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Regular Article, PCC |
a Institute of Anatomy, University of Hamburg, 20246 Hamburg, Germany
b Institute for Hormone and Fertility Research, University of Hamburg, 22529 Hamburg, Germany
ABSTRACT
Previous studies have demonstrated that cGMP is produced by nitric oxide-mediated activation of soluble guanylyl cyclase (sGC) in seminiferous tubules of the human testis. It is not known, however, whether carbon monoxide (CO), another activator of sGC, is also involved in testicular function. To address this issue, testicular probes from 65- to 75-yr-old men have been examined. The CO-generating enzyme, heme oxygenase-1 (HO-1), could be localized by immunohistochemical and immunoblot analyses to Sertoli cells. In these cells, HO-1 is detectable in adluminal cell compartments, whereas sGC immunoreactivity is distributed exclusively in basal compartments. Treatments of isolated tubules with either sodium arsenite, known to induce HO-1, or hematin, an HO substrate, resulted in 4.4- and 1.8-fold, respectively, increases in cGMP levels. ODQ, a specific sGC inhibitor, inhibited completely the sodium arsenite-stimulated cGMP production. Moreover, the HO inhibitor zinc protoporphyrin-IX and the CO scavenger hemoglobin both significantly reduced (77% or 46% of control, respectively) tubular cGMP generation. These findings, demonstrating for the first time a link between HO-1 activity in Sertoli cells and sGC-dependent cGMP production in seminiferous tubules, suggest a functional role of CO in the human testis.
cGMP, Sertoli cells, signal transduction
INTRODUCTION
For a long time, carbon monoxide (CO), generated by heme oxygenase (HO), was considered to be a degradation product without any physiological importance. In the last few years, however, CO, in analogy to nitric oxide (NO), has been implicated as a physiological messenger in the brain and cardiovascular system. Activation of soluble guanylyl cyclase (sGC) by CO was found to result in elevated levels of the second messenger cGMP [1, 2].
Heme oxygenases catalyze the oxidative cleavage of the 
-meso carbon bridge of heme b (Fe-protoporphyrin-IX), representing the initial step of heme catabolism [3]. The enzymes act in concert with NADPH-cytochrome P450 reductase and oxygen. In addition to CO, two other products of HO activity, biliverdin and Fe, are also assumed to be biologically active. Bilirubin, the reduction product of biliverdin, was shown to protect cells from oxidative stress [4], whereas Fe, in addition to its essential role for the de novo synthesis of heme, is considered also to be a gene regulator [5–7].
There are two isoforms of HO, referred to as HO-1 and HO-2. These products of separate genes [3] differ in primary structure, regulation, and tissue distribution [2]. The expression of the 32-kDa protein HO-1, also called heat shock protein 32, can be induced by a variety of endogenous (e.g., heme) and exogenous factors, such as thermal stress and chemicals like sodium arsenite and cadmium [8–11]. Highest levels of HO-1 were found in tissues with great heme catabolism, for example, liver, spleen, and bone marrow [2]. Heme oxygenase 2, a protein of 36 kDa in human [12, 13], is constitutively expressed [2]. In rat, it is present at highest levels in the brain and testis [14].
Colocalization of HOs and sGC in the brain has been demonstrated [15]. There also is evidence for an important physiological role of CO-induced cGMP [1].
In human male reproductive organs, the cGMP system has gained particular interest because sildenafil (Viagra), an inhibitor of the cGMP-specific phosphodiesterase type 5, has been established recently for the treatment of erectile dysfunction. In contrast, effects of cGMP in the human testis are still poorly characterized. The local expression of sGC and its activation by NO have been reported [16, 17]. Specifically, NO effects on Leydig [16, 18, 19] and contractile cells [17] have been demonstrated. Information is not yet available, however, about whether testicular sGC can be activated also by HO-derived CO.
MATERIALS AND METHODS
Materials
Testes were obtained from 14 patients aged 65–75 yr who were undergoing orchiectomy as the primary treatment of prostatic carcinoma. Histological evaluation of hemalaun-eosin-stained paraffin sections or of toluidin blue- and pyronin-stained semithin sections [20, 21] revealed intact spermatogenesis in all testes.
