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Molecular Endocrinology, doi:10.1210/me.2005-0347
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Molecular Endocrinology 20 (10): 2514-2527
Copyright © 2006 by The Endocrine Society

A Novel Follicle-Stimulating Hormone-Induced G{alpha}h/Phospholipase C-{delta}1 Signaling Pathway Mediating Rat Sertoli Cell Ca2+-Influx

Yuan-Feng Lin, Min-Jen Tseng, Hui-Ling Hsu, Yu-Wei Wu, Yi-Hsuan Lee and Yu-Hui Tsai

Graduate Institute of Pharmaceutical Science (Y.-F.L., Y.-W.W., Y.-H.T.), Graduate Institute of Cell and Molecular Biology (M.-J.T., H.-L.H., Y.-H.T.), and Department of Physiology (Y.-H.L.), Medical School, Taipei Medical University, Taipei, Taiwan 110, Republic of China; and Department of Life Science (M.-J.T.), National Chung Cheng University, Chia-Yi, Taiwan 621, Republic of China

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSH is known to activate Gs/cAMP signaling pathway in Sertoli cells (SCs) to support spermatogenesis. However, the molecular mechanism of FSH-induced Gs/cAMP-independent Ca2+-influx in SCs is not clear. In this study, FSH indeed induced an immediate and dose-dependent intracellular Ca2+-elevation in rat SCs. In the presence of EDTA (2.5 mM) or in the absence of extracellular Ca2+, the FSH-induced intracellular Ca2+-elevation was abolished. The confocal microscopic observation of Ca2+ image revealed that the SC cellular Ca2+ level was gradually increased after 50 sec of FSH treatment. Dantrolene, a blocker of intracellular Ca2+ release, did not affect this FSH-induced intracellular Ca2+ elevation. The pretreatment of rat SCs with phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, U73122 (3 and 10 µM), inhibited the FSH-induced Ca2+-influx in a dose-dependent manner, but treatment with Gs-specific inhibitor, NF449 (0.1 and 0.3 µM), did not. On the other hand, the activation of G{alpha}h was immediately induced by FSH in the rat SCs within 5 sec of treatment. The translocation of PLC-{delta}1 from cytosol to cell membrane and the formation of G{alpha}h /PLC-{delta}1 complexes occurred within 5 and 10 sec, respectively, of FSH exposure. The intracellular inositol 1,4,5-triphosphate (IP3) production was also detected after 30 sec of FSH treatment. The synthetic peptide of PLC-{delta}1 (TIPWNSLKQGYRHVHLL), not Gs inhibitor, predominantly inhibited the FSH-induced PLC-{delta}1 translocation, formation of G{alpha}h /PLC-{delta}1 complex, intracellular IP3 production, and Ca2+ influx. In contrast, the peptide did not interfere with FSH-induced intracellular cAMP accumulation. In conclusion, the FSH-induced immediate Ca2+ influx is unambiguously mediated by an alternative G{alpha}h /PLC-{delta}1/IP3 pathway that is distinct from the Gs/cAMP pathway in rat SCs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSH IS RELEASED from pituitary gland and primarily regulates follicular development in the ovary and spermatogenesis in the testis (1). Upon interacting with its receptor on the target cells, FSH elicits a cascade of biochemical events preceded by the activation of Gs/adenylate cyclase and the accumulation of intracellular cAMP (2). The stimulation of target cells by FSH is tightly regulated by a number of interacting mechanisms to ensure homeostasis of proper gonadal function and fertility. On the other hand, several previous studies demonstrated that the interaction of FSH with its receptor induced a rapid Ca2+ influx (3, 4, 5). This event might be associated with a phosphatidylinositol phospholipase C (PI-PLC)-dependent pathway (6), but not Gs/adenylate cyclase-dependent pathway (7) in Sertoli cells (SCs). Despite more than one decade of studies, no working mechanism has been established for the FSH-induced Ca2+ influx in SCs.

Tissue transglutaminase (tTG) has been known to possess multiple functions in various cell types (8). In addition to its transamidating activity, tTG also acts as ATPase, GTPase, and GTP-binding protein, designated as G{alpha}h (9, 10, 11, 12, 13). Recently, a 50-kDa protein, Gßh, was shown to modulate GTP-binding activity of G{alpha}h and was identified later to be calreticulin (14, 15). Calreticulin has been shown to inhibit both GTP-binding and -transamidating activities of tTG and interact with GDP-bound tTG (16). Phospholipase C-{delta}1 (PLC-{delta}1) is a member of PI-PLCs and acts as a downstream effector in the G{alpha}h-mediated intracellular signal transduction pathway (12, 17, 18). The stimulation of PLC-{delta}1 via coupling of the {alpha}1B-adrenoreceptor with tTG evokes both intracellular Ca2+ release and capacitative Ca2+ entry. The capacitative Ca2+ entry is mediated by the interaction of tTG with PLC-{delta}1 in a smooth muscle cell line, DDT1-MF2 (19).

Testicular transglutaminase was characterized and identified to be tissue-type transglutaminase in our recent report (20). It was suggested earlier to play a role in the FSH-induced activation of SCs through modulating the activities of membrane and cytosolic components (21). Polyamines and tTG substrates interfered with the fate of sequestered FSH in SCs but not the rate at which sequestration occurs (22). The administration of tTG inhibitors, bacitracin and N-ethylmaleimide, did not affect the FSH receptor binding but enhanced the dissociation of 125I-labeled human FSH from its receptor. Furthermore, the reduced tTG activity paralleled the increase in hormone-receptor dissociation (23). These authors speculated that protein cross-linking caused by tTG might be required for the stabilization of FSH-receptor complexes. Nevertheless, the involvement of tTG in FSH-elicited intracellular signaling cascades is still not clear. Recently, it was suggested that tTG acts mainly as a GTP-binding protein rather than its transamidating activity in the cells under physiological situations (24). It exchanges GTP-binding activity to transamidating activity due to the insufficient content of intracellular GTP or the excess concentration of intracellular Ca2+ in the pathological progression, such as programmed cell death (24, 25, 26). Therefore, we proposed that tTG might be the other G-protein (in addition to Gs) to mediate FSH-elicited signaling pathway in rat SCs.

