Hyperthyroidism in the developing rat testis is associated with oxidative stress and hyperphosphorylated vimentin accumulation

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Abstract

Hyperthyroidism was induced in rats and somatic indices and metabolic parameters were analyzed in testis. In addition, the morphological analysis evidenced testes maturation and intense protein synthesis and processing, supporting the enhancement in vimentin synthesis in hyperthyroid testis. Furthermore, vimentin phosphorylation was increased, indicating an accumulation of phosphorylated vimentin associated to the cytoskeleton, which could be a consequence of the extracellular-regulated kinase (ERK) activation regulating the cytoskeleton. Biomarkers of oxidative stress demonstrated an increased basal metabolic rate measured by tissue oxygen consumption, as well as, increased TBARS levels. In addition, the enzymatic and non-enzymatic antioxidant defences appeared to respond according to the augmented oxygen consumption. We observed decreased total glutathione levels, with enhancement of reduced glutathione, whereas most of the antioxidant enzyme activities were induced. Otherwise, superoxide dismutase activity was inhibited. These results support the idea that an increase in mitochondrial ROS generation, underlying cellular oxidative damage, is a side effect of hyperthyroid-induced biochemical changes by which rat testis increase their metabolic capacity.

Introduction

Thyroid hormones (TH) are essential for normal postnatal growth and development, and are known to play fundamental roles in the regulation of the energy metabolism of almost all mammalian tissues. It is well described that TH is not a major regulator of the adult testis (Barker and Klitgaard, 1952, Oppenheimer et al., 1974), nevertheless, T3 receptors are present in high quantities in neonatal Sertoli cells, indicating that the developing Sertoli cell and testis may be an important TH target (Palmero et al., 1988, Palmero et al., 1989, Jannini et al., 1990, Jannini et al., 1999). In addition, alterations in thyroid activity are frequently associated with changes in male reproductive functions, since hypothyroidism, induced or occurring soon after birth, is associated with a marked delay in sexual maturation and development (Longcope, 1991).

Vimentin is the major cytoskeletal constituent of a great variety of cells (Gyoeva and Gelfand, 1991, Shah et al., 1998), and is often expressed transiently during development (Menet et al., 2001). Both vimentin and cytokeratins are intermediate filament (IF) proteins and have been described in Sertoli cells during foetal and postnatal periods, but vimentin is the only IF described in immature and adult rat testes (Paranko et al., 1986, Romeo et al., 1995), where it plays important roles in the modifications of Sertoli cell morphology, junctional processes and cytoplasmic organization occurring during spermatogenesis (Russell and Peterson, 1985, Tanemura et al., 1994). In addition, there are several evidences in the literature pointing to phosphorylation as the most important posttranslational modification modulating both reciprocal interactions of IF proteins with other cytoskeletal components and the continuous exchange of IF subunits between a soluble pool and polymerized IF (Inada et al., 1999, Zamoner et al., 2005, Zamoner et al., 2006, Inagaki et al., 1987, Chou et al., 1996). In this context, we have recently described that TH are able to modulate IF phosphorylation in testis and cerebral cortex from rats through non-genomic mechanisms (Zamoner et al., 2005, Zamoner et al., 2006).

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that transmit signals from extracellular stimuli to multiple substrates involved in cell growth, differentiation and apoptosis. Three major subfamilies of MAPKs, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, have been identified. The outcome of MAPK activation depends on the mode of activation, cell type, and threshold of activity. The ERK MAPKs generally regulate cell growth and differentiation and there is now increasing evidences that activation of the ERK MAPKs can be induced by a variety of stimuli, including FSH and TH (Cowley et al., 1994, Papkoff et al., 1994, Cobb, 1999, Lin et al., 1999, Rama et al., 2000, Ng and Bogoyevitch, 2000, Chang and Karin, 2001, Jin et al., 2005, Yu et al., 2005). In addition, the ERK-dependent signaling is involved in the proliferation and differentiation of the Sertoli cells in a stage-specific manner modulated by FSH (Crepieux et al., 2001). However, little is known about how the ERK MAPK kinase pathway is affected in Sertoli cells after hyperthyroidism.

