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a Laboratoire de Physiologie des Régulations Cellulaires, UMR 6558, UFR Sciences, 86022-Poitiers-Cedex, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Voltage-operated (VOCCs) and voltage-independent (VICCs) calcium channels have been frequently mentioned, but not really characterized, in the membrane of Sertoli cells from immature rat testis. Their involvement in the cell biology has been suggested through quantification of the effects of various channel inhibitors (verapamil, nifedipine, cobalt, nickel, ruthenium red, gadolinium, and 
-conotoxin) on Ca2+ movements and cell products. It has been suggested that VOCCs and VICCs are either active [4–8] or inactive [9] in resting cells; are activated by high K+ depolarizing medium [4, 7, 8, 10]; and are involved in the response to FSH [4, 9, 11], cAMP [5], testosterone and dihydrotestosterone [12], ATP [13, 14], endothelin-1 [15], arginine vasopressin [6], angiotensin II [6], and retinol [8]. They seem to be implicated in the depolarization [16], hyperpolarization [17], and secretion of proteins [10] induced by FSH. It has also been suggested that thapsigargin-sensitive stores are involved in the response to FSH [6], ATP [13, 14], and endothelin-1 [15]. A capacitive calcium entry has been also demonstrated in immature Sertoli cells [18].
Recently, we have applied the whole-cell configuration of the patch-clamp technique to investigate calcium currents in Sertoli cells from immature rat testis in primary culture [19]. Voltage-dependent calcium currents were recorded that were insensitive to Bay K 8644, an L-type channel opener, but were inhibited by cobalt, nickel, isradipine, and -conotoxin GVIA, four blockers of VOCCs. We concluded that there was no L-type calcium channel in the membrane of immature Sertoli cells in primary cultures, but there were calcium channels with the biophysical and pharmacological properties of T-type calcium channels of excitable cells. Since the maximal amplitude and the activation and inactivation curves of calcium current from FSH-stimulated cells were not significantly different from those of control cells, and since the current was recorded in the presence of internal ATP and in the absence of guanosine triphosphate, we excluded the possibility that FSH controlled the activity of T-type calcium channels, directly (as FSH-operated channels) or indirectly (as G protein-operated or cAMP-dependent protein kinase A-sensitive channels). But we postulated that FSH may control the T-type channel activity by a mechanism of hyperpolarization that should set the membrane potential to the expected window current.
Faced with these conflicting results, we thought it was important to clarify the relationships between FSH and the FSH-induced Ca2+ movements. Since Ca2+ movements evoked by ATP seemed to be well established in immature Sertoli cells [13], we decided to compare the Ca2+ movements induced by ATP, FSH, and cAMP in individual cells, issuing from nonconfluent cultures of Sertoli cells, by single-cell microfluorometry with the Ca2+ probe indo-1, coupled with rapid applications of drugs onto the surface of individual Sertoli cells.
We show here that FSH, cAMP, and ATP induced a fast and biphasic rise in [Ca2+]i that was dependent on the Ca2+ release from a thapsigargin-sensitive store. The peak was followed by an entry of Ca2+ from the external medium through VOCCs, probably of the Icrac type (calcium release-activated calcium channels or currents). Because the responses to ATP, FSH, and cAMP were very similar, we speculate that FSH, by a cAMP-dependent process, either potentiated the mechanism by which the calcium was released from smooth endoplasmic reticulum by ATP or first released ATP from Sertoli cells, which then acted on cells. We also show that cells responded to FSH and cAMP by a delayed long-lasting decrease in [Ca2+]i that was never recorded in the presence of ATP. This suggests that FSH and cAMP also promote a slow redistribution of [Ca2+]i from the exchangeable pool to the bound nonexchangeable pools. We postulate that this decrease associated with FSH-induced hyperpolarization [16, 17] activates the voltage-dependent calcium channels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) except caffeine and DMSO (dimethylsulfoxide), from Merck (Darmstadt, Germany), and N-methyl-D-glucamine, sodium salt, from Fluka (Switzerland). Porcine FSH (pFSH) was a gift from Dr. Y. Combarnous (Institut National de la Recherche Agricole-INRA de Nouzilly, France). All drugs were directly dissolved in solutions except for thapsigargin and indo-1, which were dissolved in DMSO as 1 mM stock solution and then added to the external medium at final concentration of 500 nM and 3 µM, respectively. [Ca2+]i was not altered by DMSO (1%).
