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Institute of Anatomy (R.M., M.M., A.F.H., M.S.D.) and Institute for Hormone and Fertility Research (D.M., A.K.M.), University of Hamburg, Hamburg 20246, Germany
Address all correspondence and requests for reprints to: Dr. Ralf Middendorff, Institute of Anatomy, University of Hamburg, Martinistrasse 52, 20246 Hamburg, Germany. E-mail: . middendo{at}uke.unihamburg.de
Abstract
The mechanisms responsible for the initial transport of immotile sperm from the testis into the epididymis are still poorly understood. We show here by electron microscopy and immunohistochemical approaches that the tunica albuginea of the human testis contains abundantly contractile elements. This tissue is also distinguished by extraordinarily high concentrations of cyclic GMP (cGMP)-dependent protein kinase I, known to mediate cGMP-dependent relaxation. Atrial natriuretic peptide (ANP) and the nitric oxide donor sodium nitroprusside (SNP) increased cGMP production in isolated strips of the tunica, and the enzymes involved could be demonstrated by affinity cross-linking and immunological techniques. Contractile cells as well as ectopic Leydig cells were identified as sites of nitric oxide synthase expression. Physiological studies revealed spontaneous contractions exclusively in regions near the rete testis. These contractions could be attenuated but not abolished by cGMP, SNP, and ANP. Remarkably, SNP reduced only the amplitudes, whereas ANP in addition decreased the frequency of these contractions. In contrast, noradrenaline-induced contractions, detectable in all parts of the capsule, could be abolished completely by SNP. These data, demonstrating complex contraction and relaxation activities, are indicative of a major physiological role of the tunica albuginea presumably related to testicular sperm transport.
THE CAPSULE OF mammalian testes represents a complex fibrous structure consisting of various, distinct tissue layers. The major component of the capsule, the tunica albuginea, is distinguished by the presence of fibroblasts interspersed in collagen fibers (for review see Refs. 1 and 2). In addition, contractile cells have also been described repeatedly (see Refs. 3 and 4). In the human tunica albuginea, however, the localization and identity of contractile elements have not yet been elucidated unambiguously.
Earlier findings that the tunica albuginea exerts contractile properties (see Ref. 5) raised the possibility that the tunica may have physiological functions such as 1) promotion of the transport of spermatozoa out of the testis into the epididymis; 2) maintenance of the interstitial pressure inside the testis; and 3) control of the blood flow through the testis (for review see Ref. 2).
The important issue of how capsular contractility is regulated remained unclear. In particular, little attention has been directed so far to potential regional differences of contractility and to the mechanisms/agents responsible for relaxation. One agent, known to elicit smooth muscle relaxation, is cyclic GMP (cGMP). This cellular messenger molecule can be generated by different pathways, involving either soluble (cytosolic) or particulate (plasma membrane-localized) guanylate cyclase (GC) activities (see Ref. 6). Nitric oxide (NO) represents the most important stimulator of the soluble GC (sGC). Among the particulate GCs, the receptor for atrial natriuretic peptide (ANP), designated as GC-A, was found to play a pivotal role in relaxation processes (see Ref. 7).
The key enzyme in mediating the cGMP-dependent relaxation in smooth muscle cells is cGMP-dependent protein kinase I (GK I), acting on diverse cellular target proteins (see Refs. 7 and 8).
In this study, we found that the human tunica albuginea testis contains abundantly contractile elements and revealed fundamental regional differences in its contractility. These findings together with the detection of a functionally active cGMP system provide striking evidence for an autonomous physiological role of the human tunica albuginea testis.
Materials and Methods
Materials
125I-ANP (2000 Ci/mmol) was obtained from Amersham Pharmacia Biotech (Braunschweig, Germany), 32P[cGMP] (3000 Ci/mmol) from ICN Biochemicals (Eschwege, Germany). Antirabbit IgG-biotin and antimouse IgG-biotin were from DAKO Corp. (Hamburg, Germany) and the Elite avidin-biotin-peroxidase complex (ABC) was from Vector Laboratories, Inc. (Burlingame, CA). Noradrenaline (NA) was purchased from Hoechst Marion Roussel, Inc. (Frankfurt/Main, Germany), ANP and C-type natriuretic peptide (CNP) were obtained from Bachem AG (Heidelberg, Germany), and 8-Br-cGMP from Biolog (Bremen, Germany). HS 142-1 was kindly provided by Dr. Y. Matsuda (Tokyo, Japan).
