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BOR - Papers in Press, published online ahead of print July 19, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.054577
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BIOLOGY OF REPRODUCTION 75, 651–659 (2006)
DOI: 10.1095/biolreprod.106.054577
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Mechanisms Regulating Spontaneous Contractions in the Bovine Epididymal Duct1

Marco Mewe 2 3, Christiane K. Bauer 4, Jürgen R. Schwarz 4, and Ralf Middendorff 5

Institut für Anatomie II: Experimentelle Morphologie,3 Institut für Angewandte Physiologie,4 Zentrum für Experimentelle Medizin, Universitätsklinikum Hamburg-Eppendorf, Universität Hamburg, D-20246 Hamburg, Germany Institut für Anatomie und Zellbiologie,5 Justus-Liebig-Universität Giessen, D-35385 Giessen, Germany

ABSTRACT

Muscular autorhythmicity provides propulsion of spermatozoa through the epididymal duct, thereby ensuring sperm maturation. In the present study, the mechanisms underlying the bovine epididymal spontaneous phasic contractions (SCs) were analyzed by using muscle-tension recording and patch-clamp techniques. SCs were recorded from the caput, the corpus, and the proximal cauda region and found to be predominantly myogenic in origin. Removal of the luminal fluid induced a burstlike contraction pattern, and removal of the epithelium, a complete loss of SCs. Application of nifedipine, but not heparin and cyclopiazonic acid, suppressed SCs, indicating that influx of Ca2+ through L-type Ca2+ channels, but not Ca2+ release from intracellular stores, was crucial for maintaining SCs. The prostaglandin-endoperoxide synthase 2 (PTGS2) inhibitor NS-398 caused a region-dependent decrease in SCs and tone. These effects were mimicked by the mitogen-activated protein kinase (MAPK) kinase inhibitor PD-98059. Similarly, the prostaglandin F2alpha (PGF2alpha)-receptor antagonist AL-8810 reduced SC generation, whereas PGF2alpha induced SC-like activity in epithelium-denuded segments. Cell-isolation experiments revealed the existence of three morphologically different types of contractile cells, which also showed distinct biophysical properties: typical smooth muscle cells in the cauda, myofibroblast-like cells all along the duct, and atypical muscle cells (ATMs) with filament-like spurs in all regions with SCs. These data suggest that the bovine epididymal autorhythmicity is based on an epithelial PTGS2-dependent release of (an) excitatory prostaglandin(s) and a MAPK-dependent activation of L-type Ca2+ channels in the contractile cells. ATM cells may provide electrical coupling between myofibroblasts, which is essential for the generation of regular myogenic activity.

epididymis, signal transduction, sperm motility and transport

INTRODUCTION

The mammalian epididymis serves to propel spermatozoa from the testes to the epididymal cauda region. Passage of the spermatozoa through the epididymal duct is a prerequisite for epithelium-dependent sperm-maturation processes, ensuring acquisition of fertilizing potential and progressive motility [1–2], and is brought about by spontaneous phasic contractions (SCs) of periductual muscle layers (for review, see [3]). Hence, an adequate fine tuning of sperm maturation and sperm transport can be presumed. Nevertheless, the basic mechanisms and cells responsible for epididymal SC generation still have to be elucidated.

Many types of smooth muscle are characterized by autorhythmicity with underlying oscillatory depolarizations and/or spike potentials [4]. Such phasic activity is often myogenic, being generated within the smooth muscle itself or electrically coupled pacemaker cells. In general, spontaneous contractions arise from oscillations in the internal Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]i occurs as external Ca2+ enters the cell through voltage-dependent Ca2+ channels or nonselective cation channels and as Ca2+ is released from internal stores, predominantly the sarcoplasmic reticulum (SR). The SR may contribute to excitation–contraction coupling in smooth muscle by supplying Ca2+ for the myofilaments or by modulation of membrane excitability [5, 6]. In addition, mitochondria gain increasing attention as mediators of Ca2+ homeostasis and electrical activity in smooth muscle [7–9].

The epididymal peristalsis has been assumed to be myogenic in origin, because spontaneous myoelectrical activity could be recorded all along the epididymal duct [10]. In addition, sperm transport through the proximal epididymal regions is largely independent of sympathetic stimulation [11]. Several local factors like endothelin-1 [12], cGMP [13], testosterone [14], and prostaglandins [15] seem to be involved in the modulation of epididymal muscle activity. Finally, various cell types had been identified in the epididymal lamina propria [16, 17], raising the question, whether one of them exhibits pacemaker activity.

We have now investigated the origin and basic mechanisms of SC generation in the bovine epididymal duct, which exhibits a morphologic subdivision similar to that in the human [18].