This study was intended to establish whether G{alpha}h is a distinct class of G protein, other than Gs, in FSH actions in rat SCs. It also attempted to clarify the molecular mechanism regarding the FSH-induced Ca2+ influx. The obtained data unambiguously demonstrated that the G{alpha}h /PLC-{delta}1/inositol 1,4,5-triphosphate (IP3) pathway is an early event governing the FSH-induced immediate Ca2+-influx in rat SCs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of FSH-Induced Ca2+-Influx in SCs
The dose effect of recombinant human FSH on eliciting intracellular Ca2+ elevation was determined by using duel-wavelength (340 and 380 nm) fluorescence spectrophotometer after cells were loaded with the Ca2+-sensitive fluorescent dye Fura-2-AM. The intracellular Ca2+ levels of rat SCs were measured after various doses of FSH (300, 1000, and 3000 IU/liter) were administrated to Fura-2-AM (5 µM)-pretreated rat SCs. As shown in Fig. 1Go, A and B, the FSH-induced intracellular Ca2+ elevation was dose dependent. The administration of 3000 IU/liter of FSH elicited an immediate rise in the intracellular Ca2+ levels within 50 sec, reached about 2-fold elevation by 200 sec, and lasted for at least 20 min in rat SCs (Fig. 1AGo). The results agree with the observation of Gorczynska and Handelsman (27) that Ca2+ levels rise within 60 sec, reach the maximum at 180 sec, and last for at least 10 min in FSH-treated freshly isolated rat SCs.


Figure 1
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  		Fig. 1. The Characterization of FSH-Induced Cellular 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. FSH (3000 IU/liter) was administrated to the cells at 100 sec after the measurement of Ca2+-level was begun. A, SCs were treated with various doses of FSH as indicated. The peak amplitudes for each FSH dose was calculated from three independent experiments and represented as mean ± SEM (B). C, After 50 sec of FSH administration, 2.5 mM EDTA was injected into the reaction medium to chelate the extracellular free Ca2+. D, Rat SCs were incubated in the Ca2+-free phocal buffer, FSH was administrated into the phocal buffer at the designated time. E, Rat SCs were preincubated with Fura-2-AM and Dantrolene (25 or 50 µM) for 40 min at 34 C before the determination of intracellular Ca2+ levels. The peak amplitude for each experimental condition was calculated from three independent experiments and presented as mean ± SEM. F, Rat SCs were preincubated with Fluo-3 (4 µM) for 40 min at 34 C and then subjected to the observation of Ca2+ image under a confocal microscope. Upon the administration of FSH, the images were serially recorded every 10 sec. The fluorescent intensity of Fluo-3:Ca2+ complex is indicated by the inserted scale panel. The original magnification of image is 400-fold. The triplicate data of peak amplitudes were analyzed by one-way ANOVA and Duncan Multiple Range Test. Different letters above the columns indicate significant differences between the means (P < 0.05). 100s, 100 sec; IU/L, IU/liter.

 
To identify the Ca2+ source of FSH-induced immediate intracellular Ca2+ elevation, experiments were performed in the absence of the extracellular Ca2+ or after the intracellular Ca2+ release from endoplasmic reticulum was blocked. The FSH-caused intracellular Ca2+ elevation was abolished as soon as the extracellular Ca2+ was chelated with 2.5 mM EDTA (Fig. 1CGo) or removed by using Ca2+-free media (Fig. 1DGo). The pretreatment of rat SCs with Dantrolene (25 µM and 50 µM), an inhibitor of intracellular Ca2+ release, did not inhibit but significantly (P < 0.05, n = 3) enhanced the FSH-induced intracellular Ca2+ elevation (Fig. 1EGo).

To visualize the FSH-induced Ca2+ mobilization, rat SCs were pretreated with Fluo-3 (4 µM), a fluorescent indicator of Ca2+, before the FSH administration. By using a confocal microscope, the image of intracellular Ca2+ elevation occurred within 100 sec and reached a maximal level after 200 sec of FSH treatment (Fig. 1FGo). The observed Ca2+ image indicates that the influxed Ca2+ gradually streamed in from the outside of cells after 50 sec of FSH (3000 IU/liter) treatment (indicated by arrows in Fig. 1FGo). Figure 1FGo sequentially showed the elevation of FSH-induced intracellular Ca2+ levels in the cultured rat SCs.

On the other hand, FSH-induced Ca2+ influx has been demonstrated to be independent of cholera toxin (Gs) and pertussis toxin (Gi, Go)-sensitive G proteins (7), but associated with PI-PLC-dependent pathway (6) in SCs. In contrast, another study indicated that FSH-induced intracellular Ca2+ level can be mimicked by the treatment with forskolin, cholera toxin, and dibutyryl-cAMP (27). To resolve this discrepancy, we examined the possible involvement of Gs- and PI-PLC-dependent pathways in the FSH-elicited Ca2+ influx of rat SCs. NF449, an antagonist of Gs protein, and U73122, a specific inhibitor of PI-PLC, were employed to block the activities of Gs protein and PI-PLC, respectively. The pretreatment of rat SCs with NF-449 (0.1 and 0.3 µM) significantly (P < 0.05, n = 3) enhanced the FSH-induced Ca2+ influx in a dose-dependent manner (Fig. 2AGo). The pretreatment of rat SCs with 0.3 µM NF449 exhibited a 2-fold enhancement of FSH-induced intracellular Ca2+ elevation. In contrast, U73122 (3 and 10 µM) inhibited the FSH-induced Ca2+-influx in a dose-dependent manner (Fig. 2BGo). The pretreatment with 10 µM U73122 showed a 50% inhibition (P < 0.05, n = 3) of FSH-induced intracellular Ca2+ elevation.


Figure 2
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  		Fig. 2. The Effects of NF449 and U73122 on the FSH-Induced Ca2+-Influx in Rat SCs

Rat SCs were preincubated with Fura-2-AM (5 µM), and NF449 (a Gs protein-specific inhibitor, at 0.1 or 0.3 µM) (panel A), or U73122 (an inhibitor of PI-PLCs, at 3 and 10 µM) (panel B) for 40 min at 34 C before the intracellular Ca2+ determination. FSH (3000 IU/liter) was administrated at 100 sec after the onset of the Ca2+ determination process. The triplicate data of peak amplitudes were analyzed by one-way ANOVA and Duncan Multiple Range Test. The column height in panels A and B represents mean ± SEM of triplicate peak amplitudes. Different letters above the columns indicate significant differences between the means (P < 0.05). IU/L, IU/liter.