Components of the human testis
Testes were obtained from 11 patients aged 42–86 yr who were undergoing orchiectomy as the primary treatment of prostatic carcinoma. Pieces of chilled human testes were cut 1–2 h after surgery. The tissue samples were frozen in liquid nitrogen (for subsequent protein preparation and for immunohistochemical analyses) or fixed in glutaraldehyde or Bouin’s fluid for histological and immunohistochemical analyses. For isolation of components of the testis, tissue was transferred to dishes containing DMEM culture medium (Life Technologies, Inc., Eggenstein, Germany). After cutting the testes into pieces of approximately 10 mm x 15 mm x 10 mm, the testicular parenchyma and the blood vessels of the tunica vasculosa were separated from the tunica albuginea by use of fine forceps and scissors under a stereo microscope. The resulting segments of the tunica albuginea (including epiorchium and its supporting submesothelial connective tissue layer) were either frozen in liquid nitrogen (for protein preparation), transferred to 24-well microtiter plates containing the above mentioned culture medium (for cGMP measurements), or stored in DMEM at 4 C until use in tissue bath assays. Seminiferous tubules, testicular blood vessels, and Leydig cells were isolated as described elsewhere (9, 10).
Light and electron microscopic investigations
For light and electron microscopic investigations testis pieces were fixed in 5.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2, 820 milliosmoles) for 2 h and embedded in Epon 812. Semithin and ultrathin sections were prepared as described previously (11).
Immunohistochemistry
Paraffin sections, freely floating cryostat sections or cryostat sections, mounted onto chrome gelatin-coated slides and fixed by 4% paraformaldehyde in PBS for 10 min or by methanol (-20 C, 10 min) and acetone (room temperature, 5 sec), were used.
On cryostat sections the mouse monoclonal antibodies antidesmin (DAKO Corp., 1:300), antivimentin (DAKO Corp., 1:10), antismooth muscle myosin (Sigma, Deisenhofen, Germany, 1:200) and anti-
-smooth muscle actin (Sigma, 1:400) were employed. The rabbit polyclonal antisera antineuronal NO synthase (NOS) (anti-nNOS, BIOMOL Research Laboratories, Inc., Hamburg, Germany, 1:500), anti-sGC [Calbiochem (San Diego, CA) 1:500, see Ref. 12 ], anti-GK I, Calbiochem, 1:500), anticytochrome P450 side chain cleavage enzyme (P450 scc, Chemicon, Temecula, CA, 1:500) and antitestosterone (BioGenex Laboratories, Inc., San Ramon, CA, 1:40) were used on paraffin sections. Polyclonal rabbit antiendothelial NOS (anti-eNOS, BIOMOL Research Laboratories, Inc., 1:500) and antiinducible NOS (anti-iNOS, BIOMOL Research Laboratories, Inc., 1:500) were used both on cryostat and on paraffin sections. For visualization of immunoreactivity, a combination of the peroxidase antiperoxidase with the ABC procedure (13) with or without nickel glucose oxidase amplification was used (see Refs. 14 and 15). Dual staining and staining of consecutive serial sections was performed as described previously (see Ref. 15).
For negative controls, sections were used, in which primary, secondary, or tertiary antibodies were replaced by PBS. Furthermore, sections were incubated with normal rabbit or mouse serum (Sigma), as well as with purified rabbit or mouse IgGs (Sigma) instead of the primary antisera. The specificity of antidesmin, antivimentin, anti-nNOS, anti-eNOS, as well as anti-sGC antisera on sections of the human testis has been demonstrated previously (10, 11) by preadsorption of the antibodies either to the corresponding antigens (10 µg desmin/ml antidesmin 1:300; 10 µg vimentin/ml antivimentin 1:10; 20 µg nNOS/ml anti-nNOS, 1:500; 10 µg sGC/ml anti-sGC, 1:500) or, in case of anti-eNOS, to lysates from endothelial cells, known to contain eNOS and delivered by one of the manufacturers of the antibodies [Transduction Laboratories, Inc. (Lexington, KY), see below] to serve as positive control. In addition, several primary antibodies of other sources were used also to confirm reaction specificity: mouse monoclonal anti-nNOS, 1:500, anti-eNOS, 1:100 and anti-iNOS 1:500 (all purchased from Transduction Laboratories, Inc.) as well as rabbit polyclonal anti-nNOS (kindly provided by Dr. B. Mayer, Graz, Austria, 1:1000) and anti-sGC (1:200, kindly provided by Dr. D. Koesling, Bochum, Germany, 1:200).
