Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Soboloff, J.
Right arrow Articles by Tsang, B.K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Soboloff, J.
Right arrow Articles by Tsang, B.K.
Agricola
Right arrow Articles by Soboloff, J.
Right arrow Articles by Tsang, B.K.
Biology of Reproduction 60, 262-271 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Acyl Chain Length-Specific Ceramide-Induced Changes in Intracellular Ca2+ Concentration and Progesterone Production Are Not Regulated by Tumor Necrosis Factor {alpha} in Hen Granulosa Cells1

J. Soboloffa,b,d, A. Soriskyc,d, M. Désiletsb, and B.K. Tsang2,a,b,d

a Reproductive Biology Unit, Departments of Obstetrics and Gynaecology, b Cellular&Molecular Medicine, and c Biochemistry, Microbiology and Immunology, University of Ottawa, d The Ottawa Hospital (Civic Campus) Loeb Research Institute, Ottawa, Ontario, Canada K1Y 4E9


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although tumor necrosis factor {alpha} (TNF-{alpha}) has long been known to be a potent inhibitor of gonadotropin-induced cytodifferentiation in the ovaries of a variety of mammalian species, its early signal transduction events are poorly understood. We previously demonstrated that TNF-{alpha} induces a small, delayed follicular stage-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in hen granulosa cells and promotes carbachol (Cch)-induced mobilization of Ca2+ from intracellular stores in cells otherwise unresponsive to the cytokine. The focus of the current study was to examine the role of ceramide in TNF-{alpha}-induced Ca2+ regulation. Treatment with exogenous sphingomyelinase (SMase; 50 mU/ml) failed to influence basal [Ca2+]i but increased the magnitude of Cch-induced Ca2+ transients. While C8-ceramide (0.03–30 µM), but not C2-ceramide (0.03–30 µM), mimicked this effect of SMase, challenge with sphingosine (3 µM) resulted in a slow and delayed increase in basal [Ca2+]i. In order to determine whether SMase is activated by TNF-{alpha} action, changes in sphingomyelin and ceramide concentrations in F1 and F5,6 granulosa cells were determined. SMase activation was not observed after 1-, 5-, 15-, and 60-min incubations with TNF-{alpha} (1–50 ng/ml) in either F1 or F5,6 cells. Exogenous SMase and C2-ceramide both inhibited LH-induced progesterone production in F1 and F5,6 cells; however, incubation with C8-ceramide resulted in increases in both basal and LH-induced progesterone. In contrast, incubation with TNF-{alpha} had no effect on either basal or LH-induced steroidogenesis. In conclusion, our findings indicate that although ceramide regulates [Ca2+]i and progesterone secretion, the sphingolipid does not appear to play a role in the action of TNF-{alpha} in avian granulosa cells. Furthermore, ceramide-mediated responses are highly dependent on acyl chain length, potentially reflecting differences in the abilities of these ceramides to access, bind to, and/or activate ceramide-dependent signal transduction mechanisms. Nonetheless, since TNF-{alpha} did not increase the production of ceramide, the physiological regulator(s) of these responses remain unknown.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although gonadotropins have long been established as primary regulators of ovarian function, immunokines such as interleukin-1ß and tumor necrosis factor {alpha} (TNF-{alpha}) are believed to regulate the growth and differentiation of follicular cells in a paracrine and/or autocrine fashion (see [1,2]). Moreover, macrophages, leukocytes, oocytes, granulosa cells, and theca cells have all been identified as sources of TNF-{alpha} within the ovary (see [3]). The action of TNF-{alpha} in the ovary is primarily anti-gonadotropic; TNF-{alpha} inhibits gonadotropin-induced follicular production of progesterone [4], estrogen [5], and androgen [6]. Furthermore, this cytokine inhibits gonadotropin-induced follicular plasminogen activator activity and suppression of rat granulosa cell DNA synthesis [7]. In addition, in the absence of FSH or LH, TNF-{alpha} stimulates prostaglandin production in granulosa cells from preovulatory follicles [8, 9]. TNF-{alpha} has recently been shown to induce apoptosis in hen large white follicle (LWF) granulosa cells and may also have a role in follicular atresia [10]. As such, immunokines are believed to be key negative regulators of follicular development and ovulation, although they may also play a role in the induction of follicular atresia.