MATERIALS AND METHODS

Tissue Preparation

Epididymal tissue was obtained from sexually mature bulls (≥50 weeks old) in a local slaughterhouse, 10–15 min after exsanguination, and immediately placed in ice-cold Ca2+-free Hanks balanced salt solution (HBSS; Life Technologies, Inc., Karlsruhe, Germany) for transport to the laboratory. For histologic analyses, tissue samples of different regions of the duct (caput-corpus-cauda) were fixed in Bouin fluid for 24 h at 20°C. For electrophysiologic and tension studies, segments of the epididymal duct were separated in ice-cold Ca2+-free physiologic salt solution (PSS) by carefully dissecting the surrounding tissue by the use of fine forceps and scissors. For those tension studies that were performed in the absence of sperm and other intraluminal factors, the segments were perfused with Ca2+-free HBSS by the use of a Hamilton syringe. In some experiments, the epididymal epithelium of proximal duct (caput and corpus) segments was removed by perfusion with 1% Triton X-100 (Merck, Darmstadt, Germany) in PBS (3 min). All preparations were stored for at least 2 h in DMEM (Life Technologies, Inc.) at 4°C until use in tissue bath assays.

Tension Studies

For contraction studies, segments (~1.5 cm in length) of the epididymal duct were mounted with silk thread on two stainless-steel hooks in a double-jacketed 15-ml organ bath. Preparations were equilibrated in modified Krebs bicarbonate Ringer (KBR; continuously gassed with carbogen to provide oxygenation and pH of 7.3–7.4) at 33–34°C. Isometric tension was recorded by a SG4–90 force-displacement transducer (Hugo Sachs, Freiburg, Germany) and displayed on a Linseis L 6510 writer (Seeb, Germany). The output of the transducer was digitized at 1 Hz by using a Metrabyte DAS 1202 interface (Keithley Instruments, Cleveland, OH). Segments from the caudal duct were stretched to a preload tension of ~10 mN, segments from the proximal duct regions to a preload tension of ~6 mN, and 3 mN in case of Triton X-100-pretreated material. Preparations were allowed to equilibrate for 60 min and to relax to a steady-state resting tension to prevent stretch-induced contractions. The bath solution was changed once after 30 min to avoid accumulation of metabolites in the bath. Data collection was carried out by using a DOS program developed by P. Bassalay (Institute of Physiology, UKE, Hamburg, Germany). Further data processing was performed with Sigma Plot 4.01 (SPSS, Chicago, IL).

Immunohistochemistry and Histology

Immunohistochemical analyses were performed essentially as described previously [19]. In brief, paraffin sections (6 µm) were mounted on chrome gelatin-coated slides and incubated with mouse monoclonal anti-{alpha}-smooth muscle actin ({alpha}-SMA) antibodies (Sigma, Deisenhofen, Germany; 1:400) overnight. Biotinylated antimouse IgG (Dako, Hamburg, Germany) were used as secondary antibody. Peroxidase activity was visualized by the nickel glucose oxidase technique. For negative controls, the primary antibody was replaced by PBS. For histologic analyses, paraffin sections (6 µm) were stained with hematoxylin/eosin (H&E).

Cell Isolation and Identification

For enzymatic dissociation, separated and cleaned duct segments were cut into small pieces (2–3 mm) and placed in carbogenated (5% CO2 to 95% O2) Ca2+-free Krebs bicarbonate Ringer (Ca2+-free KBR; pH 7.3–7.4) for 30 min at 21°C. The pieces were then transferred to a first digestion solution (containing 1 mg/ml elastase [type I; Sigma], 3 mg/ml collagenase [type II; Sigma], and 3 mg/ml fatty acid-free bovine serum albumin [Sigma] in Ca2+-free HBSS supplemented with 0.05 mM Ca2+) and incubated for 20 min at 35°C. After three washes in carbogenated Ca2+-free Krebs Ringer (30 min), the tissue was transferred into a second digestion solution (containing 3 mg/ml collagenase [type II], 3 mg/ml hyaluronidase [Sigma], and 3 mg/ml bovine serum albumin in Ca2+-free HBSS supplemented with 0.05 mM Ca2+) for 30 min at 35°C with gentle shaking. The pieces were then triturated with glass pipettes in a trypsin-EDTA solution (Gibco BRL) for 5 min. After sedimentation, the dissociated cells were resuspended in Ca2+-free KBR and stored in DMEM up to 12 h at 4°C.

For identification of the isolated cells as smooth muscle cells, the cells were transferred into poly-D-lysine (Sigma)-coated 35-mm plastic culture dishes (Nunc, Wiesbaden, Germany) and fixed by 4% paraformaldehyde in PBS for 10 min. After blocking in normal swine serum (Rockland, Gilbertsville, PA) with 0.5% Triton X-100 in PBS, the cells were incubated with mouse monoclonal anti-{alpha}-SMA antibodies (Sigma; 1:400) for 1 h at 21°C, washed in PBS, and further processed by using Cy3-conjugated rabbit-anti-mouse-IgG (Dianova, Hamburg, Germany; 1:250) for 1 h at 21°C. Finally, the nuclei were counterstained with DAPI (Molecular Probes, Eugene, OR; 1:10 000) and the cells mounted with DABCO (Sigma; 25% DABCO in 50% glycerol-PBS).