 
The Activation of G{alpha}h and PLC-{delta}1 by FSH in Rat SCs
tTG, also known as G{alpha}h, was known to modulate the {alpha}1B-adrenoreceptor-evoked Ca2+ influx in the DDT1-MF2 smooth muscle cell line (19). Whether or not a G{alpha}h-dependent signaling pathway was also involved in the FSH-evoked Ca2+ influx of rat SCs was investigated. The fractionated membrane proteins of FSH-treated rat SCs were incubated with GTP-agarose to pull down the activated (GTP bound) G{alpha}h, and then subjected to Western blot analysis using G{alpha}h-specific antibody. The data show that the levels of activated G{alpha}h were increased by 5 sec, maintained at high levels until 30 sec, and then declined after 60 sec of FSH treatment (Fig. 3AGo, top). Rat SCs also exhibited a dose-dependent elevation of G{alpha}h levels after 30 sec of FSH treatment (Fig. 3BGo, top). According to previous studies, upon binding with GTP, tTG would act as G{alpha}h and lose its transamidating activity (24). To detect the in situ transamidating activity of tTG, rat SCs were pretreated with 5-(biotinamido)-pentylamine (1 mM), a substrate for tTG, for 40 min before the FSH treatment. The 5-(biotinamido)-pentylamine-incorporated cellular proteins were determined by the ELISA procedure as described in Materials and Methods. The data revealed that the cellular tTG activity declined significantly (P < 0.05, n = 3) after 10 sec of FSH (1000 IU/liter) treatment (Fig. 3AGo, bottom). FSH at the range of 300 IU/liter to 3000 IU/liter also showed a dose-dependent effect on the reduction of rat SC tTG activity after 10 sec of treatment (Fig. 3BGo, bottom).


Figure 3
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  		Fig. 3. The Time- and Dose-Dependent Effects of FSH on GTP-Bound G{alpha}h Activity and in Situ tTG Activity of Rat SCs

Rat SCs were incubated with FSH (1000 IU/liter) for the indicated time intervals, or treated with various doses of FSH for 10 sec. The GTP-bound G{alpha}h protein in the cell membrane fraction was analyzed by SDS-PAGE followed by Western blot analysis with a tTG-specific antibody (A and B, tops). On the other hand, the in situ tTG activity was determined by incubating SCs with 1 mM 5'-biotinamido-pentylamine for 40 min at 34 C before the treatment with FSH for designated time intervals (A, bottom), or with various doses of FSH for 10 sec (B, bottom). The tTG activity was assessed as described in Materials and Methods. The data from triplicate analysis were analyzed by one-way ANOVA and Duncan Multiple Range Test. Data shown in Fig. 1Go, A and B (bottoms), represent means ± SEM (n = 3). Different letters above the columns indicate significant differences between the means (P < 0.05). Ab, Antibody; IB, immunoblot.

 
Because PLC-{delta}1 was identified as an effector in G{alpha}h-mediated signaling pathway (18), experiments were performed to examine whether the administration of FSH activates rat SC PLC-{delta}1. The activated PLC-{delta}1 translocates from cytosol to plasma membrane. Thus, the isolated cytosolic and membrane proteins of rat SCs were subjected to Western blot analysis using PLC-{delta}1-specific antibody. The data revealed that the translocation of PLC-{delta}1 was increased at 5 sec, persisted until at least 30 sec, and then decreased by 60 sec of FSH treatment (Fig. 4AGo). Meanwhile, the formation of G{alpha}h/PLC-{delta}1 complexes was also determined by coimmunoprecipitation with G{alpha}h antibody followed by Western blotting using PLC-{delta}1 antibody. The level of G{alpha}h/PLC-{delta}1 complexes in the fractionated membrane proteins reached maximum (~3-fold) at 10 sec of FSH treatment (Fig. 4BGo). Furthermore, the formation of additional G{alpha}h /PLC-{delta}1 complexes in the SC lysate was measured in the presence of exogenous GTP-{gamma}S, which activates G{alpha}h in vitro (Fig. 4CGo). The data demonstrated that the dose effect of exogenous GTP-{gamma}S on the formation of G{alpha}h/PLC-{delta}1 complexes in a cell-free system was significant among various doses (P < 0.05; n = 3). Subsequently, the generation of IP3 mediated by FSH-activated PLC-{delta}1was examined. Figure 4EGo showed that the generation of IP3 occurred by 30 sec of FSH treatment and increased gradually up to at least 120 sec.


Figure 4
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  		Fig. 4. The Effects of FSH on PLC-{delta}1 Translocation, Formation of G{alpha}h /PLC-{delta}1 Complex, and Production of Intracellular IP3

After rat SCs incubated with FSH (1000 IU/liter) for various time intervals, the extracted proteins of cell membrane and cytosolic fractions were employed to determine the PLC-{delta}1 translocation (A). Actin was used as the internal standard for relative cytosolic protein loading. Subsequently, cell membrane proteins were immunoprecipitated with G{alpha}h antibody to detect the formation of G{alpha}h /PLC-{delta}1 complex by Western blot with PLC-{delta}1 antibody (Fig. 2BGo, top). The bottom panel of Fig. 2BGo shows the normalized intensities of PLC-{delta}1 values, relative to Ig heavy chain (IgH), which peaked at 10 sec after FSH treatment. C, SCs were preincubated with various concentrations of GTP{gamma} S to activate G{alpha}h and induce the formation of G{alpha}h /PLC-{delta}1 complexes. The complexes in the whole-cell lysates were immunoprecipitated with G{alpha}h antibody followed by ELISA with a PLC-{delta}1-specific antibody. D, After rat SCs were treated with FSH (3000 IU/liter) for indicated time intervals, the IP3 production was determined as described in Materials and Methods. Data shown in Figs. panels C and D represent means ± SEM (n = 3). Different letters above the columns indicate significant differences between the means (P < 0.05). Ab, Antibody; cPLC, cytosolic PLC; IB, immunoblot; IP, immunoprecipitation; mPLC, membrane PLC.

