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Contractile response of peritubular myoid cells to prostaglandin F2α
Molecular and Cellular Endocrinology 138 (1998) 143 – 150
Contractile response of peritubular myoid cells to prostaglandin F2a A. Tripiciano, A. Filippini, F. Ballarini, F. Palombi * Department of Histology and Medical Embryology, ‘La Sapienza’ Uni6ersity, Via A. Scarpa14, 00161 Rome, Italy Received 24 September 1997; received in revised form 5 January 1998; accepted 14 January 1998
Abstract Prostaglandin (PG) F2a, a well known agonist of smooth muscle, is produced in the male gonad. We have investigated whether PG F2a stimulates seminiferous tubule contractility through direct action on peritubular myoid cells. Myoid cells from prepubertal rats were highly purified through Percoll density gradient and cultured in vitro. Stimulation with PG F2a was observed to induce: (i) rapid and dose-dependent production of inositol phosphates; (ii) mobilization of Ca2 + from intracellular stores and (iii) cell contraction. Moreover, at a concentration of 10 mM the agonist was found to induce immediate contractile response of peritubular tissue in freshly explanted tubular fragments from both young and adult rats; the explants were examined in whole-mount preparations and the peritubular myoid cell layer was identified by selective staining for alkaline phosphatase activity. Our observations demonstrate that myoid cells are a direct target for PG F2a and suggest a role of the eicosanoid in the intragonadal control of seminiferous tubule contractility. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peritubular myoid cells (rat testis); Contractility (seminiferous tubule); Prostaglandins
1. Introduction After release from the seminiferous epithelium, testicular spermatozoa are rapidly conveyed along the lumen of the seminiferous tubules, towards the hilum of the testis. The flux of tubular fluid containing spermatozoa appears to depend upon the contractile activity of the peritubular tissue which is composed of a particular type of smooth muscle cell, the myoid cells (Clermont, 1958; Leeson and Leeson, 1963). In the rat, myoid cells are closely connected to each other in a single flat epithelioid layer (Vogl et al., 1985; Palombi et al., 1992), but contain a-smooth actin (Tung and Fritz, 1990) and desmin (Virtanen et al., 1986), which are as typical of contractile cells. In the apparent absence of neural control, seminiferous tubule contractility is likely to be regulated by local factors originating from the adjacent compartments, the seminiferous epithelium and the interstitial tissue. Recently, our laboratory has * Corresponding author. Tel: +39 6 49766585; fax: +39 6 4462854.
demonstrated that rat myoid cells contract in response to endothelin-1 (ET-1) (Filippini et al., 1993; Tripiciano et al., 1996, 1997), a vasoactive peptide (Yanagisawa et al., 1988) secreted by Sertoli cells, the somatic component of seminiferous epithelium (Fantoni et al., 1993). Moreover, a further agonist of smooth muscle contractility, vasopressin (AVP) (Penit et al., 1983), locally produced by interstitial Leydig cells (Ivell et al., 1992), has been shown to bind peritubular cells from adult rats, inducing a second messenger cascade (Howl et al., 1995) resulting in cell contraction (Tripiciano et al., 1996). Among the well known agonists of smooth muscle contractility (Schroer and Schroeder, 1994) prostaglandin F2a (PG F2a ) is produced in vitro by testicular cells originating from both the seminiferous epithelium (Jannini et al., 1994) and the interstitial compartment (Haour et al., 1979; Kern and Maddocks, 1995). Myoid cells may represent a suitable paracrine target for this eicosanoid. The hypothesis that PG F2a may contribute to the local regulation of peritubular contractility is reinforced by the early observation (Farr
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and Ellis, 1980) that at high doses (10 mM) this prostaglandin is able to increase seminiferous tubule tone and contraction frequency. In order to verify whether myoid cells are a direct target for PG F2a, we have analyzed the biochemical and morphological response of isolated myoid cells in vitro, utilizing highly purified populations of this cell type (Palombi et al., 1988; Filippini et al., 1993). Moreover, the contractile nature of peritubular response to PG F2a was also tested in whole mount preparations of seminiferous tubules.
2. Materials and methods
2.1. Materials Collagenase A from Clostridium histoliticum and DNAase-I were obtained from Boehringer Mannheim (Mannheim, Germany). Trypsin was purchased from Difco Laboratories (Detroit, MI). Eagle’s Minimum Essential Medium (MEM) was obtained from Gibco (Grand Island, NY). Percoll was purchased from Pharmacia (Uppsala, Sweden). Myo-[2-3H]inositol (16–18 Ci/mmol) was purchased from DuPont de Nemours Italiana SpA (Milan, Italy). Plastic culture dishes and multiwell plates were purchased from Falcon (Oxnard, CA). PG F2a and other reagents were purchased from Sigma (St. Louis, MO). PG F2ax was dissolved in ethanol at a concentration of 10 mM and diluted in MEM just prior to use.
2.2. Animals The animals used were adult and 3-week-old Wistar rats (Charles River, Como, Italy), fed ad libitum until killed by CO2 asphyxia or cervical disarticulation. Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
2.3. Myoid cell isolation and culture Myoid cells were isolated and purified through Percoll density gradient from 3-week old rats as previously described (Palombi et al., 1988) and plated in plastic culture dishes in MEM. The cells were cultured under serum-free conditions at a concentration of 250000 cells/well in 12-multiwell plates, unless otherwise specified, at 37°C in a water-saturated atmosphere of 95% air and 5% CO2. The assessment of myoid cell purity (Palombi and Di Carlo, 1988) was performed routinely for each preparation on the basis of the presence of alkaline phosphatase activity tested on adherent cells (as described below) and was never B96%.
