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BOR - Papers in Press, published online ahead of print October 4, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.006445
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Biology of Reproduction 67, 1975-1980 (2002)
DOI: 10.1095/biolreprod.102.006445 © 2002 Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action

Expression of the Receptor Guanylyl Cyclase C and Its Ligands in Reproductive Tissues of the Rat: A Potential Role for a Novel Signaling Pathway in the Epididymis1

Mahaboobi Jaleela, Roslyn M. Londonb, Sammy L. Eberb, Leonard R. Forteb, and Sandhya S. Visweswariah2,a

a Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India b Truman Veterans Affairs Medical Center and Department of Pharmacology, School of Medicine, University of Missouri, Columbia, Missouri 65212


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.

cyclic guanosine monophosphate, epididymis, female reproductive tract, male reproductive tract, polypeptide receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclic GMP as a second messenger is responsible for a wide variety of physiological processes, including relaxation of vascular smooth muscle [1], phototransduction in the retina [2], mucociliary clearance in the lung [3], modulation of synaptic strength in the nervous system [4], and electrolyte transport across epithelial monolayers [5]. Guanylyl cyclases are a class of enzymes that synthesize cGMP. There are two forms of guanylyl cyclase: a cytosolic form (soluble guanylyl cyclase) activatable by nitric oxide and membrane-associated forms or particulate guanylyl cyclases. Membrane-associated forms of guanylyl cyclase serve as receptors for a variety of polypeptide ligands and consist of a single polypeptide chain of molecular mass in the range of 130–160 kDa. These proteins are characterized by the presence of an extracellular ligand-binding domain, a single transmembrane domain, and a cytoplasmic domain, which is subdivided into a protein kinase-like domain and the C-terminal guanylyl cyclase domain with catalytic activity. Ligand binding to the extracellular domain leads to activation of these receptor guanylyl cyclases, which then catalyze the synthesis of cGMP.

Guanylyl cyclase C (GC-C) is the membrane-bound receptor for the heat-stable enterotoxin (ST) peptides and for the endogenous peptide ligands guanylin, uroguanylin, and lymphoguanylin. GC-C is predominantly expressed on the apical surface of epithelial cells of the intestine, and activation of GC-C by ST leads to an increase in intracellular cGMP levels [6]. Cyclic GMP cross-activates protein kinase A and protein kinase G, which phosphorylate and activate the cystic fibrosis transmembrane conductance regulator (CFTR) leading to chloride ion secretion. This secretion in turn causes secretion of water into the lumen, which results in "traveler's diarrhea" associated with the ST peptides [7, 8]. The endogenous peptides guanylin and uroguanylin have a lower affinity for GC-C than do the ST peptides and are produced within the intestinal mucosa to serve as paracrine and autocrine regulators of intestinal fluid and electrolyte secretion [9].

In immunohistochemical studies using a monoclonal antibody to GC-C [10], GC-C was expressed throughout the rat and human small intestine and was detected in the intestine of the domestic chicken [11], other avian species [12], and reptiles [13] by in situ receptor autoradiography. In addition, the GC-C transcript and/or protein have been detected in extraintestinal tissues by a variety of methods, including radioligand binding, in situ hybridization, and immunohistochemistry. For example, radiolabeled ST-binding analysis revealed that in the North American opossum, GC-C is expressed in tissues such as the proximal tubules of the kidney, airway epithelium, and seminiferous tubules of the testis [14, 15]. Northern blot analysis revealed that GC-C is expressed in the perinatal liver, placenta, and testis of the rat [16], and reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated the presence of GC-C in the adrenal glands and brain in rat, human, and bovine airway epithelia [17]. GC-C has also been detected in the hepatocytes and nonparenchymal cells during liver regeneration [18] and in clusters of cells in the stomach epithelium [19].

The distribution of guanylin and uroguanylin follows a pattern of expression similar to that of GC-C. Other than the intestine, guanylin mRNA has also been detected in several tissues such as the adrenal glands, kidney, uterus, and oviduct of the rat [17]. Guanylin bioactivity has been detected in human airway epithelium [20] and in many tissues of the opossum, including reproductive tissues and the brain [21].