Electrophysiology

Electrophysiologic recordings of dissociated contractile cells were carried out in the conventional whole-cell configuration of the patch-clamp technique [20] in standard Ringer solution. Series resistance errors were compensated as high as possible (>60%). When filled with intracellular solution, the pipette resistance varied between 3 and 5 M{Omega}. Fast and slow capacitances were compensated before the applied test-pulse sequences. The data shown have not been corrected for liquid junction potential errors (~4.5 mV). All experiments were carried out at room temperature. Stimulation, data acquisition, and analysis were carried out by using the Pulse/PulseFit 8.11 software combined with an EPC-9 patch-clamp amplifier (HEKA, Lambrecht, Germany). Further data processing was performed with Excel (Microsoft, Redmond, WA) and Sigma Plot 4.01 (SPSS).

Solutions

The nominally Ca2+-free physiologic salt solution (PSS) contained (mM): NaCl 118, KCl 6, and Hepes 10; pH was adjusted to 7.4 with NaOH. The modified Krebs bicarbonate Ringer (KBR) contained (mM): NaCl 118, KCl 4.75, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25, D-glucose 11; bubbled with carbogen to establish a pH of 7.3–7.4. In Ca2+-free Krebs bicarbonate Ringer (Ca2+-free KBR), CaCl2 was omitted. For electrophysiologic recordings, the external Ringer solution contained (mM): NaCl 135, KCl 5, CaCl2 2, MgCl2 1, Hepes 10, glucose 10; pH was adjusted to 7.4 with NaOH. The standard pipette solution contained (mM): K-gluconate 80, KCl 50, NaCl 10, MgCl2 1, EGTA 0.1, Mg2ATP 2, Hepes 5; pH was adjusted to 7.3 with KOH. The Cs+ pipette solution contained (mM): CsCl 140, MgCl2 2, EGTA 2.5, CaCl2 1 (66 nM free Ca2+, EQCAL), Hepes 10; pH was adjusted to 7.3 with CsOH. All reagents were purchased from Sigma and were of the highest analytic grade.

Chemicals

Cyclopiazonic acid (CPA), indomethacin, nifedipine, noradrenaline (NA), endothelin-1 (ET-1), gadolinium, carbonylcyanide-chlorophenylhydrazone (CCCP), atropine, yohimbine, prazosin, and {omega}-conotoxin GVIA were from Sigma. Low Molecular Weight (LMW)-heparin was purchased from Biomol (Hamburg, Germany), PGF2{alpha} from Serva (Heidelberg, Germany), tetrodotoxin (TTX) from Alomone labs (Jerusalem, Israel), NS-398 and PD-98059 from Calbiochem (Bad Soden, Germany), Al-8810 from Cayman Chemical (Ann Arbor, MI), and 8-bromo-cGMP (8-Br-cGMP) from Biolog (Bremen, Germany). Stocks of NS-398, PD-98059, CCCP, and CPA were prepared in dimethyl sulfoxide (DMSO); NA, indomethacin, and Al-8810 in ethanol; TTX, in methyl alcohol. The solvents had no effect on the epididymal contractility in their highest final concentrations in the organ bath. All other substances were dissolved in distilled water or buffer.

Statistics

Experimental data are given as mean ± SEM, where n represents the number of cells or tissue samples derived from different animals each. Student two-tailed unpaired t-test was used to assess statistical significance; P values <0.05 were considered significant.

RESULTS

Spontaneous Contractions along the Epididymal Duct

The bovine epididymal duct exhibited spontaneous rhythmic contractions (SCs) from the caput to the proximal cauda (cauda [p]) in the absence of external stimuli (see Fig. 1). The SC frequency gradually decreased from proximal to distal, accompanied by an increase in SC amplitude in the cauda (p). The mean SC frequency (contractions/min)/SC amplitude (mN) amounted to 7.2 ± 0.34/0.65 ± 0.13 in the caput, 4.3 ± 0.55/0.63 ± 0.08 in the corpus, and 2.9 ± 0.91/1.27 ± 0.11 in the cauda (p) (n = 6/region). In the mid cauda (cauda [m]) and more distally, no contractile autorhythmicity was detectable, albeit initial contractions occurred, when segments were exposed to the preload tension (most probably interpretable as a Bayliss effect). Immunohistochemical analysis revealed immunoreactivity against {alpha}-SMA in the muscle wall of the epididymal duct and associated blood vessels in all regions (Fig. 1).


