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Abstract

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements
  • 1
    Cultured epithelia of Sertoli cells from prepubertal rats were grown on Matrigel-coated millipore filters for short-circuit current (Isc) measurements. Under basal conditions, these epithelia exhibited a ‘zero’ transepithelial potential difference, a ‘zero’ short-circuit current and a transepithelial resistance of 60 Ω cm2.
  • 2
    Forskolin (100 μm) and 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) (100 μm) added to the apical side stimulated the Isc (forskolin, peak ΔIsc= 1.32 ± 0.16 μA cm−1; cpt-cAMP, peak ΔIsc= 0.88 ± 0.16 μA cm−2).
  • 3
    ATP (100 μm) added apically elicited a Isc response (peak ΔIsc= 6.45 ± 0.28 μA cm−2) which was similar in magnitude to that of 1 μm thapsigargin (peak ΔIsc= 6.09 ± 0.44 μA cm−2). The potency of the responses to other nucleotides: UTP geqslant R: gt-or-equal, slanted ATP > ADP >> AMP = adenosine indicates the involvement of a mixture of P2Y receptors.
  • 4
    Removal of extracellular Cl and HCO3 reduced the Isc response to ATP by 70 % and 40 %, respectively. Removal of K+ had no effect, whereas removal of Na+ attenuated the Isc response.
  • 5
    The response to ATP was insensitive to agents known to block anion secretion (except apical diphenylamine-2-carboxylate (DPC) and DIDS). The resistance to perturbation by pharmacological agents may be a unique property of the seminiferous epithelium.
  • 6
    Whole-cell current recordings in cultured rat Sertoli cells demonstrated a DIDS-sensitive outwardly rectifying Cl conductance with activating and inactivating characteristics at depolarizing and hyperpolarizing voltages, respectively.
  • 7
    The stimulation of electrogenic ion transport by ATP may be part of a complex mechanism regulating fluid secretion by the testis. Cultured Sertoli cell epithelia are shown to provide a useful model to investigate transepithelial transport in the seminiferous epithelium.

The Sertoli cells lining the seminiferous tubules fulfil many functions. They provide physical support to germ cells, form the blood-testis barrier and secrete protein products thought to be essential for the control and co-ordination of spermatogenesis. (For review, see Bardin et al. 1994.) The Sertoli cells are also known to secrete a fluid into the lumen (Setchell, 1970; Tuck et al. 1970; Levine & Marsh, 1971; Cheung et al. 1977). Whilst it is generally thought that this fluid secretion provides a medium to transport the newly shed immotile spermatozoa to the epididymis, the mechanisms by which fluid secretion occurs are still unknown. In this work, we have investigated the ionic basis of fluid production by Sertoli cell epithelia derived from immature rats using cell culture and short-circuit current techniques. Evidence is provided that cultured Sertoli cell epithelium transports electrolytes electrogenically when stimulated with ATP. Cultured Sertoli cell monolayers therefore provide a useful model for the investigation of electrolyte transport in the seminiferous tubules. Part of the work has been presented to The Physiological Society in a preliminary form (Ko et al. 1997).

methods

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements

Preparation of Sertoli cell suspensions

The method used followed that described by Gorczynska et al. (1994). Twenty-day-old male Sprague-Dawley rats were killed by a blow on the head followed by cervical dislocation. Testes were excised rapidly, decapsulated and cut into small fragments and washed twice in Hanks' balanced salt solution containing DNase (10 mg l−1). The fragments were then digested in 10 ml of Hanks' balanced salt solution containing 10 mg l−1 of DNase, 1 g l−1 collagenase, 1 g l−1 hyaluronidase and 1 g l−1 trypsin in a shaking water bath (37°C, 120 cycles min−1, 30 min). After digestion, cells were washed and centrifuged (22°C, 2 min, 25 ×g) three times in 50 ml of Hanks' balanced salt solution containing 10 mg l−1 of DNase. This preparation contains 97 % Sertoli cells identified morphologically by light microscopy. The Sertoli cells were then suspended in Dulbecco's modified Eagle's medium (DMEM-F12; Gibco) supplemented with epidermal growth factor (2.5 ng ml−1), insulin (10 μg ml−1), human transferrin (5 μg ml−1) and bacitracin (10 μg ml−1) and plated either on Petri dishes for patch clamping or on Matrigel (Becton Dickinson Labware) coated millipore filters for the measurement of short-circuit current. In some experiments, 2-day-old monolayers on Petri dishes were treated with a hypotonic solution (20 mm Tris, pH 7.4 at 22°C for 2.5 min) to lyse any residual germ cells before plating onto filters (Galdieri et al. 1981). However, this treatment was found not to affect the short-circuit current results.

