For ex vivo measurement data represent mean ± SEM from three to five independent replicates. The results from each experiment were analysed by Mann- PVDF–Whitney's unpaired nonparametric two-tailed test (for two point data experiments), or, for group comparison, a one-way anova, followed by Student–Newman–Keuls multiple range test.
To mimic the most likely route of human exposure to selective α1-ADRs blockers, rats were subjected to po-administration of Doxa in clinically relevant dose (5 mg/kg BW) described before (Kaye et al., 1986), for once (1×Doxa), or for two (2×Doxa) or 10 (10×Doxa) consecutive days.
Doxazosin po-application impaired levels of circulating LH/testosterone and disturbed androgens homeostasis in testis
Po-application of Doxa, widely used selective α1-ADRs blocker, for once, or two or 10 consecutive days caused significant decrease in the level of LH (Fig. 2A) and androgens (Fig. 2B) in serum. The trend of recovery of circulating LH level was registered (Fig. 2A).
The androgen levels in circulation are outcome of ‘interplay’ between LH/LHR interaction, testicular microcirculation, TIF volume/content, as well as steroidogenic machinery homeostasis within the Leydig cell influenced by plethora signals. To try to dissociate the direct effects of Doxa treatment from these paracrine and/or autocrine we followed the TIF volume and androgens contents in TIF, testicular tissue and produced in medium by testicular preparation. Results showed that only 1×Doxa treatment significantly decreased TIF volume, although repeated Doxa application did not have effect (Fig. 3A). The androgens levels in total TIF volume decreased after 1×Doxa/2×Doxa treatment, but returned to control levels after 10×Doxa application (Fig. 3B). A similar profile was registered in androgen levels extracted from pieces of testicular tissue: decrease in 1×Doxa/2×Doxa rats, but back to control levels in 10×Doxa rats (Fig. 3C). In line with these results were also basal androgens levels produced ex vivo in medium by testicular preparations isolated from control and Doxa-treated rats. The basal androgen production by testes decreased after 1×Doxa/2×Doxa treatment, but returned to control levels after 10×Doxa application (Fig. 3D, lower part). When testes were challenged with hCG to estimate steroidogenic capacity, declined androgens productions were registered in all Doxa-treated rats (Fig. 3D, upper part).
The steroidogenic activity of isolated and purified Leydig cells obtained from rats after in vivo treatment with Doxa was examined by ability of isolated Leydig cells to produce steroid hormones ex vivo. Results showed that Doxa po-application caused biphasic effect on basal progesterone (Fig. 4A) or androgen (Fig. 4B) production ex vivo compared to undisturbed control (Fig. 4A and B). 1×Doxa treatment caused significant increase, whereas 10×Doxa decreased progesterone production by Leydig cells ex vivo (Fig. 4A). In terms of testosterone, increased production was registered in Leydig cells isolated from 1×Doxa or 2×Doxa-treated rats, whereas 10×Doxa decreased androgen production (Fig. 4B). All types of Doxa treatment significantly stimulated, in time-dependent manner, estradiol production by Leydig cells (Fig. 4C).
Steroidogenic capacity of Leydig cells isolated from control and Doxa-treated rats was examined by ability of isolated Leydig cells to respond with steroids production on stimulation of LHR by hCG. Results showed similar profile like in absence of hCG i.e. LHR activation. The Doxa treatment caused biphasic effects on hCG-stimulated progesterone and androgen (T+DHT) productions ex vivo (Fig. 4A and B) compared to undisturbed control (Fig. 4A and B). Significantly increased progesterone production was registered in Leydig cells isolated from 1×Doxa, whereas 10×Doxa decreased level of progesterone produced ex vivo (Fig. 4A). The production of androgens significantly increased in Leydig cells isolated from 1×Doxa or 2×Doxa treated rats, whereas 10×Doxa decreased levels of androgens produced ex vivo (Fig. 4B). Estradiol production in presence of hCG was significantly stimulated in Leydig cells isolated from rats exposed to Doxa, independently of treatment duration (Fig. 4C).