In general, 1 to 2 h after surgery, pieces of chilled human testes were cut. These tissue samples were either frozen in liquid nitrogen (for subsequent protein preparation), fixed in Bouin fluid (for immunohistochemical analyses) or (for isolation of Sertoli cells and seminiferous tubules) transferred to dishes containing Ham F12/Dulbecco modified Eagle culture medium (Ham F12/DMEM; Sigma, Deisenhofen, Germany) supplemented with 20 mM HEPES, pH 7.4; 100 IU/ml penicillin; 100 µg/ml streptomycin; 50 µg/ml gentamycin; 0.365 mg/ml -gentamin; and 0.1% BSA.
Isolation of Seminiferous Tubules and Sertoli Cells
For isolation of Sertoli cells, 5 to 7 g of tissue was dissected and used for preparation of primary cultures according to Foucault et al. [22], resulting in approximately 70% Sertoli cell-enriched preparations. Tissue samples were washed three times with Ham F12/DMEM supplemented with 125 µg/ml amphotericin, 100 IU/ml penicillin, and 200 µg/ml streptomycin. In this medium, the tissue was minced in a Petri dish and subsequently treated enzymatically for 15 min at 36°C with 0.05% collagenase-dispase, 0.005% trypsin inhibitor (TI), and 0.001% desoxyribonuclease 1 (DNase). Dispersed seminiferous tubules were washed two times in medium, cut into small pieces, and again treated enzymatically with 0.035% collagenase-dispase, 0.0035% TI, and 0.0007% DNase for 15 min at 36°C. To remove peritubular cells, cell suspensions were washed two times in medium, followed by a third enzymatic digestion with 0.02% collagenase-dispase, 0.002% TI, and 0.0005% DNase at 36°C for 40 min. Sertoli cells were collected after filtration of the pellet through a nylon gauze (70 mesh).
Seminiferous tubules were isolated as described elsewhere [23]. They were either used for assaying cGMP production or frozen in liquid nitrogen. Sertoli cells were stored in liquid nitrogen until protein preparation.
Protein Preparation
Frozen seminiferous tubules were pulverized in a mortar, suspended (1 ml per 100 µg of tissue) in homogenization buffer (0.05 M Tris-HCl, pH 7.5, containing 10 mM EDTA, 10 mM dithiothreitol, and 0.1 mM phenyl-methyl-sulfonyl-fluoride), and homogenized by three strokes in a Potter-Elvehjem homogenizer. Isolated Sertoli cells were homogenized directly by omission of the pulverization step. After centrifugation at 3000 x g for 8 min to remove cell debris and nuclei, the supernatant protein fractions were stored at -70°C. Protein concentrations were determined with a Bio-Rad kit (Munich, Germany) using BSA (fraction V) as standard.
Immunohistochemistry
Paraffin sections (6 µm) were mounted onto chrome-gelatin precoated slides and incubated with rabbit polyclonal antibodies directed against HO-1 (purchased from Stressgen Biotech. Corp., Victoria, Canada, diluted 1:500 in PBS, pH 7.4, supplemented with 10% normal goat serum and 0.3% Triton X-100), cGMP (Biogenesis, Sandown, NH, diluted 1:1000 in PBS, pH 7.4, supplemented with 0.1% NaN3 and 0.2% BSA) or the ß1-subunit of sGC (Calbiochem, San Diego, CA, diluted 1:1000 in PBS with NaN3 and BSA). A combination of the peroxidase anti-peroxidase (PAP) technique with the avidin-biotin-peroxidase complex (ABC) method [24] was employed, visualizing peroxidase activity by the nickel-glucose oxidase approach [25].
For negative controls, primary, secondary, or tertiary antibodies were replaced by PBS. Furthermore, sections were incubated with normal rabbit serum (Sigma) or with purified rabbit IgGs (Sigma) instead of the primary antibodies. In addition, anti-HO-1 antiserum was preadsorbed to the corresponding antigen (50 µg HO-1/ml anti-HO-1, 1:500) delivered by the manufacturer of the antibody (Stressgen). The specificity of sGC staining in seminiferous tubules has been demonstrated before [17]. As a positive control for anti-cGMP-specific stainings, an additional rabbit polyclonal antibody against cGMP (Institute for Hormone and Fertility Research, Hamburg, Germany; 1:10 000) was tested on testicular sections.