 
The Requirement of G{alpha}h/PLC-{delta}1 Pathway for the FSH-Induced Ca2+-Influx
The inhibition of PI-PLC activation with U73122 verified the association of PI-PLCs with the FSH-induced Ca2+-influx in rat SCs (Fig. 2BGo). The subsequent experiments further demonstrated that the activation of G{alpha}h and PLC-{delta}1, a member of PI-PLCs, occurred in FSH-treated rat SCs (Figs. 3Go and 4Go). Consequently, experiments were designed to identify the role of the G{alpha}h/PLC-{delta}1 pathway in the FSH-induced Ca2+ influx. According to Kang et al. (19), TIPWNSLKQGYRHVHLL, a peptide corresponding to amino acid sequences of PLC-{delta}1 from 720–736, efficiently interacts with G{alpha}h and blocks the PI hydrolysis by activated PLC-{delta}1 in vitro. On the other hand, the N-terminal modification with myristic acid has been shown to facilitate protein penetrating the cell membrane (28). Therefore, this PLC-{delta}1 peptide was synthesized and myristoylated at the N terminus (Fig. 5AGo) and employed to compete with endogenous PLC-{delta}1 for the interaction with G{alpha}h in FSH-treated rat SCs. The effects of this myristoylated PLC-{delta}1 peptide (Myr-peptide) on the FSH-induced PLC-{delta}1 activation and Ca2+ influx were investigated. Rat SCs were pretreated with various concentrations (0.1, 1, and 10 µM) of the peptide for 40 min before the FSH treatment. As expected, the Myr-peptide predominantly reduced the FSH-induced translocation of PLC-{delta}1 from cytosol to cell membrane (Fig. 5BGo) and the subsequent formation of G{alpha}h/PLC-{delta}1 complexes (Fig. 5CGo) in a dose-dependent manner. The Myr-peptide also significantly (P < 0.05; n = 3) inhibited the FSH-induced IP3 generation after rat SCs were treated with FSH for 2 min (Fig. 5DGo). The FSH-induced IP3 generation was inhibited by the Myr-peptide to approximately 50% reduction at 0.1 µM and about 90% reduction at 1 or 10 µM. Furthermore, the pretreatment with various concentrations of Myr-peptide efficiently inhibited the FSH-evoked Ca2+ influx in rat SCs (Fig. 5EGo). As shown in Fig. 5EGo, the Myr-peptide exhibited significant suppression on the FSH-evoked Ca2+ influx: approximately 35% reduction at 0.1 µM and up to 65–70% reduction at 1 or 10 µM. On the other hand, myristic acid alone (10 µM) did not affect the FSH-induced translocation of PLC-{delta}1, formation of G{alpha} h/PLC-{delta}1 complexes, generation of intracellular IP3, or the occurrence of Ca2+ influx in rat SCs. These data further demonstrate the critical role of PLC-{delta}1 in the serial events underlying the FSH-induced Ca2+-influx in rat SCs.


Figure 5
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  		Fig. 5. The Essential of G{alpha}h /PLC-{delta}1 Complex Formation in the FSH-Induced SC Ca2+ Influx

Before the administration of FSH (3000 IU/liter), rat SCs were pretreated with the Myr-peptide (A) at designated concentrations for 40 min at 34 C. The FSH-induced PLC-{delta}1 translocation from cytosol to cell membrane was determined as described in Materials and Methods (B). Actin was used as the internal standard for relative cytosolic protein loading. Membrane fractions of rat SCs were subsequently immunoprecipitated with G{alpha}h antibody, followed by Western blot analysis with a PLC-{delta}1-specific antibody to reveal the relative levels of G{alpha}h /PLC-{delta}1 complexes produced (C). IgH was used as an indicator for the relative protein levels of immunoprecipitated products. On the other hand, rat SCs were preincubated with Fura-2-AM (5 µM) and various concentrations of the Myr-peptide or myristic acid (MA) (10 µM) for 40 min at 34 C before the determination of rat SC intracellular Ca2+ level in response to FSH treatment (D). The column height represents mean ± SEM (n = 3) of peak amplitudes for each experimental group. E, Rat SCs were preincubated with Myr-peptide at designated concentrations or with MA (10 µM) for 40 min at 34 C followed by the FSH treatment for 1 min, and the generation of intracellular IP3 was determined. The triplicate data in panels D and E were analyzed by one-way ANOVA and Duncan Multiple Range Test. Different letters above the columns indicate significant differences between the means (P < 0.05). Ab, Antibody; cPLC, cytosolic PLC; IB, immunoblot; IP, immunoprecipitation; IU/L, IU/liter; mPLC, membrane PLC.

 
G{alpha}h/PLC-{delta}1 Signaling Pathway Assuming a Novel Route in FSH Actions
Other studies (7) and our results (Fig. 2AGo) found that the Gs/cAMP pathway was not responsible for the FSH-induced Ca2+ influx in rat SCs. Experiments designated to distinguish the dependency of the FSH-induced activation of PLC-{delta}1 on G{alpha}h from Gs were performed. Rat SCs were pretreated with various concentrations (0.1 and 0.3 µM) of NF-449 to antagonize the endogenous Gs activity before the FSH treatment. NF449 did not inhibit, but slightly enhanced, the FSH-induced elevation of G{alpha}h in the cell membrane fraction (Fig. 6AGo, top), and significantly suppressed (P < 0.05; n = 3) the in situ activities of total cellular tTG (Fig. 6AGo, bottom) after 10 sec of FSH treatment. Simultaneous with the increased cellular G{alpha}h activity, NF-449 also enhanced the amount of PLC-{delta}1 translocation from the cytosol to cell membrane (Fig. 6BGo) and the formation of G{alpha}h /PLC-{delta}1 complexes (Fig. 6CGo) after the FSH treatment. However, the pretreatment with NF449 did not affect the FSH-induced generation of intracellular IP3 in rat SCs (Fig. 6DGo).


Figure 6
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  		Fig. 6. Two Distinct Pathways Governing the Activation of G{alpha}h /PLC-{delta}1-Mediated Ca2+ Influx and the Classical Gs-Dependent Signaling in FSH-Activated SCs

SCs were preincubated with designated doses of NF-449, a Gs protein-specific inhibitor, at 34 C for 40 min before the administration of FSH (3000 IU/liter) for 10 sec. The extracted membrane proteins (100 µg) of SCs were used to determine the levels of GTP-absorbed G{alpha}h protein (A, top). The whole-cell lysates (10 µg) were employed to measure the in situ tTG activity (A, bottom). B, The PLC-{delta}1 levels in the cell membrane (50 µg) and cytosolic (50 µg) compartments were analyzed by Western blotting with a PLC-{delta}1-specific antibody to assess the relative translocation levels of PLC-{delta}1. Actin was used as the internal standard for relative cytosolic protein loading. The extracted cell membrane proteins (100 µg) of SCs were immunoprecipitated with G{alpha}h antibody followed by Western blot analysis with a PLC-{delta}1-specific antibody to analyze for the formation of G{alpha}h /PLC-{delta}1 complexes (C). IgH was used as an indicator for the relative protein level of immunoprecipitated products. D, After rat SCs pretreated with NF449 at the designated concentrations, the cells were incubated with FSH for 1 min. The concentration of intracellular IP3 was determined as described in Materials and Methods. Data shown in the bottom portion of panels A and D represent means ± SEM (n = 3). Different letters above the columns indicate significant differences between the means (P < 0.05). Ab, Antibody; cPLC, cytosolic PLC; IB, immunoblot; IU/L, IU/liter.