2.4. Alkaline phosphatase cytochemistry Selective myoid cell identification through alkaline phosphatase cytochemistry was performed as previously described (Palombi and Di Carlo, 1988), basically following the method of Ackermann (Ackermann, 1962). Briefly, the fixed cells were incubated in an alkaline solution containing 0.5 mg/ml Fast Blue RR in water and 40 ml/ml a-naphtol phosphate (0.25% solution, pH 8.6). After 30 min incubation in the dark, a purple–blue precipitate was apparent specifically on the surface of myoid cells (Palombi and Di Carlo, 1988).
2.5. Scanning electron microscopy (SEM) analysis of cell contraction in 6itro Myoid cells were cultured in Falcon dishes, treated with 10 mM PG F2a or with vehicle on the second day of culture, immediately fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) and postfixed in 1% OsO4 in Zetterquist buffer. After dehydrating and critical point drying in ethanol, the plate was carefully fragmented with the aid of a sharp blade, the samples were coated with gold and viewed under a Hitachi (Tokyo, Japan) S-570 scanning electron microscope.
2.6. IP production To measure IP production, myoid cells were incubated with 4mCi/ml myo-[2-3H]inositol for 48 h starting from the second day of culture. After labeling, cells were extensively rinsed in Hanks’ Balanced Salt Solution (HBSS) and preincubated with 20 mM LiCl. PG F2a treatment was started 10 min after LiCl addition. After 30 min the incubation was stopped by replacing the medium with ice-cold 10% trichloroacetic acid (TCA). The TCA was removed from samples with a single extraction with 1:1 v/v of a mixture of trichlorotrifluoroethane and tri-n-octylamine (74:26, v/v). IP separation was performed by ion exchange chromatography as described elsewhere (Berridge, 1983; Adamo et al., 1985).
2.7. Measurement of [Ca 2 + ]i [Ca2 + ]i was measured by dual wavelength fluorescence in single cells loaded with the Ca2 + -sensitive indicator fura-2 (Grynkiewicz et al., 1985). Testicular myoid cells were plated onto plastic coverslips in MEM. After 4 days in culture, the cells were incubated in 1 ml MEM containing 3 mM fura-2-acetoxymethylester for 1 h at 37°C. The cells were then rinsed with Krebs-Henseleit-Hepes (KHH) buffer (140.7 mM Na + , 5.3 mM K + , 132.4 mM Cl − , 0.98 mM PO24 − , 1.25 mM Ca2 + , 0.81 mM Mg2 + , 5.5 mM glucose and 20.3 mM Hepes) supplemented with 0.2%
A. Tripiciano et al. / Molecular and Cellular Endocrinology 138 (1998) 142–150
fatty acid-free bovine serum albumin (BSA). Measurements were performed in single cells, at 340 and 380 nm excitation wavelengths, with an AR-Sm microfluorimeter (Spex Industries, Edison, NJ) connected to a Diaphot TMD inverted microscope (Nikon, Tokyo, Japan) equipped with a CF X 40 objective. Emission was collected by a photomultiplier carrying a 510 nm cut-off filter and recorded by an ASEM Desk 2010 computer (ASEM SpA, Buia, Italy), which automatically calculated real-time 340/380 ratios. Calibration of the signal was obtained at the end of each observation by adding 5 mM ionomycin to saturate the dye maximal fluorescence, followed by 7.5 mM EGTA plus 60 mM Tris –HCl, pH 10.5, to release Ca2 + from fura-2 and obtain minimal fluorescence. [Ca2 + ]i was calculated according to previously described formulas (Grynkiewicz et al., 1985). When indicated, the cells were pretreated either with thapsigargin, MnCl2 or EGTA.
2.8. Statistical analysis Data are presented as the mean9SE of results from at least three independent experiments. Student’s t-test was used for statistical comparison of means where applicable.
2.9. Preparation and treatment of seminiferous tubule whole mounts The morphological response of peritubular tissue to acute treatment with PG F2a was analyzed in tubular whole mounts. Seminiferous tubules were prepared as previously described (Tripiciano et al., 1996). Briefly, Wistar rats aged 20 – 60 days were killed by CO2 asphyxia, their testes were excised, decapsulated and digested under gentle shaking at room temperature in Eagle’s Minimal Essential Medium (MEM) containing 1 mg/ml collagenase. After digestion of the interstitium the tubular mass was rinsed in MEM, stretches of tubules were dissected by means of sharp needles, then carefully transferred to 35 mm culture dishes in 300 ml of medium. The tubules were incubated for 10 min at 32°C in a humidified chamber under an athmosphere containing 5% CO2. At the end of the incubation time, the medium was replaced by 600 ml of MEM containing 10 mM PG F2a, immediately (i.e. within 20 s) followed by addition of 4% paraformaldehyde in 0.1 M phosphate buffer (PBS). After 10 min fixation and rinsing in buffer, the samples were processed in order to highlight the site localization of alkaline phosphatase activity (Palombi and Di Carlo, 1988). After staining, single tubules were gently collected, laid onto microscopy slides, and covered with a coverslip in a drop of 50% glycerol in PBS. The tubular whole-mounts were viewed and photographed under a
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Zeiss Axioplan Microscope (Carl Zeiss, Oberkochen, Germany).
3. Results
3.1. PG F2a stimulation of inositol phosphates accumulation In order to determine whether myoid cell treatment with PG F2a is followed by phosphoinositidase-C (PLC) activation resulting in the generation of intracellular inositol 1,4,5-trisphosphate (IP3), we investigated the effect of different concentrations of PG F2a on the accumulation of IP3 derived inositol phosphates (inositol mono-, bis- and trisphosphates) in primary cultures of peritubular myoid cells. The exposure of myoid cells to PG F2a for 30 min evoked a dose-dependent increase in inositol phosphates accumulation, indicating that the agonist activates phospholipase C, which, in turn, catalyzes the breakdown of polyphosphoinositides (Fig. 1).