Medium A was a Ca2+- and Mg2+-free modified Earle's solution containing 116.3 mM NaCl, 5.4 mM KCl, 0.9 mM NaH2PO4, and 5.5 mM glucose, supplemented with 53.5 mM mannitol, 20 mM Hepes, streptomycin sulfate (100 µM), and penicillin G (100 IU/ml) (Sigma). The pH was adjusted to 7.4. Medium B was medium A supplemented with 2 mM CaCl2, 1 mM MgCl2, 4.1 mM Na2HCO3, 40 mM mannitol, 20 mM Hepes, and 0.2% BSA. The media were sterilized by filtration through a 0.22-µm-pore filter (Millipore, Bedford, MA).
Preparation of Sertoli Cells
Sertoli cells were isolated as previously described [19, 20]. Testes of immature (12–14 days old) rats (Wistar) were digested enzymatically by collagenase (0.25 mg/ml; Worthington Biochemical Corporation, Freehold, NJ; 291 U/mg) and pancreatin (0.5 mg/ml, grade VI; Sigma), then by trypsine (5 mg/ml, trypsin-EDTA mixture; Life Technologies, Paisley, Scotland) in medium A. Cells were then resuspended in medium A containing 0.1 mg/ml trypsin inhibitor (soybean-type 1S; Sigma). The cell density was measured with a hematocytometer, and viability was determined by trypan blue exclusion. The preparations contained more than 99% living cells, among which there were more than 95% Sertoli cells as identified by light microscopy.
Cell Culture
Cells were plated at 100 000 cells/cm2 in 35-mm plastic Petri dishes (Nunclon, Nunc, Roskilde, Denmark) in which the bottom had been replaced by a glued glass coverslip of 0.17-µm thickness. The culture medium (RPMI 1640; Life Technologies) was supplemented with L-glutamine (2 mM), transferrin (0.005 mg/ml), insulin (0.01 mg/ml), BSA (1 mg/ml), Hepes (10 mM), and sodium bicarbonate (20 mM) (all from Sigma), as well as streptomycin sulfate (100 µM) and penicillin G (100 IU/ml). The culture dishes were maintained at 34°C in a humidified atmosphere of 95% air:5% CO2. The medium was changed after 48 h, then every 2 days.
Intracellular Free Ca2+ Concentration Measurements
Intracellular free calcium concentrations were measured by a ratiometric fluorescence method [21] using the calcium probe indo-1 (Sigma) and an interactive laser cytometer ACAS 570 (Meridian Instruments, Okemos, MI).
Cell Loading with Indo-1/AM
Experiments were performed in 2-day-cultured Sertoli cells. After the culture medium was replaced with the physiological solution, the cells were loaded with 3 µM lipophilic form of indo-1 (indo-1/AM in DMSO) dissolved in medium B (final concentration of 0.3% DMSO). They were incubated for 50 min in the dark, at room temperature, washed three times with medium B, and then incubated 20 min at 34°C to complete the de-esterification of indo-1/AM into indo-1 by intracellular esterases.
Protocol for Ca2+ Measurements
The experiments were conducted as previously described by our laboratory [22, 23], on single Sertoli cells that were first identified by light microscopy. Briefly, the Ca2+ measurements were performed at a fixed point in the cell ("point-scan" mode). Excitation of indo-1 was performed by means of the UV rays (351–363 nm) of a 5-W pulsed argon laser. Emission was detected at 2 wavelengths: 405 nm for Ca2+-bound indo fluorescence and 485 nm for Ca2+-free indo fluorescence. The fluorescence intensities at 405 and 485 nm, and the Ca2+ transients as the resulting ratio (R) of the fluorescence signals (F405/F485), were displayed on the screen and stored online. The method for calibration in situ [21] was difficult to apply to Sertoli cells and was used in some rare cells. This procedure involved exposures to10 µM ionomycin in 0 Ca2+-10 mM EGTA medium B and 10 mM Ca2+ medium B to determine the minimal and maximal ratios of fluorescence. Experiments were performed at 30–32°C in the control solution (medium B), which flowed down from a plastic capillary positioned near the cell. Then, depending on the experiments, the test solutions were superfused.
