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Endocrinology Vol. 149, No. 3 1031-1037
Copyright © 2008 by The Endocrine Society

Follicle-Stimulating Hormone-Induced G{alpha}h/Phospholipase C-{delta}1 Signaling Mediating a Noncapacitative Ca2+ Influx through T-Type Ca2+ Channels in Rat Sertoli Cells

Tsung-Hsuan Lai, Yuan-Feng Lin1, Feng-Chang Wu and Yu-Hui Tsai1

Division of Reproduction Medicine (T.-H.L.), Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan 106, Republic of China; Fu Jen Catholic University School of Medicine (T.-H.L.), Taipei, Taiwan, 242, Republic of China; and Graduate Institute of Cell and Molecular Biology (Y.-F.L., F.-C.W., Y.-H.T.), Graduate Institute of Medical Sciences, Taipei Medical University, (Y.-F.L., F.-C.W., Y.-H.T.), Center for Reproduction Medicine & Sciences, Taipei Medical University Hospital (Y.-H.T.), Taipei, Taiwan 110, Republic of China

Address all correspondence and requests for reprints to: Yuan-Feng Lin or Yu-Hui Tsai, Ph.D., Graduate Institute of Medical Sciences, Taipei Medical University, 250, Wu-Hsing Street, Taipei, Taiwan 110, Republic of China. E-mail: cmbyht18{at}tmu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our previous study demonstrated that FSH-induced immediate Ca2+ influx in rat Sertoli cells (SCs) is mediated by the G{alpha}h/phospholipase C-{delta}1 (PLC-{delta}1) signaling pathway. As to which Ca2+ channel is responsible for such Ca2+ influx was not understood. In this study, thapsigargin triggered an in-store calcium release and evoked a 1.5-fold elevation of intracellular Ca2+ in Ca2+-free media, whereas FSH exhibited no effect. The readdition of CaCl2 (2.5 mM) to FSH-pretreated or thapsigargin-sensitized SCs in Ca2+-free media immediately elicited a rapid Ca2+ influx or a 2-fold increase of second intracellular Ca2+ elevation, respectively. The addition of Ca2+ chelator EGTA (0.2 mM) reduced the FSH-induced elevation of intracellular Ca2+ in SCs incubated with CaCl2. However, pretreatment with dantrolene (25 µM), which inhibits in-store calcium release, did not affect the FSH-induced elevation of intracellular Ca2+. NiCl2 (10 µM), a T-type calcium channel blocker, abolished the FSH-induced SC Ca2+ influx. Furthermore, mibefradil (10 and 100 µM), another specific blocker for T-type Ca2+ channels, dose-dependently suppressed the FSH-induced Ca2+ influx. In contrast, nifedipine (10 and 50 µM) or {omega}-conotoxin GVIA (100 and 500 nM), blocker of L- or N-type Ca2+ channels, respectively, did not affect the FSH-induced SC Ca2+ influx. On the other hand, FSH-induced Ca2+ influx was significantly reduced by pretreatment of SCs with myristoylated synthetic peptide (0.1 and 1 µM) of PLC-{delta}1 fragment TIPWNSLKQGYRHVHLL but not affected by 2',5'-dideoxyadenosine (3 and 15 µM), a selective inhibitor of adenylate cyclase. In conclusion, the FSH-induced G{alpha}h/PLC-{delta}1 pathway-dependent Ca2+ influx of rat SCs is mediated by T-type Ca2+ channels and independent of in-store calcium release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UPON INTERACTING WITH its receptor on Sertoli cells (SCs), FSH activates the Gs/adenylate cyclase signaling pathway to elevate intracellular cAMP levels (1). On the other hand, FSH also induces an immediate Ca2+ influx through a Gs- and adenylate cyclase-independent phospholipase C (PLC)-dependent signaling pathway in rat SCs (2, 3). Recently, we have identified a G{alpha}h/PLC-{delta}1 signaling pathway mediating FSH-induced immediate Ca2+ influx in rat SCs (4). However, the type of Ca2+ channel involved remains unknown.