Intracellular cGMP accumulation elicited by guanylin and uroguanylin influences transepithelial Cl- and HCO3- secretion by interacting with and stimulating the enzyme activities of protein kinase A and protein kinase G-II. In the present study, we demonstrated the presence of GC-C, guanylin, and uroguanylin transcripts in reproductive tissues such as the testis, epididymis, prostate, ovary, oviduct, and uterus of the rat. High levels of GC-C mRNA were detected in the epididymis, which indicates for the first time the functionality of GC-C in epididymal tissue and its cellular expression. Because CFTR, a chloride channel [22], is an important downstream effector molecule in the GC-C signaling pathway, the coordinated expression of GC-C, its ligands, and CFTR may provide the machinery for the regulation of Cl- secretion and the maintenance of ion balance in reproductive tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reverse Transcriptase and Polymerase Chain Reaction

Adult male and female Wistar rats (>60 days of age) were used for all experiments. Total RNA was prepared from whole tissues using Tri Reagent (Sigma, St. Louis, MO). The tissue was minced and homogenized in Tri Reagent (100 mg tissue in 1.4 ml of Tri Reagent), and the homogenate was incubated at room temperature for 15 min. Chloroform (280 µl) was then added, and samples were vortexed thoroughly. The homogenate was allowed to stand for 15 min at room temperature and was then centrifuged at 12 000 x g at 4°C for 15 min. The resulting aqueous solution was then processed through the RNeasy RNA purification kit (Qiagen, Valencia, CA) as per the manufacturer's instructions. Complementary DNAs were synthesized from 5 µg of total RNA using reverse transcriptase (Superscript II; Life Technologies, Rockville, MD). Primers were designed to amplify the protein kinase-like domain (PKLD) of GC-C, pre-prouroguanylin, and pre-proguanylin. The sequences of the primers used were as follows. For the PKLD of GC-C, the forward primer was RGC 1307 (5'-ATTGC-CGTCTTCACGCTCAC-3'), and the reverse primer was RGC 2195 (5'-GAAGGAGGGTTGTCAAAAATGTT-3'). For uroguanylin, the forward primer sequence was 5'-ATGTCAGGAATTCAACTGTGG-3', and the reverse primer sequence was 5'-AGGTGTCTCGAGTCATTTCAT-3'. For guanylin, the forward primer sequence was 5'-GTGCCTTGGCTGTCCTGGTAGAAG-3', and the reverse primer sequence was 5'-TCTGCAGGATCTCCTCGGCGTTG-3'.

The GC-C, uroguanylin, and guanylin primers amplified products of 888, 317, and 250 base pairs (bp), respectively. PCR was carried out for 30 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1.5 min using Taq DNA polymerase (Gibco-BRL, Rockville, MD). The PCR-generated cDNA products were visualized by agarose gel electrophoresis and ethidium bromide staining. Negative controls included RNA of equivalent concentration without an RT reaction and water blanks for PCR. Neither of these showed any amplified product.

After gel electrophoresis, the DNA was transferred to nylon membranes. The membrane was prehybridized with Express hybridization mix (Clontech, Palo Alto, CA) at 70°C for 1 h. Probes were prepared by labeling purified PCR products obtained from the intestine using the same primers. Hybridization was carried out at 70°C for 1 h, blots were washed with 2x saline-sodium citrate (SSC)/0.1% SDS (w/v) for 5 min at room temperature, then 0.2x SSC/0.1% SDS (w/v) for 15 min at 72°C. The membrane was then covered with Saran Wrap and exposed to x-ray film at -70°C to obtain an autoradiogram. The RT-PCR products were reamplified and sequenced to confirm the products obtained (data not shown).

Immunoprecipitation of GC-C

Caput epididymis and cauda epididymis tissue was homogenized in homogenization buffer (50 mM Hepes pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 5 mM EDTA, 2 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). The homogenate was sonicated for 20 sec, and lysed cells were centrifuged at 1000 x g for 5 min. The supernatant was collected and subjected to centrifugation at 30 000 x g for 1 h. The membrane pellet obtained was resuspended in resuspension buffer (50 mM Hepes pH 7.5, 10 µg/ml aprotinin and 10 µg/ml leupeptin), and membrane protein (5 mg) was solubilized in the presence of 1% SDS (w/v) and 500 mM NaCl for 1 h. The fraction was centrifuged for 20 min at 12 000 x g, and the supernatant was diluted to a final composition of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, and 0.1% SDS (w/v) (immunoprecipitation buffer) containing 5 µg/ml aprotinin and 5 µg/ml leupeptin. GC-C present in the solubilized membrane preparation was immunoprecipitated using the GC-C:4D7 monoclonal antibody raised against the PKLD at a concentration of 2 µg/ml IgG as described previously [23]. The immune complex formed was precipitated with protein G agarose, and the protein G beads were washed and subjected to SDS gel electrophoresis and Western blot analysis using GC-C:4D7, as described previously.

