Effects of testosterone on muscarinic acetylcholine receptors in the rat epididymis

Life Sciences 77 (2005) 656 – 669 www.elsevier.com/locate/lifescie

Effects of testosterone on muscarinic acetylcholine receptors in the rat epididymis Elisabeth Maro´stica, Maria Christina W. Avellar, Catarina S. PortoT Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de Sa˜o Paulo-Escola Paulista de Medicina, Rua Treˆs de maio 100, INFAR, Vila Clementino, 04044-020 Sa˜o Paulo, Brazil Received 22 July 2004; accepted 7 December 2004

Abstract The effect of testosterone on the expression of muscarinic acetylcholine receptor (mAChR) subtypes was studied in the rat epididymis, at mRNA and protein level. The rat androgen status was monitored by measuring plasma testosterone level and caput and cauda epididymis wet weight. Ribonuclease protection assay (RPA) and [3H]quinuclidinyl benzilate ([3H]QNB) binding assay were performed in the caput and cauda epididymis from control (50-day old), castrated, castrated and treated with testosterone and sexually immature (30-day old) rats. The expression of each mAChR transcript subtype differed depending on the epididymal region analyzed and rat testosterone and/or testicular factors status. In control rats, RPA showed the presence of mRNA for M1, M2 and M3 mAChR in the caput and cauda epididymis. The abundance of m2 and m3 transcripts in the cauda was higher than that in the caput epididymis. Low amount of m1 transcript was observed in both regions. Orchidectomy increased m1 mRNA amount in the caput and cauda epididymis when compared to control rats, an effect slightly modified by testosterone replacement. Although orchidectomy down-regulated the level of m2 transcript in both epididymal regions, castration significantly increased m3 mRNA amount in the caput region. These effects on m2 and m3 transcripts were prevented by testosterone replacement to castrated rats. Similar abundance of m3 transcript, however, was detected in the cauda

T Corresponding author. Tel./Fax: +55 11 5576 4448. E-mail address: [email protected] (C.S. Porto). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.12.031

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epididymis of all experimental group tested. [3H]QNB binding studies revealed that orchidectomy downregulated the number of mAChR detected in both epididymal regions, an effect also prevented by testosterone replacement. Thus, testosterone and/or testicular factors may play a role in the regulation of mAChR expression in the rat epididymis. D 2005 Elsevier Inc. All rights reserved. Keywords: Epididymis; Testosterone; Muscarinic acetylcholine receptors

Introduction The epididymis, an organ that provides an unique and specialized environment to sperm final maturation and storage prior to ejaculation (Robaire and Hermo, 1988; Cooper, 1995), is submitted to hormonal and neuronal regulation that are essential to sperm maturation. These processes, however, have not been fully understood. The epididymis receives autonomic innervation, including adrenergic and cholinergic fibers, originary from the inferior mesenteric ganglion, major pelvic ganglion and pelvic accessory ganglion. The relative abundance and arrangement of nerves vary along the epididymis, the innervation being more abundant in the cauda epididymis than in the other epididymal segments (caput and corpus) (El-Badawi and Schenk, 1967; Hodson, 1970; Robaire and Hermo, 1988; SuarezGarnacho et al., 1989; Silva et al., 2002). Adrenergic innervation in epididymis of adult animals controls excurrent duct system contraction, sperm transport through it, blood flow, electrolyte transport and protein processing (Baumgarten et al., 1968; Billups et al., 1990a,b; Chan et al., 1994; Ricker et al., 1996; Kempinas et al., 1998a,b). The involvement of the cholinergic system in the epididymis has also been described. In vitro and in vivo studies have shown that cholinergic agonists induce epididymal contraction (Laitinen and Talo, 1981; Pholpramool and Triphrom, 1984) and data from our laboratory have identified muscarinic acetylcholine receptors (mAChR) expressed along rat epididymis (Maro´stica et al., 2001). In this latter study, m2 mRNA was detected in both caput and cauda epididymis, while m3 mRNA was only identified in the cauda epididymis. Competition binding studies with [3H]QNB, a mAChR nonselective antagonist, indicated a predominance of M2 mAChR subtype in both epididymal regions (Maro´stica et al., 2001) and functional studies suggested the involvement of M3 mAChR subtype in contraction of cauda epididymis (unpublished data). Androgens have potent effects on many aspects of neuronal regulation of male reproductive organs during development and adulthood, inducing changes in soma size, dentritic arborization, synthesis of neurotransmitter and receptor expression (see Gibson, 1981; Keast and Saunders, 1998; Keast, 1999, for reviews). The androgen regulation of mAChR density has been shown in prostate, vas deferens and urinary bladder (Shapiro et al., 1985; Longhurst and Brotcke, 1989; Anderson and Navarro, 1988). The literature also indicates that orchidectomy induces reduction in the cholinesterase content (Risley and Skrepetos, 1964) and an increase in the amplitude of spontaneous contractile activity (Din-Udom et al., 1985) in the rat epididymis. In the present work, the effects of testosterone on the expression of mAChR subtypes, at mRNA and protein level, were studied in this accessory sex organ.