Representative experiments are illustrated by the ratio F405/F485 obtained with Anchored Cell Analysis and Sorting software (Meridian Instruments, Okemos, MI) and from other specific software (FIG.P6; Biosoft, Cambridge, UK). Each data point corresponded to 256 averaged measurements. Peak amplitudes and plateau levels of ratios (means ± SD) are expressed as percentages of basal ratio for n experiments obtained in different Petri dishes issuing from different cultures. Statistical significances of results were assessed by means of Student's t-test (FIG.P6; Biosoft) performed on unpaired data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Freshly dispersed Sertoli cells from testes of 12- to 14-day-old rats via a three-step enzymatic procedure were free of myoid and Leydig cells. Some rare germ cells and fibroblasts (less than 1%) were present in culture dishes. Germ cells had been removed by changing the culture medium or eliminated by their morphology. Very few fibroblasts were present. They were easily recognized by morphological criteria under phase-contrast microscopy and by their low fluorescence [20].
Once plated at low density, Sertoli cells attached themselves progressively to the glass coverslips and flattened out. Two days later, in the absence of FSH, they formed nonconfluent cultures that presented an "epithelial-like" appearance under phase-contrast microscopy, as previously illustrated [20]. When they were cultured for 1 h and more with FSH, the Sertoli cells started to take on a more rounded and thicker appearance. This transformation to "fibroblast-like" was also observed following cAMP stimulation but not after hCG treatment. The cells showed a concentration-dependent responsiveness to FSH by morphological changes [20], by an increase in the membrane potential [17], and by an increase in diffusional coupling [20]. These cells have been extensively used for electrophysiological experiments [17, 19].
Basal [Ca2+]i in Cultured Sertoli Cells
After the identification of Sertoli cells by phase-contrast microscopy, fluorescence of whole cells was generated in "image scan" mode. Emission of indo-1 was always homogenous within the cytoplasm, so [Ca2+]i measurements were performed in point-scan mode. In this mode, recordings of fluorescence emission were done approximately near the cell center. Because indo-1 excitation induced a double-emission spectrum with an isosbestic point, and because the evolution of [Ca2+]i was expressed as F405/F485, changes in cell thickness induced by FSH stimulation did not interfere with the [Ca2+]i measurements.
Under these conditions, steady state Ca2+ fluorescence was recorded over 15 min in about 350 individual Sertoli cells resulting from 34 different cultures growing on glass coverslips. When they were superfused with medium B, a basal [Ca2+]i level of 190 ± 50 nM was calculated (n = 11). Some rare, spontaneous, steep, and short peaks of [Ca2+]i were also recorded on 4 cells as shown in Figure 1. This basal level was greatly increased when 10 µM ionomycin was added to the bath (n = 11).
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Effect of Extracellular ATP on [Ca2+]i in Cultured Sertoli Cells
One hundred and ten cells resulting from 20 different cultures were stimulated with 50 µM ATP in the presence of extracellular Ca2+. All cells responded to ATP by a rise in [Ca2+]i (Fig. 2A). There was a first phase with a steep and rapid rise in [Ca2+]i and a peak that was reached within 5 sec after the addition of ATP. Then [Ca2+]i declined to a somewhat lower level (the plateau level), which remained stable within 10-min exposures to ATP. Then, after the removal of ATP, [Ca2+]i dropped and reached resting values within 1 min. The peak value of [Ca2+]i averaged a 229.0 ± 41.1% increase over the basal level and then returned to a plateau value of 91.4 ± 29.9% (n = 9) over the basal level. In control cells, ATP did not modify the cell shape (not shown).