Formation of cell monolayers

Pervious culture supports were made by sticking (using Silastic 734 RTV adhesive) silicone washers with a 0.4 cm2 hole to Matrigel-coated millipore filters (type HAWP, 0.45 μm). Such assembly forms a well into which Sertoli cells could be seeded (Cuthbert & Wong, 1986; Wong, 1988a). Freshly isolated Sertoli cells suspended in DMEM-F12 were plated into the wells at a cell density of 25 × 104 cells filter−1. Four Millipore filter assemblies were floated on 12 ml of culture medium in 30 cm2 Petri dishes and the cultures were incubated at 32°C in 95 % O2 and 5 % CO2. Sample cultures were removed, stained with haematoxylin-eosin, and examined microscopically to determine when confluence was reached. Generally, monolayers were ready for use after 2 to 3 days in culture. Confluent monolayers were used for short-circuit current recording. On a few occasions monolayers on filters were subjected to morphological examination by light and electron microscopy using standard procedures. The results confirmed Sertoli cells from immature rats formed confluent epithelial monolayers with tight junctions between cells as reported previously (Byers et al. 1986); the results are not shown herein.

Short-circuit current recording

Sertoli cell monolayers were clamped between the two halves of Ussing chambers with a 0.6 cm2 window. The method used for short-circuiting primary cultures of epithelial monolayers was as given elsewhere (Wong, 1988a). Short-circuiting was accomplished using a WPI dual voltage clamp (WP Instruments, New Haven, CT, USA) and currents were displayed continuously on pen recorders. Transepithelial resistance was measured by transiently commanding the clamp to set the voltage at 0.1 or 0.2 mV away from zero. The resulting changes in transepithelial current allowed calculation of conductance using Ohm's law. In most experiments monolayers were bathed on both sides with Krebs-Henseleit solution (20 ml), gassed with 95 % O2, 5 % CO2 and heated to 32°C.

Secretory agonists were added either to the apical or basolateral bathing solution and the change in current was usually followed until the current had reached a steady state. In most cases, the effect of transport inhibitor was tested by pretreating the monolayers with the inhibitor for 10 min before addition of the agonist (ATP). The rise in the short-circuit current (Isc) over 10 min in the presence of the inhibitor was measured and compared with a control response without the inhibitor and the results are expressed in charge transfer (microcoulombs, μC) over the 10 min period. On a few occasions, the inhibitor was added after the agonist, usually when the response has reached steady state.

Whole-cell current recording

For these studies, cells were grown in culture dishes instead of permeable supports. Current recordings were obtained using the whole-cell patch-clamp technique as described by Hamill and others (Hamill et al. 1981) with a patch-clamp amplifier (Axopatch-200 or Axopatch-1D, Axon Instruments). Patch pipettes, made from borosilicate glass (Vitrex, Modulohm I/S, Herlev, Denmark), were prepared as previously described (Huang et al. 1993). They had a resistance of 2–6 MΩ. After formation of whole-cell configuration, the series resistance and cell capacitance were compensated. The control of command voltage was carried out using an IBM-AT-compatible computer equipped with an interface (TL-1-125, Axon Instruments) and utilizing the software pCLAMP v. 6. The output current signals, after being filtered through an 8-pole Bessel filter (AI-2040, Axon Instruments) at a cut-off frequency of 1 kHz, were displayed on a chart recorder (Graphic, Yokohama, Japan).

The following pipette solution was used (mm): KCl, 140; MgCl2, 1; CaCl2, 1.3; Hepes, 10; EGTA, 2 (pH 7.2). The intracellular free Ca2+ was estimated to be 100 nm. The bath solution contained: KCl, 40; CaCl2, 1; Hepes, 10 (pH 7.4). Osmolarity of the solutions was adjusted to 300 mosmol l−1 by addition of mannitol.