On the other hand, it has been largely described that hyperthyroidism is associated with hypermetabolic state and increased oxygen consumption in tissues. In this context, Palmero et al. (1994) firstly demonstrated a TH-induced increase in oxygen consumption at testis level. In this process, oxygen can undergo univalent reduction by one-electron transfer, which allows the formation of oxygen radicals and other reactive species (ROS), such as the superoxide anion radical (radical dotO2), the hydrogen peroxide (H2O2) and the hydroxyl radical (radical dotOH) (Venditti and Di Meo, 2006). When ROS generation exceeds the antioxidant capacity of cells, oxidative stress develops (Sies, 1991). In this context, it is described that hyperthyroidism enhances ROS generation and induces changes in antioxidant defences in different tissues, including liver (Fernández et al., 1985, Huh et al., 1998), heart (Venditti et al., 1997, Asayama et al., 1987, Civelek et al., 2001, Shinohara et al., 2000), brain (Adamo et al., 1989, Das and Chainy, 2004), testis (Choudhury et al., 2003), blood (Videla et al., 1988, Bianchi et al., 1999, Bednarek et al., 2004) and muscle (Asayama et al., 1987, Shinohara et al., 2000, Zaiton et al., 1993).

In the present report we investigate some metabolic parameters of induced hyperthyroidism in rat testis. We first focused on somatic indices, morphological alterations, vimentin expression and phosphorylation and MAPK signaling activation. We also investigate some aspects of oxidative stress (oxygen consumption; lipid peroxidation, measured through thiobarbituric acid reactive substances—TBARS; oxidized glutathione—GSSG), enzymatic (glutathione reductase—GR, glutathione-S-transferase—GST, glutathione peroxidase—GPx, superoxide dismutase—SOD and catalase—CAT) and non-enzymatic antioxidant defences (total—TG and reduced glutathione levels—GSH) in immature hyperthyroid rat testis.

Section snippets

Chemicals

[32P] Na2HPO4 was purchased from CNEN, São Paulo, Brazil. 3,5,3′-Triiodo-l-thyronine (T3), benzamidine, leupeptin, antipain, pepstatin, chymostatin, anti-vimentin antibody (clone vim 13.2), peroxidase conjugated rabbit anti mouse IgG, acrylamide and bis-acrylamide were obtained from Sigma Chemical Co. (St Louis, MO, USA). The antibodies p44/42 MAP Kinase (anti ERK1/2) and Phospho-p44/42 MAP Kinase (Thr202/Tyr204) were obtained from Cell Signaling Technology (Boston, MA, USA). The

Results

In vivo treatment with T3 (80 μg/kg for 7 days) induced hyperthyroidism, resulting in increased serum concentrations of free (FT3) and total T3 (T3), associated with significantly reduced serum concentrations of free (FT4) and total T4 (T4). In addition, the TSH levels were below the detection limit of the method (Table 1). Moreover, hyperthyroid animals presented increased testicular and decreased body weights (Table 2). Light micrographs of testis sections from control and hyperthyroid groups

Discussion

The biochemical effects of TH demonstrate that the Sertoli cell is the main direct target in the testis for TH, and that the prepuberal period is the temporal frame for its action. The increased metabolic activity of the Sertoli cell caused by T3 appears to be a prerequisite for the expansion of spermatogenesis. Thus, T3 shares with FSH the role of pivotal regulator of the early phases of tubular development (Jannini et al., 1995).

The effectiveness of T3 administration (80 μg/kg body weight) in

Acknowledgments

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and PROPESq-UFRGS. The authors are indebted to the Universidade Federal do Paraná (UFPR, Curitiba-PR, Brazil) for the electronic microscope facilities.

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