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Endocrinology Vol. 145, No. 3 1392-1401
Copyright © 2004 by The Endocrine Society

Spatiotemporal Regulation of the Two Atrial Natriuretic Peptide Receptors in Testis

Dieter Müller, Amal K. Mukhopadhyay, Robert C. Speth, Gabriela Guidone, Regine Potthast, Lincoln R. Potter and Ralf Middendorff

Institute for Hormone and Fertility Research (D.M., A.K.M., G.G.), University of Hamburg, 22529 Hamburg, Germany; Veterinary and Comparative Anatomy, Pharmacology and Physiology (R.C.S.), Washington State University, Pullman, Washington 99164-6520; Department of Biochemistry, Molecular Biology, and Biophysics (R.P., L.R.P.), University of Minnesota, Minneapolis Minnesota 55455; and Institute of Anatomy (R.M.), University of Hamburg, 20246 Hamburg, Germany

Address all correspondence and requests for reprints to: Dr. Dieter Müller, Institute for Hormone and Fertility Research, University of Hamburg, Falkenried 88, D-20251 Hamburg, Germany. E-mail: mueller{at}ihf.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
By interacting with a guanylyl cyclase (GC) activity-containing receptor, termed GC-A, atrial natriuretic peptide (ANP) acts as a regulator of blood pressure and fluid volume homeostasis. High expression levels of GC-A in the testis and reported effects of ANP on testosterone secretion by Leydig cells are indicative of important local functions in this organ. Here we show, based on radioligand receptor labeling and immunological approaches, that seminiferous tubules rather than Leydig cells are the predominant GC-A expression sites in the rat testis. Functional activity was proved by ANP- induced cGMP accumulation in isolated seminiferous tubules. Although ontogenetic studies revealed a massive increase in GC-A levels during sexual maturation, the so-called natriuretic peptide clearance receptor, another type of ANP receptor proposed to locally control the availability of natriuretic peptides, was found to be expressed predominantly before puberty, exceeding the level of GC-A expression at this time. Natriuretic peptide clearance receptor also shows a distinct distribution pattern surrounding the seminiferous tubules. These findings raise the possibility of novel physiological roles for ANP and cGMP in the testis related to germ cell maturation and/or the regulation of the onset of puberty and suggest that the two ANP receptors function in a coordinated manner at this target organ.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PEPTIDE HORMONE atrial natriuretic peptide (ANP), secreted into the circulation by cardiac cells, plays an important role in the control of fluid volume homeostasis and blood pressure (1, 2). Most of the effects of ANP in target tissues appear to be mediated via binding to and stimulation of a plasma membrane receptor, which contains an intracellular guanylyl cyclase (GC) domain, generating cGMP as the second messenger (3). This receptor, designated GC-A or natriuretic peptide receptor (NPR)-A, has been shown to be differentially glycosylated, resulting in apparent molecular masses of 122 or 130 kDa, respectively (4). Another receptor for natriuretic peptides, termed NPR-C, which is approximately 60 kDa in size and devoid of GC activity, has been proposed to serve as a natriuretic peptide clearance receptor (5), although additional functions may exist (6, 7, 8, 9). Unlike GC-A, which preferentially responds to ANP, the NPR-C receptor binds ANP and other members of the natriuretic peptide receptor family (brain natriuretic peptide, BNP, and C-type natriuretic peptide, CNP) with equal affinity (6). The identification of severe defects in mice after targeted inactivation of the NPR-C gene (10) establishes the physiological significance of this receptor in regulating local activities of the natriuretic peptide system. For experimental purposes, synthetic ANP analogs such as C-ANF or des-Cys-ANP, which bind to NPR-C but not to the GC-coupled natriuretic peptide receptors (6), are available.

Besides its well-established vasorelaxant and natriuretic properties, ANP has various effects in the organism, which apparently are not related to blood pressure and plasma volume regulation (11). Consistent with this, ANP receptors are expressed in a wide variety of mammalian cell types and organs (12). Of particular interest in this context is the presence of GC-A in testis because comparative analyses among various rat tissues revealed the highest ANP-induced cGMP production by testis membranes (13). Recently two independent investigations demonstrated exceptionally strong expression of GC-A in this reproductive organ (4, 14).