 
The interrelationship between the effects of FSH on G{alpha}h /PLC-{delta}1 activation and intracellular cAMP accumulation was investigated. Rat SCs were pretreated with isobutyl-methyl-xanthine (1 mM), an inhibitor of phosphodiesterase, to block the degradation of cAMP. Upon the exposure to FSH, SC intracellular cAMP began to accumulate and reached the maximal level after 30 min of treatment (Fig. 7AGo). This effect of FSH on intracellular cAMP accumulation in rat SCs exhibited a dose-dependent manner over the range of 30–300 IU/liter (Fig. 7BGo). Furthermore, the pretreatment of rat SCs with adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (1 and 10 µM), dose-dependently reduced the FSH-induced intracellular cAMP accumulation in a dose-dependent manner. On the other hand, the pretreatment with Myr-peptide (0.1 and 1 µM) and myristic acid (10 µM) alone did not affect the FSH-induced intracellular cAMP accumulation in rat SCs (Fig. 7CGo). However, pretreating rat SCs with 10 µM of Myr-peptide significantly (P < 0.05; n = 3) enhanced the FSH-induced intracellular cAMP accumulation.


Figure 7
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  		Fig. 7. The Effects of 2',5'-Dideoxyadenosine and Myristoylated Synthetic PLC-{delta}1 Peptide on FSH-Induced Intracellular cAMP Accumulation

Rat SCs were preincubated with 1 mM phosphodiesterase inhibitor, isobutyl-methylxanthine, for 40 min at 34 C. The cells were then incubated with FSH (1000 IU/liter) for indicated time intervals (A) or with FSH at designated concentrations for 30 min (B). The determination of intracellular cAMP level was performed as described in Materials and Methods. The data in panels A and B represent means ± SEM (n = 3) of various groups of triplicate assay. C, In addition to the pretreatment with isobutyl-methylxanthine, rat SCs simultaneously pretreated with designated concentrations of adenylate cyclase inhibitor 2',5'-dideoxyadenosine (2',5'-dd-Ado), or the Myr-peptide for 40 min at 34 C. Rat SCs were then treated with FSH (1000 IU/liter) for 30 min at 34 C. The concentration of intracellular cAMP was determined as described in Materials and Methods. "MA" indicates that rat SCs were pretreated with myristic acid (10 µM) followed by FSH (1000 IU/liter) treatment. The column heights represent means ± SEM (n = 3). Different letters above the columns indicate significant differences between the means (P < 0.05).IU/L, IU/liter; MA, myristic acid.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The FSH-induced Ca2+ influx has been determined by the uptake of 45Ca2+ into SCs by other researchers (4, 29). No further study has defined the molecular mechanism(s) of FSH-induced Ca2+ influx in SCs during the past two decades. In the early 1990s, it was demonstrated that the FSH-induced Ca2+ influx is independent of cholera toxin (Gs)- and pertussis toxin (Gi, Go)-sensitive G proteins, as well as adenylate cyclase in SCs (7). FSH receptor itself was once suggested to be a Ca2+ channel in rat SCs (5); however, this suggestion has been refuted by Shibata et al. (30). On the other hand, the inositol phosphate turnover was more recently linked to the elevation of cytosolic Ca2+ induced by nucleotide receptor in rat SCs (6). Our study found that FSH induced a sustained (>20 min) intracellular Ca2+-elevation in Fura-2 preloaded rat SCs. The phenomenon was also observed by Grasso et al. (5) using 45Ca2+ uptake. We subsequently established that the FSH-induced intracellular Ca2+ elevation was caused by the extracellular Ca2+ influx. To further distinguish the role of Gs- from that of PI-PLC-dependent pathways in FSH-induced Ca2+ influx, the antagonist of Gs protein, NF449, and the PI-PLC-specific inhibitor, U73122, were employed to block endogenous Gs and PI-PLC activities, respectively. The pretreatment of SCs with NF449 did not suppress the FSH-induced Ca2+ influx. In contrast, U73122 inhibited the FSH-induced Ca2+-influx in rat SCs in a dose-dependent manner. The study using Myr-peptide fragment derived from PLC-{delta}1 amino acid sequence directly verified that the FSH-induced Ca2+ influx is mediated by G{alpha}h /PLC-{delta}1 pathway in rat SCs. This is the first study to document that a novel FSH-activated G{alpha}h /PLC-{delta}1 pathway, distinct from the classical Gs/cAMP pathway, mediates an immediate Ca2+ influx in rat SCs.

In contrast, dibutyryl-cAMP was reported to replicate the effect of FSH on the elevation of intracellular Ca2+-level in rat SCs (27, 31). Gorczynska and Handelsman (27) concluded that the FSH-induced sustained phase of Ca2+ influx may be a signal amplification of cAMP rather than an alternate primary signal transduction system for FSH in SCs. However, they did not demonstrate the source of intracellular Ca2+ mobilized by the treatment of SCs with dibutyryl-cAMP. The subsequent report by Sharma et al. (31) showed that the dibutyryl-cAMP-induced transient intracellular Ca2+ elevation was caused by the intracellular Ca2+ release from internal stores of rat SCs. Furthermore, the elevation of cAMP generated by pertussis toxin pretreatment did not affect the FSH-induced Ca2+ influx (31). This further suggests that the FSH-induced Ca2+ influx is dissociated from the effect of cAMP pathway.

tTG or transglutaminase II (TGII) is a member of the transglutaminase family. It catalyzes Ca2+-dependent cross-linking reactions by establishing irreversible {epsilon}-({gamma}-glutamyl)-lysine and N,N-bis({gamma}-glutamyl)-polyamine bonds between proteins in the absence or presence of polyamines (25, 26). In addition to its transglutaminase activity, tTG also possesses the properties of a G protein, exhibiting GTP-binding and GTPase activities, and is designated as G{alpha}h (13, 32). Its function is regulated by intracellular level of either Ca2+ or GTP; an increase in the former or a reduction of the latter stimulates its transglutaminase activity (33, 34). Based on these properties of tTG, we monitored the changes in biological functions of SC cell tTG. Our data demonstrated that the cellular tTG activity was immediately reduced within 5 sec of FSH exposure. On the other hand, the functions of tTG/ G{alpha}h molecules are also regulated by their localization within the cell. The membrane-associated form of the molecules exhibits GTP binding activity of G{alpha}h whereas the free form of the molecules in cytosol shows the tTG characteristics (10). According to the observation, we examined the activity of G{alpha}h proteins in fractionated membrane proteins of FSH-treated rat SCs. The GTP-bound G{alpha}h level was elevated in the isolated membrane proteins, as well as in cytosolic proteins (data not shown) after SCs were treated with FSH. However, the protein levels of tTG in both fractions did not change after FSH treatment of SCs (data not shown). Therefore, a portion of tTG molecules might act as G{alpha}h rather than transamidating enzyme and mediate the signal generated from the interaction of FSH with its receptor on rat SCs.