Protein preparations
Frozen tissue was pulverized in a mortar, suspended in (1 ml per 100 µg tissue wet weight) homogenization buffer [0.05 M Tris-HCl (pH 7.5) containing 10 mM EDTA, 10 mM dithiotreitol (DTT), and 0.1 mM phenylmethylsulfonyl fluoride] and homogenized by three strokes in a Potter-Elvehjem homogenizer. After centrifugation at 3,000 x g for 8 min to remove cell debris and nuclei, the supernatant fractions were centrifuged for 30 min at 100,000 x g. The resulting supernatant fractions, referred to as soluble protein fractions, were stored at -70 C. The crude membrane pellets were washed once each in homogenization buffer plus 0.6M KCl, in homogenization buffer and were finally resuspended in 100–600 µl of 0.05M Tris-HCl buffer (pH 7.5). Protein concentration was determined with a Bio-Rad Laboratories, Inc. (Munich, Germany) kit using BSA (fraction V) as standard.
Immunoblotting
After separation of proteins by SDS-PAGE in 8% acrylamide gels, the immunoblotting procedure was performed essentially as described previously (16).
Anti-eNOS (Transduction Laboratories, Inc. 1:100), anti-sGC (Calbiochem, 1:1000) and anti-GK I (Calbiochem, 1:5000) were used as primary, antimouse or antirabbit IgG, linked to peroxidase (Pierce Chemical Co., Rockford, IL, 1:2000) as secondary antibodies.
Affinity cross-linking of GC-A by 125I-ANP
Reactions were performed in 40 µl of 20 mM HEPES buffer, pH 7.5, containing 5 mM MgCl2, 125 mM NaCl, and the protease inhibitors parahydroxymercury benzoate (60 µg/ml), bacitracin (1 mg/ml), bestatin (50 µg/ml), phosphoramidon (50 µg/ml), and 1,10-phenanthroline (1 mM). Membranes were preincubated in this buffer for 5 min at 4 C before the addition of 125I-ANP (final concentration: 0.5 nM). The samples were incubated for 15 min at 22 C, and UV light cross-linking was induced as described previously (17). Samples were boiled for 3 min before analysis by SDS-PAGE under reducing conditions according to Laemmli (18) in 7% polyacrylamide separation gels. Gels were dried after staining and then exposed to Kodak (Rochester, NY) XAR-5 films between intensifying screens at -70 C for 5–10 d.
Affinity cross-linking of cGMP-binding proteins by 32P[cGMP]
Soluble or particulate fractions of tissue homogenates (50 µg of protein) were incubated for 15 min at 0 C with 32P[cGMP] (3 nM) in a total volume of 50 µl of 25 mM Tris-HCl buffer, pH 7.5, containing 100 mM KCl, 2.5 mM EDTA, 1 mM DTT, and 10 µM 3-isobutyl-1-methyl-xanthine (IBMX, Sigma). Samples were then irradiated in the dark for 12 min at room temperature on a UV table (UV transilluminator model TM-36, UVP Inc., San Gabriel, CA). Reactions were terminated by chilling and the addition of 300 µl ice-cold 10% (vol/vol) trichloroacetic acid. After 20 min at 0 C, samples were centrifuged at 4 C for 5 min at 15,000 x g, and the protein pellets were resolved in 50 µl SDS-PAGE sample buffer consisting of 0.125M Tris-HCl (pH 6.8), 66.6 mM DTT, 10% (vol/vol) glycerin, 5% (wt/vol) SDS and 0.02% (wt/vol) bromophenol blue. Samples were boiled for 3 min before analysis by SDS-PAGE under reducing conditions according to Laemmli (18) in 9% polyacrylamide separation gels. Gels were dried after staining and then exposed to Kodak XAR-5 films between intensifying screens at -70 C for 2 to 12 d.
Measurements of cGMP production
In principle, the assessment of testicular cGMP levels in response to various agents has been described earlier (10). After isolation of tunica albuginea segments (see above), the culture medium was removed and tunica preparations (testes from five patients, four different preparations from each testis) were washed twice in Locke’s salt solution (16). Pieces of the tunica were first incubated for 1 h at 34 C in 250 µl Locke’s solution containing additionally 0.25 mM IBMX in the absence and then for 1 h in the presence of either 1 µM ANP, 1 mM SNP (Sigma), or 1 mM L-nitro arginine methyl ester (L-NAME, Sigma). 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, Hamburg, Germany). The minimum detection limit was 0.14 pmol/ml and cross-reactivity with cAMP less than 0.0001%.