Statistical analysis

Results are expressed as means ±s.e.m. Comparisons between groups of data were made by using Student's unpaired t test. A P value of less than 0.05 was considered statistically significant.

Solutions

Krebs-Henseleit solution had the following composition (mm): NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.8; KH2PO4, 1.8; NaHCO3, 25; and glucose, 14. This solution had a pH of 7.3–7.4 when bubbled with 95 % O2, 5 % CO2. Cl-free Krebs-Henseleit solution was made by substitution of sodium gluconate, potassium gluconate and CaSO4 for NaCl, KCl and CaCl2, respectively. In bicarbonate-free solution, HCO3 was replaced by 25 mm Hepes and the solution was bubbled with pure O2 to maintain pH at 7.4. In Na+-free solution, NaCl was replaced by NMDG chloride and NaHCO3 by choline bicarbonate. In K+-free solution, KCl and KH2PO4 were simply omitted from the Krebs-Henseleit solution.

Materials

DMEM-F12 and Hanks' balanced salt solution were purchased from Gibco. DNase, collagenase, hyaluronidase, trypsin, bacitracin, acetazolamide, 6-ethoxyzolamide, bafilomycin A1, bumetanide, DIDS, ouabain, insulin, transferrin, epidermal growth factor, 8-(4-chlorophenylthio)-cAMP (cpt-cAMP), forskolin, ATP, UTP, ADP, AMP and adenosine were from Sigma. Since it has been shown that the responses to ADP in other tissues may be attributed to trace amounts of ATP contamination in the ADP supplied by the manufacturer, stock solutions of ADP (10 mm) were pretreated with D-glucose (22 mm) and hexokinase (Boehringer, 10 i.u. ml−1) at 37°C for 1 h to remove contaminating ATP (Harden et al. 1997). Diphenylamine-2-carboxylate (DPC) was bought from Reidel-de-Haen Chemicals (Hannover, Germany). Thapsigargin and chlorothiazide were from Research Biochemicals International, follicle-stimulating hormone (FSH) from the National Institutes of Health (Bethesda, MD, USA) and H2DIDS from Molecular Probes. Amiloride was a gift from Merck, Sharp & Dohme (HK) Ltd.

results

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements

When clamped in the Ussing chamber, cultured rat Sertoli cell epithelia exhibited a basal transepithelial potential difference of about 0 mV and a short-circuit current (Isc) of 0 μA cm−2. The transepithelial resistance calculated from the currents required to achieve small potential displacements was estimated to be 60.7 ± 8.0 Ω cm2 (mean ±s.e.m., n= 24). This value indicates that the monolayers form low-resistance epithelia.

Effects of thapsigargin, forskolin and cAMP

Since the hormonal regulation of Sertoli cell functions has been shown to be mediated by calcium and cAMP (Leung & Steele, 1992), we chose first to examine the effects of increasing cAMP and intracellular Ca2+ on the Isc. Figure 1 shows the Isc response to forskolin (100 μm), cpt-cAMP (100 μm) and thapsigargin (1 μm). All three agents caused increases in inward current when added to the apical side. Thapsigargin increased the Isc to a peak of 6.09 ± 0.44 μA cm−2 (n= 6), whereafter the current declined to about 4 μA cm−2 after 10 min (Fig. 1A). The response to forskolin (100 μm) was smaller (peak ΔIsc= 1.32 ± 0.16 μA cm−2, n= 15) and transient, with the Isc declined to the basal level after 10 min (Fig. 1B). cpt-cAMP (100 μm) elicited a Isc response similar to that of forskolin in magnitude (peak ΔIsc= 0.88 ± 0.16 μA cm−2, n= 17). However, the time course of rise of the Isc was slower and the peak current was attained only after 10 min. At the peak of the response, addition of DPC (1 mm) to the apical side completely inhibited the current to the basal level (Fig. 1C). Addition of thapsigargin (1 μm), forskolin (100 μm) or cpt-cAMP (100 μm) to the basolateral side did not affect the Isc (results not shown).

image

Figure 1. Short-circuit current (Isc) response to thapsigargin (1 μm, A), forskolin (100 μm, B) and cpt-cAMP (100 μm, C) added to the apical bathing solution

DPC (1 mm) was added to the apical bathing solution during the cpt-cAMP response (C). Horizontal line indicates zero Isc. Each point shows the mean ± s.e.m. of 6 experiments (thapsigargin), 15 experiments (forskolin) and 17 experiments (cpt-cAMP).