Based on previous findings that ANP can influence testosterone production by Leydig cells (15, 16), research activities hitherto have focused mainly on Leydig cells as the local targets for ANP in testis. In the present study, we provide evidence that seminiferous tubules represent the predominant expression sites of GC-A in the adult rat testis. In addition, we show for the first time the expression in testis of the natriuretic peptide clearance receptor (NPR-C), which has a completely different, namely peritubular, localization. The differential developmental regulation of these two receptors, resulting in a predominant occurrence of NPR-C before and GC-A after puberty, suggests a local functional interrelation to modify the activities of ANP in the testis during postnatal development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The synthetic peptides ANP (residues 1–28, rat), CNP (residues 32–53, rat) and C-ANF [des (Gln18, Ser19, Gly20, Leu21, Gly22) ANP4–23--NH2] were purchased from Bachem (Heidelberg, Germany). 125I-ANP (IM 186, rat) and 125I-labeled des-Cys-ANP = des[Cys7, Cys23]ANP6–28 (IM 256, rat), 2 kCi/mmol each, were obtained from Amersham (Braunschweig, Germany). 125I-[Tyr0]CNP32–53 (rat, human), 2.2 kCi/mmol, was purchased from Peptide Radioiodination Service Center (Pullman, WA).

Tissues and cells
Male Wistar rats were killed by decapitation and organs were quickly removed. The tissues were either immediately frozen in liquid nitrogen (if designed for membrane preparations) or chilled on dry ice (if designed for receptor autoradiography) and then stored at -80 C until use. For preparations of seminiferous tubules and Leydig cells, testes were decapsulated carefully after dissection, and the tissue was dispersed by collagenase treatment and mechanical agitation based on a previously described method (17). In brief, testes were incubated with collagenase (type 1a; 0.25 mg/ml, Sigma, Taufkirchen, Germany) in MEM (4 ml per testis; Life Technologies, Inc., Eggenstein, Germany) containing 1 mg/ml BSA for 20 min at 37 C in a shaking water bath (100 cycles/min). Another 2.0 volumes of medium were added, and the samples were agitated manually about 20 times. Then tubules were allowed to sediment and washed twice with medium. The supernatant fractions (containing Leydig cells) were filtered through gauze, and the Leydig cells in the filtrate were purified by density gradient centrifugation in Percoll as described earlier (18). The rats were used according to government principles regarding the care and use of animals with permission (G813/591-00.33) of the local regulatory authority.

Membrane preparation
Frozen tissues were pulverized in a mortar, suspended in 10 ml/g tissue of ice-cold homogenization buffer [50 mM Tris-HCl (pH 7.5), containing 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride] and homogenized by three strokes in a Potter-Elvehjem homogenizer. For Leydig cells, the pulverization step was left out, and homogenization was performed with 1 ml/107 cells. After centrifugation at 3000 x g for 8 min at 4 C to remove cell debris and nuclei, the supernatant fractions were centrifuged for 30 min at 100,000 x g at 4 C. The resulting crude membrane pellets were washed once in homogenization buffer plus 0.6 M KCl and were finally resuspended in 50 mM Tris-HCl buffer (pH 7.5). Protein concentrations were determined by using a kit from Bio-Rad Laboratories (Munich, Germany) with BSA (Sigma, fraction V) as standard.

Affinity cross-linking
The protocol for photoaffinity labeling of ANP receptors in crude membranes by 125I-ANP has been described in detail recently (4). In brief, membranes were incubated with either 125I-labeled ANP (0.5 nM), CNP (2 nM), or des-Cys-ANP (1 nM), ligand/receptor cross-links were induced by UV light irradiation, and reaction products were analyzed by SDS-PAGE under reducing conditions in 7.0% acrylamide separation gels followed by autoradiography at -70 C using XAR-5 films (Kodak, Stuttgart, Germany) and intensifying screens. The results shown are representative for three replicate assays carried out with membranes derived from at least three different animals each.

Immunoblotting
After separation by SDS-PAGE under reducing conditions in 7% acrylamide gels, proteins were transferred to nitrocellulose membranes (RPN 2020 D, Amersham) at 30 V for 12–14 h at 4 C. Immunoblots were pretreated for 2 h at room temperature with blocking solution (1096176, Roche, Mannheim, Germany), probed with polyclonal anti-GC-A (A034, kindly provided by D. L. Garbers, University of Texas Southwestern Medical Center, Dallas, TX), diluted 1:2000 in 90% TBS-Tween 20/10% blocking solution/0.005% thimerosal, and then incubated with antirabbit IgG/horseradish peroxidase conjugate (Sigma, A 4914, diluted 1:1000). Signals were detected using enhanced chemiluminescence (Amersham, RPN 2105) on x-ray films (13862 C, Fuji, Tokyo, Japan). For specificity control, we systematically examined different rat tissues distinguished by either high or low GC-A levels or a preferential expression of GC-B (e.g. pineal gland, pituitary) or NPR-C (e.g. fat tissue, lung), respectively. These studies showed a clear correlation between GC-A levels detectable by immunoblotting and those detectable by photoaffinity labeling and also ruled out any cross-reactivity of the antibody to the two other receptors.