3.2. Effect of PG F2a on [Ca 2 + ]i Fura-2-loaded myoid cells were equilibrated in KHH for at least 10 min prior to PG treatment. The basal cytosolic calcium was 8799 nM (n= 20). [Ca2 + ]i remained stable, and no rapid oscillations were observed. PG F2a stimulated a dose-dependent increase in [Ca2 + ]i in single cells loaded with the fluorescent Ca2 + indicator fura-2 (Fig. 2a). The onset of response was immediate after addition of the agonist. [Ca2 + ]i peaked within 15 s, followed by slow decay towards baseline values within 10 min (Fig. 2b). Since [Ca2 + ]i elevations in response to PG F2a occurred in myoid cells in the virtual absence of extracellular Ca2 + following chelation with 3 mM EGTA (Fig. 3a), which suggested the involvement of intracellular calcium, we analyzed a putative mechanism of mobilization of Ca2 + from internal stores. Thapsigargin increased the basal cytosolic calcium concentration to 148913 nM (Fig. 3b). Depletion of intracellular calcium stores with thapsigargin before stimulation with PG F2a abolished the PG F2a mediated calcium rise in the presence of extracellular calcium, which indicates that the increase in calcium is predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. In addition, in order to verify the non-involvement of calcium influx in PG F2a response, we performed experiments with Mn2 + quenching of the fura-2 dye to monitor the possible entry of this extracellular divalent cation. In fact, Mn2 + is known to permeate the cell through the same channels as those used by Ca2 + , thereby rapidly quenching fura-2 fluorescence by irreversible binding (Hallan et al., 1989). Addition of PG F2a to myoid cells in the presence of Mn2 + resulted in a rapid increase in fura-2
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Fig. 1. Concentration dependent production of inositol phosphates (IP1 +IP2 +IP3) in response to PG F2a, by purified myoid cells. The cells were stimulated as indicated for 30 min in the presence of 20 mM LiCl. Control samples received corresponding amount of vehicle only. Each point represents the mean 9 SE from three independent determinations.
fluorescence which stabilized at lower levels without any appreciable quenching below the basal line (Fig. 3c). This further indicates that the increase in [Ca2 + ]i induced by PG F2a in myoid cells involves Ca2 + release mostly from intracellular stores.
3.3. Analysis of the contractile response of myoid cells to PG F2a in 6itro Myoid cell shape in vitro was studied by means of SEM in control and PG F2a -stimulated conditions. As shown in Fig. 4, the cells respond to a 10 mM concentration of the agonist, with immediate contraction. The effect is less apparent with 1 mM prostaglandin (not shown) and is not appreciable at lower concentrations.
Previous analysis has in fact shown that myoid cells are polygonal squamous elements arranged in a single epithelioid layer (Vogl et al., 1985; Palombi et al., 1992). Immediately after treatment with PG F2a at a dose of 10 mM, the individual myoid cells become apparent: the central perinuclear area of the cell appears intensely stained, while only faintly visible processes appear to connect the contracted cells to one another (Fig. 5). Although the size of the alkaline phosphatase decorated profiles (hence the degree of cell contraction) may vary, the same pattern of response was constantly observed in virtually the whole peritubulum both in young and mature samples. In control samples, incubated with the vehicle alone, no cell contraction was observed.
3.4. PG F2a mediates seminiferous tubule contractility
4. Discussion
When the surface of seminiferous tubules is viewed in whole-mount preparations after selective alkaline phosphatase staining of myoid cells (Fig. 5), the distribution of the enzyme appears uniform on the cell surface and the contours of the individual cells are hardly apparent.
Among secretion products involved in short range intercellular communication, prostaglandins represent a broad family of short-lived lipids, responsible for a great variety of biological responses. A major target of these eicosanoids are smooth muscle cells in vascular
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and non-vascular tissue (Schroer and Schroeder, 1994), in which different prostaglandins control either relaxation or contraction through different signal transduction pathways coupled to specific membrane receptors (Narumiya, 1996). PG F2a has been shown to induce contraction of vessels, uterus, gastrointestinal tract, bronchi and trachea, and it is generally agreed that in most cases it contributes to the multifactorial control of smooth muscle tone (Schroer and Schroeder, 1994). Modifications of seminiferous tubule contractile activity following treatment with PG F2a have been described in the rat (Farr and Ellis, 1980). In tubular explants, micromolar doses of this eicosanoid induced a reduction in diameter and increased frequency of spontaneous contraction. Moreover, increased sperm numbers were observed in rabbit deferent ducts after PG F2a treatment (Hafs et al., 1974). In the present study, we
Fig. 2. Effect of PG F2a treatment on [Ca2 + ]i of single myoid cells loaded with fura-2. (a) [Ca2 + ]i peak response to different concentrations of PG F2a. The data are representative of three to five independent experiments for each dose. (b) Trace representing real-time fura-2 fluorescence in single myoid cell stimulated with 10 mM PG F2a.