Voltage-gated Ca2+ channels on plasma membrane are divided into T-, L-, N-, P/Q-, and R-type Ca2+ channels depending on their physiological and pharmacological properties (5). T-type Ca2+ channels are transient low-voltage-activated Ca2+ channels that control Ca2+ influx in excitable cells during small depolarizations around resting potential (6, 7). They are selectively blocked by Ni2+ and mibefradil in various cell types (8, 9, 10). L-type Ca2+ channels are long-term high-voltage-activated Ca2+ channels that are mainly found in muscle and endocrine cells where they initiate contraction and secretion (7, 11). They are blocked by the organic antagonists, including dihydropyridines, phenyl-akylamines, and benzothiazepines (12). N-type, P/Q-type, and R-type Ca2+ channels also require strong depolarization for activation (7, 13, 14, 15). They are unaffected by L-type Ca2+ antagonists but blocked by specific polypeptide toxins from snail and spider venoms, such as {omega}-conotoxin GVIA (16, 17), {omega}-agatoxin IVA (18, 19), and {omega}-conotoxin MVIIC (20, 21). They are expressed primarily in neurons of most fast synapses, where they initiate neurotransmission (13, 14).

Previous studies demonstrated that both low-voltage-activated and high-voltage-activated Ca2+ channels exist in rat SCs (22, 23, 24, 25). Electrophysiological studies demonstrated that T-type Ca2+ channels of excitable cells are located in the membrane of immature SCs in primary cultures. These channels do not appear directly sensitive to FSH (24). On the other hand, the inhibition of N-type Ca2+ current by {omega}-conotoxin GVIA was shown to reduce about 50–60% of the FSH-induced protein secretion by rat SCs after 6 h of FSH treatment (25). However, no additional evidence has verified that N-type Ca2+ channels or the others mediate the FSH-induced immediate Ca2+ influx in rat SCs. Therefore, the present study was intended to identify the type of Ca2+ channel involved in the FSH-induced immediate Ca2+ influx in rat SCs. Furthermore, the phenomenon that the activation of PLC leads to the generation of inositol 1,4,5-trisphosphate (IP3), which in turn induces a slight in-store Ca2+ release. That the in-store Ca2+ release is a prerequisite to the subsequent Ca2+ uptake through the Ca2+ channel is so-called capacitative Ca2+ influx (26, 27). Capacitative Ca2+ influx in rat SCs has been reported previously (28). Consequently, whether the FSH-induced IP3-dependent immediate Ca2+ influx (4) resulted from capacitative Ca2+ influx would also be verified.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human FSH was from the National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance, CA). Nifedipine and mibefradil were purchased from Calbiochem (Merck Biosciences, Darmstadt, Germany). Collagenase, hyaluronidase, fura-2-AM, and poly-L-lysine were purchased from Sigma-Aldrich (St. Louis, MO). Myristoylated synthetic peptide of PLC-{delta}1 myr-TIPWNSLKQGYRHVHLL was synthesized by Synpep Corp. (Dublin, CA).

Animals
Wistar rats, 21 d old, from the Animal Facility of National Taiwan University were killed in a CO2 chamber according to the National Institutes of Health Guidelines. Permission for using rodents for this study was approved by the Institutional Animal Care and Use Committee of Taipei Medical University (LAC-95-0067).

Cell culture
SCs were isolated from testes of 21-d-old rats as described previously (29). The cells from collagenase/hyaluronidase-treated testicular tubules were plated in 100-mm Corning culture dishes in DMEM/Ham’s F-12 medium (DFM) (equal parts of DMEM and Ham’s F-12 containing 100 µg/ml streptomycin, 100 U/ml penicillin, and 5 µg/ml fungizone) (all were purchased from Invitrogen, Carlsbad, CA) and cultured at 34 C in a humidified atmosphere with 5% CO2. The cells were cultured in DFM for 3 d to allow a firm attachment of SCs. The SCs were subsequently cultured in DFM-6F media (DFM plus insulin, 1.0 µg/ml; epidermal grouth factor, 10 ng/ml; vitamin A and E, 200 ng/ml each; progesterone and hydrocortisone, 10–8 mol/liter each) as described previously (29) for an additional 2–3 d and then used for the study of the effect on FSH.