Receptor autoradiography
The testes were cryostat sectioned at a thickness of 20 µm, thaw mounted onto chrome-gelatin-coated slides, dried at room temperature, and stored at -20 C. On the day of assay, the tissue sections were brought to room temperature and preincubated in 50 mM HEPES buffer (pH 7.5), containing 150 mM NaCl, 5 mM MgCl2, and 0.1% BSA for 5 min. The sections were then incubated in a humid chamber with 125I-labeled ANP, CNP, or des-Cys-ANP (1 nM each) in the same buffer (total volume per section 70 µl) for 60 min at 4 C. Nonspecific binding of the radioligands was determined by incubating adjacent tissue sections with 500 nM of the nonradiolabeled peptides in addition to the radiolabeled peptides. The sections were washed twice for 5 min each in the same buffer, dipped in distilled water, dried in air, and then exposed to RX (Fuji) films for 2–14 d at 4 C. Adjacent tissue sections were fixed with methanol (-20 C, 10 min) and acetone (room temperature, 5 sec) and finally stained with hematoxylin/eosin for subsequent histological analyses. The results shown are representative for experiments performed with testes derived from at least four different animals each.

Immunohistochemistry
For immunohistochemical analyses, rat testes were fixed in Bouin’s fixative at room temperature for 24 h, followed by dehydration in ascending ethanol concentrations and embedding in paraffin.

Antigen retrieval of sections (6 µm), mounted onto chrome-gelatin-precoated slides, and deparaffined in descending ethanol concentrations were performed using microwave cooking (7 min at 700 W, 14 min at 450 W) in 0.1 M citrate buffer (pH 6.0). After preincubation with normal swine serum, sections were incubated with purified rabbit polyclonal antibodies directed against GC-A [6326, diluted 1:1.000 in PBS (pH 7.4) supplemented with 0.1% NaN3 and 0.2% BSA] or rabbit anti-GC-A antiserum (6326, diluted 1:10,000 in PBS with NaN3 and BSA). The generation and receptor specificity of this antibody has been reported previously (19). A combination of the peroxidase antiperoxidase (PAP) technique with the avidin-biotin-peroxidase complex method was employed, visualizing peroxidase activity by the nickel-glucose oxidase approach (20). For negative controls, the primary antibodies were either replaced by PBS or sections incubated with preimmune serum (of the rabbits used for antigen generation) instead of the primary antibodies.

Measurement of GC activity in isolated seminiferous tubules
The assessment of GC activity in isolated seminiferous tubules was performed according to a previously described protocol (20). In brief, testes were cut into two pieces and transferred to dishes containing Ham’s F12/DMEM culture medium (Life Technologies, Inc.) supplemented with 15 mM NaHCO3, 20 mM HEPES (pH 7.4), 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, 10 µg/ml transferrin, 5 µg/ml hydrocortisone, and 2% fetal bovine serum. Seminiferous tubules were identified under a stereo microscope, connective tissue was removed, and isolated tubules were transferred into 24-well microtiter plates (10–20 pieces of tubular sections/well) containing the culture medium stated above. After incubation for 1 h at 34 C under 5% CO2/95% O2, the medium was removed and tubules were washed twice in Locke’s salt solution [154 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 1 mM MgCl2, 6 mM NaHCO3, 10 mM glucose, 2 mM HEPES (pH 7.4)] supplemented with 20 µg/ml bacitracin. To measure the effects of ANP on cGMP production, the tubules were first incubated for 1 h at 34 C in 250 µl Locke’s solution containing additionally 0.25 mM 3-isobutyl-1-methyl-xanthine (Sigma) in the absence and then the presence (1 µM) of ANP. Solutions were removed after each incubation, immediately frozen in liquid nitrogen, and stored at -70 C until cGMP determination (performed in triplicate) using a commercial ELISA (IHF, Hamburg, Germany) that is based on a previously described assay (21).