All measurements of cGMP were performed in duplicate. Treatment effects, based on 20 different experiments, each in the absence or presence of ANP, SNP, and L-NAME, respectively (see Fig. 6
), were assessed statistically using t test as installed in the GraphPad Software, Inc. (San Diego, CA) InStat Software with P value less than 0.05 as the criterion of significance. The results shown (see Fig. 6) are mean +SE (SEM) of treatment effects of all experiments performed with tunica preparations.
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Longitudinal strips (approximately 3 mm x 15 mm) of the tunica albuginea from areas close to or distant from the rete testis, stored in DMEM at 4C (see above), were mounted, with silk thread, on two stainless-steel hooks in an organ bath (15 ml), containing platinum electrodes. Preparations were equilibrated in modified Krebs solution (KCl 4.75 mM, NaCl 118 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, MgSO4 1.2 mM, CaCl2 2.5 mM, D-glucose 11.12 mM) at 36 C and continuously gassed with carbogen (5% CO2/95% O2). One of the two hooks was connected to a SG4–90 force-displacement transducer (Hugo Sachs, Freiburg, Germany) for isometric tension recording. The output of the transducer was displayed on a Linseis L 6510 writer and digitized at 1 Hz using a Metrabyte DAS 1202 (Keithley) interface. The preparations were stretched to a pretension of approximately 1.5 g. Preparations were equilibrated for 90 min and allowed to relax to a steady-state resting tension. The contractile activity of tunica segments from different areas was studied and the relaxant effects of the NO donor SNP (1 mM), of ANP (1 µM) and of the cell-permeable cGMP-analog 8-Br-cGMP (100 µM) on spontaneous and NA-induced (1 µM) contractions were analyzed.
Results
Structural organization and contractile elements of the human testicular capsule
Light and electron microscopy in conjunction with immunohistochemical approaches were used to characterize the structure and to identify the types and distribution of contractile elements within the capsule.
These studies revealed that the capsule consists of 1) an outer layer of visceral peritoneum (epiorchium), which is supported by a submesothelial connective tissue layer; 2) the main fibromuscular layer, the so-called tunica albuginea; and 3), the tunica vasculosa, representing a loose connective tissue layer that outlines the testicular parenchyma (see Fig. 1 for a summarizing scheme).
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).
In contrast and of particular significance, the main connective tissue layer, the tunica albuginea, is characterized by an abundant occurrence of contractile elements (see Fig. 1). These are arranged between fibroblasts and parallel-running collagen fibers. The contractile cells could be identified as myofibroblasts and smooth muscle cells both by immunological approaches (Figs. 1 and 2) and by electron microscopy (Fig. 3, A–C). Immunohistochemically (Figs. 1 and 2), myofibroblasts are characterized by immunoreactivities for vimentin, desmin and -smooth muscle actin (Figs. 1, B–D, and 2). Smooth muscle cells showed an additional staining for smooth muscle myosin (Fig. 1E). Fibroblasts, in contrast, only showed vimentin-immunoreactivity (Fig. 2). Detailed analyses of myofibroblasts by electron microscopy revealed a pronounced structural heterogeneity. Some cells are elongated and partially ramified (Fig. 3A), whereas others are flat and interposed between thick collagen fiber bundles (Fig. 3B). In addition, abundant perinuclear-located rough endoplasmic reticulum (Fig. 3B) and free ribosomes as well as glycogen deposits are often detectable within the cytoplasm. Smooth muscle cells, however, show the typical phenotype (Fig. 3C) and are in part connected by intercellular junctions.
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, based on light microscopy analyses), distinguished from each other by distinct structural and immunocytochemical features (Fig. 1). In the outer zone, numerous myofibroblasts in addition to a smaller number of smooth muscle cells are detectable. In contrast, the inner zone of the tunica albuginea is characterized by a smaller number of myofibroblasts and a predominant occurrence of smooth muscle cells (see Fig. 1) that are running in parallel. Between these zones a great number of arteries is detectable. Systematic analyses revealed that contractile cells are present within the entire circumference of the tunica albuginea. However, the number of these cells is relatively low at the caudal pole of the testis, increases toward the cranial pole and is highest at the dorso-cranial area of the mediastinum testis and the rete testis.