Measurement of intracellular calcium
SCs were cultured in six-well culture plates containing 9 x 22 mm poly-L-lysine-coated cover slides. The FSH-induced SC Ca2+ influx was determined after pretreating the cells with fura-2-AM (2 µM) as described in the previous study (4). The analysis of intracellular calcium changes was performed in a Hitachi F-4500 fluorescence spectrophotometer using an intracellular cation measurement system (Hitachi Scientific Instruments, Gaithersburg, MD).

Statistical analysis
Each data point represents the mean of three independent experiments and is presented as mean ± SEM. The statistical analysis was performed by one-way ANOVA and Duncan’s multiple range test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of FSH-induced Ca2+ influx in rat SCs
To verify whether intracellular Ca2+ release participates in the FSH-induced immediate elevation of intracellular Ca2+ levels [Ca2+]i, dantrolene was used to block the intracellular Ca2+ release from endoplasmic reticulum stores. The pretreatment of rat SCs with dantrolene (25 µM), compared with control (Fig. 1AGo), did not affect the FSH-induced elevation of [Ca2+]i (Fig. 1BGo). However, the administration of EGTA (0.2 mM) immediately abolished the FSH-induced rat SC intracellular Ca2+ elevation (Fig. 1CGo). These results are in accordance with our previous report that FSH induces an immediately inward Ca2+ mobilization in rat SCs within 100 sec under confocal microscopic observation (4).


Figure 1
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  		FIG. 1. The characterization of FSH-induced immediate Ca2+ elevation in rat SCs. Rat SCs were preincubated with fura-2-AM (5 µM) for 40 min at 34 C and then subjected to intracellular Ca2+ determination. A and B, FSH (300 ng/ml) was added to the incubation medium at the indicated time point in the absence (A) or presence (B) of dantrolene (25 µM); C, EGTA (0.2 mM) was added to medium to chelate extracellular calcium at the indicated time point after the administration of FSH.

 
To determine whether the FSH-induced SC immediate Ca2+ influx is elicited through capacitative Ca2+-entry model (28), the elevation of [Ca2+]i was performed in the absence of extracellular Ca2+. Without extracellular Ca2+, FSH (300 ng/ml) failed to elicit an intracellular Ca2+ elevation in SCs (Fig. 2AGo). However, thapsigargin (1 µM), an inhibitor of sarco-/endoplasmic reticulum ATPase, transiently evoked a 1.5-fold elevation of [Ca2+]i in the SCs and then returned to basal level by a few minutes in the absence of extracellular Ca2+ (Fig. 2BGo). In these experimental conditions, the addition of extracellular Ca2+ (2.5 mM) caused a rapid rise in [Ca2+]i (Fig. 2Go, A and B). On the other hand, the treatment of SCs with 0.1% BSA/PBS (Fig. 2CGo) or 0.1% dimethylsulfoxide (DMSO) (Fig. 2DGo), the solvents of FSH and thapsigargin stock solution, respectively, did not evoke an elevation of [Ca2+]i in the absence of extracellular Ca2+. However, supplementing with Ca2+ in these experimental conditions slightly elevated [Ca2+]i (Fig. 2Go, C and D). These results suggest that the FSH-induced rat SC Ca2+ influx did not result from capacitative Ca2+ entry.


Figure 2
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  		FIG. 2. The characterization of FSH-induced noncapacitative Ca2+ influx in rat SCs. Rat SCs were preincubated with fura-2-AM (5 µM) as stated in Fig. 1Go before intracellular Ca2+ determination. The following were added at the indicated time points to the incubation medium without extracellular Ca2+: A, FSH (300 ng/ml); B, thapsigargin (1 µM); C, BSA (0.1% dilution in PBS); or D, DMSO (0.1%). CaCl2 (2.5 mM) was added to each incubation medium at the indicated time points. The 0.1% BSA and 0.1% DMSO were used as solvent control of FSH and thapsigargin, respectively.