Figure 1
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  		FIG. 1. Basal contractile activity and {alpha}-smooth muscle actin ({alpha}-SMA) expression in the course of the epididymal duct. Shown are representative isometric force recordings of stress-relaxed duct segments from the regions indicated in the schematic illustration of the bovine epididymis. On the right, {alpha}-SMA immunoreactivity in cross sections of the caput, corpus, and cauda region is shown. Arrows point to the epididymal muscle wall, arrowheads to blood vessels. E, epithelium; bar = 100 µm

Significance of Neuronal, Luminal, and Epithelial Factors

The Na+ channel blocker TTX (2 µM) and the N-type Ca2+ channel blocker {omega}-conotoxin GVIA (1 µM) were used to assess the significance of neuronal factors for SC generation. In the corpus, combined application of these agents resulted in a slight decrease in tone and a reduction in SC amplitude by 24.0 ± 3.1% and in SC frequency by 9.8 ± 2.9% (P < 0.05, n = 3; Fig. 2A). Separate application of the muscarinic receptor–blocker atropine (10 µM) and the {alpha}1- and {alpha}2-adrenoceptor antagonists prazosin (1 µM) and yohimbine (10 µM), respectively, had no significant SC-modulatory effect (n = 3 each; data not shown).


Figure 2
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  		FIG. 2. Relevance of neuronal and luminal factors and the epithelium to spontaneous phasic contraction (SC) generation in the proximal duct. A) Effect of combined application of tetrodotoxin (TTX; 2 µM) and {omega}-Conotoxin GVIA (1 µM). B) Representative burstlike contraction pattern in HBSS-perfused proximal duct segments. C) H&E-stained cross sections of normal and Triton X-100-perfused corpus segments that lacked SCs (D and E). Typical effects of ET-1 (10 nM) and cumulatively applied 8-Br-cGMP (100 µM; D) and PGF2{alpha} (5 µg/ml; E) in epithelium-denuded duct segments are shown. Arrows point to the muscle wall; E, epithelium; bars = 100 µm

Proximal epididymal duct segments that had previously been perfused with HBSS to remove sperm and other luminal factors exhibited spontaneous burstlike instead of regular phasic contractions (Fig. 2B). In addition, a reduction in mean contraction amplitude by 20.0 ± 2.1% (P < 0.05, n = 9) was observed.

To assess the role of epithelial factors for SC generation, the epithelium of the proximal duct segments was removed by perfusion with Triton X-100. In Fig. 2C, H&E-stained cross sections of normal and Triton X-100-perfused corpus segments are shown. Epithelium-denuded proximal duct segments were devoid of SC (Fig. 2D and E), but ET-1 (10 nM, n = 4; Fig. 2D) and NA (10 µM, n = 4; data not shown) still induced contraction, verifying the intactness of the remaining muscle wall. However, only PGF2{alpha} (5 µg/ml) induced phasic activity similar to the SC profile in intact segments (n = 3; Fig. 2E). The cGMP analogue 8-Br-cGMP (100 µM), which had been shown to suppress epididymal SC [13], also caused relaxation in ET-1-precontracted epithelium-denuded segments (n = 3; Fig. 2D).

Effects of External and Internal Calcium Modulation

As shown in Figure 3A, spontaneous phasic activity in the proximal duct regions was absent in Ca2+-free medium (n = 4). In normal Ringer, the block of dihydropyridine-sensitive Ca2+ channels by nifedipine (10 µM) caused a complete and sustained inhibition of SC (n = 5; Fig. 3B). Evidence that excessive preload tension was not involved in epididymal SC generation is given by the lack of effect of gadolinium (100 µM), which blocks stretch-sensitive nonselective cation channels (n = 3; Fig. 3C).


Figure 3
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  		FIG. 3. Relevance of external Ca2+ and Ca2+ release from internal stores for generation of spontaneous contractions (SCs). Isometric force recordings showing representative effects of Ca2+-free medium (A), nifedipine (10 µM; B), gadolinium (100 µM; C), the IP3 receptor blocker LMW-heparin (200 µg/ml; D), the SERCA blocker CPA (10 µM; E), and the mitochondrial Ca2+-uptake inhibitor CCCP (5 µM; F) on SC generation in proximal duct segments. The typical effect of NA (10 µM) after application of CCCP is maintained (F)

To assess the role of Ca2+ release from intracellular stores for SC generation, tissues were exposed to 200 µg/ml of the IP3 receptor (ITPR) blocker LMW-heparin (n = 4; Fig. 3D) or 10 µM of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) blocker CPA (n = 3; Fig. 3E). Neither of these agents exerted an effect on SC frequency; however, both induced a sustained reduction in basal tone. In addition, CPA produced a significant increase in SC amplitude by 29.1 ± 3.7% (P < 0.05, n = 3), whereas LMW-heparin application caused a slight reduction in SC amplitude by 10.7 ± 2.1% (n = 3).