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Materials and methods Animals and treatments Male Wistar rats were housed in the Animal Facility at Instituto Nacional de Farmacologia, UNIFESP-EPM and maintained on a 12 h light, 12 h dark lighting schedule, at 208C, food and water ad libitum. Animal procedures were according to guidelines approved by the Research Ethical Committee from UNIFESP-EPM. Rats were divided in four groups: control (50-day old, CO), castrated (animals submitted to surgical castration at 40-day old and sacrificed on day 10 after castration, CA), castrated and treated with testosterone (animals surgically castrated and immediately treated with testosterone proprionate 3 mg/kg, s.c., daily, for 10 days, CAT) and sexually immature (30-day old, SI). The epididymis was dissected, freed 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) (Turner et al., 1990). Caput and cauda regions were used throughout the experiments. Rat body weight and wet weights of caput and cauda epididymis, expressed as relative weight (mg tissue/ 100 g body weight), were determined. Preliminary experiments with tissues from sham-operated rats or animals injected with vehicle were also tested as controls. Since no significant changes were observed when these two experimental groups were compared to control, all subsequent experiments were performed with tissues from normal rats as a control. Testosterone propionate was dissolved in ethanol and soybean oil (1:3 v/v). Measurement of plasma testosterone level Blood from abdominal aorta artery was collected from eight to eighteen animals for each experimental group, and the plasma testosterone level was measured by radioimmunoassay, using Coat-A-Count Total Testosterone kit (Diagnostic Products Co., Los Angeles, CA), according to manufacturer’s instructions. The assay detection limit was 0.04 ng/ml, and the intra-assay and inter-assay coefficients of variation were 2.4% and 2.3%, respectively. Ribonuclease protection assay (RPA) RNA isolation Total RNA was extracted from rat caput and cauda epididymis, brain and heart, using guanidine isothiocyanate followed by centrifugation through a cesium chloride gradient, as previously described (Chirgwin et al., 1979). Tissues from two to four animals from each experimental group were pooled and used for each RNA extraction. RNA samples were then quantified, using a spectrophotometer at 260/280 nm and stored at
708C for later use. RNA labeling Linearized mAChR constructs, containing rat cDNA inserts for m1, m3 and m4 in the antisense orientation, under the transcriptional control of SP6 RNA polymerase, were used to make antisense stranded RNA probes. Plasmids were kindly provided by Dr. Tom I. Bonner (Laboratory of Cell Biology, NIH, Bethesda, MD) and are described in Bonner et al. (1987). A 594 bp PCR product, including a 552 bp m2 gene fragment in the antisense orientation under T7 RNA polymerase

