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Biology of Reproduction 65, 1120-1126 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Characterization of Muscarinic Acetylcholine Receptors in the Rat Epididymis1

Elisabeth Marósticaa, Emanuele F. Guazea, Maria Christina W. Avellara, and Catarina S. Porto2,a

a Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, SP 04044-020, Brazil

ABSTRACT

The aim of the present study was to characterize the muscarinic acetylcholine receptor subtypes present in the caput and cauda of rat epididymis. The specific binding of [3H]quinuclidinyl benzilate ([3H]QNB) to epididymal membranes was time dependent, temperature dependent, and saturable. The cauda epididymis showed higher affinity to [3H]QNB and higher muscarinic receptor density when compared to the caput region. The [3H]QNB binding was tested in competition studies with different muscarinic receptor antagonists. Each antagonist tested displaced [3H]QNB bound to caput and cauda epididymal membrane with similar affinity. Correlation among the negative logarithm of inhibition constant values (pKi) for these antagonists obtained in the epididymis with their correspondent published pKi values obtained in tissues that expressed each receptor subtype (M1, M2, M3, and M4) indicated that the muscarinic receptors present in caput and cauda epididymis belong to the muscarinic M2 receptor subtype. When reverse transcription-polymerase chain reaction was used to identify muscarinic receptor mRNA subtypes in the epididymis, only m2 transcripts were detected in the caput region, while both m2 and m3 mRNA subtypes were observed in the cauda region. In conclusion, these results demonstrate that muscarinic receptors are present in the rat epididymis, with expression levels dependent on the region of the epididymis analyzed. Thus, the cholinergic neurotransmitter in the epididymis may be a factor controlling contractility and/or the luminal fluid microenvironment.

catecholamines and acetylcholine, epididymis, male sexual function

INTRODUCTION

The male reproductive tract receives abundant innervation from the autonomic nervous system. This innervation has been shown to influence vasoactivity within the testis [1], sperm transport through the excurrent duct system [2], and muscle contraction and secretion within the sex accessory glands [3].

The autonomic innervation to the epididymis, the organ in which sperm undergo final maturation and storage prior to ejaculation [4], includes the inferior mesenteric ganglion, major pelvic ganglion [5], and pelvic accessory ganglion [6, 7]. Within the epididymis, nerve fibers are localized to peritubular and subepithelial regions [8, 9], and a direct neuroepithelial connection has also been suggested [9]. The relative abundance and arrangement of nerves vary along the epididymis, the innervation being more abundant in the cauda epididymis than in the other segments (caput and corpus) [9, 10].

Spermatozoa require an optimal transit time through the epididymis to attain the ability to be motile and to fertilize mature oocytes. Transport of spermatozoa through the epididymis is mainly the result of spontaneous contractions of the smooth muscle surrounding the epididymal duct and can be modified by androgen [11, 12], prostaglandins E2 and F2{alpha} [13–15], {alpha}1-adrenoceptor agonists, and muscarinic receptor agonists [16–18]. It has also been reported that prostaglandins cause a significant potentiation of contractile responses of epididymal smooth muscle to stimulation by norepinephrine or acetylcholine [14].

Ratnasooriya et al. [19] reported that local application of the {alpha}1-adrenoceptor agonist, methoxamine, to the epididymis caused a marked reduction in the fertility of the male rats. However, {alpha}1-adrenoceptor antagonists, bunazosin and tamsulosin, increased sperm concentration and intraluminal fluid movement in the cauda epididymis, decreased the intraluminal pressure, and inhibited the fertility of male rats [20, 21]. Furthermore, the surgical removal of the inferior mesenteric ganglion alters sperm transport [22, 23] and induces changes in cauda epididymal fluid protein composition [6]. Although the rat epididymal smooth muscle has been shown to contract in response to carbachol and methacholine [17, 18, 24], no further data are available on the characteristics of the muscarinic acetylcholine receptors involved in epididymal function. Molecular cloning studies have identified five muscarinic acetylcholine receptor genes that are expressed in multiples tissues, each gene corresponding to the pharmacological subtypes M1, M2, M3, M4, and M5, as revised guidelines of the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) (see Birdsall et al. [25] and Caulfield and Birdsall [26] for reviews). The present study was, therefore, designed to characterize muscarinic acetylcholine receptor subtypes in the rat caput and cauda epididymis.