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provide the first demonstration that peritubular myoid cells are a direct target of PG F2a, to which they respond with a second messenger increase and contractile behaviour. We have observed that in vitro cultures of purified myoid cells display a dose-dependent production of IPs and an increase in [Ca2 + ]i in response to PG F2a, suggesting that binding of the eicosanoid to the cell activates phospholipase-C (PLC), which results in phosphoinositides turnover. These data are in accordance with what is known about PG F2a in other contractile cell types (Fukuo et al., 1986; Morimoto et al., 1990), as is the observation that no increase in cAMP production follows treatment with this prostaglandin (data not shown). In myoid cells, IP3 production in response to PG F2a is accompanied by a moderate but dose-dependent rapid increase in [Ca2 + ]i. As for the mechanisms underlying this increase, the relevance of Ca2 + mobilization from internal stores versus a possible contribution of Ca2 + entry through the plasma membrane is indicated by three lines of evidence: (i) the calcium response appeared to be unaffected by chelation of extracellular Ca2 + with EGTA; (ii) depletion of Ca2 + stores by means of thapsigargin, an inhibitor of endoplasmic reticulum Ca2 + -ATPase (Thastrup et al., 1990), abolished the calcium response to PG F2a ; (iii) fura-2 fluorescence induced by PG F2a undergoes no quenching in Mn2 + -enriched medium, indicating (Hallan et al., 1989) that stimulation does not significantly open Ca2 + channels. In myoid cells, therefore, PG-stimulated [Ca2 + ]i increase appears to depend mainly, if not exclusively, upon intracellular release; in other contractile cell types a contribution from both intracellular pools and calcium entry have been reported (Mene` et al., 1987; Fukuo et al., 1986). The in vitro model consisting of highly purified populations of myoid cells was fundamental for the analysis of the direct biochemical response to PG F2a. Moreover, it showed that myoid cells are the tubular target for the agonist and that the morphological response to the agonist is of a contractile nature. Myoid cells were observed to undergo immediate contraction in response to PG F2a, not only in cell cultures but also when organized in peritubular tissue in freshly-explanted seminiferous tubules. The direct visualization of contracted myoid cells through selective alkaline phosphatase cytochemistry in tubular whole-mounts suggests a physiological role for PG F2a on peritubular contractility. Several factors have been indicated in the past as possible regulators of seminiferous tubule contractile activity in the rat, but so far experimental evidence indicating a direct role in the paracrine regulation of myoid cell contractility has been produced only for ET-1 and AVP. ET-1 has been shown to be produced by Sertoli cells (Fantoni et al., 1993) and to bind high affinity receptors on isolated myoid cells (Filippini et
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Fig. 3. [Ca2 + ]i increase induced in single myoid cells by PG F2a treatment depends upon release from intracellular pools. (a) Myoid cell culture was equilibrated in KHH buffer containing 3 mM EGTA. Single cell response to 10 mM PG F2a appeared unaffected by the virtual absence of extracellular calcium. (b) Myoid cell culture was exposed to 10 mM thapsigargin. In response to this inhibitor of the endoplasmic reticulum Ca2 + -ATPase, a slow and transient [Ca2 + ]i increment was observed in single cells. Administration of 10 mM PG F2a at the time when [Ca2 + ]i was stabilized failed to elicit any further [Ca2 + ]i increment. (c) Cells were equilibrated in KHH buffer containing 100 mM MnCl2, then treated with 10 mM PG F2a. Despite the presence of Mn2 + in the extracellular medium, no appreciable quenching of the fura-2 fluorescence was induced by the agonist. Fluorescence was completely quenched by subsequent addition of 5mM ionomycin. (d) Mean values of PG F2a dependent [Ca2 + ]i increases (both peak and plateau) in controls or cells treated with 10 mM PG F2a in EGTA or thapsigargin-containing buffer. Results are from at least four independent experiments.
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prostaglandin found to be effective on myoid cells appears to be high when compared to that (10 nM) required for ET or AVP response (Filippini et al., 1993; Howl et al., 1995). In this regard, however, the following points are noteworthy: (i) other contractile cells are known to require similarly high concentrations of PG F2a (Fukuo et al., 1986; Mene` et al., 1987); (ii) it is possible that, in vivo, high local concentrations of prostaglandins form transiently within a restricted tissue compartment; (iii) it is possible that the concentration which actually acts on the cells in culture does not exactly correspond to the one that is administered.
Fig. 4. Scanning electron micrograph showing the contractile response of myoid cells to acute PG F2a stimulation. Cells have been cultured for 20 h before treatment and fixed immediately after receiving: (a) 10 mM PG F2a ; (b) vehicle only (control). Contraction induced by the treatment results in the formation of slender cytoplasmic processes. Bar: 10 mm.
al., 1993), thus stimulating a second messenger cascade (Filippini et al., 1993; Tripiciano et al., 1997) and an immediate contractile response (Tripiciano et al., 1996); contraction stimulated by ET-1 has been reported also for peritubular tissue in situ both in the prepubertal and in the adult gonad (Tripiciano et al., 1996). The presence of high affinity receptors for AVP and second messenger production in response to this peptide have been demonstrated for peritubular cells from adult rats (Howl et al. 1995). Peritubular tissue has also been shown to undergo immediate contraction in response to stimulation of tubular explants with AVP (Tripiciano et al., 1996). Moreover the peptide is known to be produced locally by Leydig cells of the testicular interstitium (Ivell et al., 1992). On the basis of the experimental evidence presented, we candidate PG F2a as a new modulator of peritubular contractility, a role for which it seems to meet all the basic requirements, i.e. direct biochemical activation, direct contractile response and local production. The physiological relevance of myoid cell responsiveness to PG F2a is suggested by the evidence that the eicosanoid is produced at high basal levels by interstitial macrophages (Kern and Maddocks, 1995) and, under gonadotropin stimulation, by Leydig cells (Haour et al., 1979) and Sertoli cells (Jannini et al., 1994). The concentration of
Fig. 5. Surface view of peritubular layer in whole-mount preparation of seminiferous tubules from prepuberal (a, b) and adult (c, d) rats. Myoid cells have been selectively stained through alkaline phosphatase cytochemistry. a and c: control samples; b and d: tubules fixed immediately after stimulation with 10 mM PG F2a. In both the young and adult, myoid cells appear to contract in response to stimulation. Bar: a, b 40 mm; c, d 100 mm.
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Whatever the precise role and the possible interplay of the different myoid cell agonists within the complex paracrine interactions characterizing the male gonad (Skinner, 1991), PG F2a appears to be one of the factors responsible for the local control on sperm and tubular output resulting from their action on myoid cell contractility.