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transcriptional control, was obtained in our laboratory. Specific m2 gene primers included a 21 nt T3 and T7 promoter sequence at the 5V end of the sense and antisense primer, respectively, as follows: m2-T3 sense and m2-T7 antisense. Linearized plasmid containing a 304 bp h-actin gene fragment in the antisense orientation under the transcriptional control of T7/SP6 promoter was obtained from AMBION, Inc. (Austin, TX). RNA probes were radiolabeled with [a-32P]UTP and specific RNA polymerase with MAXIscript in vitro transcription kit. Full length probes were purified on a denaturing 8 M urea/5% polyacrylamide gel prior to use. Sizes of full length probes and protected fragments were, respectively, m1: 430/377 nt, m2: 577/552 nt, m3: 720/687 nt, m4: 563/519 nt, and h-actin: 304/245 nt. Hybridization Hybridization of RNA probes to total RNA samples was performed with RPA IIIk assay kit, as previously described (Borges et al., 2001). Total RNA from rat brain was used as positive control for m1, m3 and m4 transcripts and total RNA from rat heart for m2 transcript. Assays were performed with total RNA (caput and cauda epididymis, 50 Ag; brain, 20 Ag; heart, 20 Ag), obtained from four to six different RNA extractions, and radiolabeled probe (106 cpm). Probe excess was confirmed in experiments with increasing amounts of total RNA. Nucleotide sizes were determined on a denaturing 8 M urea/5% polyacrylamide gel by comparison with the 0.1–1.0 Kb [32P]-RNA Century Marker Template set. Gels were dried and exposed to XAR-5 film (Kodak, Co., Rochester, NY) for 12–72 h at
708C. Autoradiographs were scanned and, within the linear range of optical density, the level of each mRNA transcript was estimated by the area as arbitrary unit using Scion Image program (Scion Co., Frederick, USA) and normalized against h-actin in each sample. [3H]quinuclidinyl benzilate ([3H]QNB) binding assay Membrane preparation The caput and cauda epididymal regions, obtained from twelve to thirty six animals from each experimental group, were pooled and used for each membrane preparation. Tissues 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 at 48C, with an Ultra-Turrax homogenizer. Membrane preparation was obtained as described by Maro´stica et al. (2001). Protein concentration of membrane preparations was determined with a protein reagent assay (Bio Rad Laboratories, Inc., Hercules, CA). Saturation binding experiments [3H]QNB binding assays were performed in rat epididymal membrane, obtained from three to six different membrane preparations. Binding assays were carried out as previously described (Maro´stica et al., 2001). Briefly, epididymal membrane (160 Ag protein/ml) was incubated with 0.02 nM to 12 nM [3H]QNB in the absence (total binding) and presence (nonspecific binding) of 1 mM atropine for 1 h at 308C. Specific binding was calculated as the difference between total and nonspecific binding. Saturation binding data were analyzed using a weighted nonlinear least-square iterative curve-fitting program GraphPad Prism (GraphPad Prism Software Inc, San Diego, CA, USA). 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 Scatchard plot (Munson and Rodbard, 1980).

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Statistical analysis Data were expressed as mean F SEM. Statistical analysis was determined by one-way analysis of variance (ANOVA) followed by Newman-Keuls test for multiple range comparisons, or by Student’s ttest to compare differences between two groups (Milliken and Johnson, 1984). P values b 0.05 were accepted as significant. Drugs and reagents [3H]Quinuclidynil benzilate (45,5–49 Ci/mmol), [a-32P]UTP (800 Ci/mmol) and Aquasol II were purchased from New England Nuclear (Boston, MA). Coomassie blue G (protein reagent) was purchased from Bio Rad Laboratories (Richmond, CA). MAXIscriptk transcription kit, RPA IIIk assay kit and RNA Century Marker Template Plus were purchased from AMBION, Inc. (Austin, TX). All other drugs and reagents were purchased from Sigma Chemical Co. (St Louis, MO) or GIBCO (Gaithersburg, MD).

Results Rat body weight, epididymis relative weight and plasma testosterone level Rat body weight significantly increased from 30-to 50-day old rats. The different animal treatments, however, did not induce any change in rat body weight (Table 1). The rat androgen status was monitored by measuring plasma testosterone level and caput and cauda epididymis relative weight (Table 1). Plasma testosterone levels were low in the immature rats (30-day old rats) and increased significantly in 50-day old rats (control). This hormone change occurred in parallel with an increase in both caput and cauda epididymis relative weight. Orchidectomy decreased the plasma testosterone level and the relative weight of both epididymal regions. Although testosterone treatment in castrated rats induced a 5-fold increase in the plasma testosterone level, the relative weight of the caput and cauda regions was partially maintained when compared to control animals (Table 1).