Immunoblotting
After separation by SDS-PAGE in 12% acrylamide gels, proteins were transferred to nitrocellulose membranes (Amersham, Braunschweig, Germany) at room temperature for 18 h at 12 V in transfer buffer containing 100 mM Tris base and 193 mM glycine. The transferred proteins were visualized by staining with Ponceau S (Sigma, Deisenhofen, Germany), and the membranes were incubated for 2 h in a solution containing 1% blocking reagent (Amersham) in 0.1 M maleic acid, 0.15 M NaCl, and 0.005% thimerosal, adjusted to pH 7.5. After washing for 5 min in TBST (20 mM Tris, pH 7.6, 137 mM NaCl, 0.05% Tween 20), the proteins were incubated with polyclonal rabbit anti-HO-1 antibodies (see earlier mention; diluted 1:500) for 1 h at room temperature, rinsed twice for 10 min in TBST, and then incubated with anti-rabbit IgG linked to peroxidase (1:5000; Pierce, Rockford, IL). Enhanced chemiluminescence fluorography (ECL), performed with ECL detection reagents from Amersham according to the manufacturer's instructions, and medical x-ray film (Fuji) were used for the detection of bound secondary antibodies.
Measurement of cGMP Production by Isolated Seminiferous Tubules
Testes from 14 patients were analyzed. From each testis, at least three different preparations of seminiferous tubules were taken. In principle, the assessment of tubular cGMP levels in response to various agents has been described earlier [17].
Tubules were preincubated for 2 h at 34°C in 250 µl Locke solution (154 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 1 mM MgCl2, 6 mM NaHCO3, 10 mM glucose, and 2 mM HEPES, pH 7.4) containing additionally 0.25 mM 3-isobutyl-1-methyl-xanthine (IBMX, Sigma) and 20 µM bacitracin. Solutions serving to assess basal cGMP production of the individual tubule preparations were withdrawn and immediately frozen in liquid nitrogen. Tubules were then incubated for an additional 2 h, using the same solution as above, in the presence of either sodium arsenite (10 µM; Sigma), established as a specific inducer of HO-1 [9, 11]; hematin (50 µM; Sigma), representing an HO substrate [26]; zinc protoporphyrin-IX (ZnPP, 50 µM; Biomol, Hamburg, Germany), an inhibitor of HO [26]; or hemoglobin (10 µM; Sigma), a CO scavenger molecule. The corresponding concentrations of hemoglobin, ZnPP, hematin, and sodium arsenite were chosen in analogy to previous studies [9, 11, 26].
In case of sodium arsenite, shorter incubation periods (1 h, 1.5 h) were also analyzed. In these cases, the preincubation time was reduced correspondingly. Basal and sodium arsenite-induced cGMP production were determined also in the presence of 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 10µM; Alexis, San Diego, CA), an sGC inhibitor [27]. Solutions were removed after each incubation, immediately frozen in liquid nitrogen, and stored at -70°C.
cGMP was measured by means of a commercial ELISA (Institute for Hormone and Fertility Research). The minimum detection limit was 0.14 pmol/ml, and cross-reactivity with cAMP was less than 0.0001%.
All measurements of cGMP were performed in duplicate. Treatment effects, each in the absence or presence of sodium arsenite, hematin, ZnPP, and hemoglobin, respectively, were assessed statistically by t-test as installed in the GraphPad InStat Software (GraphPad Inc., San Diego, CA), with P < 0.05 as the criterion of significance. The results shown in Figures 5 and 6 are mean ± SEM of treatment effects of all experiments performed with tubule preparations.
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RESULTS
Immunoblot analyses served to examine the expression of HO-1 within the human testis. These studies (Fig. 1) revealed a single protein migrating at approximately 32 kDa, consistent with the molecular mass of HO-1 [2], in protein fractions of seminiferous tubules and of isolated Sertoli cells. On the basis of densitometric measurements (data not shown) and taking into account the differences in protein loading, the relative concentration of HO-1 in Sertoli cells has been calculated to be 1.6-fold higher than that in membranes from whole seminiferous tubules.