MATERIALS AND METHODS

Membrane Preparation

Male Wistar rats, 50 days old, were maintained on a 12L:12D lighting schedule, at 20°C, with food and water ad libitum. The rats were killed using the guidelines for the care and use of laboratory animal, approved by the Research Ethical Committee from Universidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina. The epididymis was dissected, free of fat and sectioned in three different regions: caput (initial segment, proximal and distal caput), corpus (proximal and distal corpus), and cauda (proximal and distal cauda) [27, 28]. The caput and cauda regions were minced and homogenized in 25 mM Tris-HCl, pH 7.4, containing 0.3 M sucrose, 5 mM MgCl2, 1 mM EDTA, and 1 mM PMSF (Sigma Chemical Co., St. Louis, MO), with an Ultra-Turrax homogenizer. Each homogenate was centrifuged at 1000 x g for 10 min. The supernatant was filtered through two layers of gauze and then centrifuged at 100 000 x g for 1 h. The final 100 000 x g pellet was resuspended in 1 ml of binding buffer (25 mM Tris-HCl, pH 7.4, containing 5 mM MgCl2, 1 mM EDTA, and 1 mM PMSF) using a Dounce homogenizer and stored at -70°C. All procedures were carried out at 4°C and all solutions contained freshly added 1 mM PMSF to inhibit proteolysis.

Protein Determination

Protein concentration of membrane preparations was determined according to the method of Bradford [29], using BSA as a standard (Bio-Rad protein assay; Bio-Rad Laboratories, Hercules, CA).

Membrane Binding Assay

In preliminary studies, the appropriate conditions for binding assays of [3H]quinuclidinyl benzilate ([3H]QNB), a muscarinic acetylcholine receptor nonselective antagonist, were determined. According to the results, all subsequent binding studies were performed with a membrane protein concentration of 160 µg/ml and incubation time of 1 h at 30°C.

Saturation binding experiments Epididymal membrane preparations (160 µg protein/ml) were incubated with 0.02–12 nM [3H]QNB (specific activity 43.0–45.4 Ci/mmol; New England Nuclear, Boston, MA) in the absence (total binding) and presence (nonspecific binding) of atropine (atropine sulfate) (Sigma) for 1 h at 30°C. Specific binding was calculated as the difference between total and nonspecific binding. All experiments were performed in triplicate, using the binding buffer described before. After incubation, the binding reaction was stopped by addition of ice-cold PBS and rapid filtration through a GF/B glass fiber filter (Whatman International Ltd., Maidstone, UK) under vacuum. The filters were washed three times with 3 ml ice-cold PBS, partially dried under vacuum, and placed in scintillation vials containing Aquasol II (New England Nuclear), and the amount of radioactivity was determined.

Displacement binding experiments Epididymal membrane preparations (160 µg protein/ml) were incubated for 1 h at 30°C with 1 nM (caput) or 0.5 nM (cauda) [3H]QNB in the absence and presence of increasing concentrations of the following muscarinic acetylcholine receptor antagonists: atropine (atropine sulfate; Sigma), nonselective antagonist; pirenzepine (pirenzepine hydrochloride; Sigma), M1 selective; methoctramine (methoctramine tetrahydrochloride; Research Biochemicals International, RBI, Natick, MA), M2:M4 selective; 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide; RBI), M1:M3 selective; and HHSiD (hexahydro-sila-difenidol hydrochloride; RBI), M3:M1 selective (see Birdsall et al. [25], Caulfield and Birdsall [26], and Eglen et al. [30] for reviews). The reactions were stopped as described above. The binding in the presence of unlabeled competitors was expressed as percentage of control (absence of unlabeled antagonists) and plotted as a function of log molar concentration.