Table 1 Plasma testosterone, body weight and epididymis relative weight from control (50-day old) (CO), castrated (CA), castrated and treated with testosterone (CAT) and sexually immature (30-day old) (SI) rats Experimental group

Plasma testosterone (ng/ml)

Body weight (g)

CO CA CAT SI

0.53 F 0.07a (n = 14) b0.04b (n = 8) 2.89 F 0.21c (n = 18) 0.24 F 0.03d (n = 15)

158.75 F 3.78a 155.00 F 1.67a 153.33 F 2.82a 68.33 F 0.41b

Epididymis relative weight (mg/100 g) caput (n (n (n (n

= = = =

12) 6) 6) 6)

26.30 F 0.85a 7.58 F 0.18b 14.05 F 0.39c 14.83 F 0.95c

cauda (n (n (n (n

= = = =

12) 6) 6) 6)

16.25 F 6.94 F 14.17 F 11.83 F

0.32a,* (n = 12) 0.20b,* (n = 6) 0.43c (n = 6) 0.75d,* (n = 6)

Values are expressed as mean F SEM of (n) different animals. Different letters in the same column indicate significant differences among values obtained for different experimental groups ( P b 0.05; Newman-Keuls test). T Significantly different from caput region ( P b 0.05; Student’s t-test). Testosterone plasma assay detection limit was 0.04 ng/ml.

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Expression of mAChR mRNA subtypes in rat epididymis RPA studies indicated that protected fragments were visualized as a single band in the expected sizes when RNA from brain (m1: 377 nt; m3: 687 nt; m4: 519 nt) or heart (m2: 552 nt) was used as positive controls (data not shown). The presence of m1, m2 and m3 protected fragments was observed when labeled RNA probes were hybridized with total RNA from caput and cauda epididymis of the different experimental groups tested (Fig. 1). Although m4 protected fragments were not detected in these tissues, the absence of this transcript in the epididymis was not due to probe or RNA quality since this mAChR mRNA subtype was detected in the brain (data not shown). h-actin expression was similarly detected in both epididymal regions of all experimental groups (Fig. 1). The expression of each mAChR transcript subtype differed depending on the epididymis region analyzed and rat testosterone and/or testicular factors status. Low levels of m1 transcript in the caput and cauda of epididymis were detected in 50-day old rats (control) (Fig. 1). According to densitometric analysis, orchidectomy significantly increased the abundance of m1 transcript in both epididymal regions when compared to control rats. Testosterone replacement in castrated rats slightly modified this effect (Fig. 1). In the caput and cauda epididymis from immature rats, the levels of m1 transcript were higher than in epididymis from 50-day old rats (Fig. 1). The level of m2 transcript was higher in the cauda than in the caput region from control rats. This transcript subtype was downregulated by orchidectomy in the caput and cauda epididymis, an effect prevented by testosterone replacement in castrated rats (Fig. 1). In the caput epididymis from immature animals, the level of m2 mRNA was higher than that observed in epididymis from 50-day old rats. In the

m3

β-actin

Arbitrary Units

SI

CAT

CA

CO

SI

CAT

CAPUT

Arbitrary Units

m2

CAUDA

Arbitrary Units

m1

CA

CO

CAPUT

CAUDA

200

200

100

100

0

0

200

200

100

100

0

0

200

200

100

100

0

m1

m2

m3

0

CO CA CAT SI

CO CA CAT SI

Fig. 1. Ribonuclease protection assay for the identification of mAChR mRNA subtypes in caput and cauda epididymis from control (50-day old) (CO), castrated (CA), castrated and treated with testosterone (CAT) and sexually immature (30-day old) (SI) rats. Left panel: Representative autoradiogram. Protected fragments of m1 (377 nt), m2 (552 nt), m3 (687 nt) and h-actin (245 nt) mRNA were separated on 8 M urea/5% polyacrylamide gel and are indicated by respective arrows. Right panel: Densitometric analysis of the results obtained from the autoradiograms, expressed as arbitrary units. Results are representative of four to six experiments.