Acknowledgements Research supported by a grant from Italian Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST). The authors thank Antonella Zacheo for skillful technical support.
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Contractile response of peritubular myoid cells to prostaglandin F2a A. Tripiciano, A. Filippini, F. Ballarini, F. Palombi * Department of Histology and Medical Embryology, ‘La Sapienza’ Uni6ersity, Via A. Scarpa14, 00161 Rome, Italy Received 24 September 1997; received in revised form 5 January 1998; accepted 14 January 1998
Abstract Prostaglandin (PG) F2a, a well known agonist of smooth muscle, is produced in the male gonad. We have investigated whether PG F2a stimulates seminiferous tubule contractility through direct action on peritubular myoid cells. Myoid cells from prepubertal rats were highly purified through Percoll density gradient and cultured in vitro. Stimulation with PG F2a was observed to induce: (i) rapid and dose-dependent production of inositol phosphates; (ii) mobilization of Ca2 + from intracellular stores and (iii) cell contraction. Moreover, at a concentration of 10 mM the agonist was found to induce immediate contractile response of peritubular tissue in freshly explanted tubular fragments from both young and adult rats; the explants were examined in whole-mount preparations and the peritubular myoid cell layer was identified by selective staining for alkaline phosphatase activity. Our observations demonstrate that myoid cells are a direct target for PG F2a and suggest a role of the eicosanoid in the intragonadal control of seminiferous tubule contractility. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peritubular myoid cells (rat testis); Contractility (seminiferous tubule); Prostaglandins
1. Introduction After release from the seminiferous epithelium, testicular spermatozoa are rapidly conveyed along the lumen of the seminiferous tubules, towards the hilum of the testis. The flux of tubular fluid containing spermatozoa appears to depend upon the contractile activity of the peritubular tissue which is composed of a particular type of smooth muscle cell, the myoid cells (Clermont, 1958; Leeson and Leeson, 1963). In the rat, myoid cells are closely connected to each other in a single flat epithelioid layer (Vogl et al., 1985; Palombi et al., 1992), but contain a-smooth actin (Tung and Fritz, 1990) and desmin (Virtanen et al., 1986), which are as typical of contractile cells. In the apparent absence of neural control, seminiferous tubule contractility is likely to be regulated by local factors originating from the adjacent compartments, the seminiferous epithelium and the interstitial tissue. Recently, our laboratory has * Corresponding author. Tel: +39 6 49766585; fax: +39 6 4462854.
demonstrated that rat myoid cells contract in response to endothelin-1 (ET-1) (Filippini et al., 1993; Tripiciano et al., 1996, 1997), a vasoactive peptide (Yanagisawa et al., 1988) secreted by Sertoli cells, the somatic component of seminiferous epithelium (Fantoni et al., 1993). Moreover, a further agonist of smooth muscle contractility, vasopressin (AVP) (Penit et al., 1983), locally produced by interstitial Leydig cells (Ivell et al., 1992), has been shown to bind peritubular cells from adult rats, inducing a second messenger cascade (Howl et al., 1995) resulting in cell contraction (Tripiciano et al., 1996). Among the well known agonists of smooth muscle contractility (Schroer and Schroeder, 1994) prostaglandin F2a (PG F2a ) is produced in vitro by testicular cells originating from both the seminiferous epithelium (Jannini et al., 1994) and the interstitial compartment (Haour et al., 1979; Kern and Maddocks, 1995). Myoid cells may represent a suitable paracrine target for this eicosanoid. The hypothesis that PG F2a may contribute to the local regulation of peritubular contractility is reinforced by the early observation (Farr
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and Ellis, 1980) that at high doses (10 mM) this prostaglandin is able to increase seminiferous tubule tone and contraction frequency. In order to verify whether myoid cells are a direct target for PG F2a, we have analyzed the biochemical and morphological response of isolated myoid cells in vitro, utilizing highly purified populations of this cell type (Palombi et al., 1988; Filippini et al., 1993). Moreover, the contractile nature of peritubular response to PG F2a was also tested in whole mount preparations of seminiferous tubules.
2. Materials and methods
2.1. Materials Collagenase A from Clostridium histoliticum and DNAase-I were obtained from Boehringer Mannheim (Mannheim, Germany). Trypsin was purchased from Difco Laboratories (Detroit, MI). Eagle’s Minimum Essential Medium (MEM) was obtained from Gibco (Grand Island, NY). Percoll was purchased from Pharmacia (Uppsala, Sweden). Myo-[2-3H]inositol (16–18 Ci/mmol) was purchased from DuPont de Nemours Italiana SpA (Milan, Italy). Plastic culture dishes and multiwell plates were purchased from Falcon (Oxnard, CA). PG F2a and other reagents were purchased from Sigma (St. Louis, MO). PG F2ax was dissolved in ethanol at a concentration of 10 mM and diluted in MEM just prior to use.
2.2. Animals The animals used were adult and 3-week-old Wistar rats (Charles River, Como, Italy), fed ad libitum until killed by CO2 asphyxia or cervical disarticulation. Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
2.3. Myoid cell isolation and culture Myoid cells were isolated and purified through Percoll density gradient from 3-week old rats as previously described (Palombi et al., 1988) and plated in plastic culture dishes in MEM. The cells were cultured under serum-free conditions at a concentration of 250000 cells/well in 12-multiwell plates, unless otherwise specified, at 37°C in a water-saturated atmosphere of 95% air and 5% CO2. The assessment of myoid cell purity (Palombi and Di Carlo, 1988) was performed routinely for each preparation on the basis of the presence of alkaline phosphatase activity tested on adherent cells (as described below) and was never B96%.