TNF-{alpha} influences cellular responses by mechanisms that have been only partially characterized. More recently, interactions between the TNF-{alpha} receptor and cytosolic proteins—via unique cytoplasmic motifs known as death domain homology regions and/or TNF-{alpha} receptor-activated factor domains—are believed to initiate signal transduction pathways following ligand binding [11]. Furthermore, the degradation of sphingomyelin into ceramide and phosphocholine is now well demonstrated as a key early event in TNF-{alpha} action [12]. Within the ovary, ceramide-mediated TNF-{alpha} induced inhibition of P450 side-chain cleavage enzyme [13], 3ß-hydroxysteroid dehydrogenase isomerase [13], and P450 aromatase activity [14] in rat granulosa cells and TNF-{alpha}-induced apoptosis in hen LWFs [10]. Furthermore, both exogenous sphingomyelinase (SMase) and ceramide have been shown to mimic TNF-{alpha}-induced prostaglandin synthesis as well as inhibition of gonadotropin-induced progesterone production by the cytokine [13].

Several studies have demonstrated the involvement of autonomic input in the control of follicular development [15], compensatory ovarian hypertrophy [16], ovulation [17], ovarian blood flow [18], and steroidogenesis [19]. Furthermore, acetylcholine was shown to increase ovarian oxytocin [20] and progesterone [21] secretion in vivo and in vitro. We have previously demonstrated at least two mechanisms of action for muscarinic input in hen granulosa cells: increasing intracellular Ca2+ [Ca2+]i [22] and intracellular pH [23]. Furthermore, we have shown that carbachol (Cch; a muscarinic agonist)-induced Ca2+ transients are heterogeneous in nature and developmentally regulated [24]. Specifically, large inositol 1,4,5-triphosphate (IP3)-dependent increases in [Ca2+]i occurred in approximately 50% of the highly differentiated F1 granulosa cells and only 15% of the less differentiated F5,6 cells. Cch challenge resulted in small transmembrane influxes of Ca2+ in all Cch-responsive cells, although the source of this heterogeneity has not been determined. This supports the concept that IP3-mediated Cch responses are a differentiated characteristic of granulosa cells. Since the purpose of this differentiation is to prepare for ovulation, cholinergic nerves may play a role in this process via muscarinic receptor-mediated IP3-induced Ca2+ transients. The role of Ca2+, however, remains to be determined.

As indicated by our prior studies [25], TNF-{alpha} increases [Ca2+]i in hen granulosa cells in the presence and absence of Cch. Whereas TNF-{alpha} challenge increased extracellular Ca2+-dependent [Ca2+]i in approximately 50% of the undifferentiated F5,6 granulosa cells and 25% of the highly differentiated F1 cells, the cytokine facilitated slow Cch-induced Ca2+ transients in both F1 and F5,6 cells, with a more pronounced response in F1 cells. TNF-{alpha} was previously shown to increase [Ca2+]i in 30A5 preadipocytes [26], human and murine fibroblasts [27, 28], neutrophils [29, 30], anterior pituitary cells [31], and sympathetic neurons [32]. The mechanism of TNF-{alpha}-induced Ca2+ transients has never been established, although the sphingolipids sphingosylphosphorylcholine, sphingosine, and sphingosine-1-phosphate have been demonstrated to induce changes in [Ca2+]i [33, 34]. Consequently, ceramide and/or ceramide breakdown products may have a mediatory role in the observed TNF-{alpha}-induced increases in [Ca2+]i in hen granulosa cells, and perhaps other cell types.

The focus of this study was to examine the influence of TNF-{alpha} on ceramide concentration in hen granulosa cells during follicular development and to determine whether TNF-{alpha}-induced changes in [Ca2+]i were mediated by ceramide and/or ceramide breakdown products. Furthermore, the abilities of TNF-{alpha}, SMase, and ceramides to inhibit basal and gonadotropin-induced progesterone secretion were assessed. Although TNF-{alpha}-induced SMase activation was not detected, acyl chain length-dependent ceramide-induced changes in both progesterone secretion and [Ca2+]i were observed. These observations suggest distinct differences in the abilities of different ceramides to access, bind to, and/or activate ceramide-dependent signal transduction mechanisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