Assay of particulate GC activity
Incubations were performed at 37 C in total volumes of 75 µl containing 25 mM HEPES (pH 7.4), 5 mM MgCl2, 1 mM ATP, 50 mM NaCl, 0.5 mM 3-isobutyl-1-methyl-xanthine, 5 mM creatine phosphate, 5 U creatine phosphokinase, and 1 mM GTP in either the absence or presence (1 µM) of ANP. Reactions were started by addition of crude membranes (10 µg of protein) and terminated after 12 min by chilling and immediate addition of 75 µl of ice-cold E-PBS [PBS (pH 7.0), plus 5 mM EDTA, 0.2% BSA, 0.01% thimerosal]. After centrifugation at 20,000 x g for 3 min at 4 C, the supernatant fractions were carefully removed, and 10-µl aliquots were used for measurements of cGMP using the same ELISA as above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Seminiferous tubules rather than Leydig cells are the predominant expression sites of GC-A in rat testis
Recent studies, carried out by comparative analyses of GC-A expression levels in different adult rat tissues by affinity cross-linking to radiolabeled ANP (4), suggested a strikingly high abundance of this receptor in the testis. Immunoblot experiments (Fig. 1Go) confirmed that GC-A protein levels in testis are almost as high as in lung and adrenal, the latter representing well-established ANP target organs (11, 12), and also corroborated the GC-A size difference between peripheral (130 kDa) and central (olfactory bulb, cerebellum: 122 kDa) expression sites (4). Unexpectedly, a comparison between membranes derived from either whole testes or isolated seminiferous tubules (Fig. 1Go) revealed higher receptor levels in the latter membranes, suggesting that the tubular compartment may represent a predominant GC-A expression site within the testis.



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  		FIG. 1. Immunoblot analysis of GC-A expression in different rat tissues. Equal amounts (80 µg) of membrane protein, derived from the tissues indicated, were size fractionated by SDS-PAGE, blotted, and GC-A levels detected by use of a GC-A-specific antibody. The positions of GC-A (arrow) and molecular size markers (in kDa) are indicated.

 
To further address this issue, membranes derived from whole testes, isolated Leydig cells or seminiferous tubules, respectively, of adult animals were analyzed via affinity-cross-linking to 125I-labeled ANP. After SDS-PAGE and autoradiography, a radiolabeled protein of 130 kDa, consistent with the size of GC-A in peripheral tissues (4), was detectable in all three membrane preparations (Fig. 2AGo). As indicated by the different receptor band intensities, the concentration of GC-A was found to be markedly higher in seminiferous tubule than in whole testis or Leydig cell membranes. Quantitation by {gamma}-counting of the excised receptor bands revealed 4-fold higher values of radioactivity associated with seminiferous tubule compared with Leydig cell membranes. The disappearance of the 130-kDa band in assays performed in the presence of an excess of unlabeled ANP (Fig. 2BGo) confirmed the specificity of the receptor-labeling reaction. In contrast, analogous experiments carried out with 125I-labeled CNP failed to demonstrate specifically labeled proteins, suggesting the absence of a major expression in rat testis of the CNP receptor, GC-B (also known as NPR-B).



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  		FIG. 2. Analysis of GC-A expression in testis by affinity labeling. A, Crude membranes (10 µg protein each), prepared from the sources indicated, were cross-linked to 125I-ANP (0.5 nM), and reaction products were analyzed by SDS-PAGE and autoradiography. Bands at 130 kDa, marked by an arrow, represent the radiolabeled high-molecular-weight ANP receptor, GC-A. The band appearing at approximately 66 kDa is explained by unspecifically labeled BSA, usually present in 125I-labeled peptide solutions. B, Control incubations performed with testis membranes in either the presence of 0.5 nM 125I-ANP (±1 µM unlabeled ANP) or 2 nM 125I-labeled CNP (±1 µM unlabeled CNP) proved the specificity of GC-A labeling and failed to detect any specific labeling of CNP-binding proteins.

 
Receptor autoradiography performed with 125I-labeled ANP on cryostat-sectioned testis confirmed a predominant expression of GC-A in seminiferous tubules, although the labeling of individual tubules was found to be very heterogeneous (Fig. 3AGo). Certain tubules (marked by asterisks) are distinguished by a strongly labeled adluminal cell layer and a relatively weak labeling of the remaining tissue. Others appear homogenously labeled (arrows) or virtually free (arrowheads) of ANP binding sites. Note that binding of radioactive ANP to Leydig cells cannot be resolved under the conditions of this experiment. As can be inferred from morphological inspection after hematoxylin/eosin-staining (Fig. 3BGo), the differential labeling of seminiferous tubules apparently correlates with different stages of the spermatogenic cycle (e.g. see the four tubules marked by asterisks in Fig. 3Go, A and B, in which the adluminal layer of spermatids is detectable). The specificity of labeling was demonstrated by incubating sections with excess (500 nM) unlabeled ANP (Fig. 3CGo). The failure of excess unlabeled C-ANF (D) or CNP (not shown) to block radioligand binding ruled out an essential contribution of clearance receptors (NPR-C) or CNP receptors (GC-B) to the labeling reaction.