2.4. Alkaline phosphatase cytochemistry Selective myoid cell identification through alkaline phosphatase cytochemistry was performed as previously described (Palombi and Di Carlo, 1988), basically following the method of Ackermann (Ackermann, 1962). Briefly, the fixed cells were incubated in an alkaline solution containing 0.5 mg/ml Fast Blue RR in water and 40 ml/ml a-naphtol phosphate (0.25% solution, pH 8.6). After 30 min incubation in the dark, a purple–blue precipitate was apparent specifically on the surface of myoid cells (Palombi and Di Carlo, 1988).
2.5. Scanning electron microscopy (SEM) analysis of cell contraction in 6itro Myoid cells were cultured in Falcon dishes, treated with 10 mM PG F2a or with vehicle on the second day of culture, immediately fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) and postfixed in 1% OsO4 in Zetterquist buffer. After dehydrating and critical point drying in ethanol, the plate was carefully fragmented with the aid of a sharp blade, the samples were coated with gold and viewed under a Hitachi (Tokyo, Japan) S-570 scanning electron microscope.
2.6. IP production To measure IP production, myoid cells were incubated with 4mCi/ml myo-[2-3H]inositol for 48 h starting from the second day of culture. After labeling, cells were extensively rinsed in Hanks’ Balanced Salt Solution (HBSS) and preincubated with 20 mM LiCl. PG F2a treatment was started 10 min after LiCl addition. After 30 min the incubation was stopped by replacing the medium with ice-cold 10% trichloroacetic acid (TCA). The TCA was removed from samples with a single extraction with 1:1 v/v of a mixture of trichlorotrifluoroethane and tri-n-octylamine (74:26, v/v). IP separation was performed by ion exchange chromatography as described elsewhere (Berridge, 1983; Adamo et al., 1985).
2.7. Measurement of [Ca 2 + ]i [Ca2 + ]i was measured by dual wavelength fluorescence in single cells loaded with the Ca2 + -sensitive indicator fura-2 (Grynkiewicz et al., 1985). Testicular myoid cells were plated onto plastic coverslips in MEM. After 4 days in culture, the cells were incubated in 1 ml MEM containing 3 mM fura-2-acetoxymethylester for 1 h at 37°C. The cells were then rinsed with Krebs-Henseleit-Hepes (KHH) buffer (140.7 mM Na + , 5.3 mM K + , 132.4 mM Cl − , 0.98 mM PO24 − , 1.25 mM Ca2 + , 0.81 mM Mg2 + , 5.5 mM glucose and 20.3 mM Hepes) supplemented with 0.2%
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fatty acid-free bovine serum albumin (BSA). Measurements were performed in single cells, at 340 and 380 nm excitation wavelengths, with an AR-Sm microfluorimeter (Spex Industries, Edison, NJ) connected to a Diaphot TMD inverted microscope (Nikon, Tokyo, Japan) equipped with a CF X 40 objective. Emission was collected by a photomultiplier carrying a 510 nm cut-off filter and recorded by an ASEM Desk 2010 computer (ASEM SpA, Buia, Italy), which automatically calculated real-time 340/380 ratios. Calibration of the signal was obtained at the end of each observation by adding 5 mM ionomycin to saturate the dye maximal fluorescence, followed by 7.5 mM EGTA plus 60 mM Tris –HCl, pH 10.5, to release Ca2 + from fura-2 and obtain minimal fluorescence. [Ca2 + ]i was calculated according to previously described formulas (Grynkiewicz et al., 1985). When indicated, the cells were pretreated either with thapsigargin, MnCl2 or EGTA.
2.8. Statistical analysis Data are presented as the mean9SE of results from at least three independent experiments. Student’s t-test was used for statistical comparison of means where applicable.
2.9. Preparation and treatment of seminiferous tubule whole mounts The morphological response of peritubular tissue to acute treatment with PG F2a was analyzed in tubular whole mounts. Seminiferous tubules were prepared as previously described (Tripiciano et al., 1996). Briefly, Wistar rats aged 20 – 60 days were killed by CO2 asphyxia, their testes were excised, decapsulated and digested under gentle shaking at room temperature in Eagle’s Minimal Essential Medium (MEM) containing 1 mg/ml collagenase. After digestion of the interstitium the tubular mass was rinsed in MEM, stretches of tubules were dissected by means of sharp needles, then carefully transferred to 35 mm culture dishes in 300 ml of medium. The tubules were incubated for 10 min at 32°C in a humidified chamber under an athmosphere containing 5% CO2. At the end of the incubation time, the medium was replaced by 600 ml of MEM containing 10 mM PG F2a, immediately (i.e. within 20 s) followed by addition of 4% paraformaldehyde in 0.1 M phosphate buffer (PBS). After 10 min fixation and rinsing in buffer, the samples were processed in order to highlight the site localization of alkaline phosphatase activity (Palombi and Di Carlo, 1988). After staining, single tubules were gently collected, laid onto microscopy slides, and covered with a coverslip in a drop of 50% glycerol in PBS. The tubular whole-mounts were viewed and photographed under a
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Zeiss Axioplan Microscope (Carl Zeiss, Oberkochen, Germany).
3. Results
3.1. PG F2a stimulation of inositol phosphates accumulation In order to determine whether myoid cell treatment with PG F2a is followed by phosphoinositidase-C (PLC) activation resulting in the generation of intracellular inositol 1,4,5-trisphosphate (IP3), we investigated the effect of different concentrations of PG F2a on the accumulation of IP3 derived inositol phosphates (inositol mono-, bis- and trisphosphates) in primary cultures of peritubular myoid cells. The exposure of myoid cells to PG F2a for 30 min evoked a dose-dependent increase in inositol phosphates accumulation, indicating that the agonist activates phospholipase C, which, in turn, catalyzes the breakdown of polyphosphoinositides (Fig. 1).