 
Type of Ca2+ channel associated with FSH-induced noncapacitative Ca2+ influx in rat SCs
The previous electrophysiological study on Ca2+ current of rat SCs by patch-clamp technique demonstrated the presence of a voltage-dependent T-type Ca2+ channel on plasma membrane of rat SCs (24). To further identify whether the FSH-induced immediate Ca2+ influx of rat SCs is mediated by T-type Ca2+ channels, NiCl2 (10 µM), a selective blocker of T-type Ca2+ channels (<50 µM) (10), was included in the medium before the addition of FSH (300 ng/ml). As shown in Fig. 3Go, A and B, FSH failed to evoke Ca2+ influx in the presence of 10 µM NiCl2. In addition, the pretreatment of rat SCs with mibefradil, also a specific inhibitor of T-type Ca2+ channels (9, 30), reduced the FSH-induced Ca2+ influx dose dependently (Fig. 3CGo). Mibefradil at 10 and 100 µM exhibited a significant (P < 0.05) inhibitory effect on FSH-induced rat SC Ca2+ influx by 50 and 90%, respectively (Fig. 3DGo). On the other hand, the pretreatment with nifedipine (10 and 50 µM) and {omega}-conotoxin (100 and 500 nM) to specifically inhibit the activities of L- and N-type Ca2+ channels, respectively, did not affect the FSH-induced rat SC Ca2+ influx (Fig. 4Go, A and B).


Figure 3
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  		FIG. 3. The effect of NiCl2 or mibefradil on FSH-induced Ca2+ influx in rat SCs. Rat SCs were preincubated with fura-2-AM (5 µM) as described above. A and B, H2O (as solvent control of NiCl2) (A) or NiCl2 (10 µM) (B) was added into the incubation medium at the indicated time points. After 500 sec incubation, FSH (300 ng/ml) was added into the incubation medium. C, Similarly, SCs were preincubated with fura-2-AM (5 µM) with or without mibefradil [0 (H2O, as control), 10, or 100 µM] for 40 min at 34 C and then subjected to intracellular Ca2+ determination. FSH (300 ng/ml) was added to the incubation medium at the indicated time points. D, The data from three independent experiments of various mibefradil treatments were analyzed by one-way ANOVA and Duncan multiple range test. Data shown in D represent means ± SEM (n = 3). Different letters above the columns indicate significant differences between the means (P < 0.05). H2O was used as solvent control of mibefradil.

 

Figure 4
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  		FIG. 4. The effects of nifedipine and {omega}-conotoxin on the FSH-induced Ca2+ influx in rat SCs. In addition to the pretreatment with fura-2-AM (5 µM), rat SCs simultaneously pretreated with or without nifedipine [0 (H2O, as control), 10, or 50 µM] (A) or {omega}-conotoxin [0 (0.1% DMSO, as solvent control), 100, or 500 nM] (B). Peak amplitudes of intracellular Ca2+ level in response to FSH treatment were obtained from three independent experiments and analyzed by one-way ANOVA and Duncan multiple range test. Data represent means ± SEM (n = 3). Different letters above the columns indicate significant differences between the means (P < 0.05). H2O and 0.1% DMSO was used as solvent controls of nifedipine and {omega}-conotoxin, respectively.

 
The roles of Gs/adenylate cyclase- and G{alpha}h/PLC-{delta}1-signaling in FSH-induced rat SC T-type Ca2+-channel-mediated Ca2+ influx
FSH-induced rat SC Ca2+ influx was independent of the Gs/adenylate cyclase pathway (3). In our previous study (4), 2',5'-dideoxyadenosine (3 and 15 µM) was used to inactivate adenylate cyclase and resulted in a dose-dependent reduction in the FSH-induced accumulation of intracellular cAMP. However, the pretreatment of rat SCs with 2',5'-dideoxyadenosine (3 and 15 µM) did not suppress but slightly enhanced the FSH-induced rat SC Ca2+ influx (Fig. 5AGo) in the present study. Furthermore, pretreatment of rat SCs with U73122 (5 µM), a specific inhibitor of phosphatidylinositol-dependent PLCs, or with myristoylated PLC-{delta}1 peptide (1 µM) to competitively inhibit the activation of PLC-{delta}1 by G{alpha}h also significantly (P < 0.05) reduced the FSH-induced Ca2+ influx (Fig. 5BGo). These results indicate an essential role of the G{alpha}h/PLC-{delta}1 signaling pathway in this event. On the other hand, pretreatment of SCs with DMSO (0.1%), as a solvent control of U73122 or myristic acid (1 µM), did not affect the FSH-induced Ca2+ influx (Fig. 5BGo), thus excluding the off-target effect of U73122 or myristoylated PLC-{delta}1 peptide, respectively.