In addition to the contractile elements, ectopic Leydig cells were detectable in the tunica albuginea. These are found as cell groups with rich capillary supply between myofibroblasts and collagen fibers (Figs. 1 and 3D). Morphologically, these cells show the characteristic features of testicular Leydig cells (Fig. 3D), including the presence of Reinke crystalloids. In addition, they clearly exhibit P450 scc (see Fig. 7C) and testosterone (not shown) immunoreactivity. Nerve fibers, which are predominantly nonmyelinated, are located close to ectopic Leydig cells (Figs. 1 and 3D). However, there is no evidence for specialized contacts between nerve fibers and ectopic Leydig cells.
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). Components and functional activity of the cGMP signaling system
Based on the known role of cGMP as the major mediator of smooth muscle relaxation (7, 8, 19), we next examined the presence and activity of factors implicated in cGMP signaling.
Concerning cGMP-generating enzymes, cross-linking of 125I-labeled ANP to tunica membranes (Fig. 4A) revealed a protein of 130 kDa, consistent with the size of the GC activity containing ANP receptor, GC-A (20). This reaction was proved to be specific because unlabeled ANP as well as the GC-A antagonist HS-142-1, but not the related peptide, CNP, were able to block receptor labeling. Remarkably, comparative analyses between membranes derived from whole testis, seminiferous tubules and the tunica albuginea (Fig. 4B) showed that the latter tissue is a predominant expression site for GC-A in the human testis.
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). These studies revealed sGC immunoreactivity localized to myofibroblasts and predominantly smooth muscle cells of the tunica albuginea testis (Fig. 5).
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).
To examine a local activity of NO-producing enzymes, cGMP production of the tunica albuginea was assessed after incubations in either the absence or presence of the NOS inhibitor, L-NAME (Fig. 6). Coincubations with the inhibitor resulted in strongly (by 46%) reduced cGMP levels, indicating presence and activity of NO-generating enzymes in the tunica albuginea. The distribution and molecular identities of these enzymes were elucidated by immunohistochemical approaches. These studies revealed the presence of eNOS in smooth muscle cells and myofibroblasts of the tunica albuginea (Fig. 7A), whereas immunoreactivity for nNOS within the tunica albuginea was localized to ectopic Leydig cells (Fig. 7B). Remarkably, capsular nerve fibers did not show any nNOS staining. Immunoreactivity against the inducible enzyme subtype, iNOS, remained completely undetectable (data not shown).
Next, UV light-induced affinity cross-linking of [32P]cGMP to cGMP-binding proteins was used to identify potential target proteins for cGMP. To discriminate between specific binding sites for cGMP and binding sites that are characterized by higher affinities for cAMP than for cGMP, incubations with [32P]cGMP were performed in either the absence or presence (0.8 µM) of unlabeled cAMP. These studies (Fig. 8) revealed a number of proteins, the labeling of which is prevented by addition of the competing nucleotide and hence may be considered as physiological target molecules for cAMP rather than cGMP (21). In contrast, three proteins with apparent molecular masses of 47, 59, and 74 kDa, respectively, are labeled to the same extent in either the absence or presence of unlabeled cAMP, indicating a preferred affinity for cGMP. All three of these proteins are more abundantly expressed in the tunica albuginea than in seminiferous tubules (Fig. 8). In contrast, the proteins considered to represent physiological targets for cAMP are present at higher concentrations in tubules (Fig. 8).
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) confirmed a markedly higher expression of this enzyme in tunica than in tubule (or whole testis) extracts and showed that GK I levels in the tunica albuginea are almost as high as in isolated blood vessels, dissected from the human testis. In contrast, there is no detectable expression of the enzyme in extracts from isolated Leydig cells.
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). Compared with sGC analyses (see above), this study also revealed that more cells were GK I- than sGC-immunoreactive.
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By isometric tension recordings, spontaneous contractile activity was detectable in tunica tissue pieces derived from areas close to the rete testis (Fig. 11A). The frequency of contraction was 2 to 10/10 min. In contrast, tissue pieces dissected from areas opposite to the rete testis, did not show any contractile activity (Fig. 11A).
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).
We next examined whether capsular tissue strips devoid of spontaneous contractile activity are capable of generating contractions in response to exogenous agents. Noradrenaline, a constrictor of the tunica albuginea (see Ref. 2), was effective, resulting in contractions with a frequency of 6/10 min (Fig. 11C). These contractions were immediately abolished by addition of SNP (Fig. 11C), indicating the presence and functional activity in this tissue of mechanisms capable of generating and terminating contractions.