Data analysis Saturation and displacement binding data were analyzed using a weighted nonlinear least-square iterative curve-fitting program GraphPad Prism (GraphPad Prism Software Inc., San Diego, CA). A mathematical model for one or two sites was applied. The dissociation constant (KD) and the maximum number of binding sites (Bmax) were determined from a Scatchard plot [31]. The inhibition constant (Ki) was determined from displacement curves using the Cheng and Prusoff equation [32]. All antagonist affinity values were expressed as the negative logarithm of their respective Ki value (pKi). The linear regression plots were also determined using the computer program GraphPad Prism. The correlation coefficients (r) and associated P-values were calculated. The sum of squares of differences in affinity estimated for each plot ({Sigma}[y - x]2, noted ssq) defines the proximity of the data points to the line of identity (y = x) [33].

Reverse Transcriptase-Polymerase Chain Reaction Assays

Caput and cauda epididymis were removed, immediately frozen in liquid nitrogen, and stored at -70°C until use for RNA extraction. Total RNA was extracted from frozen tissues as described by Chirgwin et al. [34]. RNA samples were then quantified and stored at -70°C for later use.

Reverse transcription-polymerase chain reaction (RT-PCR) amplification was performed using a SUPERScript II RT kit preamplification system for first-strand cDNA synthesis, according to the manufacturer's instructions (Gibco BRL, Gaithersburg, MD). Reverse transcription of total RNA (1 µg), using random hexamer primers (50 ng), was performed at 55°C in a reaction volume of 20 µl. Reactions in the absence of reverse transcriptase were also included for each RNA tested in order to check for genomic contamination. PCR was performed in 25 µl total volume containing 1.5 µl cDNA, 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 3 mM MgCl2, 0.25 mM BSA, 0.4 µM each primer, 0.2 mM dNTPs, 0.3 µl [{alpha}-32P]dCTP (New England Nuclear), and 1.25 U Taq polymerase. Samples were loaded into glass microcapillary tubes, and PCR amplification was performed in an Idaho Rapidcycler as follows: one cycle of denaturation at 96°C for 10 sec, followed by 20 cycles of denaturation at 94°C for 10 sec; annealing at 60°C for 10 sec, and extension at 72°C for 45 sec. A final extension of 72°C for 3 min was performed for all samples. Under these conditions, the amplifications of the target genes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were in the linear range, as demonstrated in a series of preliminary experiments used to optmize the number of cycles (15–35 cycles) and the appropriate amount of cDNA (0.5–2.5 µl). Samples were resolved onto 5% polyacrylamide gel in Tris-borate-EDTA buffer. The PCR product was visualized by autoradiography with Kodak X-OMAT AR films. Primers against m1–m4 muscarinic receptor mRNA subtypes [35, 36] and against GAPDH [37] were synthesized in the UNC Oligonucleotide Facility, University of North Carolina at Chapel Hill and in the Department of Biophysics, UNIFESP, Escola Paulista de Medicina, Brazil. Primer sequence, corresponding base sites and the sizes of the PCR products were as follows: m1 sense, 5'-CTGGTTTCCTTCGTTCTCTG-3' (593–612) and m1 antisense, 5'-GCTGCCTTCTTCTCCTTGAC-3' (1233–1214) ({Delta}641); m2 sense, 5'-GGCAAGCAAGAGTAGAATAAA-3' (633–653) and m2 antisense, 5'-GCCAACAGGATAGCCAAGATT-3' (1184–1164) ({Delta}552); m3 sense, 5'-GTGGTG TGATGATTGGTCTG-3' (591–610) and m3 antisense, 5'-TCTGCCGAGGAGTTGGTGTC-3' (1380–1361) ({Delta}790); m4 sense, 5'-AGTGCTTCATCCAGTTCTTGTCCA-3' (543–566) and m4 antisense, 5'-CACATTCATTGCCTGTCTGCTTTG-3' (1052–1029) ({Delta}510); GAPDH sense, 5'-CGGGAAGCTTGTGATCAATGG-3' (258–277) and GAPDH antisense, 5'-GGCAGTGATGCCATGGACTG-3' (614–595) ({Delta}357). All primers and PCR conditions were tested using total RNA from rat brain, because m1–m4 muscarinic receptor mRNA transcripts are known to be expressed in this tissue [38]. The authenticity of each target gene PCR product was confirmed by direct nucleotide sequencing performed with an ABI PRISM 377 automated sequencer (Applied Biosystems, Foster City, CA) and BigDye Terminator Sequencing kit (Applied Biosystems).