Figure 5
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  		FIG. 5. The effect of 2',5'-dideoxyadenosine (2',5'-dd-Ado) on the FSH-induced Ca2+ influx in rat SCs. A, Rat SCs were preincubated with fura-2-AM (5 µM) and with or without 2',5'-dd-Ado [0 (H2O, as control), 10, or 100 µM] for 40 min at 34 C and then subjected to intracellular Ca2+ determination. B, In addition to the pretreatment with fura-2-AM (5 µM), rat SCs were simultaneously pretreated with or without U73122 [0 (0.1% DMSO) and 5 µM], myristic acid (1 µM), or myristoylated PLC-{delta}1 synthetic peptide (myr- PLC-{delta}1 peptide) (1 µM). Peak amplitudes of intracellular Ca2+ levels in response to FSH treatments were obtained from three independent experiments and analyzed by one-way ANOVA and Duncan multiple range test. Data represent means ± SEM (n = 3). Different letters above the columns indicate significant differences between the means (P < 0.05). DMSO (0.1%) was used as solvent control of U73122. Myristic acid was used as control for the off-target effect of myristoylated PLC-{delta}1 synthetic peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous study demonstrated that FSH elicits a novel G{alpha}h/PLC-{delta}1 signaling pathway, distinct from the classical Gs/adenylate cyclase pathway, which induces intracellular IP3 generation and evokes extracellular Ca2+ influx in rat SCs (4). Although the phenomenon of FSH-induced immediate rat SC Ca2+ influx was documented in other studies (3, 31, 32, 33), as to which type of Ca2+ channel was involved in this event remains unclear. In the present study, our results define that T-type Ca2+ channels are responsible for FSH-induced immediate Ca2+ influx in rat SCs. Our results also reveal that FSH-induced T-type Ca2+ current in rat SCs is independent of the mode of capacitative Ca2+ entry.

The activation of the PLC pathway results in the generation of intracellular IP3, which stimulates the release of Ca2+ store from the endoplasmic reticulum (34). This release of in-store Ca2+ is generally associated with the subsequent activation of calcium channels and leads to an increase in the Ca2+ entry across the plasma membrane. The elevated intracellular Ca2+ levels either replenish the in-store calcium or contribute to Ca2+-dependent signaling. This process of Ca2+ entry is called capacitative Ca2+ entry or store-operated Ca2+ entry (26, 27). Rossato et al. (28) reported that the depletion of internal Ca2+ stores by thapsigargin, an inhibitor of sarco-/endoplasmic reticulum ATPase, induces Ca2+ influx from the extracellular medium in rat SCs, providing evidence for the existence of capacitative Ca2+ entry in these cells. This view was supported by the observation in Fig. 2BGo of the present study. Although our previous study demonstrated that the interaction of FSH with its receptor on rat SCs induces intracellular IP3 generation (4), the FSH-induced immediate rat SC Ca2+ influx was not caused by the IP3-dependent capacitative Ca2+ entry. This claim was supported by evidence that FSH failed to evoke the elevation of [Ca2+]i in the absence of external Ca2+ and that FSH induced Ca2+ influx after the pretreatment of SCs with dantrolene, an inhibitor of intracellular Ca2+ release. Furthermore, pretreatment with U73122 or synthetic myristoylated PLC-{delta}1 peptide to inhibit the activity of PLC-{delta}1 resulted in the reduction of FSH-induced intracellular IP3 generation (4) and Ca2+ influx in rat SCs. These results indicate that FSH-induced Ca2+ influx is mediated by intracellular IP3-dependent signaling but not through capacitative Ca2+ entry in rat SCs.