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Corresponding immunohistochemical investigations showed HO-1-immunoreactivity (-IR) localized primarily to seminiferous tubules (Fig. 2a). Extratubular HO-1-IR was detectable only in some testicular blood vessels (Fig. 2a), whereas Sertoli cells were found to represent the tubular expression sites (Fig. 2b). Staining specificity of HO-1 was demonstrated using anti-HO-1 antisera preincubated with an excess of the corresponding antigen (Fig. 2c).
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As demonstrated in Figures 2b and 3a, HO-1-IR in Sertoli cells is strictly localized to the adluminal cell areas. In contrast, a quite distinct subcellular distribution, confined to the basal cell compartments (Fig. 3b), is discernible for sGC, the target protein for HO-produced CO. Because consecutive serial 6-µm sections were used for staining with the two different antisera, the subcellular pattern of HO-1- and sGC-IR, as demonstrated in Figure 3, a and b, represents analyses of the same Sertoli cells. On the basis of these findings, it was of particular interest to examine the cellular and subcellular distribution of cGMP, the common product of both soluble (sGC) and particulate (membrane receptors) guanylyl cyclases. Immunohistochemical studies revealed that cGMP-IR in Sertoli cells (Fig. 4) is restricted to the basal cell compartments, apparently reflecting the location of sGC. In addition, both sGC- and cGMP-IR could be localized to myofibroblasts of the lamina propria, as described previously [17].
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To prove the functional activity of HO-1 and to examine whether HO-1-produced CO is capable of stimulating sGC, we analyzed the effects of 1) sodium arsenite, which specifically induces HO-1 expression [9, 11] and of 2) hematin, representing a HO substrate [26], on cGMP production by isolated seminiferous tubules. Consistent with the presence of sGC in Sertoli and peritubular lamina propria cells [17], both agents significantly increased cGMP levels in all tubule preparations investigated. The effect of sodium arsenite (Fig. 5a) was time dependent, resulting in 4.4-fold higher nucleotide levels after a 2-h incubation. Induction of cGMP production by the HO substrate hematin was 1.8-fold, as compared with basal levels (Fig. 5b). In agreement with its role as CO scavenger, hemoglobin created significantly reduced (by 54%) cGMP concentrations (Fig. 5b). Direct inhibition of HO activity by ZnPP (Fig. 5b) also resulted in reduced (77% of control) cGMP levels.
To prove the functional involvement of sGC, additional experiments were used to examine the effects of ODQ, a specific sGC inhibitor. This agent was found to suppress most (85.6%) of the basal cGMP production and to inhibit completely the sodium arsenite-induced accumulation of cGMP (Fig. 6).
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DISCUSSION
This report provides evidence for a potential role of carbon monoxide (CO), produced by HO-1, in seminiferous tubules of the human testis.
CO has been described repeatedly as an activator of sGC (reviewed in [2]). In confirmation, our functional studies revealed increased cGMP levels after incubation of isolated seminiferous tubules with sodium arsenite, an agent known to induce HO-1 expression [9, 11]. Because sGC is present in seminiferous tubules [17], this functional link between testicular HO-1 induction and elevated cGMP levels appears reasonable. The real involvement of sGC was proved by experiments in which the presence of the sGC inhibitor, ODQ, resulted in strongly reduced cGMP levels and inhibited completely the sodium arsenite-induced accumulation of the cyclic nucleotide. Moreover, addition of the HO substrate hematin was effective in elevating cGMP concentrations, whereas the presence of the HO inhibitor zinc protoporphyrin-IX (ZnPP) resulted in a significant reduction of basal cGMP levels (Fig. 5b). The latter finding, obtained in the absence of manipulations to increase artificially CO levels, is indicative of an endogenous generation of the signaling molecule. Consistently, the CO scavenger, hemoglobin, was found to be effective in inhibiting basal cGMP production (Fig. 5b).
In vascular smooth muscle cells, cGMP-accumulating effects of sodium arsenite have been observed by Christoduolides et al. [11], and the extent of augmentation of the signaling molecule described in their study was similar to that reported here. Compared with NO-induced increases in cGMP production in seminiferous tubules [17], the stimulatory effect of CO on sGC, as assessed in this study, seems low. However, CO has been shown to be capable of stimulating (purified) sGC to a degree similar to that of NO in the presence of the benzyl indazole derivative YC-1 [28]. In analogy to the possible role of endogenous benzodiazepine-like substances acting on the GABAA receptor [29], endogenous YC-1 equivalents could alter the sensitivity of sGC toward CO [28].