[{gamma}-32P]ATP, [14C]choline, Diacylglycerol Reagents Assay System, and [3H]thymidine were purchased from Amersham Life Science (Oakville, ON, Canada). Lysophosphatidic acid standard was obtained from Avanti Polar Lipids Inc. (Alabaster, AL). Acetone, ammonium formate, chloroform, formic acid, iodine, and methanol were obtained from BDH Chemicals (Toronto, ON, Canada). D-Sphingosine, N-acetyl-sphingosine (C2-ceramide), and N-myristoyl-sphingosine (C8-ceramide) were obtained from BioMol Research Laboratories (Plymouth Meeting, PA). Acetic acid, hydrochloric acid, and scintillation fluid (Scintiverse BD) were from Fisher Scientific (Ottawa, ON, Canada). TNF-{alpha} was purchased from Genzyme Diagnostics (Cambridge, MA). Fetal bovine serum (FBS), Medium 199 (M199), and minimum essential medium were obtained from Gibco Laboratories (Grand Island, NY). Ionomycin was obtained from Calbiochem (La Jolla, CA). Fura-2-acetoxymethyl ester (fura-2AM) and pluronic F-127 were obtained from Molecular Probes Inc. (Eugene, OR). Acetylcholine, Cch, ceramide, collagenase (type 1A), diethylenetriamine-pentaacetic acid (DETAPAC), dithiothreitol, dimethylsulfoxide, EGTA, fungizone, ITS supplement (insulin [5 mg/L]-transferrin [5 mg/L]-selenite [5 µg/L]), imidazole/HCl, lithium chloride, lysophosphatidylcholine standard, N-hexanoyl-sphingosine (C6-ceramide), penicillin-streptomycin, phosphatidic acid, SMase (Bacillus cereus), SM-phosphatidylcholine standard, and trypsin inhibitor (type II-s) were obtained from Sigma Chemical Co. (St. Louis, MO). Hepes was obtained from VWR Canada Ltd. (Ottawa, ON, Canada). Progesterone anti-serum was a gift from Dr. D.T. Armstrong (University of Western Ontario, London, ON, Canada).

Hen Granulosa Cell Isolation

White leghorn hens caged individually in a windowless, air-conditioned room with a 14L:10D cycle were killed by cervical dislocation 10–14 h before the expected time of ovulation. Granulosa cell layers from the largest (F1) and smallest (F5,6) follicles recruited into the follicular hierarchy were removed and dispersed by incubation at 37°C in M199 containing collagenase (type 1A; 170 U/ml) and trypsin inhibitor (type II-s; 0.01% w:v) for 10, 20, and 25 min, respectively, as described by Asem et al. [35]. The M199 was supplemented with Hepes (6.0 mg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and fungizone (0.625 µg/ml).

Sphingomyelin Assay

Granulosa cell sphingomyelin content was assessed using a modification of the sphingomyelin assay [36]. Granulosa cells (3 x 105) were plated in 24-well plates (Falcon Plastics, Los Angeles, Ca) in minimum essential medium containing NaHCO3 (2.2 mg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and fungizone (0.625 µg/ml) (hereafter referred to as MEM) with FBS (10%) for 4–6 h and subsequently maintained in serum- and choline-free MEM supplemented with ITS and [14C]choline chloride (2 µCi/ml) for 66 h. Cells were then treated with TNF-{alpha} (0.1–50 ng/ml) or exogenous SMase (50 mU/ml) for 1–60 min without medium change, since changing medium is known to induce sphingomyelin hydrolysis [37]. At the end of the culture period, medium was removed. The cells were scraped in 0.5 ml HCl (2.4 N) with a rubber policeman and then transferred to polypropylene tubes. The wells were rinsed with 125 µl NaCl (1.0 M), which was then added to the HCl extracts. The samples were then separated using a modified Bligh-Dyer extraction procedure [38]. Briefly, 0.75 ml chloroform:methanol (1:2) followed by 0.5 ml chloroform was added to the samples, which were subsequently vortexed (~5 sec) and centrifuged (207 x g; 5 min). The chloroform layer was then transferred to new polypropylene tubes, replaced with 0.75 ml chloroform, and vortexed (~5 sec). After centrifugation (207 x g; 5 min), the organic (lower) phase was collected and added to the original chloroform extract. The extracts were then dried under N2, resuspended in 25 µl chloroform:methanol (2:1), spotted onto thin layer chromatography (TLC) plates (Fisher Scientific), and developed in chloroform:methanol:acetic acid:water (50:30:8:5 [36]). The sphingomyelin and phosphatidylcholine spots were located using a phosphorimager (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada) and by comparison to unlabeled lipid standards (after iodination). The amount of radioactivity in the spots was quantified with two-dimensional (2D) densitometry (Molecular Analyst Software, Bio-Rad Laboratories Ltd.). To correct for possible variation in loading between samples, sphingomyelin concentrations were normalized by total phosphatidylcholine.