Statistical Analysis

Data were expressed as mean ± SEM. Statistical analysis was carried out using ANOVA, followed by Bonferroni test for multiple comparisons, or by two-tailed Student t-test to compare a response in two groups [39]. P-values <0.05 were accepted as significant.

RESULTS

Binding of [3H]QNB in the Rat Epididymal Membranes

The binding of [3H]QNB to caput (Fig. 1A) and cauda (Fig. 1B) epididymal membranes was specific and saturable. Scatchard analysis of specific binding fitted best a one-site model in membranes from both regions of the epididymis (Fig. 1, A and B), suggesting the presence of a single class of high-affinity sites. An analysis of five to six experiments, performed in triplicate, yielded dissociation constant (KD) and maximum number of binding sites (Bmax) summarized in Table 1. The comparison of the binding parameters indicated that the cauda epididymis shows higher affinity to [3H]QNB and higher receptor density when compared to the caput epididymis (Student t-test, P < 0.05).



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  		FIG. 1. Saturation curves (left panel) and Scatchard plot (right panel) of [3H]QNB binding in caput (A) and cauda (B) epididymal membranes. Results are representative of five experiments, performed in triplicate


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  		TABLE 1. [3H]QNB saturation binding parameters in the rat caput and cauda epididymal membranes.a

Effect of Competitive Muscarinic Acetylcholine Receptor Antagonists on the Specific [3H]QNB Binding to Rat Epididymal Membranes

The displacement curves of [3H]QNB bound to membrane preparations from caput and cauda epididymis, induced by muscarinic receptor antagonists, are shown in Figure 2. Analysis of the displacement curves induced by pirenzepine (M1 selective antagonist), methoctramine (M2:M4 selective antagonist), and HHSiD (M3:M1 selective antagonist) indicated a statistical preference for a one-site rather than a two-site fit (F-test, GraphPad Prism program). On the other hand, atropine (nonselective antagonist) and 4-DAMP (M1:M3 selective antagonist), defined two muscarinic binding sites with high (pKiH) and low (pKiL) affinity in both regions of the epididymis. The pKi values obtained from the analysis of antagonist displacement curves by one- or two-sites fit and their respective Hill slopes (nH) are summarized in Table 2. The pKi values obtained from all one-site fit antagonists in the caput epididymis were compared with the pKi values obtained in the cauda epididymis. A highly significant correlation (r = 0.95, P < 0.01) was obtained, close to the line of identity (ssq = 0.95) (Fig. 3). Similar results were observed when the analysis was performed with the pKi values of high-affinity sites obtained from the two-sites fit for atropine and 4-DAMP. Thus, each antagonist tested displaced [3H]QNB bound to caput and cauda membrane with similar affinity.



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  		FIG. 2. Displacement of specific [3H]QNB binding in epididymal membrane from caput (A) and cauda epididymis (B) by unlabeled muscarinic antagonists. Each point and vertical line represent mean ± SEM of three to six experiments, performed in triplicate


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  		TABLE 2. Muscarinic acetylcholine receptor antagonist affinities (pKi values) and Hill slopes (nH) calculated from displacement curves of [3H]QNB bound to rat caput and cauda epididymal membranes.a



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  		FIG. 3. Correlation of pKi values for atropine (1), pirenzepine (2), 4-DAMP (3), methoctramine (4), and HHSiD (5) obtained in membrane from caput and cauda epididymis. The solid line is a linear regression fit through all the points, and the dashed line has a slope equal to unity, passing through the origin. r, Correlation coefficient; ssq, sum of square of differences values