To determine whether mitochondria play a role as mediator of local Ca2+ signals, the mitochondrial protonophore and Ca2+-uptake inhibitor CCCP was used. The effect of CCCP was concentration dependent: 1 µM CCCP increased SC frequency by 9.8 ± 2.1% (P < 0.05, n = 3; data not shown), whereas 5 µM CCCP markedly inhibited SC generation (n = 3; Fig. 3F). After CCCP (5 µM) application, NA (10 µM) was still able to induce contraction, indicating intactness of the contractile apparatus.

Role of Prostanoids and MAPK-dependent Mechanisms

The prostaglandin-endoperoxide synthase PTGS1 (herein referred to the cyclooxygenase 1) inhibitor indomethacin (20 µM) induced a mean reduction in SC amplitude by 20.3 ± 2.4% (P < 0.05) and a decrease in muscle tone in both the caput and corpus region (n = 4; Fig. 4A). The PTGS2 (herein referred to the cyclooxygenase 2) inhibitor NS-398 (10 µM) exerted regionally different effects. In the caput, the blocker induced a long-lasting suppression of SC (n = 4; Fig. 4B), whereas in the corpus, spontaneous activity recovered with reduced SC frequency and amplitude after 3 to 5 min of complete inhibition (n = 4; Fig. 4C). In addition, NS-398 induced a sustained decrease in basal tone in both regions. To investigate the functional contribution of PGF2{alpha} to SC generation, the selective prostaglandin F-receptor (FP receptor, PTGFR) antagonist Al-8810 was used. AL-8810 (10 µM) induced a reduction in tone, a decrease in SC amplitude by 33.4 ± 6.1%, and a significant decrease in SC frequency by 21.8 ± 4.3% (P < 0.05) in the proximal duct regions (n = 5; Fig. 4D). The region-dependent effects of NS-398 were greatly mimicked by the MAPK kinase (MAP2K) blocker PD-98059 (20 µM; Fig. 4E and F), suggesting a functional link between PTGS2- and MAPK-dependent signaling pathways.


Figure 4
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  		FIG. 4. Role of prostaglandin-endoperoxide synthase 2 (PTGS) products and mitogen-activated protein kinase (MAPK)-dependent mechanisms in SC generation. Representative effects of the PTGS1 inhibitor indomethacin (20 µM; A), the specific PTGS2 blocker NS-398 (10 µM; B and C), the PTGFR antagonist Al-8810 (10 µM; D), and the MAP2K blocker PD-98059 (20 µM; E and F) on SC generation in the indicated proximal duct regions

Characteristics of Epididymal Contractile Cells

Three morphologically different epididymal contractile cell types obtained by enzymatic dissociation are described here for the first time. The three cell types also exhibited different biophysical properties. Electrophysiologic investigation of the cells turned out to be difficult, which might be due to the strong dissociation procedure necessary to remove the substantial amount of connective tissue present in the epididymal lamina propria. Possibly this also contributed to the relatively low resting membrane potential (RMP) values measured in all cell types.

Myofibroblasts (MFs) Most frequently found in all regions of the duct were cells of a thin, elongated spindle shape, 50–100 µm long, 5–10 µm in diameter, with bulges at the level of the nucleus (Fig. 5A and B). The cell bodies (cell capacity, ~25 pF) often revealed spines, most probably constituting cell-to-cell contacts. Thus, this cell type has characteristics similar to fibrocytes, and it presumably corresponds to the epididymal myofibroblasts described in the literature [17]. The cells exhibited immunoreactivity against {alpha}-SMA throughout the cell body (Fig. 5B), identifying them as contractile cells.


Figure 5
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  		FIG. 5. Biophysical properties of contractile cells isolated from different regions of the epididymal duct. Phase contrast (A and F) and fluorescence (B and G) photomicrographs of myofibroblasts (MFs) and atypical muscle cells (ATMs). The fluorescence staining indicates immunoreactivity for {alpha}-SMA; the nuclei (white arrows) are counterstained with DAPI; black arrows point to filament-like spurs of the ATM cell bodies. Families of membrane currents elicited by depolarizing test pulses of 200 ms duration in the voltage clamp mode in MF and ATM before (C and H) and after (D and I) application of 100 µM 8-Br-cGMP as well as on the use of intracellular Cs+ (E and J); the corresponding pulse protocols are indicated. K, L) I–V plots of the relative current amplitudes (means ± SEM) at the end of 200-ms test pulses before and after application of 8-Br-cGMP in MFs and ATMs; current amplitudes were normalized to their maximal amplitude before cGMP application. M) Time constants of outward current activation ({tau}act) before (black columns) and after (white columns) application of 8-Br-cGMP (means ± SEM, **P < 0.01); number of experiments indicated. N) Phase contrast photograph of a SM; white arrow points to the nucleus. O) Outward currents in SMs elicited as indicated in the pulse protocol. P) I–V plot of the relative current amplitudes (means ± SEM) in SMs, calculated as described earlier

In the current-clamp mode, a mean RMP of –39.5 ± 3.0 mV (n = 4) was recorded in MF. The MF turned out to be very stretch-sensitive, complicating the patch-clamp procedure. Membrane currents were elicited with depolarizing test pulses of 200 ms duration in steps of 20 mV to potentials between –60 and 80 mV from a holding potential of –60 mV. The variable test pulses induced sustained, strongly fluctuating outward currents, which did not reach saturation within the examined voltage range (n = 6; Fig. 5C). The I–V plot (Fig. 5K) shows an activation threshold of the outwardly rectifying currents between 0 and 20 mV. With 140 mM Cs+ in the pipette solution, outward currents were completely blocked (Fig. 5E), revealing their nature as K+ currents.