The recent identification of IP3 receptors on plasma membrane and the association of those receptors with Ca2+ channels (35, 36, 37) may unveil the mechanism of FSH-induced IP3-dependent, noncapacitative Ca2+ entry in rat SCs. Further efforts are required to demonstrate the existence of IP3 receptors on rat SC plasma membrane. Consequently, the direct action of IP3 in the activation of transient Ca2+ channels needs to be further substantiated.

In addition to T-type Ca2+ channels, L-, N-, and P/Q-type Ca2+ channels were also found on rat SC plasma membrane (22, 23, 24, 25). However, this study demonstrates that T-type Ca2+ channels are essential for the FSH-induced immediate Ca2+ influx in rat SCs. NiCl2 (10 µM) was shown to block the membrane depolarization-induced T-type Ca2+ current in rat SCs (24). The similar inhibitory effect of NiCl2 (10 µM) was observed in the FSH-induced rat SC Ca2+ influx in the present study. In addition, mibefradil, a T-type Ca2+-channel antagonist, dose-dependently reduced the FSH-induced rat SC Ca2+ influx. These data indicate a critical role of T-type Ca2+ channels in the FSH-induced immediate Ca2+ influx of SCs.

The pretreatment of rat SCs with {omega}-conotoxin (4 µM) caused a 50–60% reduction of FSH-induced protein secretion after 6 h of treatment (25). At high concentrations (>1 µM), {omega}-conotoxin suppressed the activities of both L- and T-type Ca2+ channels in neuronal cells (38) and rat SCs (23, 24), whereas {omega}-conotoxin at low concentration blocked N-type Ca2+ channels. In this study, a low dose of {omega}-conotoxin (100 and 500 nM) did not affect the FSH-induced rat SC Ca2+ influx. Similarly, the FSH-induced rat SC Ca2+ influx was not affected by the L-type Ca2+-channel inhibitor nifedipine. All the above findings indicate that the T-type Ca2+ channel plays a crucial role in mediating the FSH-induced immediate Ca2+ influx in rat SCs.

FSH-induced immediate Ca2+ influx in rat Sertoli cells has been well demonstrated in our laboratories and others. Our previous study identified the involvement of the G{alpha}h/PLC-{delta}1signaling pathway in this event (4). According to our previous study (4), the occurrence of the G{alpha}h/PLC-{delta}1 signaling pathway is more prominent at a FSH level greater than 300 IU/liter (3 ng/ml). This might imply that the activation of G{alpha}h/PLC-{delta}1 signaling is associated with the feedback mechanism of SCs, through releasing inhibin, at elevated circulating FSH level (25). This speculation needs to be further substantiated. In conclusion, as summarized in the scheme presented in Fig. 6Go, the data in this study further demonstrate that FSH-induced G{alpha}h/PLC-{delta}1-dependent Ca2+ influx in rat SCs is mediated by T-type Ca2+ channels. It is activated independent of in-store calcium release, via a noncapacitative calcium-entry model.


Figure 6
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  		FIG. 6. Schematic diagram summarizes signaling pathways of FSH-induced Ca2+-channel activation and calcium influx. The T-type Ca2+ channel is activated directly by IP3 instead of capacitative Ca2+ entry through IP3-induced in-store Ca2+ release. AC, Adenylate cyclase; 2',5'-dd-Ado, 2',5'-dideoxyadenosine; ER, endoplasmic reticulum; PKA, protein kinase A.

 


   Footnotes
 
This study was supported by Grants NSC96-2314-B-038-015, 94-CGH-TMU-06, and 96-CGH-TMU-15.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 6, 2007

1 Y.-F.L. and Y.-H.T. made equal contributions to this work. Back

Abbreviations: DMSO, Dimethylsulfoxide; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; SC, Sertoli cell.

Received September 10, 2007.

Accepted for publication November 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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