Measurement of cGMP Accumulation Following Uroguanylin Addition to Epididymal Tissue

The caput and cauda epididymis were dissected out and minced finely in Dulbecco modified Eagle-F12 medium containing 1 mM isobutylmethylxanthine (IBMX) and buffered with 50 mM Hepes, pH 7.5. Medium containing uroguanylin (10-5 M) and IBMX (1 mM) was added to 100 mg of tissue in 200 µl (w/v), and incubation was continued for 1 h at 37°C. After addition of 10 µl perchloric acid, the tissues were homogenized, neutralized with 10 M KOH, and centrifuged. After acetylating the samples as described previously [24], cGMP produced in the extract was measured by RIA.

Immunohistochemical Localization of GC-C in Epididymis

The caput epididymis and cauda epididymis were dissected out from an adult male Wistar rat and fixed in Bouin fixative for 12 h. The tissue was processed as described previously [10], and 5-µm serial sections were cut and spread on Vectabond-coated slides (Vector Laboratories, Burlingame, CA). The sections were deparaffinized in xylene for 30 min and hydrated in various grades of alcohol (absolute, 70%, 50%, 30% v/v) in PBS, pH 7.2, for 30 min each. The sections were treated with 90% (v/v) methanol containing 0.03% H2O2 (v/v) for 30 min and rehydrated for another 30 min in PBS. Sections were treated with 5% (v/v) goat serum in a humidified chamber for 1 h at room temperature and washed with PBS containing 0.1% (v/v) Tween 20 followed by PBS alone. GC-C:4D7 monoclonal IgG or antibody preadsorbed with a fusion protein consisting of the PKLD fused to glutathione S-transferase (GST) [23] (5 µg/ml) diluted in PBS containing 5% (v/v) goat serum was added, and incubation was continued in a humidified chamber at 4°C for 10 h. The sections were washed with PBS containing 0.1% (v/v) Tween 20 and then with PBS alone and incubated with 1:500 diluted anti-mouse horseradish peroxidase conjugate (Sigma) for 2 h. Sections were washed as above, and color was developed using 0.6 mg/ml solution of 3,3'diaminobenzidine containing 0.03% (v/v) H2O2. The sections were counter stained with Meyer hematoxylin for 1 min, dehydrated in alcohol and xylene, mounted in DPX mountant, and observed under bright light with a Zeiss microscope (Carl Zeiss Jena GmBH, Jena, Germany). Images were procured using Axiovision software (Carl Zeiss Jena GmBH).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution of GC-C, Guanylin, and Uroguanylin Transcripts in Male and Female Reproductive Tissues of the Rat

The anticipated low level of expression of GC-C in extraintestinal tissues prompted us to look for its mRNA using RT-PCR analysis. PCR products were then subjected to Southern blot analysis using as a probe the product obtained from RT-PCR of total intestinal RNA, which amplified the PKLD of rat GC-C. An 888-bp product was amplified from the ovary, oviduct, and uterus (Fig. 1). In addition, RT-PCR of testis, epididymis, seminal vesicle, and prostate gland tissue amplified an 888-bp GC-C product. The products from the intestine, ovary, and epididymis obtained by RT-PCR were visible after ethidium bromide staining, suggesting that GC-C is highly expressed in these tissues (Fig. 1). Individual products obtained by RT-PCR were cloned and sequenced, and analysis revealed that they were identical to the sequence of rat GC-C cloned from the intestine (data not shown).