Ceramide Assay

Cell culture and lipid extraction Granulosa cell ceramide content was assessed using a modification of the diaclyglycerol (DAG) kinase assay [36]. Granulosa cells (7 x 105) were plated for 4–6 h in 12-well plates (Falcon Plastics) in MEM containing FBS (10%) and subsequently maintained in serum-free MEM supplemented with ITS for 66 h. Cells were then treated with TNF-{alpha} (0.1–50 ng/ml) or exogenous SMase (50 mU/ml) for 1–60 min without medium change. At the end of the culture period, medium was removed and cells were scraped in 0.6 ml NaCl (1.0 M) with a rubber policeman. The wells were then rinsed with 200 µl NaCl (1.0 M), which was added to the original sample. The lipids were then separated using a standard Bligh-Dyer extraction [38]. Briefly, 3 ml chloroform:methanol (1:2) was added to the samples, which were subsequently vortexed (~5 sec). Chloroform (1 ml) and NaCl (1 ml; 1.0 M) were then added and the samples were vortexed (~5 sec) and centrifuged (207 x g; 5 min). The chloroform layer was then replaced with 2 ml of fresh chloroform and vortexed (~5 sec). After centrifugation (207 x g; 5 min), the lower phase was removed and added to the original chloroform extracts.

DAG kinase assay The Diacylglycerol Assay Reagents System was used to radiolabel lipids in extracts to quantify cellular ceramide. This kit included diacylglycerol kinase, which phosphorylates diacylglycerol, ceramide, and monoacylglycerol to produce phosphatidic acid, ceramide-phosphate, and lysophosphatidic acid, respectively. Samples collected as described in the previous section were dried under N2 and resuspended in 20 µl of a detergent solution (n-octyl-ß-glucopyranoside [7.5% w:v], cardiolipin [5 mM] in DETAPAC [1 mM]; vortexed [~10 sec] and sonicated [2 min prior to use]) by mixing (vortexing; ~5 sec) and sonication (2 min). The resulting suspension was incubated (30 min; room temperature [RT]) with 70 µl of "reagent mix" (10 µl of DAG kinase preparation [a suspension of E. coli membranes enriched in diacylglycerol kinase of undefined activity] dissolved in a potassium phosphate buffer [5 mM] containing glycerol [10%], imidazole/HCl [5 mM], DETAPAC [0.5 mM], and mercaptoethanol [1 mM], pH 6.8; 50 µl of Diacylglycerol Reagents Assay System Assay Buffer [Amersham Life Sciences] containing imidazole/HCl [0.1 M], NaCl [0.1 M], MgCl2 [25 mM], and EGTA [2 mM], pH 6.6, and 10 µl of dithiothreitol [0.02 M]) and 10 µl of a tracer solution (ATP [5 mM], imidazole/HCl [100 mM], DETAPAC [1 mM], and [{gamma}-32P]ATP [1.0 µCi]). Twenty microliters of perchloric acid (PCA; 1% v:v) and 450 µl of chloroform/methanol (1:2 v:v) were added; the tubes were incubated (10 min; RT), centrifuged (2000 x g; 2 min), mixed with 150 µl chloroform and 150 µl PCA, and vortexed (three 5-sec bursts). After centrifugation (2000 x g; 2 min), the aqueous (upper) phase was removed, replaced with 1 ml PCA, and vortexed (three 5-sec bursts). This washing step was again repeated. The chloroform extracts were then dried under N2, resuspended in 25 µl chloroform:methanol (95:5), spotted onto TLC plates (Fisher Scientific), and developed in chloroform:methanol:acetone:acetic acid:water (10:4:3:2:1 [36]). The ceramide-phosphate, phosphatidic acid, and lysophosphatidic acid spots were located using a phosphorimager (Bio-Rad Laboratories Ltd.) and by comparison to unlabeled lipid standards (after iodination). The amount of radioactivity in the spots was quantified with 2D densitometry (Molecular Analyst Software; Bio-Rad Laboratories Ltd.).