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The mechanism by which ATP increases [Ca2+]i was first investigated in Sertoli cell suspensions [13]. We decided to reexamine this mechanism and, in order to determine whether ATP mobilized [Ca2+]i from intracellular stores, studied the effect of 50 µM ATP in Ca2+-free medium containing 0.1 mM EGTA. Figure 2B shows that when Ca2+ was omitted from the extracellular medium, ATP was still able to induce a rise in [Ca2+]i. Under these conditions, the basal [Ca2+]i level was unchanged, and subsequent applications of 50 µM ATP induced a [Ca2+]i response whose plateau phase was abolished. The peak was more transient than in the presence of extracellular calcium, and the level returned to basal values within 1 min. The amplitude of peak response was unchanged when compared to that recorded in the presence of a Ca2+-containing medium. It averaged a 220.8 ± 22.7% increase over the basal level (not significant [n.s.] vs. value in Ca2+-containing medium; n = 4). Figure 2B also shows that the plateau-phase response was restored on return to Ca2+-containing medium. [Ca2+]i averaged a 86.5 ± 13.5% increase over basal (n.s. vs. value in Ca2+-containing medium; n = 4).
To further test the hypothesis that ATP was able to mobilize Ca2+ from intracellular stores, the effects of caffeine, an activator of ryanodine receptors, and thapsigargin, an inhibitor of the Ca2+-ATPase from smooth endoplasmic reticulum, were investigated [24]. Figure 3 illustrates the effects of ATP in the presence of 1 mM caffeine. Caffeine failed to prevent an ATP-evoked change in [Ca2+]i. The response to ATP was, however, attenuated and more transient. The peak value of [Ca2+]i averaged a 177.0 ± 14.3% increase over the basal level (p < 0.01 vs. ATP alone; n = 9). [Ca2+]i then returned to basal levels. A plateau value of [Ca2+]i averaging a 65.2 ± 33.8% increase over the basal level (n.s. vs. ATP alone; n = 9) was recorded when caffeine was removed.
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Thapsigargin selectively inhibits the Ca2+-ATPase from smooth endoplasmic reticulum [24] and therefore depletes the Ca2+ stores in the smooth endoplasmic reticulum, elevates [Ca2+]i, and induces no response with messengers acting by the phospholipase C-inositol triphosphate pathway. Thapsigargin (500 nM) dissolved in Ca2+-free medium induced a transient increase in [Ca2+]i (n = 4; Fig. 4A). When Ca2+ stores were depleted by a 20-min incubation in the presence of 500 nM thapsigargin, the peak response and the plateau phase induced by ATP were totally suppressed (n = 4; Fig. 4B). Taken together, these results suggest that there are no caffeine-sensitive Ca2+ stores in Sertoli cells and that external ATP mobilizes Ca2+ from thapsigargin-sensitive intracellular stores.
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Figure 2B shows that the plateau phase was restored on return to Ca2+-containing medium, indicating that the sustained elevation of [Ca2+]i was dependent on a Ca2+ influx from the extracellular medium. Sodium dependence of the sustained phase induced by ATP has been found in Sertoli cells in suspension [13]. So, we substituted N-methyl-D-glucamine for Na+ in the extracellular medium to suppress a potential Na+ influx. Fifty micromolar ATP still elicited the well-characterized biphasic response obtained in Ca2+-containing medium (Fig. 5A). The peak value of [Ca2+]i averaged a 244.2 ± 38.0% increase over basal level and then returned to a plateau value of 102.8 ± 32.6% (n = 5), very close to the control values (see Fig. 2A). The same results were obtained when mannitol was substituted for sodium (n = 5; not shown).
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Nickel is an inhibitor of voltage-dependent calcium channels and of sodium/calcium exchanger. In the presence of 1 mM Ni2+, the peak value of [Ca2+]i averaged a 267.3 ± 18.9% increase over the basal level and then returned to a plateau value of 81.0 ± 19.2% (Fig. 5B; n = 4), very close to the control values with ATP alone.
Depolarization by 50 mM KCl failed to increase the basal [Ca2+]i and to modify the biphasic response in [Ca2+]i induced by ATP (Fig. 5C). The peak value of [Ca2+]i was, however, reduced to a 177.5 ± 23.3% increase over basal level (p < 0.05 vs. ATP alone; n = 4). The plateau value was 61.5 ± 29.9% (n.s. vs. ATP alone (n = 4).
Effects of FSH and cAMP on [Ca2+]i in Cultured Sertoli Cells
The responsiveness of individual Sertoli cells to pFSH, ranging from 10 to 1000 ng/ml, was investigated on 60 Sertoli cells issuing from 15 different cultures. There were three distinct patterns of response. One group of cells (12 of 60 cells) presented no detectable change in [Ca2+]i. Nevertheless, these cells responded to FSH by characteristic changes in the cell morphology and to ATP by a typical biphasic rise in [Ca2+]i. One group (19 cells) responded to the various FSH concentrations with a delayed, long-lasting decrease in [Ca2+]i (Fig. 6B). Another group of 27 cells responded to FSH with an early transient rise in [Ca2+]i (Fig. 6C) followed by a delayed decrease in [Ca2+]i.