As all types of Doxa treatments affected steroidogenic activity and capacity of Leydig cells, a transcriptional analysis of elements involved and/or related with steroidogenic function of Leydig cells was performed.
Doxazosin po-application disturbed the transcription profile of steroidogenic machinery elements and transcription factors in Leydig cells
The purified Leydig cells obtained from controls and Doxa-treated rats were a source of mRNA for transcriptional analysis (for primers sequences see Table S1 in the Supplemental Materials and methods) and proteins for Western blot analysis.
Results of the real time PCR analysis showed that 2×Doxa and 10×Doxa po-application increased the level of Lhr transcript in Leydig cells (Fig. 5A) compared to undisturbed control (Fig. 5A). The expression of Scarb1, Cyp11a1 and Cyp17a1 transcripts was reduced in Leydig cells of rats exposed to Doxa independently of the treatment duration. Only 10×Doxa po-application increased the level of Tspo and Hsd3b5 transcript in Leydig cells, whereas Hsd3b1 remained unchanged. In the same cells, the expression of steroid dehydrogenase specific for androgen production (Hsd17b3, Hsd17b4) was stimulated (Fig. 5A). Transcription of StAR and Cyp19a1 in Leydig cells from Doxa-treated rats remained unchanged (Fig. 5A). The similar transcription profiles of steroidogenic enzymes were registered in testes under the basal or hCG-stimulated conditions (Supplemental Figure S1A), just Ct values were higher except for Cyp19a1 (see Table S1A in the Supplementary Materials and methods). Because the StAR and HSD3B are the main markers of Leydig cells, protein expression analysis was performed. Although level of Star transcript remained unchanged in Leydig cells from Doxa-treated rats, StAR protein level significantly increased in Leydig cells from 2xDoxa and 10×Doxa groups. Only 10×Doxa po-application stimulated the expression of HSD3B protein in Leydig cells (Fig. 5C). Similar patterns were registered in testes after basal or hCG-stimulated conditions and it seems that hCG potentiated an effect of Doxa on StAR protein expression level (Supplemental Figure S1C).
Real time PCR analysis for some transcription factors involved in regulation of steroidogenic genes expression showed significant increase of Dax1 transcript in Leydig cells of rats exposed to 10×Doxa, whereas Sf1 (transcriptional antagonist of Dax1) remained unchanged. In the same cells, level of Esr1 transcript increased. The expressions of transcripts for Jak2 or Stat5a (known regulators of Hsd3b promoter), Arr19 and Ar were not affected by Doxa-treatment (Fig. 5B). Only transcription profile of Dax1 was different in whole testes than in Leydig cells i.e. remained unchanged Dax1 (Supplemental Figure S1B).
Doxazosin po-application disturbed cAMP signalling pathway in Leydig cells
Considering the importance of cAMP-PRKA signalling pathway for steroidogenesis (reviewed in Dufau, 1998; Hansson et al., 2000; Payne & Hales, 2004; Tsai & Beavo, 2011, 2012), the real time PCR analysis for elements of this signalling in Leydig cells from control and Doxa-treated rats was performed by using the specific primers (see Table S2 in the Supplemental Materials and methods).
Results showed significant increase in the expression of soluble adenylyl cyclases Adcy10 in Leydig cells from rats exposed to 10×Doxa (Fig. 6A) compared to undisturbed control (Fig. 6A). Doxa po-application did not change the levels of Adcy3, Adcy5, Adcy6, Adcy7 and Adcy9 transcripts in Leydig cells (Fig. 6A).
10×Doxa po-application significantly increased the expression level of the gene encoding PRKA catalytic subunit A, Prkaca, although all other types of Doxa po-application significantly reduced regulatory subunit Prkar2b. The transcriptional levels of Prkacb, Prkar1a and Prkar2a in Leydig cells were not considerably affected by Doxa po-application (Fig. 6B).
In the same cells, transcription of cAMP-specific PDEs in Leydig cells from Doxa-treated rats was disturbed (Fig. 6C). The level of transcripts for Pde7b and Pde8b were significantly reduced in Leydig cells from rats exposed to 1×Doxa or 2×Doxa. On the contrary, 10×Doxa po-application significantly increased the Pde4d. The levels of Pde4a, Pde4b, Pde4c, Pde7a and Pde8a were not significantly affected by Doxa po-treatments (Fig. 6C).