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Effect of ATP

Many secretory epithelia, including the rat epididymis (Wong, 1988b) are shown to possess functional purinoceptors which affect transepithelial transport. It is of interest to see if the rat Sertoli cell epithelia also express these receptors. The effect of ATP is shown in Fig. 2. ATP (100 μm) added to the apical side caused a biphasic response. The Isc rose rapidly from zero to a peak level of 6.45 ± 0.28 μA cm−2 (n= 28). It then fell to a stable plateau at about 3 μA cm−2. The sustained plateau current after ATP was completely reduced by apical DPC (1 mm) or apical DIDS (0.25 mm) (n= 7) to the basal level (Fig. 2 inset).

image

Figure 2. Isc response to ATP (100 μm) added apically at time zero

DPC (1 mm) was added to the apical solution during the plateau phase of the response. Each point shows the mean ± s.e.m. of 28 experiments. Inset shows a monolayer challenged with apical ATP followed by DIDS (0.25 mm) added apically.

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The effects of analogues of ATP are shown in Fig. 3. In these experiments, responses were measured over 10 min following drug additions. UTP, ATP and ADP stimulated Isc in a dose-dependent manner. The EC50 values for UTP, ATP and ADP were 2.08 μm, 5.89 μm and 147.54 μm, respectively. AMP and adenosine had little effect.

image

Figure 3. Concentration-response curves for UTP (○), ATP (•), ADP (□), AMP (▪) and adenosine (▵) in stimulating Isc in cultured rat Sertoli cell epithelia

The responses were measured over 10 min following drug additions. Each point shows the mean ± s.e.m. of 6–10 experiments. The stock solutions of ADP were treated with glucose and hexokinase to convert contaminating ATP to ADP before use.

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Identity of the current-carrying species

Of the agents which affected short-circuit current (forskolin, cAMP, thapsigargin and ATP), all produced an increase in inward-flowing current. The effect of DPC, a known blocker of anion channels, on the responses to cpt-cAMP and ATP suggested that anions (Cl and HCO3) were important current-carrying species. In subsequent experiments, ion substitution experiments were performed using ATP as the agonist, since it produced the largest and most sustained response among the agents studied.

Removal of Cl from the bathing solution reduced the Isc response to apical ATP (100 μm) by 70 %, whilst removal of HCO3 (solution buffered by 25 mm Hepes gassed with pure O2) reduced the Isc by 40 % (Fig. 4). In the absence of both anions, a small current (24 % of the total current) remained. This residual current was insensitive to Na+ removal and apical amiloride (0.01 mm). Removal of K+ had no effect (ΔIsc values over 10 min in normal Krebs-Henseleit and K+-free solution were 570 ± 49 μC and 554 ± 97 μC, respectively, P > 0.05), whereas removal of Na+ (replaced by NMDG+) attenuated the Isc response to ATP (Fig. 5). The ΔIsc measured (over 10 min upon ATP addition) in normal Krebs-Henseleit and Na+-free solutions were 417 ± 63 μC and 163 ± 19 μC, respectively, P < 0.05.

image

Figure 4. Effect of ion substitution on Isc response to apical ATP (100 μm)

Responses were measured over 10 min following ATP addition. Each column shows the mean ± s.e.m. with the number of experiments shown in parentheses. KH, normal Krebs-Henseleit bathing solution.

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image

Figure 5. Effect of Na+ removal on the Isc response to apical ATP

ATP (100 μm) added at time zero. Each point represents the mean ± s.e.m. of 6 experiments. ○, normal Krebs-Henseleit solution; •, Na+-free solution.