Strips derived from regions close to the rete testis were used to examine the influence of noradrenaline treatment on the regular, endogenous contractile activity. The agent elicited an increased (by 25%) tonic tension and a 3-fold higher frequency of contraction (Fig. 11D). The addition of either SNP (Fig. 11D) or ANP (not shown) abolished the NAinduced effect and resulted in a by 40% reduced basal contractile tension.
Differential regional distribution of GK I
To investigate whether the region-specific contractile activities of tissue strips derived from different areas of the capsule (Fig. 12) are correlated with distinct expression patterns of components of the cGMP signaling system, protein extracts of regions distant from or close to the rete testis were analyzed separately.
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) revealed markedly elevated expression levels of GK I in areas located in close proximity to the rete testis (3-fold higher than distant from the rete testis as demonstrated by densitometric analyses), whereas eNOS, sGC, and GC-A levels were found to be similar in all regions examined. Discussion
Contractile elements and structural organization of the capsule
This study revealed a striking occurrence of contractile cells within the tunica albuginea of the human testis. Although present within the entire circumference of the tissue layer, these cells were found to be much more abundant in regions close to the rete testis. Our studies also demonstrate that the tunica albuginea consists of two sublayers, an outer and an inner zone. Myofibroblasts (see Ref. 11) represent the predominant contractile cell type within the outer zone of the tunica albuginea, although smooth muscle cells and fibroblasts are detectable also by electron microscopy and immunohistochemistry. In contrast, the inner zone of the tunica is characterized by a predominant but not exclusive occurrence of smooth muscle cells. Thus, all phenotypes of contractile cells, although at different quantities, are present in both layers of the tunica.
The existence of large plaques of Leydig cells within the inner zone is indicative of a second production site for testosterone within the human testis. In fact, immunohistochemical analyses revealed the presence of cytochrome P450 scc, the rate limiting enzyme of steroidogenesis, as well as of testosterone (see also Ref. 22) in these cells. Interestingly, there is evidence that testosterone is able to promote myoid cell differentiation in the testis (23). Thus, local effects of testosterone, released from ectopic Leydig cells, could explain the higher abundance of smooth muscle cells in the inner compared with the outer zone of the human tunica albuginea. Rather than reflecting a proposed (4) transformation stream of fibroblasts from the outer layer into myofibroblasts and smooth muscle cells in the inner layers of the tunica, our findings suggest that the cellular heterogeneity results from differentiation processes (11, 24) that take place within the individual zones of the tunica separately.
Components and functional activity of the cGMP signaling system
Several enzymes known to be involved in cGMP signaling were found to be present and active in the human tunica albuginea testis. As far as comparative analyses between different structural components of the testis were performed (in case of GC-A, GK I and other cGMP-binding proteins), the tunica was found to be distinguished by exceptionally high expression levels of these proteins.
Two cGMP-generating enzymes, GC-A and sGC, could be identified in the capsule by affinity cross-linking or immunohistochemistry, respectively. Both enzymes were found to be functionally active because ANP (the natural ligand for GC-A) and SNP (releasing the sGC stimulator, NO) strongly enhanced the production of cGMP during incubations with isolated pieces of the capsule. The relatively high concentrations of GC-A in the capsule, a tissue that is highly vascularized (see Fig. 2) and, therefore, has proper access to circulating ANP, suggest a major local role for this guanylate cyclase. In this context, it is worthwhile to mention that our cross-linking studies did not reveal any significant expression of the so-called natriuretic peptide clearance receptor, a protein that is involved in metabolic clearance of ANP (25).
The cellular distribution of sGC, detectable in smooth muscle cells and myofibroblasts, also refers to a direct functional involvement of this enzyme in relaxation processes. Based on the observed expression in the same cell types, eNOS appears to represent the principle local enzyme responsible for delivering of the sGC activator, NO. Another potential source of NO in the tunica albuginea is indicated by the expression of nNOS in ectopic Leydig cells of the tunica. The latter observation is consistent with a presumed neuronal phenotype of this cell type (14, 26).
The high abundance of cGMP-binding proteins in the capsule supports the idea of an important local role of cGMP-mediated effects. In contrast to the cGMP-binding proteins, presumed physiological target proteins for cAMP were found to be expressed at relatively low levels in the tunica albuginea. By 32P[cGMP]-cross-linking, three different cGMP-binding proteins with apparent molecular masses of 47, 59, and 74 kDa, respectively, were detectable in the soluble fractions of tunica homogenates, and all three proteins were found to be expressed at markedly lower levels in seminiferous tubule (Fig. 8) or whole testis (data not shown) extracts. Surveying published molecular masses of known cytosolic cGMP target proteins such as cGMP-dependent protein kinases (27) and cGMP-regulated phosphodiesterases (28) did not reveal any indication for the identities of the 47- and 59-kDa species. These proteins may represent novel, hitherto undescribed, proteins. The 74-kDa cGMP-binding protein could be identified by corresponding immunoblot approaches as GK I, a key mediator of smooth muscle relaxation (8, 19, 27). Consistently, the enzyme was found to be localized to myofibroblasts and smooth muscle cells. Further analyses, demonstrating protein levels as high as in blood vessel extracts, corroborated an extraordinarily high expression of GK I in the human testicular capsule.