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  		FIG. 3. Autoradiographic localization of GC-A in testis. Cryostat tissue sections were incubated with 125I-labeled ANP (1 nM) in either the absence (A) or presence of an excess (500 nM) of unlabeled ANP (C) or C-ANF (D) and then washed, dried, and exposed (4 d) to x-ray film. An adjacent section (B) was fixed and stained with hematoxylin- eosin. Note the heterogeneous labeling by 125I-ANP of individual tubules (A, D). Distinctive patterns of tubular binding sites are indicated in panel a by arrows, arrowheads, or asterisks, respectively. The four tubule sections marked by asterisks appear to represent the same stage of the spermatogenic cycle, recognizable in B. Magnification, x100.

 
Immunohistochemical investigations (Fig. 4Go) further confirmed the predominantly tubular distribution of GC-A in testis (Fig. 4AGo) and identified germ cells (mainly elongated and round spermatids, see Fig. 4Go, C and D) as the intratubular sites of GC-A expression. The examination of various postnatal developmental stages revealed a similar localization in tubules of peripubertal (d 27, see Fig. 4FGo) animals, whereas GC-A immunoreactivity is virtually undetectable at prepubertal (d 10, Fig. 4EGo) stages.



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  		FIG. 4. Immunohistochemical detection of GC-A in testis. Paraffin sections were incubated with polyclonal anti-GC-A (A, C–F) or preimmune serum (B), and immunoreactivity was visualized as described (19 ). Note the predominantly intratubular distribution of GC-A (A) and its expression in elongated (C) and round (D) spermatids. The expression of GC-A in Leydig cells is indicated by arrows. During development, intratubular GC-A immunoreactivity is not yet detectable at postnatal d 10 (E) but clearly present at d 27 (F).

 
GC-A levels in testis are up-regulated during puberty
Affinity cross-linking experiments with membranes from whole testes of different ontogenetic stages were used to analyze the expression of GC-A during postnatal development. Figure 5AGo demonstrates that this receptor, migrating at 130 kDa, is only weakly expressed at the prepubertal stages (d 15 and 20) examined. Receptor levels, however, increase strongly during the functional maturation of the organ (see d 24 and 26), attaining maximal (adult) values by d 45 on. Note in addition the appearance of a second radiolabeled protein (marked by an arrow) at 60 kDa, the size of which is consistent with that of NPR-C (5). This protein is detectable only at early (until d 26) developmental stages.



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  		FIG. 5. Postnatal developmental regulation of ANP receptors. A and B, Testis. Testicular membranes (20 µg protein) from different developmental stages were cross-linked to 125I-ANP, and reaction products were visualized by SDS-PAGE and autoradiography (A). The developmental up-regulation of GC-A, migrating at 130 kDa, is evident. Note in addition the appearance of a radiolabeled protein at about 60 kDa (marked by an arrow), which is detectable only at early ontogenetic stages. Positions of molecular weight markers are indicated in kilodaltons. B, GC-A levels at the developmental stages indicated were assessed also by immunoblotting (60 µg of membrane protein each). C and D, Lung. Analogous experiments as in A and B, respectively, were performed with lung membranes. The GC-A- containing sections (size range 100–150 kDa) of the autoradiogram (C) or Western blot (D) are shown.

 
Western blot analyses (Fig. 5BGo) demonstrated that the increase in GC-A labeling during postnatal development is correlated with and hence due to increases in GC-A protein levels. Comparative investigations performed with lung membranes (Fig. 5Go, C and D) did not show corresponding changes in the expression of GC-A in this organ.

To assess the functional activity of GC-A during development, pieces of seminiferous tubules isolated from either young (postnatal d 21) or adult animals were examined (for experimental details, see Materials and Methods). ANP-induced elevations of cGMP were barely detectable with tubule preparations (n = 4) from the prepubertal rats. In contrast, ANP elicited strong increases in cGMP production in all adult tubule preparations (n = 6) examined. The extent of ANP-induced cGMP formation varied considerably among different tubule preparations, ranging from 4- to 26-fold, compared with basal values. We in addition performed GC assays with membranes from pools of isolated Leydig cell and seminiferous tubules of adult animals. These studies revealed amounts of 62.5 ± 8.4 (tubules) and 24.6 ± 5.3 (Leydig cells) pmol cGMP per milligram of membrane protein in the presence of ANP (Fig. 6Go). Compared with basal activities, ANP induced 5.1-fold (tubules) or 2.6-fold (Leydig cells) increases.



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  		FIG. 6. ANP-dependent cGMP production by seminiferous tubule and Leydig cell membranes. Crude membranes (10 µg of protein) prepared from seminiferous tubules or Leydig cells of adult animals were incubated for 12 min in either the absence (basal) or presence (1 µM) of ANP, and the generation of cGMP was determined by ELISA. The data presented are mean ± SE of duplicate measurements of three separate experiments.