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cauda epididymis, however, the expression of this transcript was less abundant in the immature rats when compared to 50-day old rats (Fig. 1). The amount of m3 transcript was also more abundant in the cauda than in the caput epididymis from control rats. In the caput region, orchidectomy induced an increase in the m3 transcript levels, an effect prevented by testosterone replacement in castrated rats. High levels of m3 transcript were detected in the caput epididymis from immature rats when compared to 50-day old rats. In the cauda epididymis, however, the abundance of m3 transcript was similar among all the experimental groups (Fig. 1). Binding of [3H]QNB to rat epididymal membrane The binding of [3H]QNB to caput and cauda epididymal membranes was specific and saturable in all experimental groups (Fig. 2). Scatchard analysis of [3H]QNB specific binding fitted best a one-site model in membranes from both regions of the epididymis, suggesting the presence of a single class of high affinity sites. Analysis of three to six experiments, performed in triplicate, yielded dissociation constant (KD) and maximum number of binding sites (Bmax) summarized in Table 2. Although the receptor affinity showed statistical difference depending on epididymal region analyzed and treatment used, the range of KD values (0.47 to 1.5 nM) obtained in the caput and cauda epididymis from all experimental groups was within the values reported by the literature in different tissues of the male reproductive tract (0.02 to 5 nM). Orchidectomy down-regulated the number of mAChR in both regions,

.

Fig. 2. Scatchard plot of [3H]QNB specific binding in the caput ( ) and cauda (o) epididymal membranes obtained from control (50-day old) (A), castrated (B), castrated and treated with testosterone (C) and sexually immature (30-day old) (D) rats. Results are representative of three to six different experiments, performed in triplicate.

Epididymal COy region KD Caput Cauda

1.12 (n = 0.47 (n =

CA Bmax a

F 0.13 6) F 0.07a,* 6)

CAT

KD a

59.16 F 6.61 (n = 6) 76.18 F 3.39 a,* (n = 6)

1.50 (n = 1.05 (n =

Bmax c

F 0.14 5) F 0.06b 3)

SI

KD b

25.94 F 1.85 (n = 5) 50.18 F 4.89b,* (n = 3)

Bmax b

0.56 F 0.11 (n = 3) 1.00 F 0.15b (n=4)

KD a

45.74 F 5.14 (n = 3) 72.91 F 8.62a,* (n = 4)

Bmax b

0.63 F 0.07 (n = 5) 1.36 F 0.08b,* (n = 5)

49.00 F 4.46a (n = 5) 79.62 F 8.00a,* (n = 5)

Values are expressed as mean F SEM of (n) different experiments. Different letters in the same row indicate significant differences among values obtained for different experimental groups ( P b 0.05; Newman-Keuls test). T Significantly different from caput region ( P b 0.05; Student’s t-test). y Data published in Biol. Reprod. 65: 1120, 2001.

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Table 2 Dissociation constant (KD) (nM) and maximum number of [3H]QNB binding sites (Bmax) (fmol/mg protein) in caput and cauda epididymal membranes from control (50-day old) (CO), castrated (CA), castrated and treated with testosterone (CAT) and sexually immature (30-day old) (SI) rats

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an effect prevented by testosterone replacement in castrated rats (Fig. 2, Table 2). The number of mAChR detected in both epididymal regions from immature animals was similar to 50-day old rats.