The product of ADCYs and PDEs ‘interplay’, cAMP, followed transcriptional profile. The levels of cAMP production significantly increased in medium and in content of Leydig cells from rats exposed to 1×Doxa or 2×Doxa, but ‘back’ to normal level in Leydig cells from 10×Doxa rats (Fig. 6D).
Transcriptional profiles of cAMP signalling elements in testes (Supplementary Figure S2) were different than in Leydig cells, since all cells poses cAMP signalling elements (Supplemental Table S2A).
Doxazosin po-application disturbed cGMP signalling pathway in Leydig cells
Considering the importance of ‘cross talk’ between cAMP and cGMP signalling pathways for cell homeostasis and the role of cGMP-PRKG-PDE signalling in steroidogenesis (reviewed in Tsai & Beavo, 2011), the real time PCR analysis for elements of NO-cGMP signalling in Leydig cells from control and Doxa-treated rats was performed by using the specific primers (see Table S3 in the Supplemental Materials and methods).
Results suggested that Doxa po-application more affected transcriptional profile of cGMP (Fig. 7) than cAMP (Fig. 6) signalling pathway elements in Leydig cells. All types of Doxa-treatment significantly increased the level of transcript for Nos1 and Nos3. Oppositely, in the same cells, the level of Nos2 was reduced (Fig. 7A) compared to undisturbed control (Fig. 7A).
10×Doxa po-application significantly increased the expression level of the genes encoding GUCY1 subunits α1 (Gucy1a1), β1 (Gucy1b1), and β2 (Gucy1b2), whereas Gucy1a2 remained unchanged (Fig. 7A).
The expression of the transcript for soluble form of PRKG, Prkg1, was significantly stimulated in Leydig cells from rats exposed to 10×Doxa. The level of transcript for membrane form of PRKG, Prkg2, increased in Leydig cells from 1×Doxa and 2×Doxa rats (Fig. 7B). Because the PRKG is the main effector of cGMP signalling, protein expression analysis was performed. Results showed the significant increase of the PRKG protein level in Leydig cells isolated from rats exposed to 10×Doxa po-application (Fig. 7B, inset).
In the same cells, transcriptional profile of cGMP-specific and dual-specific PDEs in Leydig cells from Doxa-treated rats were not disturbed in a great fashion (Fig. 7C). The only changes were the increased level of the transcripts for cGMP-specific Pde6a and dual specific Pde2a in Leydig cells from rats exposed to 10×Doxa po-application. The levels of other cGMP-specific (Pde5a, Pde6d, Pde9a) and dual-specific (Pde1a, Pde1b, Pde1c, Pde3a, Pde3b, Pde10a) PDEs were not significantly affected by Doxa po-treatments (Fig. 7C).
The product of GUCYs and PDEs ‘interplay’, cGMP, was also measured ex vivo in Leydig cells from Doxa-treated animals. The levels of cGMP released in medium or stayed in content significantly increased in Leydig cells from rats exposed to 1×Doxa, but ‘back’ to normal level in Leydig cells from 2×Doxa and 10×Doxa rats (Fig. 7D).
Because transcription of NOS in Leydig cells from all Doxa-treated groups was affected in a great fashion, the product of NOS activity, NO was followed ex vivo by measuring the nitrites (the stable oxidative product of NO). Results showed significant increase of NO level released in medium by Leydig cells from rats exposed to 1×Doxa or 2×Doxa, but ‘back’ to normal level in Leydig cells from 10×Doxa rats (Fig. 7E).
Transcriptional profiles of some of the elements of NO-cGMP signalling in testes (Table S3A in the Supplementary Materials and methods) were different than in Leydig cells all testicular cells poses NO-cGMP signalling elements (Supplementary Figure S3).