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  		FIG. 1. Amplification of cDNA of GC-C by RT-PCR using RNA from various rat tissues and PKLD-specific primers. PCR products were resolved on a 1% (w/v) agarose gel stained with ethidium bromide and visualized under ultraviolet light. The RT-PCR product is visible only in the intestine, ovary, and epididymal tissue. PCR products were separated on an agarose gel and subjected to Southern blot analysis using as a probe the radiolabeled PCR product obtained from the rat intestine

Ligands for GC-C act in an autocrine, paracrine, or endocrine manner. We examined the expression of guanylin and uroguanylin in various reproductive tissues by RT-PCR and Southern blot analyses, and the data are shown in Figure 2. PCR products of 250 bp for guanylin and 317 bp for uroguanylin were detected in a number of tissues, and the sequences of these products were identical to those reported for rat guanylin and uroguanylin reported previously. These results therefore suggest that signaling mediated by GC-C and its ligands could play a role in reproductive tissues.



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  		FIG. 2. Amplification of guanylin and uroguanylin cDNA by RT-PCR. Southern blot analysis of RT-PCR products of guanylin and uroguanylin using probes prepared by labeling PCR products of guanylin and uroguanylin obtained by RT-PCR from the rat intestine. Guanylin and uroguanylin primers amplify 250- and 317-bp amplicons, respectively

It was important at this stage to show the expression of a functional form of GC-C, composed of the extracellular domain linked to the PKLD and the catalytic domain. In addition, activation of GC-C by its ligand uroguanylin would indicate that the protein expressed in these tissues was responsive to ligand stimulation. Because the epididymis showed the maximum expression of GC-C mRNA, we attempted to detect expression of GC-C protein in this tissue by Western blot analysis and to characterize the cell types in the epididymis that expressed GC-C by immunohistochemistry using a monoclonal antibody to the receptor.

Expression of GC-C in Epididymis

Membranes were prepared from the cauda and caput epididymides, and GC-C was immunoprecipitated from solubilized membrane preparations using the monoclonal antibody GC-C:4D7 generated against the PKLD of GC-C. In human cell lines, this antibody reacts with two proteins of 145 and 130 kDa, representing glycosylated forms of GC-C. Bands of similar sizes were detected following immunoprecipitation from membranes prepared from both caput and cauda epididymides (Fig. 3A), indicating that the full-length GC-C protein is expressed in this tissue. These bands were the only ones detected on Western blotting because an immunoprecipitation step was included prior to analysis.



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  		FIG. 3. GC-C expression in the rat epididymis. A) For Western blot analysis, 5 mg of membrane protein from caput and cauda epididymides was solubilized in RIPA buffer, and GC-C was immunoprecipitated using GC-C:4D7 monoclonal antibody. Two specifically reactive bands,130 and 145 kDa, were detected. B) Stimulation of cGMP production by uroguanylin in caput and cauda epididymal tissue. Minced tissue was treated with uroguanylin, and extracts were prepared for measurement of cGMP as previously described. Data shown are the mean ± SEM of duplicate determinations of each experiment performed twice. Asterisks represent P > 0.05

To determine whether GC-C expressed in the epididymis was responsive to ligand stimulation, we prepared minced tissues from the caput and cauda epididymides, added either the ST peptide or uroguanylin, and measured cGMP produced. A 3- to 5-fold stimulation of cGMP levels was seen (Fig. 3B), indicating that GC-C expressed in the epididymis was functional in terms of ligand-stimulatable cGMP production.

To study the cellular localization of GC-C in epididymal tissue, we performed immunohistochemical analysis with paraffin-embedded sections, using GC-C:4D7 monoclonal antibody. GC-C was expressed in the tubular epithelial cells of both caput epididymis and cauda epididymis (Fig. 4). The specificity of antibody binding was shown by incubating sections with antibody preadsorbed with excess GST-PKLD fusion protein or an equivalent concentration of normal mouse IgG (data not shown). Our results therefore suggest a robust expression of GC-C in the epididymis, perhaps indicating a role for this signaling pathway in this tissue in regulating fluid composition suitable for sperm maturation.