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Effects of transport inhibitors

As shown in the earlier experiments, DPC (1 mm) and DIDS (0.25 mm) added apically completely inhibited the plateau phase of the Isc response to cAMP and ATP (Figs 1 and 2). Other transport inhibitors with presumed actions on anion transport were also tested. The results are shown in Table 1. Addition of amiloride (0.01 mm), a blocker of epithelial sodium channels, to the apical side had no effect on the Isc response to ATP. The following agents added to the basolateral side were also without effect: acetazolamide (45 μm) and 6-ethoxyzolamide (1 mm), inhibitors of carbonic anhydrase; amiloride (1 mm), an inhibitor of the Na+-H+ exchanger; chlorothiazide (100 μm), an inhibitor of the Na+-Cl cotransporter; H2DIDS (300 μm), an inhibitor of the Na+-HCO3 cotransporter; and bafilomycin A1 (0.8 μm), a vacuolar H+-ATPase inhibitor. Basolateral application of bumetanide (100 μm), a Na+-K+-2Cl symport inhibitor, affected the Isc response to ATP slightly but the difference was not statistically significant (Fig. 6). The ATP-stimulated Isc was also insensitive to ouabain (2 mm) added to the basolateral side. A combination of the inhibitors also had no effect on the ATP-stimulated Isc (cocktail; Table 1).

Table 1.  Effect of transport inhibitors on ATP-stimulated ISC
TreatmentDoseControl (μC)Experimental (μC)
  1. Responses were measured over 10 min after apical addition of ATP (100 μm). Inhibitors were added either to the apical (ap) or basolateral (bl) side of the epithelium prior to ATP. *Cocktail contains bumetanide (100 μm), acetazolamide (100 μm), chlorothiazide (100 μm), H2DIDS (250 μm), ouabain (1 mm) and amiloride (100 μm). Each value shows the mean ±s.e.m.of 4–6 experiments. None of the inhibitors tested caused a significant change in the ATP response (P > 0.05).

Acetazolamide45 μm (bl)591.55 ± 77.20514.35 ± 101.33
6-Ethoxyzolamide1 mm (bl)986.23 ± 87.82838.59 ± 26.06
Amiloride0.01 mm (ap)902.28 ± 157.30937.98 ± 138.96
Amiloride1 mm (bl)635.94 ± 82.99606.02 ± 252.83
H2DIDS300 μm (bl)551.02 ± 87.82581.90 ± 45.36
Bafilomycin A10.8 μm (bl)892.63 ± 84.92784.55 ± 114.84
Chlorothiazide100 μm (bl)1059.57 ± 51.15877.19 ± 74.31
Ouabain2 mm (bl)551.02 ± 72.38524.96 ± 53.08
Cocktail*(bl)777.79 ± 159.23723.75 ± 161.16
image

Figure 6. Effect of bumetanide on Isc response to ATP

A, ATP (100 μm) was first added to the apical side to stimulate Isc followed by basolateral application of bumetanide (100 μm). Transient current pulses were the result of intermittently clamping the potential at 0.1 mV. Horizontal line indicates zero Isc. This record is representative of 4 different experiments. B, effect of pretreating tissues with basolateral bumetanide before ATP. The Isc responses were measured over 10 min upon ATP addition. Each column shows the mean ± s.e.m. of 4 experiments.

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Whole-cell patch-clamp study

ATP-stimulated whole-cell current from single voltage-clamped Sertoli cells plated on Petri dishes was also recorded. Current was elicited by voltage pulses from a holding potential of −30 mV to potentials between −120 and 120 mV with 20 mV increments. As shown in Fig. 7A, the ATP-stimulated whole-cell current exhibited time-dependent activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. The I-V relationship showed outward rectification (Fig. 7B). The reversal potential of the ATP-activated current was dependent on extracellular Cl concentrations, with measured values of −4.2 ± 3 mV (n= 3) and 22.1 ± 1.2 mV (n= 3) for 140 and 40 mm of extracellular Cl (pipette, 140 mm), respectively, which was close to the respective calculated Cl equilibrium potentials of 0 and 30 mV. The time course of the ATP response shown by the current amplitudes elicited by ± 80 mV was recorded alternately, with a 800 ms interval (Fig. 7C). A gradual decline in the ATP-stimulated whole-cell current similar to that observed in the Isc was also recorded. However, there was a time lag for the ATP whole-cell current response which was probably related to the time required for ATP to reach the cells. As with the Isc response, the ATP-stimulated whole-cell current was also inhibitable by DIDS (1 mm, n= 6; Fig. 7). The characteristics of the ATP-stimulated whole-cell current observed in rat Sertoli cells are consistent with a Ca2+-activated Cl current, previously found in other Cl secreting epithelia (Chan et al. 1992; Huang et al. 1993).