Discussion The presence of androgen receptors and mAChR in the rat epididymis has been shown in the literature (Pujol and Bayard, 1979; Sar et al., 1990; Zhu et al., 2000; Maro´stica et al., 2001). In this organ, histological and functional differentiation of the caput region during postnatal development precedes that of the cauda region (Sun and Flickinger, 1979; Rajalakshmi, 1985; Limanowski et al., 2001), mainly due to testicular fluid influence and the age-dependent and segment-specific role of testosterone during epididymal development (Sun and Flickinger, 1982; Viger and Robaire, 1994). The epididymis receives rete testis fluid that has 10-to 12-fold higher androgen concentrations than normal serum (Pujol et al., 1976). Luminal and tissular androgen concentration in the caput region is also higher than in the cauda epididymis (Turner et al., 1984; Jean-Faucher et al., 1986). In the present study, bilateral orchidectomy and testosterone replacement in castrated rats were used to determine whether the steady state of mAChR mRNA is under androgen control. Orchidectomy decreased the plasma testosterone level and the relative weight of caput and cauda epididymis. Testosterone replacement in castrated rats resulted in highly elevated serum testosterone concentration and prevented the decrease induced by castration in the relative weight of rat epididymis. (Table 1). This effect was more effective in the cauda (87%) than in the caput (53%) epididymis as previously reported by the literature (Brooks, 1979). Molecular studies have identified five mAChRs genes that are expressed in multiples tissues, each gene corresponding to the mAChR pharmacological subtypes M1 to M5 (see Birdsall et al., 1998; Caulfield and Birdsall, 1998; Eglen et al., 1999; 2001 for reviews). Reverse transcriptase-polymerase chain reaction studies, performed previously in our laboratory, revealed the presence of m2 transcript in the caput region, while m2 and m3 mRNA were observed in the cauda epididymis from 50-day old rats (Maro´stica et al., 2001). Ribonuclease protection assays, performed in the present study, confirmed these results. These studies further revealed the presence of low levels of m1 transcript in the caput and cauda epididymis as well as m3 mRNA in the caput region (Fig. 1). Ribonuclease protection assays also revealed that the abundance of mAChR transcripts differed depending on the epididymal region analyzed and rat testosterone and/or testicular factors status. In the epididymis from immature rats, the levels of m1 transcript were higher than in epididymis from 50-day old rats. Interestingly, both orchidectomy and testosterone replacement in castrated rats increased the amount of m1 transcript in the caput and cauda epididymis when compared to control rats (Fig. 1). These data suggest that testicular factors are probably involved in the regulation of m1 transcripts, since testosterone was not able to prevent all the effects induced by orchidectomy. Components within rete testis fluid may act to activate or repress the expression of certain epididymal genes and/or the synthesis and secretion of proteins (see Robaire and Viger, 1995; Kirchhoff, 1999; Hinton et al., 2000 for reviews). The abundance of m2 and m3 transcripts in the cauda region was higher than that observed in caput epididymis. Although orchidectomy down-regulated the level of m2 transcript in both epididymal regions, the m3 transcript was dependent on the region analyzed. While orchidectomy significantly increased the levels of mRNA for M3 mAChR in the caput epididymis, the abundance of this transcript

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was not different in the cauda epididymis among all experimental groups. Testosterone replacement in castrated rats prevented the effects induced by castration on m2 and m3 transcripts (Fig. 1). In immature animals, the abundance of m2 transcript in both caput and cauda epididymis was higher than that observed with orchidectomy. Similar pattern of detection was also observed for this transcript in tissues from rats castrated and treated with testosterone (Fig. 1). It is important to emphasize that the plasma testosterone concentration was low in sexually immature rats, below the assay detection limit in the castrated rats and high in rats castrated and treated with testosterone. The relative weights of caput and cauda epididymis from immature rats, however, were similar to those obtained after testosterone replacement in castrated rats despite of the difference in plasma androgen concentration between these two groups (Table 1). Taken together, these data suggest that the levels of m3 mRNA in the caput and m2 mRNA in caput and cauda region depend on the effective concentration of testosterone present in epididymal tissue. Recent studies have also identified genes for which expression is regulated after orchidectomy throughout the epididymis, in a segment-specific dependent pattern (Ezer and Robaire, 2003). [3H]QNB saturation binding assays of the present study indicated the presence of one class of highaffinity sites for QNB in caput and cauda epididymis from all experimental groups, as previously described for 50-day old rats (Maro´stica et al., 2001). Receptor affinity was different depending on the epididymal region analyzed and rat androgen status. The range of KD values obtained in caput and cauda epididymis was comparable to other tissues of male reproductive tract (0.02 to 5 nM) that express mAChR such as prostate (Lepor and Kuhar, 1984a,b; Yazawa et al., 1994), vas deferens (Lepor and Kuhar, 1984b), bladder (Levin et al., 1982; Batra et al., 1987), Sertoli cells (Borges et al., 2001) and corpus cavernosum (Traish et al., 1990). Moreover, the discovery of the family of Regulators G-protein Signaling (RGS) proteins (Brady and Limbird, 2002; Hur and Kim, 2002) and the relevance of homo- or heterodimerization of these receptors (Rios et al., 2001) bring up several possibilities to the regulation of coupled G-protein receptors function, including changes in the receptor affinity in response to agonists. Thus, the analysis of the changes in mAChR affinity observed in the different experimental groups tested in the present study is complex. The number of mAChR in both epididymal regions in immature animals was similar to 50-day old rats (Table 2), indicating that the presence of mAChR in rat spermatozoa (Young and Laing, 1991; Baccetti et al., 1995) did not interfere in mAChR number observed in the 50day old rats, since in 30-day old rat the spermatozoa was not present in epididymis (data not shown). Orchidectomy down-regulated the number of mAChR in both regions of the epididymis, although this effect was more evident in the caput than in the cauda region. Testosterone replacement in castrated rats prevented this effect (Fig. 2, Table 2). There is no direct correlation between the decrease of mAChR number after orchidectomy and the atrophy of tissue, since testosterone replacement in castrated rats partially maintained caput epididymis relative weight (Table 1) and protein content (3.93 F 0.31, 0.98 F 0.02 and 1.81 F 0.04 mg/organ in CO, CA and CAT groups, respectively) although mAChR density was similar to control rats (Fig. 2, Table 2). It is a common finding that M3 mAChR mediates smooth muscle contraction in several tissues despite of the predominance of M2 mAChR observed in radioligand binding studies (Eglen et al., 1996). Our previous competition binding study with [3H]QNB also indicated a predominance of M2 mAChR subtype, at protein level, in both epididymal regions (Maro´stica et al., 2001). Thus, the down-regulation of mAChR number induced by orchidectomy in [3H]QNB saturation binding studies performed in the present work may be reflecting the M2 mAChR subtype. Since a down-regulation of m2 transcript was also detected in RPA studies after orchidectomy, these results suggest that the testosterone level regulates