Contraction and relaxation
The identification of distinct contractile activities, localized to different areas of the testicular capsule, represents a crucial finding of this study. Most significantly, capsular regions located close to the rete testis were found to be characterized by spontaneous, rhythmic contractions. In contrast, this activity is completely absent in areas distant from the rete testis. The regions exhibiting spontaneous contractions are marked also by a relatively high abundance of smooth muscle cells and myofibroblasts. This pronounced heterogeneity in contractility suggests differences in the underlying mechanisms of activation, myogenic vs. neuronal ones. In analogy to other smooth muscle cell-containing organs with spontaneous contractility (29), a myogenic triggering of contractions might predominate in regions close to the rete testis. Neuronal input, however, might be the primary source for contractions in regions distant from the rete testis. Here, nerve fibers were shown to derive from fundamentally different sources than those reaching the other parts of the capsule (30).
Relaxation processes mediated by cGMP-dependent mechanisms were detectable in all regions of the tunica albuginea. However, the effects observed were quite distinct. Noradrenaline-induced contractions were found to be inhibited completely and rapidly by SNP in all segments of the capsule examined. In contrast, the spontaneous contractility of tissue close to the rete testis could be only partially reduced by all agents and doses tested.
These studies in addition revealed a qualitative difference between NO- and ANP-induced effects. Administration of the NO donor, SNP, to spontaneously contractile tissue selectively reduced the amplitude, whereas ANP in addition decreased the frequency of contractions. This striking difference between activators of sGC (NO) and GC-A (ANP) may be due to an expression of GC-A but not of sGC in pace maker cells, usually found in spontaneously contractile tissues, e.g. many gastrointestinal organs, to trigger the frequency of contractions (29). In this context, it might be significant that our immunohistochemical studies revealed a higher number of GK I- than sGC-positive cells. As expected, the cGMP analog 8-Br-cGMP, which exerts its influence downstream of guanylate cyclases, was found to diminish both the amplitude and frequency of spontaneous contractions.
Generally, components of the cGMP signaling system such as cGMP-generating enzymes (sGC, GC-A), NOS and GK I were detectable in all areas of the capsule. However, comparative analyses between regions distant from and close to the rete testis revealed a selective difference in the expression of GK I, showing markedly higher levels close to the rete testis. Since this kinase represents the key factor for cGMP-dependent relaxation (8, 18, 27), our findings suggest a predominant role for cGMP-elicited effects in areas near the rete testis and a local regulation primarily at the level of GK I.
It is reasonable to assume that the observed differences in contractility reflect a functional heterogeneity of the tunica. Contractions at regions distant from the rete testis, which are not spontaneous and which might be triggered in vivo by neuronal input, could play a role in maintaining the interstitial pressure inside the testis and in controlling the flow of blood through the testis because the diameter of blood vessels between the inner and outer zone of the tunica (see Fig. 1) should be affected dramatically by contractions (see Ref. 2).
On the other hand, the spontaneous contractility close to the rete testis might be directly involved in the transport of immotile sperm from the testis into the epididymis. Contractions in this region might prevent a piling up of spermatozoa after their release in distal parts of seminiferous tubules and/or in the small area of the rete testis. Consistent with such a function, spermatozoa are not detectable in tissue sections of the human rete testis (Holstein, A. F., unpublished results), albeit all sperm cells have to cross this region. In contrast to other regions, application of 8-Br-cGMP, whose relaxing potency is well established (19), was unable to inhibit completely contractile activity in these areas. Thus, the contractility close to the rete testis might represent an extremely robust mechanism. In alignment with a functional involvement of capsular contractions in sperm transport, previous studies have shown that contractile cells in the capsule are not yet present at birth but appear in rats shortly before the time of puberty (31), when the secretion of tubular fluid and the production of mature sperm begin, and that capsulotomy in rats appears to result in sperm retention within the testis (32).