Because 8-Br-cGMP exerted direct relaxation of ET-1-induced contraction in the tissue-bath assays, the cGMP-analogue was applied to the isolated muscle cells. Application of 100 µM 8-Br-cGMP induced an increase in the maximal outward current amplitude. At 60 mV, the current increase amounted to 29.3 ± 6.2% (P < 0.05, n = 6; Fig. 5D and K). In addition, the cGMP analogue significantly accelerated the activation kinetics of the outwardly rectifying K+ channels. The current traces were fitted with single exponential functions to obtain the time constants of activation ({tau}act). At 60 mV, a mean decrease in {tau}act from 21.3 ± 2.4 ms in the control to 14.2 ± 1.7 ms after 8-Br-cGMP was determined (P < 0.01, n = 6; Fig. 5M).

Atypical muscle cells (ATMs): The second cell type in the epididymal muscle wall was detected in spontaneously contractile regions only. It was characterized by a distinct irregular morphology, a relatively large nucleus, and the presence of filament-like sarcolemmal projections of the cell body (cell capacity, ~15 pF), which enhanced the irregular appearance of the cells (Fig. 5F and G). Fluorescence immunostaining with anti-{alpha}-SMA revealed an inhomogeneous reactivity of the cell body, qualifying them as "contractile" cells (Fig. 5G). These cells, which have not been described before, comprised about 5% of the whole number of contractile cells in the proximal duct. They were designated as atypical muscle cells (ATMs) following the morphologically similar atypical smooth muscle cells in the proximal renal pelvis [21].

ATMs and myofibroblasts revealed distinct differences with regard to their biophysical properties. Contrary to the MF, the ATMs never rounded up during mechanical access with the patch pipette. They exhibited a mean resting membrane potential (RMP) of –23.0 ± 2.9 mV (n = 3). The most negative holding potential tolerated by ATMs was –40 mV. Clamping the cell membrane to potentials more negative than –20 mV –mostly resulted in increased leak conductance, explaining the small number of evaluated experiments.

Application of depolarizing test pulses of 200-ms duration to potentials between –20 and 80 mV from a holding potential of –40 mV induced sustained outward currents (n = 3; Fig. 5H). Despite a smaller cell capacity, the maximal outward-current amplitudes at 80 mV were about 2.7-fold higher in the ATM (4.2 ± 0.5 nA) than in the MF (1.5 ± 0.3 nA), resulting in a current density of 276 ± 26 and 75 ± 13 pA/pF, respectively. The activation threshold of the outwardly rectifying channels, which also lacked inactivation during the 200-ms test pulses, ranged between –20 and 0 mV (Fig. 5L). The 140 mM Cs+ in the pipette solution again completely blocked the outward currents (Fig. 5J). The 8-Br-cGMP (100 µM) induced a mean increase in the maximal outward current amplitude by 87.1 ± 22.7% (n = 3) at 60 mV (Fig. 5, I and L). In addition, the cGMP analogue significantly accelerated the activation kinetics of the outwardly rectifying channels. Again the initial current increase was fitted with a single exponential function to obtain {tau}act. At 60 mV, {tau}act decreased from 18.6 ± 1.8 ms in the control to 13.0 ± 0.8 ms after 8-Br-cGMP application (n.s., n = 3; Fig. 5M).

Typical smooth muscle cells (SMs): The third type of isolated contractile cells was that of typical smooth muscle cells (SMs). They were found in the cauda region only, where they comprised about 10% of the whole amount of isolated contractile cells. The SMs showed the characteristic features of smooth muscle cells like a spindle-shaped cell body (cell capacity, ~30 pF) and a central, elliptic nucleus (Fig. 5N). In the SM, a mean RMP of –41.0 ± 3.0 mV (n = 3) was recorded. Application of depolarizing test pulses in steps of 10 mV to potentials between –60 and 80 mV from a holding potential of –60 mV induced outward currents, which reached saturation at about 60 mV (Fig. 5, O and P). At voltage steps to 50 mV or more positive potentials, the currents slightly decreased during the 200-ms test pulses (Fig. 5O), suggesting the involvement of delayed-rectifier currents (IDR). The I-V curve in Fig. 5P shows an activation threshold of the channels between –40 and –20 mV. In comparison with the MF and ATM, the SMs exhibited a considerably faster time course of current activation, with a mean time constant of 4.1 ± 0.4 ms at 60 mV (n = 3).