Ten times repeated Doxa po-application changed the transcriptional ‘signature’ of ADRs and β-adrenergic receptor kinase 1 (Adrbk1) in Leydig cells
As Doxa is a selective α1-ADRs blocker, it was interesting to analyse the effects of acute (1×Doxa) or repeated (2×Doxa, 10×Doxa) po-application on the transcription profile of all α- and β- ADRs in Leydig cells, especially because some of them activate cAMP signalling.
A rather unexpected and interesting finding was a significant increase in the levels of transcripts for α-ADRs (Adra1a, Adra1b, Adra1d) and β-ADRs (Adrb1, Adrb2, Adrb3) in Leydig cells from rats exposed to 10×Doxa. The level of ADRs transcripts remained unchanged after 1×Doxa/2×Doxa (Fig. 8A,B).
The expression level of the gene encoding β-ADRs kinase 1 (Adrbk1) was inhibited in Leydig cells from rats exposed to 2×Doxa or 10×Doxa, whereas Adrbk2 remained unchanged (Fig. 8C).
The physiological significance of Doxa-induced changes in the transcriptional ‘signature’ of ADRs was proven by β-ADRs agonist, isoproterenol, which stimulated ex vivo androgen production by testicular preparations and this effect was more pronounced in testes from Doxa-treated rats (Fig. 8D).
In this study, we have demonstrated that acute (1×Doxa) and repeated (2×Doxa, 10×Doxa) po-application of Doxa: (i) decreased the circulating LH and testosterone (T+DHT) levels; (ii) differently and biphasically affected steroidogenic activity/capacity of testes and Leydig cells; (iii) disrupted the steroidogenic machinery homeostasis; (iv) the cAMP signalling; (v) the cGMP signalling and (iv) 10×Doxa significantly changed the transcriptional milieu of ADRs in Leydig cells.
The reduced levels of LH in serum of rats exposed to Doxa are in accordance with findings showing that local application of Doxa into the preoptic/anterior hypothalamic area caused a reduction of average LH secretion and reduced markedly LH pulsatility (Jarry et al., 1990). In parallel, the decrease in the level of circulating testosterone in Doxa-treated rats was registered. These results are in line with findings showing that the in vivo administration of Doxa (10 mg/kg BW for 15 days) highly decreases circulating levels of testosterone in rats through a mechanism involving the testicular renin–angiotensin system and that the changes identified are not a consequence of changes in gonadotropin secretion (de la Chica-Rodríguez et al., 2008). On the contrary, combination Finansteride+Doxa (25 mg/kg BW/day for 3 of 30 days) induced a transient increase in plasma testosterone and a permanent reduction in DHT (Justulin et al., 2010). Novelty of the results presented in this study is the finding that levels of circulating LH and testosterone are affected even after the first or second application. On the basis of the above-mentioned finding and the fact that Doxa does not penetrate the blood-brain barrier, we could hypotothesize that the reduction of LH level could be, at least in part, the consequence of changed pituitary microcirculation by Doxa-induced α1-ADRs blockade. Doxa-treatment might change blood flow in the testes. If present, this phenomenon would decrease the amount of LH delivered to Leydig cells and impair testosterone release from the testes and together with reduced circulating LH level can decrease circulating testosterone level. The picture about systemic Doxa effect could be even more complicated as it has been shown that endothelial cells in the testes of rats carried the same receptors for LH/hCG as the receptors found on the Leydig cells. The significance of the functional endothelial LH receptor is in the response of the Leydig cells to rapid changes in LH concentrations in blood plasma through a local paracrine mechanism (Setchell et al., 2002). In addition, it was found that the topical administration of phenylephrine (selective α1-ADRs agonist) gel rapidly and significantly decreased testicular blood flow, an observation in agreement with results obtained after the intratesticular injection of epinephrine. In addition, testicular blood flow exhibits vasomotion. The mechanisms controlling vasomotion are complex and incompletely resolved, but in the rat testis, it is known that vasomotion is testosterone-dependent, is not seen prior to puberty, and is responsive to catecholamines whether peripherally infused, or microperfused around restricted fields of the testicular microvasculature (for references please see Damber et al., 1987; Damber & Bergh, 1992; Damber et al., 1992). The numerous papers (Sharpe, 1979, 1980, 1981, 1984; Setchell & Sharpe, 1981; Sharpe & Cooper, 1983; Bergh et al., 1986; Veijola & Rajaneimi, 