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  		FIG. 4. Immunohistochemical localization of GC-C in rat epididymis. Caput and cauda epididymal sections were treated with GC-C:4D7 monoclonal antibody or antibody preadsorbed with excess GST-PKLD fusion protein [23] (5 µg/ml), and bound antibody was detected as described previously. Immunostaining is visible in tubular epithelial cells in both caput and cauda epididymides. Magnification x40


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Guanylin and uroguanylin are hormones that control CFTR-dependent salt and water secretion in apical epithelia of the intestine via activation of GC-C. Although GC-C is primarily expressed in the intestine, studies have shown that it is also expressed in extraintestinal tissue, but the role of GC-C in these tissues is not clearly understood. Extraintestinal tissues are not exposed to the bacterial enterotoxin, but the endogenous ligands guanylin and uroguanylin may stimulate the receptor in an autocrine or endocrine manner. Guanylin mRNA has been detected in the uterus and oviduct [17]. Our results also indicate a coexpression of ligands for GC-C and the receptor in various reproductive tissues. CFTR is predominantly expressed in the epithelium of the epididymis [25], where it plays the role of a cAMP-activated chloride channel. CFTR is essential for functioning of the epididymis, and the high expression of GC-C in this tissue suggests a role for regulation of CFTR in the epididymis along similar lines to that in the intestine. It remains to be seen whether cGMP activates this chloride channel to provide a specialized fluid environment for sperm maturation.

Immunohistochemical data indicated that GC-C is localized in the tubular epithelial cells of both the initial segment of the caput epididymis and the cauda epididymis. In addition, some staining is observed in sperm. In the sea urchin, a sperm-associated guanylate cyclase is activated by speract and resact, the egg peptides, and increase in cGMP is known to initiate changes in sperm motility such as kinesis and chemotaxis [26]. Our preliminary evidence suggests the presence of GC-C message in round spermatids and pachytene spermatids (data not shown), raising the possibility that GC-C could regulate sperm chemotaxis in mammals.

The role of cGMP in the epididymis is open to speculation. Signaling through cAMP has been demonstrated in the epididymis [27]. The production of cGMP by particulate GC in membranes of the epididymis stimulated by atrial natriuretic peptide, brain nutriuretic peptide (BNP), and C-type nutriuretic peptide (CNP) was observed in Amyda japonica (a freshwater turtle). In vitro autoradiography studies revealed that another member of the membrane-associated guanylyl cyclase family, the natriuretic peptide receptor, is localized in the smooth muscle cell layer of the duct of the epididymis and therefore may be involved in the control of the transport of sperm in this turtle [28].

In the epididymis, secretion of electrolytes and fluid is controlled by neurohumoral factors such as bradykinin, angiotensin, endothelin, vasopressin, and 5-hydroxytyptamine, which stimulate anion secretion through the formation of prostaglandins [29]. In transgenic animals with impaired CFTR genes, uroguanylin-stimulated anion secretion is reduced in the proximal duodenum, but anion secretion responses to uroguanylin are not completely lost in the duodenum of the CF mouse. This finding indicates that uroguanylin regulates anion secretion in this segment of the intestine via CFTR-independent as well as CFTR-dependent mechanisms [30, 31]. Uroguanylin may therefore have similar physiological functions in the regulation of fluid and electrolyte transport in the epididymis. Other than CFTR, there are Na+/H+ and Cl-/HCO3- exchangers in the epididymis, and guanylin/uroguanylin may also act as regulators of channels and transporters of other Cl- channels, K+ channels, and epithelial Na+ channels [32].

GC-C knockout mice are resistant to STa-induced diarrhea and to enterotoxigenic bacteria that produce ST [33, 34]. Because natriuretic responses to uroguanylin and ST in vivo are retained in knockout mice, uroguanylin responses may also be retained in the epididymis and hence GC-C knockout mice remain viable and fertile and may develop normally. The information presented in this communication extends the cGMP signal transduction pathway to reproductive tissues in which the molecular machinery potentially exists for an intrinsic mechanism for regulation of target cell function by guanylin- and uroguanylin-mediated activation of GC-C. GC-C could be involved in the regulation of transepithelial, electrogenic secretion of chloride through cGMP-dependent protein kinase-mediated phosphorylation and activation of apical CFTR molecules in the epithelial cells of the epididymis.


   FOOTNOTES
 
1 This work was supported by a grant under the India-Japan Inter-Governmental Science and Technology Cooperation program funded by the Department of Science and Technology, Government of India. S.S.V. was the recipient of a Short Term Associateship funded by the Department of Biotechnology, Government of India. Back

2 Correspondence. FAX: 91 80 3600999; sandhya{at}mrdg.iisc.ernet.in Back

Received: 17 April 2002.

First decision: 17 May 2002.

Accepted: 1 July 2002.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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