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Departments of Experimental and Clinical Medicine and Pharmacology,2
Medical and Surgical Pediatric Sciences,3
Human Pathology,4 University of Messina, Messina 98124, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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kinases, Leydig cells, male reproductive tract, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These proteins belong to the extracellular signal-regulated kinase family and are serine/threonine protein kinases activated by a variety of cell surface receptors [6, 7]. The MAPKs are of vital importance for signal transduction pathways, and two of these in particular were investigated for their role: MAPK3/MAPK1, and MAPK8. Activation of MAPK3/MAPK1 and MAPK8 requires phosphorylation of both tyrosine and threonine residues by upstream dual-specific kinases. Active MAPKs are responsible for the phosphorylation of a variety of effector proteins, including several transcription factors [8].
Experimental evidence suggested that ischemia-reperfusion of the murine testis stimulates the activation of MAPK8 [9]. Activation of MAPK8 is correlated with the production of proinflammatory cytokines, such as tumor necrosis factor (TNF), after reperfusion of the testis [9]. The TNF is a multifunctional cytokine with effects not only in the proinflammatory response [10] but also in the immunoregulatory response [11] and apoptosis [12]. This cytokine has a significant role in certain testicular pathologies [13].
The role of MAPK3/MAPK1 in TI/R, however, has not yet been investigated fully. Therefore, in the present study, we examined the activation pattern of this kinase using an experimental model characterized by 1 h of occlusion followed by 24 h of reperfusion. In addition, we investigated MAPK8 to understand whether these two MAPKs are associated. Finally, we explored the effect of PD98059, a inhibitor of MAPK3/MAPK1 in the model of TI/R, to confirm kinase involvement in the pathogenesis of testicular torsion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD). Male Sprague-Dawley rats (weight, 250–300 g; Harlan Italy, Udine, Italy) fed a standard diet, given tap water ad libitum, and kept under a 12L:12D photoperiod were used.
Experimental Protocol
All animals were anesthetized with an i.p. injection of 50 mg/kg of pentobarbital sodium. The TI/R was induced by torsion of the left testis, with a 720° twisting of the spermatic cord to produce total occlusion of the testis for 1 h. The same testis was then detorsioned. The temperature of the animals was maintained at approximately 37°C using an overhead lamp during the surgical procedures. Following 0, 10, 20, and 30 min as well as 1, 2, 3, and 24 h of reperfusion, the rats were killed with an overdose of pentobarbital sodium, and bilateral orchidectomies were performed. Sham-operated rats were subjected to the same surgical procedures as TI/R rats except for the testicular torsion. The animals were randomized to receive either PD98059 (10 mg/kg i.p. administered immediately after detorsion) or its vehicle (dimethyl sulfoxide/0.9% NaCl [1:10, v/v] administered i.p. at 1 ml/kg).
Isolation of Cytoplasmic Proteins
Briefly, pulverized testis samples were homogenized in 1 ml of lysis buffer (25 mM Tris/HCl [pH 7.4], 1.0 mM EGTA, 1.0 mM EDTA, 0.5 mM phenyl methylsulfonyl fluoride, 10 µg/ml of aprotinin, and 10 µg/ml of leupeptin) with a Dounce homogenizer (Fisher Scientific, Springfield, NJ). The homogenate was subjected to centrifugation at 15 000 x g for 15 min. The supernatant was collected and used for protein determination using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA)
Determination of MAPK3/MAPK1, MAPK8, and TNF by Western Blot Analysis
Protein samples (50 µg) were denatured in reducing buffer (62 mM Tris [pH 6.8], 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.003% bromophenol blue) and separated by electrophoresis on an SDS (12%) polyacrylamide gel. The separated proteins were transferred onto a nitrocellulose membrane using the transfer buffer (39 mM glycine, 48 mM Tris [pH 8.3], and 20% methanol) at 200 mA for 1 h. The membranes were stained with ponceau S (0.005% in 1% acetic acid) to confirm equal amounts of protein and blocked with 5% nonfat dry milk in TBS-0.1% Tween for 1 h at room temperature, washed three times for 10 min each time in TBS-0.1% Tween, and incubated with a primary phosphorylated antibody for MAPK3/MAPK1 and MAPK8 (catalog nos. 9101 and 9251, respectively; Cell Signaling) and TNF (catalog no. AB2148P; Chemicon) (diluted 1:1000) in TBS-0.1% Tween overnight at 4°C. After being washed three times for 10 min each time in TBS-0.1% Tween, the membranes were incubated with a secondary antibody peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G (diluted 1:20 000, catalog no. 31402; Pierce) for 1 h at room temperature. After washing, the membranes were analyzed by the enhanced chemiluminescence system according to the manufacturer's protocol (Amersham). The MAPK3/MAPK1, MAPK8, and TNF protein signals were quantified by scanning densitometry using a bio-image analysis system (Bio-Profil; Celbio, Milan, Italy). The results from each experimental group were expressed as relatively integrated intensity compared with control normal testis (sham operated) measured with the same batch. Equal loading of protein was assessed on stripped blots by immunodetection of ß-actin with a rabbit monoclonal antibody (diluted 1: 500, catalog no. 4967; Cell Signaling) and peroxidase-conjugated goat anti-rabbit IgG (diluted 1:15 000, catalog no. 31402; Pierce). All antibodies were purified by protein A and peptide-affinity chromatography.