1986; Widmark et al., 1989) showed that measurement of the volume of TIF, the rate of its formation (i.e. capillary permeability) and clearance is a valid parameter of testicular vasculature, because not only this controls the rate of transport of LH and other compound to the Leydig cells but it may also affect the concentration of testosterone in TIF by altering the volume of fluid into which the androgen is secreted. The collection of TIF is of considerable potential value, both as an indicator of changes in capillary wall permeability (i.e. by measurement of interstitial fluid volume) and as a means of assessing changes in the interstitial hormonal environment of the testis (Setchell & Sharpe, 1981). The gonadotropins cause large increase in the volume of interstitial fluid within the testis (Sharpe, 1979, 1980, 1981, 1984), because of an increased permeability of testicular capillaries (Sharpe & Cooper, 1983). TIF formation, like testosterone secretion, is a major regulatory aspect of testicular function and is affected by testicular suppressants such as alcohol, morphine, isosorbide dinitrate, imidazoles (for references please see Adams et al., 1998). TIF volumes have been measured to assess TIF formation and vascular perfusion inside the testes that control access of important substances to testicular cells and structures. Possible vascular effects of Doxa may also be involved in TIF formation effects. Altered blood flow appears to be related to TIF formation, but it is not clear how the possible effects of Doxa on the testicular vasculature and the larger vascular beds of other organs interact to alter TIF formation (Sharpe & Cooper, 1983). Doxa may exert important vascular effects (Dell'Omo et al., 2005) that could alter TIF volumes because TIF volumes reflect testis vascular perfusion and TIF formation.
The androgen levels in circulation are outcome of ‘interplay’ between LH/LHR interaction, testicular microcirculation, TIF volume/content, as well as steroidogenic machinery homeostasis within the Leydig cell influenced by plethora signals. To try to dissociate the direct effects of Doxa treatment from these paracrine and/or autocrine, we followed the TIF volume and androgens contents in TIF, testicular tissue and produced in medium by testicular preparation and isolated Leydig cells. Results showed that only single Doxa treatment significantly decreased TIF volume, whereas repeated Doxa application did not have effect. The 1×Doxa was found to cause significant decreases in TIF volumes, which have been validated as a measure of TIF formation. Decreases in TIF volumes are indicators of decreases in testicular vasopermeability and testicular blood flow and of limited access of blood cells, hormones and nutrients to their sites of action within the testes. Just as the TIF-suppressant effects of ethanol, morphine and nitric oxide suggest important testicular effects of those agents, the TIF-suppressant effects of 1×Doxa/2×Doxa suggest another mechanism for potential adverse effects of Doxa on male reproductive function and fertility, indicating that the TIF volume parameter is not a superfluous parameter in studies focusing on the testicular effects of Doxa. TIF testosterone levels directly reflect testosterone secretion because testosterone is secreted into TIF before it is transported to the blood stream. The androgens levels in total TIF volume, extracted from testicular tissue and released in medium decreased after 1×Doxa treatment, but returned to control levels after 10×Doxa application. Discrepancy between androgens levels in TIF and circulation could be explained by the possibility that Doxa application could eventually affect entrance of androgens from TIF to testicular microvasculature by restriction in movement of testosterone across the endothelial barrier (Setchell et al., 2002), especially because the rat TIF contains mediators of vasopermeability (Tapanainen et al., 1990). In addition, although there is no published data about effect of Doxa on androgens/steroids metabolic clearance it is possible that systemic in vivo Doxa application could affect metabolic rate of testosterone as Doxa affected vasculature, metabolism and hepatic vascular resistance (Dell'Omo et al., 2005). Accordingly, decrease in circulating androgens after 10×Doxa treatment could be the consequence of some ‘extra-testicular’ effect(s) i.e. systemic effect(s): either changes in permeability of testicular vasculature resulting in decreased entrance of androgens from TIF to circulation, and/or disturbance in ABP level and/or change in the androgens metabolic clearance or all those mentioned.