The FSH-induced elevation of testicular transglutaminase activity was due mainly to the FSH-stimulated tTG activity in the SC (21). The activation of tTG was reported to be required for FSH action through stabilizing the cross-linking of FSH-receptor complexes in the purified testicular light membrane (23, 35). However, we could not detect the existence of covalently bound FSH-receptor complexes. On the other hand, we have identified the purified testicular transglutaminase to be a tissue transglutaminase in nature by examining its biological functions and by Western blot analysis using monoclonal antibody of rat liver tTG (20). The association of tTG/G{alpha}h activation with FSH-induced intracellular signaling cascades in SCs and granulosa cells has never been reported. In this report, we clearly demonstrated that a novel G{alpha}h-dependent pathway is involved in the FSH-elicited intracellular signal transduction pathway of rat SCs. According to Yoo et al. (36), the cellular tTG activity was elevated in response to the prolonged increase of intracellular Ca2+ levels. In this regard, our preliminary results indicate that the cellular in situ tTG activity was gradually returned to the basal level after the occurrence of FSH-induced Ca2+ influx (data not shown). Although the conversion of a portion of tTG to become active G{alpha}h is required for transducing part of the FSH-elicited signaling, the cellular transamidating function of tTG in SCs remains to be studied.

The association of PI-PLC with the induction of Ca2+ influx in SCs was reported earlier (6). In this study, PI-PLC-specific inhibitor U73122 showed a dose-dependent effect on FSH-induced Ca2+ influx in rat SCs. An additional study on which subtype of PI-PLC was involved in the FSH-induced Ca2+-influx in rat SCs was undertaken. The G{alpha}h /PLC-{delta}1 pathway has been identified to modulate the {alpha}1B-adrenoreceptor-evoked Ca2+-influx in the DDT1-MF2 smooth muscle cell line (19). The authors also defined a peptide fragment, TIPWNSLKQGYRHVHLL, corresponding to PLC-{delta}1 amino acid sequence from 720–736 that exhibits a high affinity for G{alpha}h (19). Under the Streptolysine O-induced transient permeabilization of cell membrane, 100 µM of this segment of PLC-{delta}1 peptide inhibited 50% of endogenous PLC-{delta}1 activity in hydrolyzing phosphatidylinositol (19). Therefore, this segment of the PLC-{delta}1 peptide was synthesized to block the interaction between G{alpha}h and PLC-{delta}1 in rat SCs. However, the permeabilized cells are not suitable for the Ca2+ influx study in our system. To facilitate the ability of this synthetic peptide-penetrating plasma membrane, the N terminus of this peptide was myristoylated (28). As expected, this myristoylated PLC-{delta}1 peptide (Myr-peptide), but not myristic acid itself, efficiently inhibited the FSH-induced PLC-{delta}1 activation as verified by the reduced translocation of PLC-{delta}1. This peptide fragment also suppressed the formation of a G{alpha}h /PLC-{delta}1 complex and the generation of intracellular IP3. Furthermore, the pretreatment of rat SCs with the Myr-peptide predominantly blocked the FSH-induced Ca2+ influx but showed no inhibitory effect on intracellular cAMP accumulation. These results conclude that the G{alpha}h/PLC-{delta}1 signaling pathway is a novel molecular mechanism of FSH actions in SC functions. This is the first study to document that tTG exerts its G protein capacity for mediating the immediate Ca2+ influx of rat SCs in response to a gonadotropic peptide hormone, FSH, through a nonclassical FSH-induced intracellular signaling pathway.

This study further demonstrated that the FSH-induced activation of PLC-{delta}1 resulted in the generation of IP3 production. However, the use of Dantrolene (25 µM and 50 µM), an inhibitor of intracellular Ca2+ release from endoplasmic reticulum store, did not significantly block the FSH-induced intracellular Ca2+ elevation. In addition, FSH also did not induce intracellular Ca2+ elevation in the absence of extracellular Ca2+. Furthermore, the data obtained from the confocal microscopic Ca2+ image revealed that the FSH-induced intracellular Ca2+ elevation was obviously due to the extracellular Ca2+ entry. These results seem to argue against the original IP3-Ca2+ signaling model (37, 38). The relation between IP3 and Ca2+ entry is based on the earlier theory, so-called "capacitative Ca2+-entry model" (39). The generation of intracellular IP3 through the activation of PI-PLCs causes an IP3 receptor-evoked intracellular Ca2+ releasing from the endoplasmic reticulum store and leads to the activation of subsequent extracellular Ca2+-entry (40). According to more recent studies, the IP3 receptor (41) and the IP3 receptor-like protein (42) exist in plasma membrane and might be involved in the ligand-evoked extracellular Ca2+ entry, which is independent of intracellular Ca2+ release. Furthermore, an IP3-independent mechanism of extracellular Ca2+ entry has also been reported in MDCK epithelial cells (43). In the present study, the synthetic peptide of PLC-{delta}1 significantly reduced the FSH-induced IP3 production and Ca2+ influx. It is likely that FSH-induced Ca2+ influx in rat SCs is dependent on the PLC-{delta}1-mediated IP3 production but independent of the intracellular Ca2+ release.