Histology and Evaluation of Testicular Edema
The excised testes were divided longitudinally into two parts and individually fixed in formalin. They were then dehydrated and embedded in paraffin. Serial sections (thickness, 5 µm) were obtained, deparaffinized, and stained with hematoxylin-eosin.
Light microscopy was performed without knowledge of the previous treatment. Histological lesions were evaluated in both the tubular and extratubular compartments employing an original, semiquantitative method account for interstitial extravasations and tubular and extratubular cell features. Germ cell changes (focal sloughing, coagulative necrosis, and depletion) also were evaluated. Each finding (including germ cell changes) was quantified. Histological grading was based on the following scale: 0, absent; 1, mild; 2, moderate; and 3, severe. The statistical analysis of the histological grading was performed using the method of the mode, which identifies the predominant value in each group.
To measure testicular edema, a piece from the testis body was rapidly removed, weighed, and plotted dry on filter paper. The extent of testicular edema was calculated by measuring water content: Testicular tissue was weighed before and after desiccation at 95°C for 24 h. The difference between the wet and dry tissue weights was calculated, and the degree of edema was expressed as a percentage of the tissue wet weight.
Statistical Analysis
All data are expressed as the mean ± SD. Data were analyzed by analysis of variance for multiple comparison of results; the Duncan multiple-range test was used to compare group means. In all cases, a probability error of less than 0.05 was selected as the criterion for statistical significance.
Drugs
PD98059 was obtained from Calbiochem. The compounds were administered i.p. in dimethyl sulfoxide/0.9% NaCL (1:10, v/v) solution. All substances were prepared fresh daily and administered in a volume of 1 ml/kg.
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RESULTS |
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We investigated the time course of MAPK3/MAPK1 after 1 h of testicular ischemia followed by 24 h of reperfusion. During the ischemic period, MAPK3/MAPK1 was not significantly changed (results not shown). Furthermore, very low levels of MAPK3/MAPK1 expression were observed in both the torsioned and the contralateral testes immediately after the beginning of reperfusion and in sham-TI/R animals at any time of reperfusion (Fig. 1). Expression of MAPK/MAPK1 in both testes increased at 10 min of reperfusion and reached its maximum increase at 30 min (Fig. 1). Thereafter, expression of MAPK slowly decreased, and no significant expression for this kinase was observed following 2 h of reperfusion in either TI/R testes and contralateral ones (Fig. 1).
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Administration of PD98059, an inhibitor of MAPK3/ MAPK1, markedly reduced the expression of MAPK in both testes (Fig. 1). The same pharmacological treatment did not affect MAPK3/MAPK1 expression in sham-TI/R animals (Fig. 1).
Time Course of MAPK8 in TI/R
During the ischemic period, MAPK8 expression was not significantly changed (results not shown). In addition, very low levels of MAPK8 were found in both torsioned and contralateral testes immediately after the beginning of reperfusion and in sham-TI/R animals at any time of reperfusion (Fig. 2). Expression of MAPK8 in both testes was raised after 10 min of reperfusion and reached its maximum increase after 30 min (Fig. 2). Thereafter, the expression of MAPK8 slowly decreased, and no significant change for this kinase was evidenced following 2 h of reperfusion in either TI/R and contralateral testes (Fig. 2).