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mAChR expression in the rat epididymis. Similar effect of androgen was also observed in other male reproductive tissues, such as rat prostate (Shapiro et al., 1985), rat vas deferens (Longhurst and Brotcke, 1989) and rabbit urinary bladder (Anderson and Navarro, 1988). Despite emerging evidences that testosterone may have multiples effects on autonomic neurons (Keast, 2000), the role of androgen modulation on the number of mAChR in the epididymal function remains unexplored. Besides the involvement with smooth muscle contraction, it has been also shown that M1 and M3 mAChRs are also involved, directly or indirectly, in protein secretion in other tissues such as in the prostate (Shapiro et al., 1985; Ruggieri et al., 1995; see Ventura et al., 2002 for review), seminal vesicle (Ehre´n et al., 1997), salivary glands (Nakamura and Masuhara, 1989), pancreas (Iwatsuki et al., 1989) and airway glands (Haddad and Rousell, 1998). Furthermore, functional studies have suggested that activation of M3 mAChR present in airway smooth muscle and mucosal glands can be modulated by M2 mAChR localized to the cholinergic nerve terminals in human and guinea pig trachea, modulating acetylcholine release via functional negative feedback (autoreceptors) (Patel et al., 1995; Haddad and Rousell, 1998). In the epididymis, epithelial cells lining the tubule are actively engaged in the transport of electrolytes and water and protein secretion. These processes lead to the formation of a specialized fluid milieu in which spermatozoa acquire their fertilizing capacity and motility (Leung and Wong, 1993; Cooper, 1995; Chow et al., 2004). Neurotransmitters and humoral agents produced by and acting on the epithelium may influence the fluid secretion in the epididymis. Thus, the modulation of mAChRs subtypes by testosterone observed in the present study may be a regulatory mechanism of luminal microenvironment, essential to sperm maturation. Further experimental approaches, such as immunohistochemical studies using specific antibodies against the different mAChR subtypes detected in the present study, will be important to clarify these events at protein level in the different cells along rat epididymis. In conclusion, the present results provide evidence that the expression of mAChR, at mRNA and protein level, differs depending on the region of epididymis analyzed and rat androgen and/or testicular factors status. Thus, the cholinergic neurotransmitter in the epididymis may be a factor controlling contractility and/or luminal fluid microenvironment. Since receptors are targets for the pharmacological manipulation of the physiological process and represent a direction for the development of selective therapeutic agents, the characterization, regulation and function of mAChR subtypes in the epididymis can be important to new approaches in (in)fertility and/or male contraception.

Acknowledgments We thank Espedita M. de Jesus Silva Santos and Maria Damiana Silva for technical assistance. This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, FAPESP (Grant 97/ 2056-9), Brazil. Doctoral fellowship (E.M.) and research fellowship (M.C.W.A., C.S.P.) supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq, Brazil.

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