When testes were challenged with hCG to estimate steroidogenic capacity, declined androgens productions were registered in all Doxa-treated rats. These results could support the idea that in addition to effects on testicular circulation, Doxa affects the testosterone production. It is important to emphasize that the structural organization of the testis may account for the integration of autocrine and paracrine factors to regulate testicular steroidogenesis and TIF characteristics and/or content. Although testosterone is an exclusive product of Leydig cells, secretory products originated from seminiferous tubule and/or Sertoli cells and/or non-steroidogenic cells in the testicular interstitium (Klinefelter et al., 1987; Saez, 1994; Hutson, 2006) could take place in regulation of TIF characteristics. Accordingly, reported changes in the TIF and testosterone content in testis could be the consequence of the complex effects of secretory contribution of the different cell types including, besides HSD3B-positive Leydig cells, also macrophages, endothelial cells, fibroblasts, peritubular cells, Sertoli cells etc. Results of this study on Leydig cells isolated from control or Doxa-treated rats showed different effects: 1×Doxa/2×Doxa applications significantly increased basal/hCG-stimulated androgens production by Leydig cells ex vivo, whereas 10×Doxa decreased. The discrepancy between levels of androgens in serum, TIF, testes, and those produced ex vivo by Leydig cells could be explained by the fact that isolation/purification/stimulation of Leydig cells takes approximately 12 h when isolated Leydig cells are removed from the biologically active inhibitory and/or stimulatory paracrine compounds released from the seminiferous tubules and/or macrophages, endothelial cells, fibroblasts, peritubular cells, Sertoli cells (Klinefelter et al., 1987; Saez, 1994; Hutson, 2006). In summary, the complex structural organization of the testes and coexistence of multiple regulatory mechanisms that control testicular cells and microvasculature could provide a degree of redundancy in the maintenance of testicular steroidogenesis, a crucial component of the reproductive process. It is also conceivable that this multifactorial system could reflect the gradation from simple to more complex neuroendocrine control systems for regulating hypothalamo-pituitary function and gonadal activity. The increased steroids productions and hCG response of Leydig cells from 1×Doxa/2×Doxa rats were followed by the increased production of the main stimulator of steroidogenesis, cAMP, whereas cGMP increased only after 1×Doxa treatment. The discrepancy between androgen production by testicular and Leydig cells cultures could be explained by the complex structural organization of the testes and coexistence of multiple regulatory mechanisms that control testicular cells. Removed from the testes and after purification, isolation and plating, the Leydig cells are not any more under the influence of plethora extracellular neural, endocrine and paracrine signals. In this particular case, recovery of androgen production by testicular preparation from 10×Doxa group could be the consequence of preponderance stimulatory vs. inhibitory paracrine/autocrine signals from surrounding cells. Although steroids productions and hCG response of Leydig cells from 10×Doxa decreased, levels of cAMP/cGMP remained unchanged. This results of ex vivo production could be compared with findings that the in vitro Doxa-application inhibited aldosterone and corticosterone release in a cell suspension of porcine adrenocortical cells and changed the release of nine steroids, indicating interference with enzymes of the aldosterone biogenesis pathway involved in C18-oxidation/C21beta-hydroxylation (Jager et al., 1998). The progesterone is rapidly metabolized by Leydig cells and there is possibility that systemic in vivo Doxa application could affect metabolism of progesterone and/or each of the steroid precursors (cholesterol, OH-cholesterol, pregnenolone, progesterone, androstenedione) or all of them as it is well known that Doxa has direct and indirect effects on lipid metabolism (Pool, 1991). In this study, the inhibitors of progesterone metabolism were not used to monitor the real picture/situation without disturbing the homeostasis of the Leydig cells by preventing the metabolism of progesterone. Same condition applies for other steroid precursors (cholesterol, OH-cholesterol, pregnenolone, androstenedione).