3.2. Effect of PG F2a on [Ca 2 + ]i Fura-2-loaded myoid cells were equilibrated in KHH for at least 10 min prior to PG treatment. The basal cytosolic calcium was 8799 nM (n= 20). [Ca2 + ]i remained stable, and no rapid oscillations were observed. PG F2a stimulated a dose-dependent increase in [Ca2 + ]i in single cells loaded with the fluorescent Ca2 + indicator fura-2 (Fig. 2a). The onset of response was immediate after addition of the agonist. [Ca2 + ]i peaked within 15 s, followed by slow decay towards baseline values within 10 min (Fig. 2b). Since [Ca2 + ]i elevations in response to PG F2a occurred in myoid cells in the virtual absence of extracellular Ca2 + following chelation with 3 mM EGTA (Fig. 3a), which suggested the involvement of intracellular calcium, we analyzed a putative mechanism of mobilization of Ca2 + from internal stores. Thapsigargin increased the basal cytosolic calcium concentration to 148913 nM (Fig. 3b). Depletion of intracellular calcium stores with thapsigargin before stimulation with PG F2a abolished the PG F2a mediated calcium rise in the presence of extracellular calcium, which indicates that the increase in calcium is predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. In addition, in order to verify the non-involvement of calcium influx in PG F2a response, we performed experiments with Mn2 + quenching of the fura-2 dye to monitor the possible entry of this extracellular divalent cation. In fact, Mn2 + is known to permeate the cell through the same channels as those used by Ca2 + , thereby rapidly quenching fura-2 fluorescence by irreversible binding (Hallan et al., 1989). Addition of PG F2a to myoid cells in the presence of Mn2 + resulted in a rapid increase in fura-2
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Fig. 1. Concentration dependent production of inositol phosphates (IP1 +IP2 +IP3) in response to PG F2a, by purified myoid cells. The cells were stimulated as indicated for 30 min in the presence of 20 mM LiCl. Control samples received corresponding amount of vehicle only. Each point represents the mean 9 SE from three independent determinations.
fluorescence which stabilized at lower levels without any appreciable quenching below the basal line (Fig. 3c). This further indicates that the increase in [Ca2 + ]i induced by PG F2a in myoid cells involves Ca2 + release mostly from intracellular stores.
3.3. Analysis of the contractile response of myoid cells to PG F2a in 6itro Myoid cell shape in vitro was studied by means of SEM in control and PG F2a -stimulated conditions. As shown in Fig. 4, the cells respond to a 10 mM concentration of the agonist, with immediate contraction. The effect is less apparent with 1 mM prostaglandin (not shown) and is not appreciable at lower concentrations.
Previous analysis has in fact shown that myoid cells are polygonal squamous elements arranged in a single epithelioid layer (Vogl et al., 1985; Palombi et al., 1992). Immediately after treatment with PG F2a at a dose of 10 mM, the individual myoid cells become apparent: the central perinuclear area of the cell appears intensely stained, while only faintly visible processes appear to connect the contracted cells to one another (Fig. 5). Although the size of the alkaline phosphatase decorated profiles (hence the degree of cell contraction) may vary, the same pattern of response was constantly observed in virtually the whole peritubulum both in young and mature samples. In control samples, incubated with the vehicle alone, no cell contraction was observed.
3.4. PG F2a mediates seminiferous tubule contractility
4. Discussion
When the surface of seminiferous tubules is viewed in whole-mount preparations after selective alkaline phosphatase staining of myoid cells (Fig. 5), the distribution of the enzyme appears uniform on the cell surface and the contours of the individual cells are hardly apparent.
Among secretion products involved in short range intercellular communication, prostaglandins represent a broad family of short-lived lipids, responsible for a great variety of biological responses. A major target of these eicosanoids are smooth muscle cells in vascular
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and non-vascular tissue (Schroer and Schroeder, 1994), in which different prostaglandins control either relaxation or contraction through different signal transduction pathways coupled to specific membrane receptors (Narumiya, 1996). PG F2a has been shown to induce contraction of vessels, uterus, gastrointestinal tract, bronchi and trachea, and it is generally agreed that in most cases it contributes to the multifactorial control of smooth muscle tone (Schroer and Schroeder, 1994). Modifications of seminiferous tubule contractile activity following treatment with PG F2a have been described in the rat (Farr and Ellis, 1980). In tubular explants, micromolar doses of this eicosanoid induced a reduction in diameter and increased frequency of spontaneous contraction. Moreover, increased sperm numbers were observed in rabbit deferent ducts after PG F2a treatment (Hafs et al., 1974). In the present study, we
Fig. 2. Effect of PG F2a treatment on [Ca2 + ]i of single myoid cells loaded with fura-2. (a) [Ca2 + ]i peak response to different concentrations of PG F2a. The data are representative of three to five independent experiments for each dose. (b) Trace representing real-time fura-2 fluorescence in single myoid cell stimulated with 10 mM PG F2a.
