Couto et al. Reproductive Biology and Endocrinology 2010, 8:13 http://www.rbej.com/content/8/1/13 RESEARCH Open Access Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: In vivo and ex vivo studies Janaína A Couto1, Karina LA Saraiva2, Cleiton D Barros3, Daniel P Udrisar4, Christina A Peixoto2, Juliany SB César Vieira4, Maria C Lima3, Suely L Galdino3, Ivan R Pitta3, Maria I Wanderley4* Abstract Background: The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats. Methods: Twelve adult male Wistar rats were treated with rosiglitazone (5 mg/kg) administered by gavage for 15 days. Twelve control animals were treated with the vehicle. The ability of rosiglitazone to directly affect the production of testosterone by Leydig cells ex vivo was evaluated using isolated Leydig cells from rosiglitazone- treated rats. Testosterone production was induced either by activators of the cAMP/PKA pathway (hCG and dbcAMP) or substrates of steroidogenesis [22(R)-hydroxy-cholesterol (22(R)-OH-C), which is a substrate for the P450scc enzyme, and pregnenolone, which is the product of the P450scc-catalyzed step]. Testosterone in plasma and in incubation medium was measured by radioimmunoassay. The StAR and P450scc expression was detected by immunocytochemistry. Results: The levels of total circulating testosterone were not altered by rosiglitazone treatment. A decrease in basal or induced testosterone production occurred in the Leydig cells of rosiglitazone-treated rats. The ultrastructural and immunocytochemical analysis of Leydig cells from rosiglitazone-treated rats revealed cells with characteristics of increased activity as well as increased StAR and P450scc expression, which are key proteins in androgen biosynthesis. However, a number of rosiglitazone-treated cells exhibited significant mitochondrial damage. Conclusion: The results revealed that the Leydig cells from rosiglitazone-treated rats showed significant reduction in testosterone production under basal, hCG/dbcAMP- or 22 (R)-OH-C/pregnenolone-induced conditions, although increased labeling of StAR and P450scc was detected in these cells by immunocytochemistry. The ultrastructural study suggested that the lower levels of testosterone produced by these cells could be due to mitochondrial damage induced by rosiglitazone. Background bodily glycemia and lipid control and reduces the con- Rosiglitazone is a PPARg synthetic activator from the centration of plasma androgen in patients with PCOS group of thiazolidinediones (TZDs) often used in the [1-5]. Besides their well-known effects on insulin sensi- treatment of chronic diseases such as type 2 diabetes tivity and energy metabolism, TZDs have also been and other forms of insulin resistance, as seen in polycys- reported to modulate steroid production in gonad tis- tic ovary syndrome (PCOS). Activation of PPARg by sues. For example, TZDs stimulate progesterone secre- TZDs improves insulin sensitivity and, consequently, tion in MA-10 Leydig tumor cells [6,7] and ovarian cells [8]. Opposing effects of TZDs on androgen levels and/ or production in male humans [9-11] and animal mod- * Correspondence: [email protected] 4Department of Physiology and Pharmacology, Universidade Federal de els have been described [7,12-20]. An inhibitory effect of Pernambuco, Recife, 50.670-901, Brazil © 2010 Couto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Couto et al. Reproductive Biology and Endocrinology 2010, 8:13 Page 2 of 9 http://www.rbej.com/content/8/1/13 rosiglitazone on the production of testosterone has been decapsulated testes were incubated in an enzyme solution demonstrated in healthy men [11], while in obese male of 0.5 mg/ml collagenase (Sigma), 0.2 mg/ml soybean tryp- Zucker rats [19] plasma testosterone was not affected by sin inhibitor (Sigma) and 5 μg/ml leupeptin (Sigma) in this TZD. TZDs have also been reported to directly PBS (136.9 mM NaCl, 2.68 mM KCl, 8.1 mM Na2H- inhibit steroidogenic enzymes and steroid secretion in PO4.7H2O, 1.47 mM KH2PO4) containing 0.1% bovine vitro, as evidenced by a decrease in the activity of 17a- serum albumin (PBS/BSA) (BSA, fraction V, ICN Biome- hydroxylase, 17,20-lyase, aromatase and 3b-hydroxyster- dical, California, CA, USA), pH 7.4, in a shaking water oid dehydrogenase [7,12-18]. Although there is evidence bath (20 min, 90 Hz, 34°C). The dispersed testes were sus- of the modulation of testosterone action and/or produc- pended in 50 ml (final volume) PBS/BSA and the disso- tion by TZDs, the effects of oral rosiglitazone treatment ciated tubules were allowed to settle (5 min). The on circulating plasma testosterone and/or production supernatant was filtered and washed with 5 ml