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The concentration dependence of the response to FSH was investigated in two cultures. FSH at 10 ng/ml failed to induce a significant rise in [Ca2+]i (n = 5). The peak value of [Ca2+]i averaged a 21.0 ± 6.8% increase over the basal level in the presence of 100 ng/ml pFSH (n = 6) and 45.9 ± 16.2% (n = 8) (p < 0.005) in the presence of 1000 ng/ml pFSH. In these cells, the peak phase was less steep and rapid than after an application of ATP. In contrast, the decrease in [Ca2+]i did not appear to be concentration dependent.
It is well known that FSH binds to specific receptors on the membrane of Sertoli cells, resulting in stimulation of adenylate cyclase and a rise in intracellular cAMP [2]. To investigate whether the hormone modifies [Ca2+]i by a cAMP-dependent process, 30 indo-1-loaded cells issuing from 5 different cultures were locally exposed to rapid applications of 1 mM CPT-cAMP (8-[4-chloro-phenylthio]-adenosine 3':5'-cyclic monophosphate, monosodium salt), a membrane-permeant analogue of cAMP. The application of CPT-cAMP induced a delayed decrease in [Ca2+]i (n = 24) (Fig. 7A), which was preceded by a peak increase in [Ca2+]i identical to that induced by FSH (n = 22; Fig. 7B). The peak value of [Ca2+]i averaged a 63.6 ± 41.4% increase over basal level in the presence of 1 mM CPT-cAMP (n = 22). In control cells from different dishes issuing from the same cultures, CPT-cAMP induced the characteristic change in the cell morphology.
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Because CPT-cAMP mimicked the effects of FSH, we examined the mechanism by which CPT-cAMP triggered [Ca2+]i changes. First we were curious to know whether CPT-cAMP mobilized [Ca2+]i from intracellular stores. The effects of cAMP were examined in Ca2+-free medium containing 0.1 mM EGTA (Fig. 8A). When Ca2+ was omitted from the extracellular medium, CPT-cAMP was still able to induce a rise in [Ca2+]i (n = 4). Under these conditions, CPT-cAMP induced a [Ca2+]i response whose plateau phase was abolished. The peak was more transient than in the presence of calcium medium, and the level returned to basal values within 1 min. The amplitude of the peak response was unchanged when compared with the peak response recorded in the presence of a Ca2+-containing medium. It averaged a 70.5 ± 45.4% increase over basal [Ca2+]i (n = 4). The plateau-phase response was restored on return to a Ca2+-containing solution (Fig. 8A). It was still recorded in the presence of 1 mM Ni2+ (n = 3) (not shown). This suggests that the sustained elevation of [Ca2+]i induced by CPT-cAMP was dependent on a Ca2+ influx from the extracellular medium, mediated by the opening of VICCs.
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Since we found no caffeine-sensitive stores in Sertoli cells, we investigated the effects of cAMP in cells with thapsigargin-depleted stores. The peak response and the plateau phase induced by CPT-cAMP were both suppressed by 500 nM thapsigargin (n = 5; Fig. 8B). Taken together, these results suggest that cAMP is able to mobilize Ca2+ from thapsigargin-sensitive intracellular stores in cultured Sertoli cells.
Effects of CPT-cAMP and ATP on [Ca2+]i in Cultured Sertoli Cells
The kinetics of [Ca2+]i rises induced by FSH and CPT-cAMP are typical for activation of receptors that induce Ca2+ release from thapsigargin-sensitive stores, and a consecutive, voltage-independent Ca2+ influx from extracellular medium. Since ATP also induced [Ca2+]i changes by this double mechanism, we investigated whether ATP and CTP-cAMP were able to involve the same Ca2+ stores to increase [Ca2+]i.
