Department of Urology, Yamaguchi University School of Medicine, Japan
1 To whom correspondence should be addressed at: Department of Pharmacology, University of Iowa, 2-310 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242, USA. Email: koji-shiraishi{at}uiowa.edu.
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Abstract |
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Key words: haem oxygenase-1/human/oxidative stress/varicocele
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Introduction |
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In response to oxidative stress, haem oxygenase (HO) system, which contains 32 kDa inducible form (HO-1) and two constitutive forms (HO-2 and -3), plays an important role in cell protection (Maines, 1997). Carbon monoxide (CO), biliverdin and Fe, which are haem degradation products generated by HO, have antioxidant activities (Vogt et al., 1995
). Besides oxidative stress, HO-1 is induced by heavy metals, inflammatory mediators and oxidized low density lipoproteins (Otterbein and Choi, 2000
). To date, there have been few reports focused on the expression and the role of HO-1 in testis. Using rat, HO-1 activity in testis has been shown to increase after heat shock (Maines and Ewing, 1996
). Ozawa et al. (2002)
reported that CO generated by Leydig cell HO-1 triggered apoptosis of germ cells and modulated spermatogenesis under exposure of cadmium chloride (Ozawa et al., 2002
). In humans, Middendorff et al. (2000)
observed the expression of HO-1 in Sertoli cells and suggested a functional role of CO on soluble guanyl cyclase-dependent cGMP production in seminiferous tubule. The functional significance of HO-1 in testis is not fully understood, because HO-1 activity can result in either cell protection or injury depending on the experimental setting.
A consequence of oxidative stress is membrane peroxidation, primarily involving polyunsaturated fatty acids. The levels of lipid peroxidation products are increased in testis and semen in patients with varicocele (Sharma and Agarwal, 1996; Köksal et al., 2002
). A specific and stable end-product of lipid peroxidation, the aldehyde 4-hydroxy-2-nonenal (4-HNE), can diffuse within, or even escape from, the cell and attack targets far from the site of the original free radical event (Esterbauer et al., 1991
). 4-HNE is a potent alkylating agent that reacts with DNA and proteins, generating various forms of adducts (cysteine, lysine and histidine residues) (Esterbauer et al., 1991
) that are capable of inducing specific cellular stress responses such as cell signalling and apoptosis (Uchida, 2003
).
Using testicular biopsy specimens from the patients with or without varicocele, expression of 4-HNE-modified proteins was analysed as a parameter of oxidative stress. We further examined the effects of varicocele on the expression and localization of HO-1. Then, we specifically studied the effects of the expressions of 4-HNE-modified proteins and HO-1 on the clinical parameters of spermatogenesis to elucidate the role of HO-1 in varicocele testes.
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Materials and methods |
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SDSPAGE and western blotting
The frozen testes were homogenized in 10 volumes of sucrose-Tris-EGTA buffer (pH 7.4) with protease inhibitors (-mercaptoethanol, phenylmethylsulphonyl fluoride, pepstatin A, leupeptin) with a homogenizer three times each for 30 s at the maximum speed. Equal amounts of protein (20 µg) were electrophoresed on 12% gels for HO-1 or 7.5% for HNE-modified protein and transfered to polyvinylidene difluoride membranes. These membranes were blocked with 5% non-fat dried milk in Tris-buffered saline for one hour. The membranes were incubated with rabbit polyclonal anti-HO-1 antibody (at a dilution of 1:800; StressGen Biotechnologies Corp., Victoria, BC, Canada) or mouse monoclonal anti-HNE-modified protein (at a dilution of 1:1000; Japan Institute for the Control for Aging, Shizuoka, Japan) in 1% bovine serum albumin overnight at room temperature. Then the membranes were reacted with secondary antibody at room temperature for one hour. The antigens were visualized with an ECL western blotting detection kit (Amersham Pharmacia Biotech. Piscataway, NJ, USA) and quantified using an image analyzer (Densitograph AE-6900M; Atto Co., Tokyo, Japan). The data were calculated as percentage of control and expressed as mean±SE. For the evaluation of HNE-modified protein, the sum of total bands per lane between 247 and 43 kDa were calculated.
Immunohistochemistry
The testicular specimens were fixed in Bouin's solution for 2 h and embedded in paraffin. Two serial 4 µm thick sections were cut and stained with haematoxylineosin staining for Johnsen's score count or immunostaining for HO-1. The sections for immunohistochemistry were mounted on silan-coated glass slides (Dako Japan, Kyoto, Japan), deparaffinized in xylene and rehydrated in graded ethanol. After inhibition of endogenous peroxidases in hydrogen peroxide solution, antigen retrieval was performed by heating in citrate buffer (pH 6) at 98°C for 30 min. Overnight incubation was carried out using the same rabbit anti-HO-1 polyclonal antibody (at a dilution of 1:100) at 4°C. Goat anti-rabbit immunoglobulin was used as a secondary antibody (1:100; Dako, Kyoto, Japan) for one hour. Sections were counterstained with hematoxylin. Omitting the first antibody served as a negative control and revealed no signal.
Statistical analysis
The Statview J 4.02 program (Abacus Concepts, Inc., Berkeley, CA, USA) was used for statistical analysis. Data are expressed as the mean±SE. The significance of differences was evaluated by the unpaired t-test to compare with the control group. To examine the association between expression of HO-1, generation of 4-HNE-modified proteins, HO-1/4-HNE-modified protein ratio and clinico-histological parameters, Spearman rank correlation coefficients were carried out. P<0.05 is considered statistically significant.