[Ca2+]i Measurement

Granulosa cells (1 x 105) were plated for 4–6 h on glass coverslips in 1 ml of MEM and FBS (10%) and cultured subsequently for 17–48 h in MEM without FBS for an additional 17–48 h. Cells were loaded with fura-2 (30 min; 37°C) in a normal buffered solution (NBS; 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.1 mM MgCl2, 2.6 mM dextrose, and 10 mM Hepes) containing pluronic F-127 (0.00125% w:v) and fura-2AM (2.5 µM) according to Morley et al. [22]. After three rinses with NBS, cells were incubated for an additional 10–60 min in NBS to ensure full hydrolysis of fura-2AM. Excitation spectra (320–400 nm), run to verify proper loading, were monophasic and peaked around 355 nm.

Experiments were conducted on single cells or groups of 6 cells or less. Plated cells were placed in a water bath custom-fitted to the stage of an inverted epifluorescence microscope (Nikon Diaphot; Nikon Instr., Garden City, NY) equipped with a x40 objective. The cells were superfused (0.5 ml/min; RT) through a pipette tip held in place above and behind the cell being examined (< 0.1 ml dead space) for rapid exchange of solution. Measurements were performed using 350-nm and 380-nm excitation wavelengths at a sampling rate of 10 Hz. Fura-2 fluorescence was monitored through a photomultiplier tube with emissions centered at 505 nm. Background fluorescence, determined from unloaded cells, was about one tenth that of fura-2-loaded cells and was subtracted prior to [Ca2+]i determination.

The concentration of intracellular free Ca2+ was calculated according to the following formula [39]:

where R is the ratio of the fluorescence intensities measured at 350 nm and 380 nm during the experiments and F is the fluorescence intensity measured at 380 nm. Rmin, Rmax, Fmin, and Fmax were determined from in situ calibration of unlysed cells using 4 µM ionomycin in the absence (Rmin and Fmin; 10 mM EGTA in Ca2+-free NBS) and presence (Rmax and Fmax) of Ca2+. Kd (135 nM) is the dissociation constant for fura-2 at room temperature. Rmin and Rmax were found to be 0.98 ± 0.047 and 11.03 ± 0.50, respectively, while Fmin/Fmax was found to be 7.46 ± 0.27. In addition to in situ calibration, cells were lysed with digitonin (20 µM) to determine the extent of fura-2 sequestration. Consistent with that observed in other systems [40], a decrease of 82.9 ± 1.6% (n = 7) was observed within 10 min of exposure to the detergent. These values were not significantly different between stages of maturation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of Basal and Cch-Induced [Ca2+]i by Sphingomyelin Metabolites

The primary action of SMase is to degrade sphingomyelin to produce ceramide. The typical effects of exogenous SMase, ceramide analogues, and sphingosine on [Ca2+]i in granulosa cells are depicted in Figure 1. While exogenous SMase had no significant effect on basal [Ca2+]i (SMase-induced Ca2+ transients were observed in only 1 of 24 cells), Cch-induced Ca2+ transients in the presence of SMase were larger than those observed in the absence of this enzyme in both F1 (5 of 11 cells studied) and F5,6 cells (6 of 13 cells studied). Unlike TNF-{alpha}, which only increased the magnitude of small Cch-induced Ca2+ transients, SMase appeared able to increase the magnitude of both small (< 250 nM; Fig. 1A) and large (> 250 nM; Fig. 1B) Cch-induced Ca2+ transients. As ceramide itself is too hydrophobic to be delivered to cells in aqueous solution, the short-chain ceramide analogues C2-ceramide (3 µM) and C8-ceramide (3 µM) were used to determine whether exogenous ceramide may mimic the actions of TNF-{alpha} or TNF-{alpha}-Cch on [Ca2+]i. While C2-ceramide had no apparent effect on basal or Cch-induced [Ca2+]i (Fig. 1C; n = 12), pretreatment with C8-ceramide enhanced Cch-induced Ca2+ transients in a manner similar to exogenous SMase, although changes in basal [Ca2+]i were not observed (Fig. 1D; n = 7).