Receptor-mediated responses are known to be decreased in response to repetitive applications at short intervals. In Sertoli cells, [Ca2+]i transients, which were induced by FSH, CPT-cAMP, and ATP, exhibited some heterogeneity in their shape and magnitude among different cells issuing from the same or different cultures. However, when two repetitive applications of ATP (n = 5; Fig. 9A) or CPT-cAMP (not shown), separated by 10-min rinse periods, were performed on the same cells, the peak [Ca2+]i responses were decreased. In different experiments in which both ATP and CPT-cAMP were successively applied to cells, we observed that the ATP-induced [Ca2+]i response was followed by a weak response to CTP-cAMP (n = 4; Fig. 9B), and the CPT-cAMP response was followed by a weak response to ATP (n = 5; Fig. 9C). On the other hand, in the absence of response to FSH or CTP-cAMP, ATP was always able to induce a great rise in [Ca2+]i (n = 12; Fig. 9D). Such preliminary results suggest that ATP and CTP-cAMP were able to act on the same Ca2+ stores from the smooth endoplasmic reticulum to increase [Ca2+]i.
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Origin of the Refractory Response to FSH and CTP-cAMP
Of 60 Sertoli cells, 12 did not respond to FSH, whereas 21 of 48 cells responded to FSH only by a decrease in [Ca2+]i. On the other hand, 6 of 30 Sertoli cells did not respond to CPT-cAMP, whereas 2 of 24 cells stimulated with CPT-cAMP did not show a transient rise in [Ca2+]i. With a basal [Ca2+]i of 2.10-7 M, a value consistent with the concentration at which the cAMP-dependent phosphodiesterases (PDE) are activated [1], we thought that activation of the enzyme might be responsible for the lack of response to FSH and cAMP. Incubation of cells was thus performed in the presence of the broad PDE inhibitor isobutylmethylxanthine (0.5 mM, n = 5). This inhibitor failed to restore the response to the messengers (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transient Rises in [Ca2+]i Induced by ATP
ATP induced a fast, biphasic rise in [Ca2+]i with a peak response dependent on a release of Ca2+ from internal stores. ATP failed to release Ca2+ when 1) the thapsigargin-sensitive stores were depleted and 2) caffeine, an activator of ryanodine-sensitive internal Ca2+ stores, failed to induce a rise in [Ca2+]i. Therefore, we think that Ca2+ stores are located in the smooth endoplasmic reticulum and released by an inositol 1,4,5-triphosphate (IP3)-dependent mechanism.
Besides Ca2+ release from intracellular stores, ATP induced a long-lasting Ca2+ influx from the extracellular medium that was abolished in EGTA-supplemented Ca2+-free medium. This plateau phase was suppressed by an external application of Ca+-free medium and by Ni2+, an inhibitor of voltage-dependent calcium channels and Na+/Ca2+ exchanger. We conclude that the sustained phase in [Ca2+]i induced by ATP is associated with a Ca2+ influx through the activation of a calcium release-activated calcium current (Icrac), whose presence has been shown in Sertoli cells [18]. Therefore, these results differ from those obtained in suspensions of Sertoli cells [13]. The mechanism by which ATP induces Ca2+ movements in Sertoli cells is identical to that found in other cell types expressing purinergic P2 receptor subtypes [25, 26].
Current opinion is that the release of Ca2+ by ATP from intracellular compartments (as for other messengers that act on cells by increasing the activity of phospholipase C) is primarily triggered by IP3, which activates VICCs in the membrane of smooth endoplasmic reticulum. The release of Ca2+ may induce a consecutive Ca2+ release from stores by a mechanism termed CICR (calcium-induced calcium release) that is activated by ryanodine and caffeine and inhibited by ruthenium red. Since caffeine had no direct effect on the basal [Ca2+]i of unstimulated cells, the present study failed to reveal a CICR in Sertoli cells. However, caffeine attenuated the ATP-evoked peak of [Ca2+]i and completely suppressed the consecutive plateau phase. We thus speculate that caffeine prevents the IP3-induced Ca2+ release by a partial blockade of the IP3 receptor located on smooth endoplasmic reticulum as in hepatocytes [25], or inhibits the IP3 production as in granulosa cells and pancreatic acinar cells [26, 27].