NF449 is an established selective antagonist of Gs protein. It suppresses the activation of Gs and blocks the coupling of ß-adrenergic receptor to Gs (44). In this study, the pretreatment of rat SCs with NF449 enhanced, instead of inhibited, the effects of FSH on the immediate Ca2+ influx, and the activations of G{alpha}h as well as PLC-{delta}1. On the other hand, the pretreatment with the myristoylated PLC-{delta}1 peptide did not affect or slightly enhanced (at high doses) the FSH-induced intracellular cAMP accumulation. These results indicate that the signaling pathways of FSH-induced Gs-dependent cAMP accumulation and G{alpha}h-dependent Ca2+ influx are somehow interrelated. Therefore, two possibilities were considered: 1) Gs and G{alpha}h share one FSH receptor in rat SCs. The dysfunction of Gs-dependent signaling pathway could switch to additively enhance the effect of FSH on G{alpha}h-dependent signaling pathway; 2) FSH has more than one receptor in rat SCs. According to other studies (45, 46, 47), there are three differentially spliced FSH receptors in ovine testis: designated as R1 (Gs-coupled FSH receptor), R2 (similar to R1 having a shorter carboxyl terminus), and R3 (alternative splicing converts the G protein-coupled FSH receptor gene product into a growth factor type 1-like receptor). FSH-R3, unlike R1 and R2, lacks the seven-transmembrane region and utilizes a single putative transmembrane segment to mediate the actions of FSH on both the ovine testis and ovary (48). The recent study on FSH-R3-transfected human embryonic kidney cells showed that R3 is competent for FSH-evoked Ca2+ influx. However, based on the general structure of a G protein-coupled receptor, FSH-R3 with a nature of growth factor type I receptor should not couple to any G proteins (3, 49). It is speculated, therefore, that FSH may bind to an atypical receptor that couples with G{alpha}h to promote Ca2+ influx in rat SCs. The nature of this G{alpha}h-coupled receptor is currently not known; it might be either the R2 form of the FSH receptor if Gs and G{alpha}h proteins do not share the R1 classical FSH receptor or another, as yet unidentified, novel FSH receptor in the rat SCs.

In summary, the FSH-induced Ca2+ influx is mediated by a novel G{alpha}h /PLC-{delta}1 pathway that is distinct from the classical Gs/adenylate cyclase pathway in rat SCs. In addition, the FSH-induced Ca2+ influx is dependent on intracellular PLC-{delta}1-mediated IP3 generation and independent of the intracellular Ca2+-release. Furthermore, the molecular manipulation of G{alpha}h /PLC-{delta}1 pathway by using the myristoylated PLC-{delta}1 peptide antagonist might provide a means to study the unknown FSH actions in SC biological functions and the events of SC-supported spermatogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
Wistar rats (21 d old) were purchased from the Animal Facility of National Taiwan University. The animals were killed in a CO2 chamber according to the NIH Guidelines. Permission for using rodents for this study was approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University.

Chemicals
Dantrolene, NF449, U73122, isobutyl-methylxanthine, and 2',5'-dideoxy-adenosine were purchased from Calbiochem (Merck Biosciences, Darmstadt, Germany). GTP{gamma}S and myristic acid were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture
SCs were isolated from testes of 21-d-old Wistar rats as described previously (50). The cells prepared from minced, collagenase/hyaluronidase (Sigma-Aldrich)-treated testicular tubules were plated onto 100-mm dishes in DFM (equal parts of DMEM and Ham’s F-12 containing 100 µg/ml streptomycin; 100 U/ml penicillin; and 5 µg/ml fungizone, pH 7.2–7.4) (all were purchased from Invitrogen, Carlsbad, CA) and cultured at 34 C. The cells were cultured for 3 d to allow a firm attachment of SCs. The majority of the contaminating germinal cells floating in the media were removed. The remaining contaminating germinal cells were burst by treatment for 2 min with hypotonic solution (10 mM Tris buffer, pH 7.4). The enriched SCs conditioned in DFM-6 F media (DFM supplemented with insulin, 1.0 µg/ml; epidermal growth factor, 10 ng/ml; vitamin A and C, 200 ng/ml each; progesterone and hydrocortisone, 10–8 mol/liter each) (all supplements were purchased from Sigma-Aldrich) for 2–3 more days were used for studying the effect of FSH.

Measurement of Intracellular Calcium
SCs were cultured in six-well culture plates containing poly-L-lysine (Sigma-Aldrich)-coated 9 x 22 mm cover slides. After 5-d culture, SCs were pretreated with 5 µM Fura-2-AM (Sigma-Aldrich) for 40 min at 34 C. Subsequently, each SC-grown cover slide was transferred into a 4-ml quartz cuvette containing 2 ml focal buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 2.6 mM D-glucose). The analysis of intracellular calcium oscillation was performed in a Hitachi F-4500 Fluorescence Spectrophotometer (Hitachi Scientific Instruments, Tokyo, Japan) using an Intracellular Cation Measurement System. A designated amount of FSH was added using a microinjection syringe when the base line (380 nm) is stable. The levels of intracellular calcium are presented by the ratio of 340:380-nm excitation wavelength-induced fluorescent intensities emitted at 510 nm.

Calcium Image
SCs were seeded on the poly-L-lysine-coated round cover slide (22 mm in diameter and 0.17 mm in thickness, purchased from Sigma-Aldrich). After 5 d of culture, cells were preincubated with 4 µM Fluo-3 dye (Molecular Probes, Inc., Eugene, OR; Invitrogen) at 34 C for 40 min. After incubation, cells grown on the slide were transferred to a slide chamber containing 0.4 ml of Hank’s balanced saline solution and subjected to image observation by using a confocal microscope (Olympus, Tokyo, Japan) with an Argon laser (488 nm). Upon adjusting proper field, 0.1 ml of FSH (3000 IU/liter) was carefully added into the slide chamber and started to observe the changes of intracellular Ca2+ levels.

In Situ tTG Activity Assay
The in situ tTG activity assay was performed according to Zhang et al. (34). Briefly, before treatment with recombinant human FSH (Organon, BH Oss, The Netherlands), SCs were preincubated with 1 mM 5-(biotinamido)-pentylamine (Pierce Biotech, Rockford, IL) for 40 min. An aliquot of cell homogenate proteins (10 µg) in 50 µl of coating buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 5 mM EGTA) was added to each well of 96-well ELISA plate (Nunc, Apogent Com., Roskilde, Denmark) and incubated at 4 C overnight. To block the coated well, an aliquot of 200 µl of 5% BSA, 0.01% Tween 20, 0.01% sodium dodecyl sulfate (SDS) in borate buffer saline (BBS) was added. After an additional 2 h of incubation at 37 C, the plate was rinsed once with 1% BSA, 0.01% Tween-20 in BBS. To each well, 100 µl of horseradish peroxidase-conjugated streptavidin (Southern Biotech, Birmingham, AL) (1:1000) in 1% BSA, 0.01% Tween 20 in BBS was added, followed by incubating for another 1 h at room temperature (RT). After being rinsed four times with 1% BSA, 0.01% Tween 20 in BBS, the specimen in each well was incubated with 200 µl of TMB substrate solution (Sigma-Aldrich) for 10–20 min at RT. The reaction was stopped with 50 µl of 3 N HCl, and the absorbance at 450 nm was measured in a microplate spectrophotometer (Hitachi Scientific Instruments, Japan).