DISCUSSION

The results of the present study demonstrate that the bovine epididymal duct exhibits autorhythmicity, characterized by regular phasic contractions of distally decreasing frequency. Neither inhibition of TTX-sensitive Na+ channels and N-type Ca2+ channels to prevent neuronal transmitter release nor inhibition of muscarinic and {alpha}-adrenergic receptors blocked SC generation, indicating that it is predominantly myogenic in nature. In accordance, only a sparse innervation was found in the bovine caput and corpus region [22]. Epididymal SCs could be observed only up to the cauda (p), suggesting a predominantly neurogenic control of the musculature in the cauda (m). The occurrence of epididymal SC in vivo and in vitro has already been reported in the guinea-pig, rat, rabbit, and mouse [14, 15, 23–25]. However, in contrast to the present findings in the bull, spontaneous contractions were described to occur throughout the cauda [24–26]. This discrepancy may be species dependent or involve methodologic reasons. The present force recordings were carried out on stress-relaxed duct segments, as this mostly fits the natural conditions. An activation of mechanosensitive channels as a cause for SC could thereby be excluded, as shown by the inability of gadolinium to affect SC (see [27]).

Perfused caput and corpus segments showed a burstlike contraction pattern and a reduced contraction amplitude in comparison to the control. Previously, the synthesis of angiotensin II (Ang II) has been considered as a possible means of how sperm may exert direct influence on epididymal muscle activity [28]. Other possible SC-modulatory luminal factors might include interleukins [29] and androgens, which crucially influence epididymal PG synthesis [30].

Because removal of extracellular Ca2+ as well as application of nifedipine totally abolished epididymal SC generation, extracellular Ca2+ entry through L-type Ca2+ channels can be considered essential for phasic muscle activity. By contrast, calcium release from the sarcoplasmic reticulum (SR) plays a minor role in epididymal peristalsis, as indicated by the only tone-reducing effects of the SERCA blocker CPA and the ITPR blocker LMW-heparin in proximal duct segments. In the PRP and the urinary bladder, generation of spontaneous phasic activity also depends on the influx of Ca2+ through L-type Ca2+ channels, whereas Ca2+ release from ryanodine-/IP3-sensitive stores only modulates electrical activity [31, 32]. By contrast, in most other organs, cyclic release of internal Ca2+ forms an essential mechanism underlying electrical and contractile autorhythmicity [33–37].

Contrary to the SR, mitochondria appear to play a crucial role as mediators of SC-relevant local Ca2+ signals. Whereas low concentrations of the mitochondrial protonophore CCCP (1 µM) may enforce contractile activity by increasing the [Ca2+]i, higher concentrations (5 µM) induced a long-lasting inhibition of SC. Because NA was still able to induce contraction, membrane-associated effects of the mitochondrial Ca2+-uptake inhibitor can be assumed. CCCP also suppresses spontaneous contractions in gastric smooth muscle, most probably by affecting the activity of L-type Ca2+ channels via mitochondrial Ca2+ handling [9]. A similar mechanism is discussed in cardiac myocytes [38].

The lack of SC in epithelium-denuded proximal duct segments and the SC-suppressive effect of NS-398 indicate that an epithelial PTGS2-dependent prostaglandin (PG) synthesis is crucial for triggering epididymal SC. By contrast, selective blockade of PTGS1 had only marginal effects on SC generation. Endogenous PGs have already previously been considered as local regulators of epididymal SC (for review, see [5]). In rat, PTGS2 expression had been localized to the epithelial principal cells, and the predominant PG synthesized by this PTGS isozyme seems to be PGF2{alpha} [39]. In conjunction with the present findings, demonstrating a marked reduction in contractility by the PTGFR antagonist Al-8810 and induction of SC-like activity in epithelium-denuded proximal duct segments by PGF2{alpha}, a PTGS2-mediated PGF2{alpha} synthesis underlying epididymal SC can be assumed. Interestingly, endogenous PGs also play a crucial role in maintaining peristalsis in the upper urinary tract [40–42], and activation of PTGFR after complete suppression of autorhythmicity by PTGS inhibition resulted in a reconstitution of the initial contraction pattern [43, 44].