 
Differential distribution and developmental regulation of NPR-C
The appearance in cross-linking assays of a radiolabeled protein with an apparent molecular mass of 60 kDa (see Fig. 5AGo) suggested a local expression in testis, at least until postnatal d 26, of NPR-C. To prove the identity of this receptor, testis membranes derived from young (postnatal d 20) animals were cross-linked to 125I-ANP in either the absence or presence of an excess of unlabeled C-ANF, a selective ligand for NPR-C (6). This ligand specifically inhibited labeling by 125I-ANP of the 60-kDa protein (Fig. 7Go), confirming the testicular expression of NPR-C at that period of postnatal development.



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  		FIG. 7. Expression of NPR-C in the prepubertal testis. A, Membranes (25 µg of protein) from postnatal d 20 testes were incubated with 125I-ANP in either the absence (none) or presence (1 µM) of the NPR-C-specific ligand, C-ANF, or ANP, respectively. After cross-linking, reaction products were analyzed by SDS-PAGE and autoradiography. Bands representing the 60-kDa (NPR-C) and 130-kDa (GC-A) ANP receptors are marked by arrows. The migration of reference proteins (in kilodaltons) is shown. B, An analogous experiment, performed with membranes from a tissue (fat) in which NPR-C is highly expressed, is shown for comparison.

 
The failure to detect NPR-C in testis membrane preparations from adult animals (see Fig. 5AGo) could be explained by either a total lack of the receptor or relatively low expression levels, which cannot be visualized due to resolution limits of the assay employed. Because trapping of the common ligand, 125I-ANP, by the excess of GC-A expression at that developmental stage could interfere with detection of low NPR-C levels, we used a radiolabeled NPR-C-specific ligand, 125I-des-Cys-ANP, in further cross-linking experiments. These studies, performed with a relatively high amount (80 µg) of membrane protein, were capable of identifying NPR-C expression in testes of adult animals (Fig. 8Go).



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  		FIG. 8. Identification of NPR-C in membranes prepared from adult testes by affinity cross-linking to 125I-des-Cys-ANP. Testis membranes (80 µg of protein) from 3-month-old animals were incubated with the 125I-labeled NPR-C-specific ligand, des-Cys-ANP, in either the absence or presence (1 µM) of C-ANF, and cross-linking was induced by UV light irradiation. Reaction products were analyzed by SDS-PAGE and autoradiography. The migration of the radiolabeled receptor (arrow) and molecular size markers (in kilodaltons) are shown.

 
We next used 125I-labeled des-Cys-ANP, selectively binding to the clearance receptor (6), to examine the localization of NPR-C in testis by receptor autoradiography. These experiments revealed that, unlike GC-A (see Fig. 3Go), NPR-C has a remarkably regular, pitted distribution, apparently surrounding the seminiferous tubules and without any detectable heterogeneity throughout the section (Fig. 9AGo). Although the specific binding sites are less resolved on sections from prepubertal (postnatal d 15) animals (Fig. 9DGo), the principal arrangement pattern, indicating a peritubular localization, is of the same kind. The specificity of receptor binding was ascertained by incubations performed in the presence of an excess of unlabeled ANP (Fig. 9Go, B and E) or C-ANF (Fig. 9Go, C and F), respectively. As deduced from comparative analyses of hematoxylin/eosin-stained sections (see insets in Fig. 9Go, A and D), the less pronounced resolution of the 125I-des-Cys-ANP binding sites at the prepubertal stage is explained by the smaller tubular diameters and an approximately 4-fold higher number of tubules per unit area, compared with adult tissue sections. Together these findings indicate that the amount of intratubular cells (predominantly germ cells that apparently do not express NPR-C) massively increases during puberty, whereas a comparable increase in peritubular cells (expressing NPR-C) is not the case. Thus, the concentration of NPR-C in membranes prepared from whole testes will decrease significantly by this alteration. This essentially explains why NPR-C expression is hardly detectable by photoaffinity labeling in whole-testis membranes during and after puberty (Fig. 5AGo).



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  		FIG. 9. Autoradiographic localization of NPR-C in testis. Cryostat tissue sections, derived from either adult (A–C) or prepubertal (D–F) testes, were incubated with 125I-labeled des-Cys-ANP (1 nM) in either the absence (A, D) or presence of an excess (500 nM) of unlabeled ANP (B, E) or C-ANF (C, F) and then washed, dried, and exposed (12 d) to x-ray film. Certain sections were subsequently fixed and stained with hematoxylin-eosin (see insets in A and D). Magnification, x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GC-A and possible roles for cGMP in testis
The strikingly high levels of GC-A in testis, as assessed in the present and two recent (4, 14) studies, suggest major physiological functions for ANP and cGMP in this organ of the male reproductive tract. Based on early findings (15, 16) that ANP stimulates, via accumulation of cGMP, the synthesis and release of testosterone, the androgen-producing Leydig cells of the testis have been considered hitherto to represent the predominant local sites of ANP activity. Corresponding studies and their proposed physiological significance are discussed comprehensively in several recent reports (22, 23, 24, 25). Our present data, showing preferential expression and functional activity of GC-A in rat seminiferous tubules, refer to a significant role in a different testicular compartment without direct effects on steroidogenesis. This compartment serves to produce spermatozoa and contains somatic (Sertoli) cells of regulatory relevance in addition to the germinal cells.