Our results showed that the most sensitive components of Leydig cells steroidogenic machinery on Doxa po-application are Scarb1 and the key CYP enzymes (Cyp11a1, Cyp17a1). The expression of these transcripts was inhibited in response to all types of Doxa po-application. The decreased Cyp11a1/Cyp17a1 transcription could be the consequence of the decreased level of the circulating LH after Doxa treatment. The rather unexpected finding of this study was that 10×Doxa induced increase of Tspo and Hsd3b5 transcripts, without changing the Hsd3b1 or Star transcripts in Leydig cells. In the same cells, the protein expressions for the main regulators of steroidogenesis, StAR and HSD3B, were also stimulated. The expression of some transcription factors involved in regulation of steroidogenic gene expression showed significant increase of Dax1 transcript in Leydig cells of rats exposed to 10×Doxa, whereas Sf1 (transcriptional antagonist of Dax1) remained unchanged. As a nuclear receptor, DAX1 has been shown to function as a transcriptional repressor, particularly of pathways regulated by other nuclear receptors, such as SF1. The exact mechanism of DAX1 action during adulthood and critical stages of development are not fully understood (Jadhav et al., 2011). It is well known that regulation of steroidogenic genes transcription is quite complicated, multifactorial, tissue-specific, constitutive, cAMP-dependent/independent and it includes a broad range of different transcription factors. It was demonstrated that optimal steroidogenic capacity is achieved if transcription pressure is applied on all steroidogenic genes (reviewed in Lavoie & King, 2009; Martinez-Arguelles & Papadopoulos, 2010; Midzak et al.,2011; Simard et al., 2005). Although transcription of steroidogenic genes is regulated by cAMP-dependent transcription factors, like CRE/CREB (cAMP response element-CRE and CRE-binding protein-CREB), each steroidogenic gene itself requires specific cAMP responsive sequences in the promoter region of each gene. These sequences will bind precisely defined and very specific transcription factors, as well as co-activators for the regulation of particular steroidogenic genes expression. In Leydig cells, STAR, CYPs, and HSD3B, cAMP-regulated promoter activity primarily involves SF1, CREB/CREM, GATA4 and is modulated by other transcriptional cofactors. The orphan nuclear receptor and transcription factor SF1 (now termed NR5A1) is expressed in all steroidogenic tissues. SF1 has been shown to increase expression of the steroidogenic machinery by binding to its response element site found in the promoter regions of the genes encoding for STAR, CYPs and HSD3B (reviewed in Lavoie & King, 2009; Martinez-Arguelles & Papadopoulos, 2010; Midzak et al., 2011; Simard et al., 2005). Although our results demonstrated that Doxa differently affect steroidogenic genes expression we were not able to detect changes in Sf1 transcript. The lack of Doxa to change Sf1 expression in purified Leydig cells suggest that other transcription factors/signalling pathways, alone or with cAMP- CREB/CREM are more affected by Doxa. It has been shown that the po-application of Doxa (30 mg/kg BW/day) for 4 weeks attenuated urogenital expression of Creb1 in spontaneously hypertensive rats (Yono et al.,2009). Although our results showed that Doxa-treatment did not change Arr19 transcript, it is noteworthy to mention that recent studies showed disturbance of Leydig cells steroidogenesis by ARR19, an anti-steroidogenic factor negatively regulated by LH-cAMP signalling (for references please see Qamar et al., 2010).
Considering the importance of cAMP-PRKA signalling pathway for Leydig cells steroidogenesis, it is important to point out that 1×Doxa/2×Doxa treatment increased cAMP production in Leydig cells, whereas 10×Doxa did not have any effect. Maybe it is note worthy that the inhibitor(s) of cAMP metabolism, i.e. PDEs inhibitors, were not used because there are many complex cross couplings between signalling pathways (PI3-AKT, Ras-MAPK, PLC-PKC) activated from ADRs and signalling from adenosine receptors (Fenton et al., 2010) that could affect PDEs (Komatsu et al., 2012), so culturing the Leydig cells in media with PDEs inhibitor(s) will more complicate the already complex systemic in vivo effects of Doxa and potentially mask effects of Doxa application. There is not much evidence about cAMP homeostasis after Doxa treatment. The eventual explanation could be that because α1-ADRs are blocked by Doxa, there are more available ligands to occupy β-ADRs, G protein coupled receptors able to activate AC and to increase cAMP production. The physiological significance of Doxa-induced changes in the transcriptional ‘signature’ of ADRs was proven by β-ADRs agonist, isoproterenol. The application β-ADRs agonist stimulated ex vivo androgens production by testicular preparations and this effect was more pronounced in testes from Doxa-treated rats.