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 1. The effect of exogenous SMase and sphingomyelin metabolites on basal and Cch-induced Ca2+ transients in hen granulosa cells. A) Influence of SMase on slow Cch-induced Ca2+ transients (changes in [Ca2+]i less than 250 nM; n = 22). B) Influence of SMase on fast Cch-induced Ca2+ transients (changes in [Ca2+]i greater than 250 nM; n = 11). C) Influence of C2-ceramide on slow Cch-induced Ca2+ transients (changes in [Ca2+]i less than 250 nM; n = 12). D) Influence of C8-ceramide on slow Cch-induced Ca2+ transients (changes in [Ca2+]i less than 250 nM; n = 7). E) Sphingosine (3 µM) induced an approximately 343 ± 83 nM increase in [Ca2+]i at a rate of 7.83 ± 2.58 nM/sec in both F1 and F5,6 granulosa cells (n = 9). Ca2+ transients illustrated within each panel were recorded from the same cell separated by a minimum of 4-min washout. Exposures of cells to 50 mU/ml SMase and 3 µM concentrations of C2-ceramide, C8-ceramide, and 0.2 mM Cch are indicated by their respective horizontal bars.

Sphingosine, a product of ceramide degradation, has been shown to increase [Ca2+]i in several different systems [33, 34]. In F1 and F5,6 granulosa cells, sphingosine (3 µM) induced large increases in [Ca2+]i {Delta}[Ca2+]i = 343 ± 83 nM; n = 9) with a relatively slow rate of rise (d[Ca2+]i/dt = 7.83 ± 2.58 nM/sec; n = 9) and a characteristic delay (Fig. 1E). Although the magnitude of sphingosine-induced Ca2+ transients was much greater than observed in response to TNF-{alpha} [34], [Ca2+]i increased at a similar rate in response to both factors. This supports the concept that sphingosine, if produced locally at appropriate concentrations, could mediate TNF-{alpha}-induced Ca2+ transients.

Effect of TNF-{alpha} on SMase Activity in HenGranulosa Cells

After a preincubation period of 66 h in the presence or absence of [14C]choline, F1 granulosa cells were challenged with vehicle or TNF-{alpha} (0.1, 1, 10, or 50 ng/ml) to determine whether the cytokine activates SMase. No significant changes in sphingomyelin or ceramide concentration were observed either at 60 min of exposure to TNF-{alpha} (Fig. 2) or during shorter incubation periods (1, 5, and 15 min; data not shown). In contrast, these cells responded to exogenous SMase with a significant decrease in sphingomyelin content (28 ± 6.6%; n = 3) and a concomitant rise in ceramide concentration (1138 ± 206%; n = 7; Fig. 2). Since TNF-{alpha}-induced Ca2+ transients were developmentally regulated, the influence of TNF-{alpha} on sphingomyelin and ceramide contents were also determined in F5,6 cells (data not shown). Neither sphingomyelin (TNF-{alpha} = 10 ng/ml; 105 ± 28% over control, n = 3) nor ceramide content (TNF-{alpha} = 1 and 50 ng/ml; 90 ± 16% and 83 ± 29% compared to control, respectively; n = 3) was significantly affected by the presence of the cytokine. Finally, since the sphingomyelin protocol required a lengthy preincubation, the possibility that TNF-{alpha} (10 ng/ml) could induce ceramide production was assessed in cells preincubated for only 24 h (data not shown). After a 60-min incubation, TNF-{alpha}-induced ceramide production was 119 ± 22% (n = 2) in F1 cells and 90 ± 14% (n = 2) in F5,6 cells. As TNF-{alpha} and Cch interact in the regulation of [Ca2+]i, the possibility that Cch potentiates the TNF-{alpha}-induced SMase activation was investigated with F1 granulosa cells after Cch (0.2 mM) challenge. No significant changes in sphingomyelin or ceramide content were observed irrespective of the duration of incubation (1, 5, or 15 min; data not shown).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2. TNF-{alpha} did not activate a SMase in F1 granulosa cells after 60-min incubation. A) The effect of TNF-{alpha}. B) The effect of exogenous SMase. * Indicates a significant difference.

Effect of TNF-{alpha} on Granulosa Cell Progesterone Production during Follicular Development