image

Figure 7. ATP-activated whole-cell currents in rat Sertoli cells

A, whole-cell current recordings obtained prior to (a) and 2 min after ATP (100 μm) stimulation (b), and after addition of DIDS (1 mm; c). Currents were elicited by voltage pulses from a holding potential of −30 mV to potentials between −120 and 120 mV with 20 mV increments. B, corresponding I-V curves measured at 220 ms after voltage pulses: ○, control; •, ATP; ▪, DIDS. C, current amplitudes at ±80 mV, recorded alternately with a 800 ms interval. Letter-designated dashed lines (a, b and c) represent the time periods during which whole-cell current recordings were taken to generate the results in A. Cells were subject to a transmembrane Cl gradient of 140 : 40 mm (cell : bath). The intracellular Ca2+ was buffered to 100 nm.

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discussion

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements

Although previous studies employing efferent duct ligation in intact rat testes (Setchell, 1967) and micropuncture and microcannulation of the rat seminiferous tubules in vivo (Tuck et al. 1970; Levinè & Marsh, 1971) and in vitro (Cheung et al. 1977) have provided evidence that the seminiferous tubules elaborate a watery secretion into the tubular lumen, the mechanism of secretion and its regulation have not been fully explored. This study represents a first attempt to unravel secretory mechanisms by measuring electrogenic ion transport across cultured monolayers of rat Sertoli cells. Immature rat Sertoli cells grown on filters coated with basement membrane matrices have been shown to form confluent cell monolayers which assume an epithelial-like appearance reminiscent of Sertoli cells in vivo (Byers, 1986; Dym, 1994). These epithelia have been used to study protein biosynthesis and secretion (Onoda & Djakiew, 1990; Grima et al. 1992), FSH-induced cAMP production (Dym et al. 1991) and Sertoli cell differentiation (Hadley et al. 1985), etc. However, the use of cultured Sertoli cell epithelia to study ion transport has not been attempted.

When clamped in the Ussing chamber, cultured immature rat Sertoli cell epithelia did not exhibit a measurable transepithelial potential. The low transpithelial resistance of 60 Ω cm2 probably reflects a leaky intercellular pathway. Measurement of transtubular potential difference in intact rat seminiferous tubules in vivo revealed a value of 4 mV, lumen negative (Levinè & Marsh, 1971). However, direct comparison between the two sets of data is difficult since the seminiferous tubule fluid contains a high concentration of K+ ions (Tuck et al. 1970; Levinè & Marsh, 1971) which would have depolarized the luminal membrane of the tubular cells in vivo. The short-circuit current (Isc) is virtually zero at rest but could be increased by thapsigargin and forskolin, which are known to increase intracellular Ca2+ and cAMP, respectively. It transpires that, as in other secretory epithelia, transepithelial ion transport in the Sertoli cells is under the control of a dual intracellular signalling pathway involving Ca2+ and cAMP. The magnitude of the response to thapsigargin was greater than that to forskolin and cpt-cAMP (Figs 1 and 8), indicating that the Ca2+ pathway predominates in the control of transepithelial ion secretion by Sertoli cells. Although forskolin and cpt-cAMP produced Isc responses of similar magnitude, the time courses were very different (Fig. 1). Forskolin, being more lipid soluble than cpt-cAMP, would elicit a faster response. In rat Sertoli cells, cAMP was found to raise intracellular Ca2+ (Gorczynska et al. 1994). A possibility therefore exists that the stimulation of Isc by forskolin and cpt-cAMP could be mediated by a rise in intracellular Ca2+.

image

Figure 8. Responses to ATP (•), thapsigargin (○) and cpt-cAMP (▴) plotted on the same graph

Results were taken from Figs 1 and 2.