Purification of Membrane Proteins
Membrane protein fraction was purified using a commercial kit (Pierce Biotech) according to the manufacturer’s instructions. Briefly, SCs from each 10-cm Petri dish were collected in a 1.5-ml Eppendrof tube with 100 µl of buffer A, supplied in the kit. After 10-min incubation at RT, 200 µl of buffer B/C (diluted 1 part of reagent B with 2 parts of reagent C), supplied in the kit, were added and followed by incubation for another 30 min on ice with vortexing every 5 min. After centrifugation at 10,000 x g for 2 min at 4 C, the supernatant was transferred into a new Eppendrof tube and incubated at 37 C for 10 more min. Finally, the supernatant was further centrifuged at 10,000 x g for 2 more min at RT to separate the membrane fraction from cytosolic fraction in the hydrophobic and hydrophilic phases, respectively.

Western Blotting
The protein concentrations of cell lysates were determined by the Bradford method (51) using BSA as a standard. Proteins boiled for 5 min in SDS sample buffer [62.5 mM Tris (pH 6.7), 1.25% SDS, 12.5% glycerol, and 2.5% ß -mercaptoethanol]. Proteins were separated by SDS-PAGE on 10% gels. After being transferred to PVDF membrane, the proteins on the membrane were incubated with antibodies against G{alpha}h/tTG (NeoMarker, Newmarket, Suffolk, UK), PLC-{delta}1 (BD Transduction Laboratories, Ontario, Canada) or actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoreactive bands were visualized using the enhanced chemiluminescence system (Amersham Bioscience, Tokyo, Japan) or peroxidase chromogen kit (Biomeda Corp., Foster City, CA).

Immunoprecipitation
The membrane protein (100 µg) was diluted with 500 µl of 5-fold diluted buffer B (prepared as instructed by the manufacturer of membrane extraction kit) containing 1 mM sodium orthovanadate, 1 mM dithiothreitol (DTT), and 4% protease inhibitor cocktail (Merck Biosciences, San Diego, CA). Membrane proteins were then incubated with 2 µg of anti-G{alpha}h antibody for 2 h at 4 C followed by precipitation with 20 µl of protein A-agarose beads (Sigma-Aldrich) for 1 h at 4 C. On the other hand, the membrane proteins (100 µg) were absorbed to GTP-agarose (Sigma-Aldrich) overnight at 4 C. The immunoprecipitated or GTP-agarose-absorbed complexes/proteins were subsequently analyzed by SDS-PAGE/Western blotting using PLC-{delta}1 or G{alpha}h antibodies. Densitometric analysis of immunoreactive band intensities was performed using Imageplus software (IBM, NY).

Measurement of IP3 Production
SCs grown in wells of the six-well culture plates were treated with FSH (103 U/liter) for designated time periods or in the presence of various inhibitors. The production of intracellular IP3 was determined using HitHunter IP3 Fluorescence Polarization Assay Kits (Amersham Bioscience) according to manufacturer protocol. Briefly, the action of FSH was terminated by placing SCs on ice, and adding 0.2 N perchloric acid (0.5 ml) to lyse the cells after the removal of culture media. The culture plates were then shaken for 5 min at 650 rpm on the ELISA shaker. Twenty microliters of the cell lysate or IP3 standards ranged from 1.33 x 10 –6 to 6.7 x 10 –1 M were pipetted into 96-well black ELISA plate. IP3 tracer (20 µl) was subsequently added into each well. After shaking the plate for 5 min at 650 rpm, the IP3 binding protein (40 µl) was finally added into the plate. The fluorescence polarization of IP3 tracer (fluorescein) was detected in a microplate reader with fluorescence polarization filter, using the excitation wavelength at 485 nm and the emission wavelength at 530 nm. The intracellular IP3 concentration of each specimen was calculated from the plotted standard curve.

Peptide Synthesis
TIPWNSLKQGYRHVHLL, a peptide corresponding to PLC-{delta}1 amino acid sequence from 720–736 was synthesized at Synpep Corp. (Dublin, CA). The TIPWNSLKQGYRHVHLL peptide was myristoylated at the N terminus and purified by HPLC (purity ~ 86%). The 10 mM synthetic peptide was dissolved in one part of 0.1% trifluoroacetic acid in deionized/distilled water mixed with one part of 0.1% trifluoroacetic acid in acetonitrile, aliquoted, and kept at –70 C as stock.

Determination of Intracellular cAMP
Cell lysates in lysis buffer (provided in the cAMP ELISA kit) were collected from SC cultures after various FSH treatments. Total protein concentration of each specimen was adjusted to 0.5 mg/ml. The intracellular cAMP levels in 100 µl of cell lysates were assessed using cAMP ELISA kits (Amersham Bioscience) according to manufacturer protocol.

Statistical Analysis
Each datum obtained from three independent experiments or an experiment of triplicate assay was presented as mean ± SEM. The statistical analysis was performed by one-way ANOVA and Duncan’s Multiple Range Test.


   ACKNOWLEDGMENTS
 
We thank Dr. Haw-Ming Huang for his generosity in granting us access to his microplate reader equipped with fluorescence polarization filter.


   FOOTNOTES
 
This work was supported by NSC-89-2314-B038-022 (to Y.-H.T.), NSC-89-2314-B038-040 (to Y.-H.T.), NSC-90-2314-B038-012 (to Y.-H.T.), and NSC-93-2314-B038-027 (to Y.-H.T.), Taiwan, Republic of China.

Y.-F. Lin, M.-J. Tseng, H.-L. Hsu, Y.-W. Wu, Y.-H. Lee, and Y.-H. Tsai have nothing to declare.

First Published Online May 18, 2006

Abbreviations: BBS, Borate buffer saline; IP3, inositol 1,4,5-triphosphate; PI-PLC, phosphatidyl inositol phospholipase C; PLC, phospholipase C; RT, room temperature; SC, Sertoli cell; SDS, sodium dodecyl sulfate; tTG, tissue transglutaminase.

Received for publication August 29, 2005. Accepted for publication May 8, 2006.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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