In conclusion (Fig. 13), this study elucidates an abundant occurrence of different contractile elements in the tunica albuginea of the human testis. The complex and regionalized contractility, regulated by cGMP-mediated processes, refers to important physiological activities such as the constant promotion of sperm transport within the rete testis.
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Acknowledgments
We are grateful to M. Köhler, J. Lübberstedt, A. Salewski, and E. Schäfer for excellent technical assistance.
Footnotes
The present study was supported by grants from the Deutsche Forschungsgemeinschaft (Mi 637/1-1, GRK 336, and Da 459/1-1).
Abbreviations: ABC, Avidin-biotin-peroxidase complex; ANP, atrial natriuretic peptide; anti-eNOS, antiendothelial NOS; anti-iNOS, antiinducible NOS; anti-nNOS, antineuronal NOS; cGMP, cyclic GMP; CNP, C-type natriuretic peptide; DTT, dithiothreitol; GC, guanylate cyclase; GC-A, receptor for ANP; GK I, cGMP-dependent protein kinase I; IBMX, 3-isobutyl-1-methyl-xanthine; L-NAME, L-nitro arginine methyl ester; NA, nonadrenaline; NO, nitric oxide; NOS, NO synthase; sGC, soluble GC; P450 scc, P450 side chain cleavage enzyme; SNP, sodium nitroprusside.
Received September 5, 2001.
Accepted April 4, 2002.
References
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  D. Muller, B. Hida, G. Guidone, R. C. Speth, T. V. Michurina, G. Enikolopov, and R. Middendorff Expression of Guanylyl Cyclase (GC)-A and GC-B during Brain Development: Evidence for a Role of GC-B in Perinatal Neurogenesis Endocrinology, December 1, 2009; 150(12): 5520 - 5529. [Abstract] [Full Text] [PDF]
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D. Muller, L. Cortes-Dericks, L. T. Budnik, B. Brunswig-Spickenheier, M. Pancratius, R. C. Speth, A. K. Mukhopadhyay, and R. Middendorff Homologous and Lysophosphatidic Acid-Induced Desensitization of the Atrial Natriuretic Peptide Receptor, Guanylyl Cyclase-A, in MA-10 Leydig Cells Endocrinology, June 1, 2006; 147(6): 2974 - 2985. [Abstract] [Full Text] [PDF]
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M. Mewe, C. K. Bauer, D. Muller, and R. Middendorff Regulation of Spontaneous Contractile Activity in the Bovine Epididymal Duct by Cyclic Guanosine 5'-Monophosphate-Dependent Pathways Endocrinology, April 1, 2006; 147(4): 2051 - 2062. [Abstract] [Full Text] [PDF]
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 F. C.L. Banks, G. E. Knight, R. C. Calvert, M. Turmaine, C. S. Thompson, D. P. Mikhailidis, R. J. Morgan, and G. Burnstock Smooth Muscle and Purinergic Contraction of the Human, Rabbit, Rat, and Mouse Testicular Capsule Biol Reprod, March 1, 2006; 74(3): 473 - 480. [Abstract] [Full Text] [PDF]
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 A. Shafik, O. El Sibai, and A. A. Shafik Electric Activity of the Testicular Tunica Albuginea During Ejaculation: A Canine Study Experimental Biology and Medicine, September 1, 2005; 230(8): 569 - 572. [Abstract] [Full Text] [PDF]
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 M. S. Davidoff, R. Middendorff, G. Enikolopov, D. Riethmacher, A. F. Holstein, and D. Muller Progenitor cells of the testosterone-producing Leydig cells revealed J. Cell Biol., December 6, 2004; 167(5): 935 - 944. [Abstract] [Full Text] [PDF]
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 B. Willipinski-Stapelfeldt, J. Lubberstedt, S. Stelter, K. Vogt, A. K. Mukhopadhyay, and D. Muller Comparative analysis between cyclic GMP and cyclic AMP signalling in human sperm Mol. Hum. Reprod., July 1, 2004; 10(7): 543 - 552. [Abstract] [Full Text] [PDF]
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D. Muller, A. K. Mukhopadhyay, R. C. Speth, G. Guidone, R. Potthast, L. R. Potter, and R. Middendorff Spatiotemporal Regulation of the Two Atrial Natriuretic Peptide Receptors in Testis Endocrinology, March 1, 2004; 145(3): 1392 - 1401. [Abstract] [Full Text] [PDF]
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0.05) from controls. SNP- and ANP-induced as well as L-NAME-inhibited cGMP productions are indicated as "% of control".