A correlation analysis among the different muscarinic antagonist pKi values obtained in the caput and cauda epididymis with the pKi values obtained by Doods et al. [40] using the same antagonists against guinea-pig cortical tissue (M1), cardiac tissue (M2), and submandibular gland (M3) and Chinese hamster ovary (CHO) cells (m4) was carried out. The correlation plots and respective correlation coefficients (r) are shown in Figure 4. The analysis indicated that the muscarinic acetylcholine receptor detected by [3H]QNB binding has a highly significant correlation to the M2 receptor subtype (r = 0.98, ssq = 1.11, P < 0.003 in the caput; r = 0.99, ssq = 0.29, P < 0.002 in the cauda epididymis).



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  		FIG. 4. Correlation of pKi values for atropine (1), pirenzepine (2), 4-DAMP (3), methoctramine (4), and HHSiD (5) published by Doods et al. [40] with pKi values obtained for the same antagonists in membrane from caput (A) and cauda (B) epididymis. The solid line is a linear regression fit through all the points and the dashed line has a slope equal to unity, passing through the origin. r, Correlation coefficient; ssq, sum of square of differences values

Identification of Muscarinic Acetylcholine Receptor mRNA Subtypes in Epididymis by RT-PCR

The effectiveness of the m1–m4 muscarinic acetylcholine receptor subtype specific primers was checked by amplifying all four muscarinic receptor sequences from rat brain (Fig. 5). Each PCR product was of the predicted size and nucleotide sequence. No PCR products were detected when reverse transcriptase was omitted from the RT-PCR reaction, demonstrating that the amplified products are indeed from cDNA and not from genomic DNA (data not shown). When RT-PCR was performed with total RNA from caput and cauda epididymis, only one DNA band corresponding to the m2 muscarinic receptor mRNA subtype was amplified in the caput epididymis (Fig. 5). On the other hand, m2 and m3 bands were detected when total RNA from the cauda epididymis was tested (Fig. 5). PCR products corresponding to m1 and m4 mRNA transcripts were not detected in rat epididymis.



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  		FIG. 5. Representative autoradiogram from a RT-PCR experiment showing amplification of m1, m2, m3, m4, and GAPDH mRNA targets in caput and cauda epididymis. Rat brain mRNA was used as a positive control for all from muscarinic receptor transcripts

DISCUSSION

The biochemical changes that sperm undergo during the transit to the proximal cauda epididymis are important to confer upon them the ability to fertilize a mature oocyte [41]. However, the mechanisms underlying these changes are not yet understood. Studies using both chemical and surgical denervation have shown that adrenergic innervation affects epididymal functions [6, 7, 22, 23, 42, 43], but the specific role of autonomic innervation of the epididymis on the fertility of epididymal sperm remains unclear.

We now report, by using radioligand binding assays, the presence of muscarinic acetylcholine receptors in the rat caput and cauda epididymis. The [3H]QNB bound to a single class of high-affinity sites in both regions of the epididymis, the cauda epididymis exhibiting higher affinity and containing higher receptor density when compared to the caput region. These data are in agreement with previous neuroanatomic studies showing higher cholinergic innervation density within the cauda than in other segments of the rat epididymis [5, 9, 10].