Transient Rises in [Ca2+]i Induced by FSH and cAMP
Fifty-six percent of cells that responded to FSH and 73% of cAMP-stimulated cells showed the typical Ca2+ response profile of ATP-stimulated cells. Since the plateau phase was suppressed in the presence of Ca2+-free medium, we conclude that a Ca2+ release from internal stores was responsible for the pattern of the transient response and that a transmembrane Ca2+ influx maintained the FSH-evoked plateau. The peak of calcium induced by cAMP was suppressed in Sertoli cells exposed to thapsigargin, whereas the sustained phase was insensitive to Ni2+. We conclude that the stimulation by cAMP elicits Ca2+ movements that do not differ qualitatively from those evoked by ATP. Sertoli cells, like other cells [28], exhibited a refractoriness to repetitive stimulations by ATP, which mainly reflected the time required for the replenishment of intracellular Ca2+ stores. In cells stimulated by ATP and then by cAMP, or inversely, the magnitude of the second [Ca2+]i peak response was always inversely proportional to that of the first response. Thus we believe that ATP, FSH, and cAMP trigger Ca2+ from the same stores. We conclude that FSH, by a cAMP-dependent mechanism, induces an early phase of [Ca2+]i that probably results from the release of Ca2+ from the smooth endoplasmic reticulum, and a second phase that is dependent on a Ca2+ influx through VICCs (Icrac). It is well established that FSH does not activate the phosphatidyl inositol-calcium-dependent pathway in Sertoli cells [29, 30] and that, once activated by serum, this pathway is inhibited by FSH [29]. These results and likewise the fact that the slope of Ca2+ rising, which is generally steeper with ATP than with cAMP or FSH, strongly suggest that the mechanism by which FSH and cAMP release Ca2+ from the smooth endoplasmic reticulum differs from that for ATP. Thus, we think that cAMP, through the activation of protein kinase A, either increases the sensitivity of IP3 receptors to basal levels of IP3 as observed in liver [31], or releases ATP that in turn acts on Sertoli cells by an autocrine/paracrine stimulation. However, such a proposed release and autocrine/paracrine role for ATP in Sertoli cells are only speculative at this stage.
Of FSH-stimulated cells, 44%, and of cAMP-stimulated cells, 27% failed to respond by a transient rise in [Ca2+]i, despite the fact that they responded to these messengers by changes in cell shape. We observed that refractoriness was specific to FSH and cAMP, since the cells always responded with the typical [Ca2+]i peak and sustained phases when they were later stimulated by ATP. We postulated that the high basal [Ca2+]i could activate cytosolic phosphodiesterases [3], leading to lower cAMP concentration. However, the PDE inhibitor isobutylmethylxanthine was unable to modify the basal level of [Ca2+]i. Therefore, the refractoriness to FSH and cAMP, if any remains to be determined, may be associated with the stimulation of ATP-depleted Sertoli cells.
Long-Lasting Lowering of [Ca2+]i Induced by FSH and cAMP
With concentrations of 10–1000 ng/ml FSH and in the presence of 1 mM cAMP, 80% of Sertoli cells responded to FSH with a slow, delayed, long-lasting decrease in the basal [Ca2+]i. These changes paralleled those observed in the cell shape. By contrast, ATP failed to lower [Ca2+]i under the basal level and to modify the cell shape. These results corroborated what has been observed in FSH-stimulated Sertoli cells in culture [3], when [Ca2+]i was first raised by depolarization [7], or when the cells were spread on Petri dishes [32]. It is well established that the sequestration of Ca2+ in the smooth endoplasmic reticulum occurs by activation of membrane-bound Ca2+-ATPase. Indeed, inhibition of this pump by thapsigargin depletes the Ca2+ stores and allows Ca2+ to leak from the pools back into the cell cytoplasm. Since we observed that thapsigargin increased the basal [Ca2+]i and suppressed the response to cAMP as well as ATP, we think that [Ca2+]i could be lowered by the binding of Ca2+ to the cytoskeleton and/or by the sequestration of Ca2+ into the smooth endoplasmic reticulum.