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ATP (100 μm) added to the apical side caused a relatively large and sustained increase in Isc. The inward current could be wholly inhibited by apically applied 1 mm DPC or 0.25 mm DIDS (Fig. 2), suggesting that the current could be attributed to chloride secretion. The lack of effect of apical amiloride (0.01 mm) argues against Na+ reabsorption being responsible for the inwardly flowing current (Table 1). Ion substitution experiments also supported anion secretion as the basis of the Isc response to ATP. Removal of chloride from the external medium greatly attenuated the Isc, suggesting that the current is likely to be due to chloride secretion. However, removal of HCO3 also reduced the Isc by 40 %. This reduction in current after HCO3 removal could be due to loss of HCO3 secretion per se or HCO3-dependent Cl secretion. The former seems unlikely for the following reasons. First, acetazolamide and 6-ethoxyzolamide, inhibitors of carbonic anhydrase (CA) did not affect the Isc response to ATP (Table 1). Rete testis fluid collection following efferent duct ligation has shown no acute effect of acetazolamide on fluid production by the rat testis (Setchell, 1967). Using a histochemical method to detect CA, Cohen et al. (1976) and Ekstedt & Ridderstrale (1992) localized CA in the interstitial cells but not in the seminiferous tubules of the rat and rabbit testes. We too were unable to detect CA in the primary cultures of immature rat Sertoli cells (authors' unpublished observation). This negative staining for CA in the Sertoli cells contrasts sharply with the abundant CA expressed by the rat epididymal cells which have been shown to secrete HCO3 copiously (Chan et al. 1996). Secondly, amiloride (1 mm), bafilomycin A1 (0.8 μm) and H2DIDS (300 μm), inhibitors of the Na+-H+ exchanger, H+-ATPase and Na+-HCO3 cotransporter, respectively, added basolaterally did not affect the Isc response to ATP (Table 1). This indicates that these transporters and pump could not have contributed to the Isc, and by inference HCO3 secretion, on account of the fact that the Na+-H+ exchanger, H+-ATPase, and Na+-HCO3 cotransporter have been implicated in HCO3 secretion in other epithelia (see Gleeson, 1992; Raeder, 1992). Thirdly, contrary to the earlier work of Tuck et al. (1970), a more recent micropuncture study by Caflisch & DuBose (1990) showed that the rat seminiferous tubular fluid contains a very low concentration of HCO3. These results led these authors to argue that active HCO3 secretion is not an important factor in the formation of the seminiferous tubular fluid.

It would seem highly probable that the Isc response to apically applied ATP is due to transepithelial Cl secretion. However, basolaterally applied bumetanide at a concentration which has been shown to abolish Cl secretion in other Cl-secreting epithelia produced only a small inhibition of the ATP-induced Isc in cultured Sertoli cell epithelia (Fig. 6). Bumetanide added in combination with the other inhibitors also did not affect the Isc response to ATP (Table 1). Removal of extracellular K+ also produced no noticeable effect although Na+ removal significantly reduced the Isc response to ATP. However, the dependence on Na+ may reflect a requirement of Na+ in Ca2+ influx (see below). Ouabain (2 mm), a Na+-K+-ATPase inhibitor, applied basolaterally did not affect the Isc and this may be related to the well-known phenomenon that rat tissues are rather insensitive to ouabain. The present study did not show conclusively how Cl is taken up into the cell across the basolateral membrane. What is clearer, however, is the exit pathway at the apical membrane. The complete inhibition of the Isc by apical DPC (1 mm) and DIDS (0.25 mm) (Fig. 2) supports the notion that apical anion channels are the pathway for Cl exit. The identification of an ATP-activated Cl conductance in cultured rat Sertoli cells lends further support to this contention (Fig. 7).