Besides cAMP signalling, NO-cGMP pathway is also involved in the regulation of Leydig cell steroidogenesis (reviewed in Ducsay & Myers, 2011). Results of this study showed that NO-cGMP signalling was more affected by Doxa-treatment independent of its duration. The results about stimulated transcription of constitutive nitric oxide synthases (Nos1, Nos3) are in line with recent findings that administration of Doxa (30 mg/kg BW/day for 4 weeks) to the spontaneously hypertensive rats caused an up-regulation of NOS1 in the bladder and penis and NOS3 in the penis (Yono et al., 2007).
Our results clearly showed that Leydig cells responded to Doxa po-application by developing the ‘adaptive’ response on the transcriptional level (Fig. 9). 10×Doxa po-application stimulated cGMP signalling and increased the level of transcripts for ADRs in Leydig cells. The expressions of Adcy10 (the soluble cAMP ‘maker/provider’ in Leydig cell) were stimulated. In the same cells, increased transcription for the cAMP ‘remover’ in Leydig cell (Pde4d) was registered. By this transcriptional signalling scenario, it is possible that the Leydig cell is trying to ‘accelerate’ cAMP-signalling and to switch off the adrenalin-occupied receptors from Gs to Gi (Daaka et al., 1997) to control both the intracellular bursts of cAMP (through the adrenalin-β2ADR activation of Gs and increased Adcy) and the basal cAMP (through the adrenalin-β2ADR/PDE4d Gi-mediated braking effect). Thus, results present in this article provide new molecular/transcriptional base for the Leydig cells response to the systemic blockade of α1-ADRs and could be of possible interest to further investigation in light of post-traumatic stress disorders, hypertension, obesity, metabolic syndrome, LUTS/BHP, ED and disrupted sexual health.
So far, only evidences about effects of Doxa on androgenesis are the levels of circulating androgens. There is no any evidence about status of transcripts, proteins, enzyme activities in any steroid-producing cells. According to our best knowledge, results of this study are the first to be reported presenting the effect of any α1-selective ADRs antagonist on the transcriptional profile of steroidogenic machinery elements (including transcription factors), cAMP and cGMP signalling, as well as, ADRs and β-ADRs kinases in Leydig or any other steroidogenic cells. Eventually, this study could be the solid base for the evaluating Doxa pharmacogenomic data in human reproductive health risk assessment. However, there are many questions that should be answered concerning the effects of systemic Doxa treatment on androgens and/or steroid hormones homeostasis. Some of the questions are how Doxa affects the following: ABP level; metabolic clearance of androgens; transport of steroids from TIF to vasculature; metabolism of steroids precursors; conversion of particular steroid substrate to the next one in steroidogenic chain; activities of steroidogenic enzymes; macrophages (it is well known that macrophages are significant source of 25OH-cholesterole, the steroid precursor able to enter to mitochondria without StAR and Doxa could affect macrophages). Accordingly, to obtain a more complete and accurate picture of the impact of the systemic Doxa application requires additional research.
This work was supported by the Autonomic Province of Vojvodina  and Serbian Ministry of Education and Science . We are very grateful to Professor Douglas Stocco (Texas Tech University) and Professor Ian Masson (University of Edinburgh) for very kind donation of HSD3B antibody for generous and continuous donation of StAR antiserums. We appreciate Professor Gordon Niswender (Colorado State University) for supplying antibodies for RIA analysis.
N.J.S. – helped design the study, performed experiments, analysed data and performed all the statistical analysis, drafted the figures and text, edited the manuscript and approved the final version. M.M.J. – performed experiments, edited the manuscript and approved the final version. T.S.K. – helped design the study, made final figures, helped with data interpretation, critically reviewed and corrected the manuscript for intellectual content and approved the final version. S.A.A. – design the study, analysed and interpreted data, critically reviewed and corrected the manuscript for intellectual content and approved the final version.