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Administration of PD98059, an inhibitor of MAPK3/ MAPK1, markedly reduced MAPK in both testes. In contrast, the MAPK3/MAPK1 inhibitor did not significantly modify the expression of MAPK8 in sham-TI/R animals (Fig. 2).
Time Course of TNF in TI/R
No significant TNF expression was observed during the ischemic period. The time course of the inflammatory cytokine TNF was investigated at 60 and 180 min of reperfusion in the torsioned testis and in the contralateral one (Fig. 3). Very low levels of the inflammatory cytokine were observed in both the torsioned and the contralateral testes immediately after the beginning of reperfusion in TI/R and sham-TI/R animals (results not shown). Expression of TNF in both testes was increased after 60 min of reperfusion and reached its maximum increase after 180 min (Fig. 3). Levels of TNF returned to its basal value within 5 h of reperfusion (results not shown); this in close agreement with its role as an early, permissive, and triggering cytokine in the reperfusion injury. Administration of PD98059, an MAPK3/MAPK1 inhibitor, markedly reduced TNF in both testes but did not modify the inflammatory cytokine in sham-TI/R testes (Fig. 3).
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Tissue Edema
Tissue edema was measured to characterize the severity of the testicular ischemia. The TI/R resulted in significant organ edema (Fig. 4). Administration of PD98059 markedly reduced testicular edema. Thus, inhibition of MAPKs ameliorated the severity of TI/R (Fig. 4).
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Histology
Testes of sham-TI/R rats, treated with either vehicle or PD98059, showed no changes in histological appearance either in the tubular and the extratubular compartments (Table 1).
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On the other hand, histological observation following ischemia-reperfusion displayed a severe, plasma-rich, interstitial effusion together with venular blood vessel and lymphatic vessel dilation in the TI/R testes. Loosening of Leydig cells and an extensive detachment of germ cells also were seen, consisting of focal sloughing, coagulative necrosis, and depletion (Fig. 5A and Table 1). Contralateral findings showed mild testicular damages as a consequence of mild interstitial expansion and moderate lymphatic/venular vessel dilation (Fig. 5B and Table 1).
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Administration of PD98059 reduced the histological changes in both ipsilateral and contralateral testes (Fig. 5, C and D, and Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A similar time course of activation for both kinases was observed in both ipsilateral and contralateral testes. This would suggest that the mechanisms activating the MAPKs in both testes are either similar or, if different, operate at the same time.
Recent data have demonstrated an increase in TNF expression after reperfusion of the testis [9]. In agreement with this finding, we found a marked TNF expression in the TI/R testis that peaked at 3 h of reperfusion. As observed for the MAPKs, the contralateral testis also showed a robust increase in the proinflammatory cytokine content, thus confirming that TI/R also causes a marked bilateral impairment.
The histological features observed in the present study agreed with the observed biochemical changes. They involved both tubular and extratubular testicular compartments and, to a lesser extent, the contralateral testis. Thus, our data underline the evidence of a marked activation of both MAPK3/MAPK1 and MAPK8 in the contralateral testis. Indeed, previous work on the involvement of MAPKs in TI/R did not investigate this issue. More specifically, Lysiak et al. [9] gave no information regarding MAPK pathway activation in the contralateral testis; therefore, it might be speculated that even under those experimental conditions, activation of the two MAPKs would have been likely to occur. However, the contralateral testis showed very mild damage, thus indicating the presence of a lack of correlation between biochemical changes and pathological alterations. The reason for this discrepancy cannot, at this time, be identified fully. In myocardial ischemia-reperfusion or the "phenomenon" of preconditioning, early activation of MAPKs may contribute to cardioprotection in the so-called reperfusion injury salvage kinase (RISK) pathway [14]. Therefore, for similarity, we might hypothesize that activation of both MAPK3/MAPK1 and MAPK8 in the contralateral testis represents a protective mechanism. However, this hypothesis deserves further examination in future experiments.
Contralateral involvement has been recognized previously by investigating other pathogenetic mechanisms. Indeed, acute testicular torsion causes oxidative stress and irreversible damage in both the torsioned, ipsilateral testis and the contralateral testis [15–17].
The mechanism of this contralateral damage is unclear. One theory postulates a decrease in contralateral testicular blood flow as a reflex to an afferent stimulus [18, 19]. However, whatever the triggering mechanism, the cascade of pathological events occurring in both testes is similar. This experimental evidence has important clinical correlates. In fact, use of the same approach to protect against damage in both testes can be proposed.