With regard to the examination of material from only elder (65- to 75-yr-old) men, certain aging-related effects are conceivable. It should be noted, however, that the spermatogenic activity of all of the testicular probes used in this study has been proved morphologically to be sufficient.
Our studies demonstrate that Sertoli cells represent the major—if not exclusive—sites of expression of HO-1 within seminiferous tubules. Immunohistochemical investigations provided evidence for the presence of sGC in Sertoli cells (Fig. 3) and peritubular myofibroblasts [17]. Therefore, Sertoli cells themselves and neighboring contractile cells of the lamina propria may represent the physiological targets for HO-1-produced CO. Heme oxygenase 2, the constitutively expressed HO isoform, is present in germ cells of rat [16] and human [30] testis. However, neither sGC nor cGMP are detectable in these cells [17]. Hence, germ cells might not contribute directly to sGC-mediated cGMP production in human seminiferous tubules [17], but effects of germ cell-produced CO in neighboring Sertoli and peritubular cells are conceivable. In these cells, as described recently for human placental tissue [31], a combined functional role of both isoforms of HO might exist.
Peritubular lamina propria cells express filaments characteristic of fibroblasts and smooth muscle cells [20, 21]. Paracrine influences of CO on these myofibroblasts are conceivable in the context of CO effects on smooth muscle cells in other organs [11]. It can be postulated that CO mediates relaxation of myofibroblasts, thereby participating in the regulation of the peristaltic activity of the tubules, which in turn is necessary for sperm transport [32].
A physiological role of the CO-sGC-cGMP pathway within Sertoli cells appears less obvious. Interestingly, HO-1 (Fig. 3a) is present exclusively in the adluminal compartments of these cells, whereas both sGC (Fig. 3b) and cGMP (Fig. 4) are localized in the basal parts, indicating a subcellular separation of the sites of production and possible action of CO.
Enhanced cGMP levels in isolated rat Sertoli cells in response to activators of particulate (atrial natriuretic peptide) and soluble (sodium nitroprusside) guanylyl cyclases, respectively, have been described [33]. In the same study, the testicular paracrine factor PmodS, known to regulate the secretion of androgen-binding protein and transferrin in Sertoli cells [34, 35], was found to act via enhanced cGMP concentrations. Therefore, HO-elicited increases in cGMP levels could influence basic secretory functions of Sertoli cells. Another possible role of cGMP in Sertoli cells may concern functions related to changes in cell structure. It is known that Sertoli cells show very different phenotypes, each depending on the stage of spermatogenesis [36, 37]. In this regard, it is interesting to note that changes in the assembly of Sertoli cells' intermediate filaments, which represent target proteins for cGMP-dependent kinase activity [38], have been demonstrated [37, 39].
Induction of HO-1 activity by heat shock in various rat tissues, including the testis, has been reported previously [10, 40]. The authors interpreted this effect in the context of a general defense strategy of cells against thermal stress. The adluminal localization of HO-1 would be consistent with such a role in protecting germ cells against oxidative stress.
Taken together, the data presented here demonstrate a link between HO-1 activity in Sertoli cells and cGMP production in seminiferous tubules of the human testis (Fig. 7), thereby providing initial evidence for a functional role of CO as part of the testicular cGMP signaling system.
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ACKNOWLEDGMENTS
We are grateful to M. Köhler for her excellent technical assistance.
FOOTNOTES
First decision: 16 August 1999.
1 The present study was supported by grants from the Deutsche Forschungsgemeinschaft (Mi 637/1-1). 
2 Correspondence: Ralf Middendorff, Institute of Anatomy, University of Hamburg, Martinistr. 52, 20246 Hamburg, Germany. FAX: 49 40 42803 4966; middendo{at}uke.uni-hamburg.de
Accepted: March 10, 2000.
Received: June 24, 1999.
REFERENCES
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 R. Middendorff, D. Muller, M. Mewe, A. K. Mukhopadhyay, A. F. Holstein, and M. S. Davidoff The Tunica Albuginea of the Human Testis Is Characterized by Complex Contraction and Relaxation Activities Regulated by Cyclic GMP J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3486 - 3499. [Abstract] [Full Text] [PDF]
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