MAP2K-MAPK signaling cascades also seem to play a key role in epididymal SC generation. PD-98059, a blocker of the non-receptor-coupled tyrosine kinase MAP2K, caused strong suppression of SC and reduction in tone, particularly at the level of the caput. The region-dependent relaxation pattern of the MAP2K blocker was very similar to that of NS-398, pointing to a functional interplay between a PTGS2-mediated (epithelial) prostanoid synthesis and MAPK activation. In smooth muscle, the action of PGF2{alpha} [45] was found to be assisted by a G-protein-coupled receptor (GPR)-dependent MAPK activation. Inhibition of the MAP2K even abolished PGF2{alpha}-induced contraction in iris sphincter smooth muscle [46]. Possibly, the latter finding also mirrors the principles in the epididymal duct. Contrary to nifedipine, PD-98059 also affected epididymal muscle tone, suggesting that endogenous MAP2K activity brings about not only a (SC-relevant) regulation of L-type Ca2+ channels, but also a (tonus-relevant) sensitization of the contractile apparatus. An activation of L-type Ca2+ channels via tyrosine kinases or MAPK signaling in smooth muscle has already been discussed [47–51].

The cell-isolation studies revealed the existence of three different contractile cell types with distinct morphologic and biophysical properties in the epididymal duct. The cell type most frequently found in the proximal duct regions shared many morphologic characteristics of fibrocytes, like bulges at the level of the nucleus (see [52]). Because of the phenotype and numeric dominance, these cells were suspected to be the myofibroblasts (MFs) described in the literature [16, 17]. Contrary to the MFs, the ATMs, which are described for the first time in the epididymal duct, lacked a clear contractile behavior and were detected only in regions exhibiting SCs. In addition, the ATM could be voltage-clamped only to maximal –40 mV, indicating membrane properties different from those of MFs. The third cell type showed morphologic features of typical smooth muscle cells (SMs) and could be clearly distinguished from the much smaller vascular smooth muscle cells (see [52]). In accordance with earlier studies [16, 17], SMs were found only in the cauda region.

On depolarization, K+ outward currents were recorded in all contractile cell types. A comparison of MFs and ATMs revealed a considerably higher K+ current density in ATMs. Because of the strong fluctuations and the high activation threshold of the outward currents in the MFs, an involvement of Ca2+-activated K+ currents through maxi-channels (BK channels, KCNMA1) seems likely (see [5]). By contrast, outward-current traces in the ATMs were comparatively smooth, pointing to voltage-dependent Kv channels.

MFs and ATMs also differed with respect to the effects of 8-Br-cGMP. We have recently demonstrated the significance of the cGMP system in the local control of epididymal contractility [13]. In the present experiments, 8-Br-cGMP reduced ET-1-induced contraction in epithelium-denuded proximal duct segments, indicating the presence of cGMP-dependent signaling pathways in the contractile cells. This fact was confirmed by our finding that 8-Br-cGMP accelerated the activation kinetics of the outwardly rectifying K+ channels in both ATMs and MFs. In addition, 8-Br-cGMP induced an increase in the current amplitudes, which was especially pronounced in ATMs. Both Ca2+-activated [53] and voltage-dependent K+ channels [54] have been determined as putative targets of cGMP in smooth muscle.

With regard to the mechanisms and structures underlying epididymal SC, interesting parallels with the proximal renal pelvis (PRP) can be observed. Within the cauda, where the gap-junction protein connexin43 was found [55], electrical activity is conducted over a relatively long distance of about 1 cm [56]. By contrast, in the proximal duct, areas with synchronous electrical activity turned out to be much smaller [10], and the nexus has not been detected yet, pointing to a regionally different coordination of muscle activity. In the PRP, so-called atypical muscle cells seem to generate pacemaker currents, which spread electrotonically to the usually quiescent typical smooth muscle cells [21]. Interstitial cells of Cajal (ICC)-like cells seem to coordinate synchronous muscle activity. Because the ATMs could be detected in regions with SCs only and, in addition, share morphologic characteristics like filament-like processes of the cell body with the atypical and ICC-like cells in the PRP [21], it is tempting to speculate that they also play a role in the initiation or orchestration of epididymal phasic activity.

In summary, the present study provides a detailed characterization of the structures and mechanisms triggering SCs in the bovine epididymal duct (see Fig. 6). We demonstrate that epididymal SCs are predominantly myogenic in origin and depend on the presence of an intact epithelium as well as PTGS2 and MAPK activation. In addition, Ca2+ entry through L-type Ca2+ channels and local Ca2+ buffering by mitochondria turned out to be crucial for SC generation. At present, no evidence exists for pacemaker cells in the epididymal duct, but the newly described ATMs with their long cell processes may undergo extensive coupling to the contractile MFs to serve this function.


Figure 6
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  		FIG. 6. Hypothetical model of factors crucial for the generation of epididymal spontaneous phasic contractions (SCs). Mit., mitochondria; GPR, G-protein-coupled receptor

FOOTNOTES

2 Correspondence: Marco Mewe, Institut für Angewandte Physiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. FAX: 49 0 40 42803 9127; mewe{at}uke.uni-hamburg.de Back

1 Supported by grants from the Deutsche Forschungsgemeinschaft (Mi 637/1–1). Back

Received: 9 June 2006.

First decision: 29 June 2006.

Accepted: 12 July 2006.

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