While TNF-{alpha} is believed to inhibit LH-induced steroidogenesis in rat granulosa cells via ceramide production [14], the role of TNF-{alpha} and the possible involvement of the SMase pathway in the regulation of hen progesterone concentration have not been studied. In the present studies, F1 and F5,6 granulosa cells were incubated for 24 h with TNF-{alpha} (10 ng/ml) or SMase (50 mU/ml) in the absence or presence of LH (1, 5, 10, 50, 100, or 500 ng/ml), and progesterone secretion was assessed (Fig. 3). Although TNF-{alpha} (10 ng/ml) had no influence on basal or LH-induced granulosa cell progesterone production, exogenous SMase (50 mU/ml) inhibited this response at low concentrations (1–10 ng/ml or 5–50 ng/ml; F1 or F5,6 cells, respectively) but not high concentrations (50–500 ng/ml or 100–500 ng/ml; F1 or F5,6 cells, respectively) of the gonadotropin. The lack of effect of TNF-{alpha} on basal and LH-stimulated granulosa cell steroid production was independent of the concentration of the cytokine and the stage of follicular maturation (Table 1).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 3. SMase, but not TNF-{alpha}, inhibited LH-induced progesterone production in hen granulosa cells during follicular development. F1 (A, C) and F5,6 (B, D) granulosa cells (2.5 x 105) were cultured with vehicle, SMase, or TNF-{alpha} in the presence or absence of LH (1–500 ng/ml) for 24 h (n = 5). Progesterone secretion was determined by RIA of spent medium; % inhibition (C, D) was calculated by dividing the TNF-{alpha}- and SMase-induced progesterone content in the presence of LH (1–500 ng/ml) by the quantity of progesterone secreted in response to each respective concentration of LH. Asterisk represents significant difference from control (p < 0.05).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Granulosa cell progesterone production in the presence or absence of varying concentrations of TNF-{alpha} and optimal concentrations of LH.a

Since exogenous SMase inhibited LH-induced progesterone production, the abilities of ceramides of different acyl chain length (C2-, C6-, and C8-ceramide) and of sphingosine to inhibit LH (10 ng/ml)-induced progesterone production were also assessed to confirm that the effects of SMase were mediated via ceramide and not through nonspecific actions of the enzyme (Fig. 4). At concentrations of 3 µM, neither ceramide nor sphingosine influenced granulosa cell progesterone secretion irrespective of the presence of LH (10 ng/ml) or the stage of follicular development. At a higher concentration (30 µM), however, C2-ceramide inhibited LH (10 ng/ml)-induced progesterone production in F5,6 but not F1 cells. Moreover, exposure of either F1 or F5,6 granulosa cells to 30 µM C6- or C8-ceramide resulted in increases in basal progesterone production in a manner similar to LH (Fig. 4). Furthermore, C8-ceramide enhanced LH-induced progesterone production in F5,6 but not F1 cells, while C6-ceramide had no effect on LH-induced steroidogenesis. Sphingosine at 30 µM was not studied as this concentration was highly cytotoxic, resulting in immediate cell death (unpublished results).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4. The effect of ceramide analogues and sphingosine on basal and LH-induced progesterone production in hen granulosa cells during follicular development. Progesterone production was assessed by RIA of spent medium from F1 or F5,6 cells (2.5 x 105) cultured 24 h in the presence of vehicle, C2- C6- or C8-ceramide (3 or 30 µM), or sphingosine (Sph; 3 µM). A) Basal progesterone production in F5,6 granulosa cells (n = 3). B) LH (10 ng/ml)-induced progesterone production in F5,6 granulosa cells (n = 3). C) Basal progesterone production in F1 granulosa cells (n = 3). D) LH (10 ng/ml)-induced progesterone production in F1 granulosa cells (n = 3). a: Significant difference (p < 0.05) from control; b: significant difference (p < 0.05) from the corresponding 3 µM ceramide concentration.

Changes in [Ca2+]i induced by TNF-{alpha}, SMase, or C8-ceramide required the presence of Cch. Consequently, the possibility that interactions between these agonists may also regulate progesterone secretion was assessed (Fig. 5). Consistent with results from prior studies [22], exposure of granulosa cells to Cch (0.2 mM) had no effect on granulosa cell progesterone production in F1 or F5,6 cells irrespective of the presence of LH. Furthermore, unlike interactions related to [Ca2+]i, the presence of Cch had no influence on progesterone secretions in the presence of TNF-{alpha}, SMase, C2-, C6-, C8-ceramide, or sphingosine as assessed by two-way ANOVA (Fig. 5).