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Results |
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Discussion |
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ROS act as central mediators of pathologies including inflammation, infection, alcohol toxicity, neurodegenerative disease, ischaemiareperfusion injury, cryptorchidism, endocrine disruption by environmental stress, damage from UV radiation and male infertility (Sharma and Agarwal, 1996). Spermatogenesis per se requires extensive restructuring in the seminiferous epithelium. Stage-specific expression of superoxide dismutase and glutathione peroxidase play important antioxidant roles (Mruk et al., 2002
). On the other hand, association between HO-1 activity in Sertoli cells and CO was suggested to act on the cyclic guanosine 3', 5'-monophosphate signalling system in human testis (Middendorff et al., 2000
). Ample evidence supports the notion that HO-1 serves to provide potent cytoprotective effects in many in vitro and in vivo models of oxidant-induced cellular and tissue injury (Otterbein et al., 2000
). Up-regulation of HO-1 in varicocele testes (Figure 2) may have a role in conferring cytoprotection to vulnerable testicular cells in the face of sustained oxidative stress by producing the three major catalytic by-products; CO, ferritin, and bilirubin. Different from HO-2 or -3, HO-1 is an inducible type of HO and a small amount of HO-1 was detected in normal condition (Figure 2 and 4A). As shown by Middendorff et al. (2000)
, HO-1 is considered to play a role in normal spermatogenesis.
Concerning the observation that thermal stress per se can induce HO-1 expression (Maines and Ewing, 1996), it is unclear whether increased oxidative stress results in the expression of HO-1. Testicular macrophages are known to produce ROS during inflammation or infection (Wei et al., 1988
). We observed that inflammatory cells produced massive nitric oxide (NO), one of the ROS, in an inducible NO synthase (iNOS)-dependent manner (Shiraishi et al., 2001
). In addition, endothelial NADPH oxidase contributes as a source of increased ROS (Shiraishi et al., 2003b
). Inflammatory cells and endothelium are considered as sources of ROS; however, these observations do not characterize the pathopysiology of varicocele. It is therefore necessary to investigate the mechanisms of ROS on the expression of HO-1.
Due to the limitation of small samples obtained from testicular biopsy, it is hard to apply the methods widely used for the evaluation of oxidative stress in animals. 4-HNE, the most abundant product of lipid peroxidation that induces ROS production by disrupting the mitochondrial membrane potential, is a highly reactive molecule and can pass among subcellular compartments; thereby it has the potential to interact with many cell proteins (Uchida, 2003). The stability and abundance of 4-HNE make it a commonly used parameter of oxidative stress. Using anti-4-HNE-modified protein antibody, we detected many proteins modified by 4-HNE, which suggests an increased oxidative stress in varicocele testes (Figure 1). The pattern of 4-HNE-modified proteins on our western blots differed from those seen in the experimental rat vasectomy (Shiraishi et al., 2003a
). This could reflect species differences or could be a defect of the rat model. We were unable to obtain accurate and conclusive results about localization of 4-HNE-modified protein by immunohistochemistry because of high cross-reactivity of the antibody to other molecules (Uchida, 2003
). We have therefore tried to elucidate molecules that are vulnerable to the modification by 4-HNE and which will ultimately provide a therapeutic target. By N-terminal amino acid sequence and immunoprecipitation, we identified a 53 kDa protein as albumin (unpublished data). Several molecules have been found to be modified by 4-HNE (Uchida, 2003
).
Intense expression of HO-1 in Leydig cells (Figure 4B) is in agreement with the function of the cells as a major source of HO-1 in the testis; however, there are several controversies in the distribution of HO-1 between rat and human in normal condition. Maines and Ewing (1996) observed the detectable levels of HO-1 protein only in the Sertoli cells in rats and its localization suggests a functional role of CO as a part of the normal testicular cGMP signalling system (Middendorff et al., 2000
). Ozawa et al. (2002)
reported that HO-1 was expressed mostly in the intersitial macrophages, but not in Leydig cells. We observed the increased expression of HO-1 in Leydig cells (Figure 4B), in agreement with Maines and Ewing (1996)
. After treatment with CdCl2, increased HO-1 immunoreactivity was seen localized to Leydig cells, while there was no change in Sertoli cells. Diemer et al. (2003)
, using Leydig tumour cells, reported that oxidative stress resulted in pertubed function of steroidogenic acute regulatory protein through a loss of mitochondrial membrane potential. There is no correlation between expression of HO-1 and serum testosterone concentration (Table I), nevertheless intratesticular testosterone, dihydrotestosterone or estradiol should be determined to elucidate the effect of HO-1 expression on steroidgenic function of Leydig cells.
We should be aware of the many aspects of HO-1 to understand its role in physiology and pathphysiology of testes. Schipper et al. (1999) reported that free ion and CO generated from HO-1-mediated haem catabolism caused the abnormal patterns of iron deposition in brain and mitochondrial insufficiency in various human neurodegenerative disorders. It is of interest to note that germ cell apoptosis is paradoxically promoted after induction of HO-1 (Ozawa et al., 2002
). In this study, HO-1 is considered to act as cytoprotection, contributing to the improvement of spermatogenesis in varicocele testes (Figure 5). Usefulness of antioxidants in treating men subject to oxidative stress has been reported (Mruk et al., 2002
). As well as varicocelectomy, antioxidant therapy may open a new therapeutic strategy against varicocele.
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Acknowledgements |
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References |
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Submitted on December 22, 2004; resubmitted on April 6, 2005; accepted on April 8, 2005.
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