It has been suggested that in other experimental models of organ damage, these MAPK pathways may, at least in part, be overactivated, inducing cell damage such as apoptotic or necrotic cell death [20, 21]. However, the exact role of MAPK3/MAPK1 and MAPK8 for testicular torsion remains unclear.
The pathophysiological mechanisms involved in organ damage caused by testicular torsion seem to be linked strictly to the ischemia during torsion and to a secondary event, reperfusion, during untwisting of the spermatic cord. In fact, although the restoration of testicular flow to the ischemic gonad is essential for tissue salvage, organ reperfusion also may have detrimental effects. Our experimental evidence suggests that reperfusion plays a key role in activation of the two MAPKs.
In the present study, both MAPKs were not significantly expressed during the ischemic period but, instead, were activated within 5 min, peaked at 30–40 min, and decreased at 1 h after detorsion. Furthermore, MAPK3/MAPK1 and MAPK8 could not be detected after 2 h of reperfusion.
These results suggest that MAPKs are early mediators of short-term testicular reperfusion damage and may represent early signals able to trigger an inflammation cascade, finally leading to the organ injury. Activation of MAPK3/ MAPK1 occurs primarily in response to growth factors or mitogens [22], whereas activation of MAPK8 depends on ultraviolet irradiation, protein synthesis inhibitors, osmotic stress, and inflammatory cytokines [23–25]. Both kinases also are triggered by the oxidative stress-lipid peroxidation phenomenon, as shown previously in another experimental model [26]. In addition, TI/R is characterized by a strong oxidative stress [27]. Therefore, we can speculate that oxidative stress-lipid peroxidation, under these experimental conditions, also may cause MAPK3/MAPK1 and MAPK8 activation. However, this hypothesis deserves to be investigated with further experimental studies.
To confirm the key role of MAPK3/MAPK1 in the pathogenesis of TI/R, we carried out the experiment with PD98059, a well-known inhibitor of this kinase. The MAPK3/MAPK1 is upstream-activated by the MAPK kinase 1 (MAP2K1). PD98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] is a flavonoid that potently inhibits MAP2K1 [28]. The usefulness of this agent stems from its solubility, its specificity for the MAP2K1 that activates MAPK3/MAPK1, and its ability to cross the cell membrane [29].
Administration of PD98059 resulted in a marked reduction in MAPK3/MAPK1. Surprisingly, PD98059 also causes a profound decrease in MAPK8. Because the flavonoid inhibitor also blunts protein kinase C, which is involved in the activation of MAPK8, it can be speculated that the dose used in our experiment also produces an antagonist effect against the other kinase that plays a pivotal role in the development of TI/R. Alternatively, it could be hypothesized that MAPK8 activation is regulated by MAPK3/MAPK1 [30].
Collectively, these data indicate that the flavonoid would represent a good candidate for the management of testicular torsion. In agreement with this idea, our results clearly show that the compound afforded dramatic protection in both gonads, almost completely reversing the morphological changes and biochemical parameters. These data are even more impressive considering that the pharmacological intervention was initiated after the onset of reperfusion, thus mimicking real clinical situations in which the patient comes to the clinical observation before the surgical procedures of detorsion.
In conclusion, MAPK3/MAPK1 plays a fundamental role in TI/R, and its blockade might represent a new, therapeutically active approach to the treatment of unilateral testicular torsion.
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FOOTNOTES |
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Received: 9 February 2005.
First decision: 28 February 2005.
Accepted: 18 May 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interleukin-I in the mammalian testis and their involvement in testicular torsion and autoimmune orchitis. Reprod Biol Endocrinol 2004 2:1-10[CrossRef][Medline]This article has been cited by other articles:
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  H. Zhang, S. M. Moochhala, and M. Bhatia Endogenous Hydrogen Sulfide Regulates Inflammatory Response by Activating the ERK Pathway in Polymicrobial Sepsis J. Immunol., September 15, 2008; 181(6): 4320 - 4331. [Abstract] [Full Text] [PDF]
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 T. Genovese, E. Esposito, E. Mazzon, C. Muia, R. Di Paola, R. Meli, P. Bramanti, and S. Cuzzocrea Evidence for the Role of Mitogen-Activated Protein Kinase Signaling Pathways in the Development of Spinal Cord Injury J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 100 - 114. [Abstract] [Full Text] [PDF]
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P < 0.01 vs. TI/R plus vehicle