Four approaches, namely immunoblotting, affinity cross-linking, receptor autoradiography, and immunohistochemistry, consistently revealed a major tubular expression of GC-A in the adult rat testis. It is obvious that the pronounced expression in this compartment, which represents more than 80% of testicular tissue (26), is responsible for the observed relatively high levels of GC-A in testis membranes. In contrast to lung (Fig. 5Go, C and D) and other organs (kidney, liver; data not shown) examined, the expression of GC-A in testis is low until puberty, suggesting a less important local role for ANP before the functional maturation of this reproductive organ. Measurements of ANP-induced cGMP production in tubules isolated from either adult or prepubertal animals were consistent with a peripubertal onset of a substantial GC-A expression within the tubular compartment. Provided that this onset of receptor expression is a tubule-specific phenomenon, Leydig cells could represent the major sites for ANP actions in the immature testis. Recent findings demonstrating a stimulation of steroidogenesis by ANP in fetal rat Leydig cells (25) at least indicate an expression of the receptor in these cells at early developmental stages.

In vitro functional assays with isolated mature seminiferous tubules demonstrated tubule-associated GC-A activity, and immunohistochemical analyses identified germ cells as the sites of GC-A expression. The significant question concerning the local availability of ANP in vivo represents a complex issue. Based on the existence of the blood-testis barrier that develops in the rat at about 18 d of age (26), the systemically delivered peptide hormone should have free access to intratubular GC-A only at early developmental stages. However, there is considerable evidence for a local synthesis in the rat testis of ANP (27), and the peptide has been localized immunohistochemically to round and elongating spermatids (28). A recent immunocytochemical study confirming the presence of ANP in seminiferous tubules revealed a distinct spatial and temporal distribution during testicular development (29).

A prerequisite for the generation of physiological effects following ANP/GC-A interactions is the presence of cGMP target proteins. Among these, cGMP-dependent protein kinase (PKG, also abbreviated cGK) is thought to represent the key mediator of cellular cGMP activities (30). In this context, a recently identified male germ cell-specific cGK-anchoring protein, designated as GKAP42 (31), is noteworthy. This protein, expressed exclusively and in a stage-dependent manner in spermatocytes and round spermatids, colocalizes and interacts specifically with PKG I{alpha} and has been proposed to function, together with the kinase, to regulate germ cell development (31). Thus, the similar expression of these two enzymes and GC-A in testis might refer to a functional interaction of cGMP-generating GC-A and the cGMP target complex consisting of PKG I{alpha} and GKAP42. Moreover, recent studies demonstrated that GC-A can act as a regulator of cell size (32) and proliferation (33), so growth regulation appears to represent an important aspect of the natriuretic peptide system (34, 35).

Based on previous reports that human spermatozoa can respond in a cGMP-dependent manner to ANP (36) and that the peptide is capable of inducing acrosome reactions in spermatozoa (37), there is also a possibility for a role of GC-A in mature sperm cells. However, affinity cross-linking experiments performed in our laboratory failed to detect GC-A in membranes of spermatozoa collected from the rat epididymis (data not shown). Remarkably, GKAP42, present in spermatocytes and spermatids, was also undetectable in sperm prepared from the epididymis (31).

NPR-C in testis
The physiological significance of NPR-C, originally proposed to mediate metabolic clearance of ANP from the circulation (5), has been corroborated recently by studies with mice after targeted inactivation of the NPR-C gene (10). The half-life of ANP in the circulation of homozygotes lacking NPR-C was found to be enhanced, compared with wild-type animals, and the cardiovascular (reduced blood pressure) and renal (increased urine output) effects observed were consistent with a diminished systemic clearance of ANP in the mutant animals. Unexpected findings of striking skeletal deformities and an increased bone turnover in the homozygous mutants provided evidence for a perhaps more important role of NPR-C in regulating specific effects in other organs, including a modulation of autocrine/paracrine activities of locally produced natriuretic peptides (10).