PBS/BSA. have not been reported in experimental animal models. The filtered cell suspension was centrifuged (150 × g, 15 The aim of the present study was to determine min, 20°C). The pellet was re-suspended in 5 ml PBS/BSA, whether oral rosiglitazone treatment influences testicular loaded onto the top of a discontinuous Percoll (HE production of testosterone using an ex-vivo model of Healthcare Bio-Sciences AB, Uppsala, Sweden) density Leydig cells isolated from rosiglitazone-treated adult gradient (20%, 35%, 43%, 68% and 90%) and centrifuged at male rats. Ultrastructural and immunocytochemical ana- 800 × g for 30 min at 20°C. Cells in the 43-68% interface lysis of Leydig cell was performed to assess the cellular (specific gravity: 1.0640-1.0960 g/ml) were collected, integrity and the expression of StAR and P450scc, which washed twice with medium 199 (M199) (Gibco, Grand are key proteins in androgen biosynthesis. Island, NY, USA) containing 0.1% BSA, re-suspended in M199/0.1% BSA and used immediately for the experi- Methods ments. Cells (0.3 × 106 cells/0.5 ml) were treated (incu- Animals and experimental procedures bated) for 2 h with M199 (basal testosterone), hCG Twelve male Wistar-Albino rats (aged 8-9 weeks and (Sigma) (1 mIU/ml), dbcAMP (Sigma) (1 mM), 22-hydro- weighing 200-250 g) were used and obtained from the xycholesterol (Sigma) (10 μM) or pregnenolone (Sigma) (1 Bioterium of the Department of Antibiotics, Universi- μM) (stimulated/induced testosterone) in a shaking water dade Federal de Pernambuco, Brazil. The rats were kept bath (60 Hz, 34°C) in an atmosphere of 95% O2 and 5% in a small colony in the animal house at a temperature CO2. At the end of incubation, the cells were centrifuged. of 22 ± 3°C, with a 12:12 hour light/dark cycle, receiving The supernatant was collected and stored at -20°C until standard feed (Purina®) and water as required. The testosterone measurement by radioimmunoassay (RIA). experimental procedure was approved by the local To assess the effects of chronic treatment with rosiglita- Ethics Committee for Animal Experimentation. The rats zone on cell viability the trypan blue exclusion was used. were randomly divided into two groups of six animals The trypan blue exclusion is widely used screening each. The test group received rosiglitazon (GlaxoS- method to measure plasma membrane integrity. The try- mithKline, Aranda de Duero, Spain) prepared using 1% pan blue assay was performed after the period of 2 h incu- (v/v) Tween-80 (Sigma Chemical Co., St. Louis, MO, bation of cells. Cells were incubated with trypan blue USA) and administered daily via oral gavage at 5 mg/ (0,5%) for 20 min and the percentage of blue cells, indicat- kg/d. The control group received 1% vehicle. Both ing a capture of the colorant due to plasma membrane groups were treated for 15 consecutive days. After treat- rupture, were counted. 90-95% of no colored cells was ment, the rats were sacrificed with carbon dioxide. Fol- considered normal cell viability. lowing decapitation, truncal blood was collected in Light microscopy heparinized tubes and kept on ice until the plasma was The testes were fixed in Bouin’s solution for eight hours, obtained by centrifugation (600 × g, 15 min. 6°C). The then dehydrated in an alcohol series and embedded in plasma was aliquoted and stored at -20°C until analysis paraffin wax. Serial sections of 4 μmwerecutwitha for testosterone concentration. The testes and seminal microtome (Leica RM 3125RT) and stained with hema- vesicles were removed and weighed. The testes were toxylin-eosin for histological analysis [22]. used for ex vivo assays and morphological analysis. The Electron transmission microscopy dry weight of the seminal vesicles was determined by For routines procedures, the fragments of testes were drying these tissues in an oven overnight at 110°C. fixed overnight in a solution containing 2.5% glutaralde- Ex vivo study hyde (Sigma) and 4% paraformaldehyde (Sigma) in 0.1 For the ex vivo study, the testes from rosiglitazone-treated M cacodylate (Sigma) buffer. After fixation, the samples or control rats were decapsulated and the Leydig cells were washed twice in the same buffer and post-fixed in were isolated and purified as described in Wanderley and a solution containing 1% osmium tetroxide (Sigma), 2 Negro-Vilar [21], with slight modifications. Briefly, the mM calcium chloride and 0.8% potassium ferricyanide Couto et al. Reproductive Biology and Endocrinology 2010, 8:13 Page 3 of 9 http://www.rbej.com/content/8/1/13 in 0.1 cacodylate buffer, pH 7.2, dehydrated in acetone medium by a charcoal-dextran RIA [25] that employs and embedded in SPIN-PON resin (Embed 812).