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provide the first demonstration that peritubular myoid cells are a direct target of PG F2a, to which they respond with a second messenger increase and contractile behaviour. We have observed that in vitro cultures of purified myoid cells display a dose-dependent production of IPs and an increase in [Ca2 + ]i in response to PG F2a, suggesting that binding of the eicosanoid to the cell activates phospholipase-C (PLC), which results in phosphoinositides turnover. These data are in accordance with what is known about PG F2a in other contractile cell types (Fukuo et al., 1986; Morimoto et al., 1990), as is the observation that no increase in cAMP production follows treatment with this prostaglandin (data not shown). In myoid cells, IP3 production in response to PG F2a is accompanied by a moderate but dose-dependent rapid increase in [Ca2 + ]i. As for the mechanisms underlying this increase, the relevance of Ca2 + mobilization from internal stores versus a possible contribution of Ca2 + entry through the plasma membrane is indicated by three lines of evidence: (i) the calcium response appeared to be unaffected by chelation of extracellular Ca2 + with EGTA; (ii) depletion of Ca2 + stores by means of thapsigargin, an inhibitor of endoplasmic reticulum Ca2 + -ATPase (Thastrup et al., 1990), abolished the calcium response to PG F2a ; (iii) fura-2 fluorescence induced by PG F2a undergoes no quenching in Mn2 + -enriched medium, indicating (Hallan et al., 1989) that stimulation does not significantly open Ca2 + channels. In myoid cells, therefore, PG-stimulated [Ca2 + ]i increase appears to depend mainly, if not exclusively, upon intracellular release; in other contractile cell types a contribution from both intracellular pools and calcium entry have been reported (Mene` et al., 1987; Fukuo et al., 1986). The in vitro model consisting of highly purified populations of myoid cells was fundamental for the analysis of the direct biochemical response to PG F2a. Moreover, it showed that myoid cells are the tubular target for the agonist and that the morphological response to the agonist is of a contractile nature. Myoid cells were observed to undergo immediate contraction in response to PG F2a, not only in cell cultures but also when organized in peritubular tissue in freshly-explanted seminiferous tubules. The direct visualization of contracted myoid cells through selective alkaline phosphatase cytochemistry in tubular whole-mounts suggests a physiological role for PG F2a on peritubular contractility. Several factors have been indicated in the past as possible regulators of seminiferous tubule contractile activity in the rat, but so far experimental evidence indicating a direct role in the paracrine regulation of myoid cell contractility has been produced only for ET-1 and AVP. ET-1 has been shown to be produced by Sertoli cells (Fantoni et al., 1993) and to bind high affinity receptors on isolated myoid cells (Filippini et
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Fig. 3. [Ca2 + ]i increase induced in single myoid cells by PG F2a treatment depends upon release from intracellular pools. (a) Myoid cell culture was equilibrated in KHH buffer containing 3 mM EGTA. Single cell response to 10 mM PG F2a appeared unaffected by the virtual absence of extracellular calcium. (b) Myoid cell culture was exposed to 10 mM thapsigargin. In response to this inhibitor of the endoplasmic reticulum Ca2 + -ATPase, a slow and transient [Ca2 + ]i increment was observed in single cells. Administration of 10 mM PG F2a at the time when [Ca2 + ]i was stabilized failed to elicit any further [Ca2 + ]i increment. (c) Cells were equilibrated in KHH buffer containing 100 mM MnCl2, then treated with 10 mM PG F2a. Despite the presence of Mn2 + in the extracellular medium, no appreciable quenching of the fura-2 fluorescence was induced by the agonist. Fluorescence was completely quenched by subsequent addition of 5mM ionomycin. (d) Mean values of PG F2a dependent [Ca2 + ]i increases (both peak and plateau) in controls or cells treated with 10 mM PG F2a in EGTA or thapsigargin-containing buffer. Results are from at least four independent experiments.
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prostaglandin found to be effective on myoid cells appears to be high when compared to that (10 nM) required for ET or AVP response (Filippini et al., 1993; Howl et al., 1995). In this regard, however, the following points are noteworthy: (i) other contractile cells are known to require similarly high concentrations of PG F2a (Fukuo et al., 1986; Mene` et al., 1987); (ii) it is possible that, in vivo, high local concentrations of prostaglandins form transiently within a restricted tissue compartment; (iii) it is possible that the concentration which actually acts on the cells in culture does not exactly correspond to the one that is administered.
Fig. 4. Scanning electron micrograph showing the contractile response of myoid cells to acute PG F2a stimulation. Cells have been cultured for 20 h before treatment and fixed immediately after receiving: (a) 10 mM PG F2a ; (b) vehicle only (control). Contraction induced by the treatment results in the formation of slender cytoplasmic processes. Bar: 10 mm.
al., 1993), thus stimulating a second messenger cascade (Filippini et al., 1993; Tripiciano et al., 1997) and an immediate contractile response (Tripiciano et al., 1996); contraction stimulated by ET-1 has been reported also for peritubular tissue in situ both in the prepubertal and in the adult gonad (Tripiciano et al., 1996). The presence of high affinity receptors for AVP and second messenger production in response to this peptide have been demonstrated for peritubular cells from adult rats (Howl et al. 1995). Peritubular tissue has also been shown to undergo immediate contraction in response to stimulation of tubular explants with AVP (Tripiciano et al., 1996). Moreover the peptide is known to be produced locally by Leydig cells of the testicular interstitium (Ivell et al., 1992). On the basis of the experimental evidence presented, we candidate PG F2a as a new modulator of peritubular contractility, a role for which it seems to meet all the basic requirements, i.e. direct biochemical activation, direct contractile response and local production. The physiological relevance of myoid cell responsiveness to PG F2a is suggested by the evidence that the eicosanoid is produced at high basal levels by interstitial macrophages (Kern and Maddocks, 1995) and, under gonadotropin stimulation, by Leydig cells (Haour et al., 1979) and Sertoli cells (Jannini et al., 1994). The concentration of
Fig. 5. Surface view of peritubular layer in whole-mount preparation of seminiferous tubules from prepuberal (a, b) and adult (c, d) rats. Myoid cells have been selectively stained through alkaline phosphatase cytochemistry. a and c: control samples; b and d: tubules fixed immediately after stimulation with 10 mM PG F2a. In both the young and adult, myoid cells appear to contract in response to stimulation. Bar: a, b 40 mm; c, d 100 mm.
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Whatever the precise role and the possible interplay of the different myoid cell agonists within the complex paracrine interactions characterizing the male gonad (Skinner, 1991), PG F2a appears to be one of the factors responsible for the local control on sperm and tubular output resulting from their action on myoid cell contractility.
Acknowledgements Research supported by a grant from Italian Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST). The authors thank Antonella Zacheo for skillful technical support.
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