In this regard, our findings of an expression of NPR-C suggest that it may control the concentration of systemically delivered and/or locally synthesized natriuretic peptides in the testis. As assessed by affinity cross-linking experiments, demonstrating higher amounts of radioactivity associated with NPR-C than with GC-A in testis membranes at postnatal d 15 (Fig. 5AGo), the clearance receptor appears to represent the predominant natriuretic peptide receptor in testis before the onset of puberty. Consistent with this, receptor autoradiography performed on postnatal d 5 testis sections (data not shown), revealed that the presence of an excess of (the NPR-C-specific) C-ANF was more effective in blocking 125I-ANP binding sites than that of the GC-A antagonist, HS-142–1. Thus, the clearance receptor may play a particularly effective role in testis during the early postnatal development, i.e. before the formation of the blood-testis barrier.

Whereas a peritubular localization of NPR-C is obvious from our receptor autoradiography studies, the specific cell type(s) expressing the receptor remained undetectable by this approach. However, based on strikingly equal anatomical distribution patterns (compare Fig. 9AGo of this study and Fig. 30 in Ref. 26), the microvasculature surrounding the seminiferous tubules represents a reasonable candidate structure for the local sites of NPR-C expression, consistent with previous reports indicating a wide expression of the receptor in vascular tissues (38). This kind of localization would favor a functional role in regulating the local availability of systemically delivered natriuretic peptides, and the peritubular distribution of the receptor suggests that it serves primarily to reduce natriuretic peptide exposure to GC- coupled receptors (i.e. GC-A) within the seminiferous tubules. However, effects on natriuretic peptide exposure to Leydig cells as well as potential activities elicited by NPR-C-mediated inhibition of cAMP formation (7, 8) cannot be excluded.

Our present findings indicate that the cellular (peritubular) localization of the clearance receptor remains preserved but that the ratio of NPR-C/GC-A is dramatically altered during postnatal development in testis. In this context, it is important to note that the relatively high abundance of NPR-C before the formation of the blood-testis barrier correlates temporally with strongly elevated ANP plasma levels in the neonatal period in rats (39, 40) and human (41). Whereas the enhanced secretion of ANP is thought to reflect and be important for the cardiovascular changes taking place at this ontogenetic stage, it might be deleterious to local physiological roles elicited by ANP in other organs. An appropriately localized and regulated expression of the clearance receptor would represent a suitable protection mechanism. In fact, the kidney apparently is an example. This organ is less responsive to ANP after birth than in the adult (42) due to an enhanced expression and activity of NPR-C during early postnatal developmental stages (43). Our present findings suggest that NPR-C functions during early postnatal development in testis as a local protective mechanism acting by modulating the availability of ANP within the seminiferous tubules. In the mature organ, NPR-C may still serve to inhibit the access of systemically delivered ANP to intratubular GC-A, thereby favoring autocrine and/or paracrine activities elicited by the locally produced peptide.

Because NPR-C has equal affinity also to the two other members of the natriuretic peptide family, BNP and CNP, possible activities associated with these peptides have to be considered. A local role for the receptor in regulating CNP effects appears unlikely, because our cross-linking and receptor autoradiography experiments did not reveal detectable expression levels of the CNP receptor (GC-B) in rat testis, whereas, by the same approaches, GC-B expression in other tissues could be demonstrated (44). BNP, on the other hand, has been thought hitherto to exert its biological effects, like ANP, through GC-A (3). However, there is now increasing evidence that BNP can signal in a tissue-specific manner through a receptor other than GC-A (45). Of particular interest, recent findings demonstrate the presence in testis of a novel GC-linked receptor that prefers BNP over ANP (14), but the identity, biological significance, and cellular localization of this receptor remain to be elucidated.

In conclusion, our present findings raise the possibility of a novel role for ANP in testis, mediated by GC-A-generated cGMP in seminiferous tubules. The GC-A expression pattern and its postnatal developmental regulation is indicative of functional implications in both pubertal and adult spermatogenesis. The completely different distribution and developmental regulation of the NPR-C suggest that it acts prepubertally to control the local availability of circulating ANP and represents a notable example for tailoring the expression of components of the natriuretic peptide system to specific requirements at his target organs.


   Acknowledgments
 
We thank Dr. D. L. Garbers for kindly providing an anti-GC-A antibody and Susanne Giehler and Monika Kistler for excellent technical assistance.


   Footnotes
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Iv 7/4-3-8 to D.M. and A.K.M.; Mi 637/1-1 to R.M.) and Bundesministerium für Forschung und Technologie (01 KY 9103/0 to D.M.).

Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; GC, guanylyl cyclase; NPR, natriuretic peptide receptor; NPR-C, natriuretic peptide clearance receptor; PKG, cGMP-dependent protein kinase.

Received June 5, 2003.

Accepted for publication November 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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