It is likely that the Isc response of the Sertoli cells to ATP is mediated by an increase in cell Ca2+. The Isc response to ATP is mimicked by thapsigargin (Fig. 8), which increases intracellular Ca2+ (Rossato et al. 1996). Extracellular ATP has been shown to increase intracellular Ca2+ via a G-protein-coupled phosphatidyl inositol pathway in rat Sertoli cells (Filippini et al. 1994; Rudge et al. 1995; Foresta et al. 1995). The EC50 value for ATP-induced increase in Ca2+ (10–15 μm) (Filippini et al. 1994) is in close agreement with the ATP-stimulated Isc (Fig. 3). The present study has therefore ascribed a functional meaning to the ATP-induced Ca2+ response. The potency of stimulation of Isc by ATP and its analogues is in the order UTP geqslant R: gt-or-equal, slanted ATP > ADP >> AMP = adenosine. This order of potency is also observed for the stimulation of inositol phosphate accumulation in prepubertal rat Sertoli cells (Filippini et al. 1994; Rudge et al. 1995). Current research in the field of P2Y receptors has led to the identification of four receptor subtypes, viz. P2Y1, P2Y2, P2Y4 and P2Y6 with different sensitivities towards the adenine and uridine nucleotides. By matching the known nucleotide selectivity of these receptor subtypes (Nicholas et al. 1996) with the potencies of the nucleotides obtained in our present study, it transpired that there could be a mixture of P2Y receptors, for instance, P2Y2 and/or P2Y4 (with selectivity to UTP and ATP but not to ADP) and P2Y6 receptors (with selectivity to UTP and ADP but not to ATP) to account for the UTP, ATP and ADP effects on anion secretion by the Sertoli cells. However, support for this notion requires more detailed pharmacological studies. As with other anion-secreting epithelia, a capacitative Ca2+ entry process (Putney, 1986) has been identified in rat Sertoli cells (Rossato et al. 1996). Gorczynska & Handelsman (1993) show that Ca2+ influx following depletion of the intracellular Ca2+ store with thapsigargin is dependent on membrane depolarization secondary to Na+ influx. This may have a bearing on the observed reduction of the Isc response to ATP, where in the absence of extracellular sodium, the plateau phase of the response was apparently affected to a greater extent than the peak response (Fig. 5). The former is thought to be associated with Ca2+ influx.

The physiological significance of the effect of apically applied ATP on transepithelial ion transport remains to be elucidated. High-affinity purine receptors that bind adenosine and ATP are identified in the rat Sertoli cells (Monaco et al. 1984). In the intact seminiferous epithelium, germ cells are held in close association with the Sertoli cells. It is conceivable that ATP released from germ cells interacts with apical purinoceptors in the Sertoli cells to regulate electrolyte and fluid secretion. In this way ATP serves as a paracrine factor that mediates ‘cross-talk’ between the developing germ cells and the Sertoli cells. Such a proposition is not untenable in view of the recent reports that germ cells in vitro activate the phosphatidyl inositol pathway (probably through ATP release) in rat Sertoli cells (Welsh & Ireland, 1992). Furthermore, the detection of cystic fibrosis transmembrane conductance (CFTR) gene expression in germ cells in vivo (Trezise et al. 1993) gives added weight to this hypothesis, for there is now a growing body of evidence that CFTR mediates ATP efflux in a number of cell systems (Rotoli et al. 1996; Cantiello et al. 1997). Alternatively, ATP may be released by the Sertoli cells in the testis and act on the same or adjacent Sertoli cells to control fluid secretion. However, such a proposed autocrine role for ATP in the Sertoli cells is only speculative at this stage.

In conclusion, the present work is the first attempt to explore electrolyte transport by Sertoli cells. It demonstrates an increase in Isc by ATP acting from the apical side of the epithelium. These effects are mediated by an increase in Ca2+. The increase in Isc is likely to be caused by anion secretion, although the process is quite insensitive to inhibitors which are known to affect Cl and HCO3 secretion in other epithelia. The resistance of the Sertoli cell epithelium to perturbation by pharmacological agents can be construed as an advantage to the seminiferous epithelium, whose major function is to create a favourable environment for sperm production to ensure the perpetuation of the species. The stimulation by ATP of transepithelial transport may be part of the complex mechanism regulating fluid secretion by the mammalian testis.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements

We are grateful to Dr C. Y. Cheng for advice on Sertoli cell culture. The excellent technical assistance of Y. W. Chung, C. Y. Yip and Ms P. Y. Leung is gratefully acknowledged. This work was supported by the Research Grant Council and the Chinese University of Hong Kong.