Electrophysiological Approach to Calcium Movements in Sertoli Cell
Since the pioneering study by Means et al. [3] showing that FSH alters the level and distribution of Ca2+ within Sertoli cells, many attempts have been made to clarify the changes in [Ca2+]i induced by FSH. The hormone induced either no response [7], a sustained rise in [Ca2+]i [4, 5, 9,11, 33], or a decrease [3, 7, 32]. The increase in [Ca2+]i was concentration dependent [4, 9], or it involved a progressive recruitment of responsive cells [11]. The response to FSH was independent of cAMP [9, 33] or mimicked by the nucleotide [4, 5, 11], abolished by EGTA-supplemented Ca2+-free medium [4, 9, 11], and inhibited by verapamil [4, 9,11], nifedipine [4, 9], cobalt and nickel [4, 11], ruthenium red [4, 9], and gadolinium [9]. Based on the sensitivity to inhibitors, VOCCs activated by depolarization and VICCs have been claimed to be involved in the response to FSH and cAMP.
The present study shows that voltage-dependent calcium channels are not involved in the fast rise in [Ca2+]i induced by FSH, cAMP, and ATP. On the contrary, the data strongly suggest the role of VICCs (termed Icrac), or capacitive calcium entry, that are activated by depletion of the smooth endoplasmic reticulum. However, the transient effect of FSH and cAMP that mimicked the response to ATP led us to think that this mechanism is not the main pathway by which the hormone controls the Ca2+ movements in Sertoli cells but one that participates in the modulation of the basic Ca2+ movements induced by FSH.
It is not easy to compare and integrate the various data on FSH-induced Ca2+ movements that have been obtained by different methods on individual Sertoli cells or populations of Sertoli cells either in primary culture or freshly isolated from immature rat testis. Moreover, 45Ca2+ uptake and fluorescence dyes do not give identical information on cell Ca2+ movements. Nevertheless, we may select two studies from the abundant literature on FSH-induced Ca2+ movements that are representative of two opposite results obtained with Fura-2. The study by Gorczynka and Handelsman [4] shows that FSH increases the [Ca2+]i level dose-dependently, whereas Ravindranath et al. [32] show that FSH decreases the [Ca2+]i, as in our study. In the former, the cells were freshly isolated, whereas in the latter they were cultured and spread on glass. The [Ca2+]i level is low in cell suspension (80–100 nM in [4] and 40–80 nM in [32]), and high in cultured cells (120–160 nM in [32] and 140–240 nM in our study). Thus, we think that the Ca2+ movements in immature Sertoli cells are dependent on the [Ca2+]i level. When the level is high, FSH induces a progressive decrease in [Ca2+]i by redistribution of calcium from cytosol to a bound nonexchangeable pool, as shown previously [3]. Consequently, the decrease in [Ca2+]i associated with the FSH-induced hyperpolarization [16, 17] progressively increases the driving force for Ca2+, while membrane hyperpolarization per se may set the membrane potential close to the window current of T-type calcium channels. Together they allow the influx of Ca2+ through the T-type calcium channels, sensitive to some VOCC blockers. Moreover, it is important to associate the protein secretion by Sertoli cells, which is sensitive to -conotoxin [10], and the sensitivity of T-type calcium channels to this toxin [19] (Fig. 10).
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This study and others lead us to consider that the basic Ca2+ movements induced by FSH and cAMP may be modulated by myoid cells, through some components of the basal membrane [32], and by germ cells that may either secrete ATP [34] and increase the calcium level, or, in opposition, release some inhibiting factors from spermatids [35] and thus lower the Ca2+ level. Further experiments will be required to clarify this important point. Moreover, the Ca2+ movements in Sertoli cells may now be studied in either a coculture system or in situ in the seminiferous tubule.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: Nathalie Lalevée, IGH, UPR 1142 CNRS, 141 rue de la Cardonille, 34 396 Montpellier, Cedex 5, France.
Accepted: March 4, 1999.
Received: December 22, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Conotoxin-sensitive Ca2+ voltage-gated channels modulate protein secretion in cultured rat Sertoli cells. Mol Cell Endocrinol 1997; 26:117–123.This article has been cited by other articles:
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  B. Sommersberg, A. Bulling, U. Salzer, U. Fröhlich, R. E. Garfield, A. Amsterdam, and A. Mayerhofer Gap Junction Communication and Connexin 43 Gene Expression in a Rat Granulosa Cell Line: Regulation by Follicle-Stimulating Hormone Biol Reprod, December 1, 2000; 63(6): 1661 - 1668. [Abstract] [Full Text]
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