In order to characterize the muscarinic acetylcholine receptor subtypes present in the rat epididymis, five different muscarinic receptor antagonists were examined for their ability to compete with [3H]QNB for its binding sites. The muscarinic acetylcholine receptor antagonist affinities (pKi values) obtained in the caput and cauda epididymis were compared with those obtained by Doods et al. [40]. Furthermore, a correlation analysis among the pKi values obtained in epididymis was also carried out with the pKi values, for the same antagonists above, obtained from CHO cells expressing human recombinant M1–M5 muscarinic acetylcholine receptor subtypes [44, 45] (data not shown). Both analyses indicated that muscarinic acetylcholine receptors detected in the epididymis have a highly significant correlation with the M2 receptor subtype. In addition, the pKi values for methoctramine obtained in the present study in the caput (7.80 ± 0.15) and cauda epididymis (7.26 ± 0.11) were comparable to those reported in tissues that express muscarinic M2 receptors, such as rat vas deferens (pKi = 8.4 [46]), smooth muscle cell culture from human prostate (pKi = 8.1 [47]), and guinea pig uterus (pKi = 7.5 [40], and pKi = 8.1 [48]). Futhermore, the displacement curve for 4-DAMP revealed two components and the Hill slope differed significantly from the unit in both regions of epididymis. The pKi values for the high-affinity site defined by 4-DAMP in caput and cauda epididymis were similar to that found in binding studies on cloned muscarinic m1 and m3 receptors [44] or to that obtained in glandular tissue (muscarinic M3) [40]. However, the low affinity obtained for most selective compounds, pirenzepine (pKi = 5.65 and 6.06, in the caput and cauda, respectively) and HHSiD (pKi = 6.09 and 6.66, in the caput and cauda, respectively), suggests that muscarinic M1 and M3 receptors are not present in rat epididymis when compared with literature data (pKi = 7.5–7.9 for pirenzepine at the M1 receptor; pKi = 7.8–8.2 for HHSiD at the M3 receptor [25, 26, 30, 40, 44]). Taken together, the radioligand binding studies indicate that caput and cauda epididymis present a predominant population of M2 acetylcholine muscarinic receptors.

RT-PCR assays further substantiated competition binding studies, as the presence of m2 transcript was detected in both caput and cauda epididymis. RT-PCR assays also indicated the amplification of m3 gene product in the cauda region. It is important to emphasize that m3 muscarinic receptor mRNA is present in cauda epididymis, despite the fact that M3 receptor protein was not detected by radioligand binding studies. It is worth mentioning at this point that, although human and rat epididymis contracts in response to cholinergic muscarinic agonists [16, 18, 24, 49], the muscarinic receptor subtype affecting epididymal smooth muscle tone has not been described yet. Regarding muscarinic receptors, it is a common finding that M3 receptors mediated smooth muscle contraction in rat vas deferens, uterus, and ileum, despite a predominant population of M2 muscarinic receptors detected by radioligand binding assays in membrane preparations from these same tissues [30, 46, 50–54]. Thus, discrepancies in receptor characterization may reflect the different sensibility of experimental molecular and pharmacological approaches to detect receptors differently expressed in one or more cells in a given tissue. Considering that different cells are present in the rat epididymis [55], further studies will be necessary to understand the relative contribution of m2 or m3 transcripts in the epididymis. Thus, functional studies using moderately selective drugs coupled to immunological techniques to localize and determine the relative levels of M2/M3 muscarinic receptors will be an important tool to better characterize muscarinic receptor subtypes present in rat epididymis. These studies will indicate if the cholinergic neurotransmitter may be a factor controlling contractility and/or the luminal fluid environment that is essential for the functional preservation of spermatozoa.

In conclusion, the present study demonstrates the presence of muscarinic acetylcholine receptors in the caput and cauda epididymis, the cauda exhibiting higher affinity and higher receptor density than the caput epididymis. A predominant population of M2 muscarinic receptors was detected by competitive binding studies in the caput and cauda epididymis. RT-PCR assays indicated that, besides the presence of m2 transcript in both regions, an m3 gene product was also detected in the cauda epididymis

ACKNOWLEDGMENTS

We thank Espedita M. de Jesus Santos and Maria Damiana Silva for technical assistance.

FOOTNOTES

First decision: 31 October 2000.

1 This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (grant 97/2056-9), Brazil. T.W. Fogarty International (grant 5R37HDO4466-26, subcontract UNC 5-53284). Master fellowship supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (E.M.) and scholarship supported by FAPESP (E.F.G.); research fellowship supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (M.C.W.A. and C.S.P.). Back

2 Correspondence: Catarina Segreti Porto, Section of Experimental Endocrinology, Department of Pharmacology, Rua Três de maio 100, São Paulo, SP 04044-020, Brazil. FAX: 55 115 576 4448; porto.farm{at}infar.epm.br Back

Accepted: May 22, 2001.

Received: September 22, 2000.

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