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 5. The effect of Cch on progesterone production in the presence and absence of LH, TNF-{alpha}, SMase, or sphingomyelin metabolites. Progesterone production was assessed by RIA of spent medium from F1 or F5,6 cells (2.5 x 105) cultured 24 h in the presence of vehicle or various treatments. A) Progesterone production in F5,6 granulosa cells (n = 3). B) Progesterone production in F1 granulosa cells (n = 3). a and b: Significant difference from control and from each other (p < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary purpose of this study was to determine whether SMase mediates TNF-{alpha}-induced changes in [Ca2+]i and progesterone production in hen granulosa cells. Exogenous SMase, C8-ceramide, and sphingosine mimicked TNF-{alpha}-induced changes in [Ca2+]i, while C2-ceramide was ineffective. Nonetheless, TNF-{alpha}-induced SMase activity was not detected either early or late in follicular development. This concept that TNF-{alpha} action is independent of SMase activity in hen granulosa cells was further supported by the fact that exogenous SMase and C2-ceramide inhibited LH-induced progesterone production and that C6- and C8-ceramide stimulated progesterone production, while TNF-{alpha} challenge failed to influence granulosa cell steroidogenesis. Consequently, it can be concluded that although sphingolipids mimic TNF-{alpha}-induced changes in [Ca2+]i, TNF-{alpha} does not activate a SMase in this cell type. Consequently, the physiological modulator of SMase in hen granulosa cells remains to be determined.

In the current study, neither F1 nor F5,6 granulosa cells responded to TNF-{alpha} challenge with increases in ceramide production or steroidogenesis. Nonetheless, a prior study from this laboratory showed that granulosa cells from both developmental stages respond to TNF-{alpha} with increases in both basal and Cch-induced [Ca2+]i [25]. Moreover, additional studies of TNF-{alpha}-transforming growth factor {alpha} interactions in the regulation of F5,6 granulosa cell survival and integrin production have been performed (unpublished results). Consequently, we are confident that the lack of effect of TNF-{alpha} on either ceramide or progesterone production is not related to either a lack of TNF-{alpha} receptors or the quality of TNF-{alpha} preparations.

Ceramide is known to result in mutually contradictory changes in cell survival, cell cycle progression, and differentiative character in several different cell types. Ceramide is produced after exposure of U937 cells to TNF-{alpha}; and both C2-ceramide [41] and natural ceramide [42] induce apoptosis in this cell line in a manner similar to the cytokine, suggesting a mediatory role for ceramide in TNF-{alpha}-induced apoptosis. C8-ceramide and exogenous SMase induce apoptosis in granulosa cells of F1 and LWF hen follicles [10], while C2-ceramide induced apoptosis in early antral rat follicles [43]. In contrast, ceramide induces cell cycle arrest in HL-60 cells [44] and Molt-4 leukemia cells [45], as well as in fibroblasts [46], where the sphingolipid is also associated with cellular senescence [47]. Finally, ceramide also induces proliferation via mitogen-activated protein kinase activation in HL-60 cells [48] and fibroblasts [49] or via activation of NF-{kappa}B and protein kinase C{varsigma} in Jurkat T cells [50]. These findings support the concept that the ceramide-induced responses are highly variable and may differ depending not only upon the cell type but also upon the physiological conditions of each independent study, and may reflect the involvement of different effector systems in ceramide signaling.

TNF-{alpha}-induced ceramide production has been studied previously in both mammalian and avian ovaries. In rat preantral granulosa cells, the cytokine induces ceramide production [14], while Fas, a member of the TNF-{alpha} family, induced ceramide production in theca cells [51]. Although TNF-{alpha}-induced ceramide production has never been reported in the hen, TNF-{alpha} and C8-ceramide induced apoptosis in LWF granulosa cells, while only C8-ceramide induced apoptosis in F1 granulosa cells [10]. Moreover, preliminary data from our laboratory demonstrate that pharmacological concentrations of TNF-{alpha} (50 ng/ml) do induce a small increase in ceramide production in LWF granulosa cells (138 ± 8.7%, n = 2; data not shown). Since TNF-{alpha}-induced ceramide production in F5,6 and F1 cells was not observed even at 50 ng/ml (Fig. 2), this suggests the presence of a developmentally regulated switch in either TNF-{alpha} receptor subtype or the expression of SMase. Although additional study would be required to confirm this hypothesis, this concept is consistent with current developmental models, since a large proportion of LWFs become atretic in vivo, while follicles within the follicular hierarchy are committed to ovulation.

Our finding that both C6- and C8-ceramide stimulated basal progesterone production was the first demonstration of ceramide-induced steroidogenesis in any system. In contrast, prior studies showed that C8-ceramide induced apoptosis in hen granulosa cells [10], suggesting that apoptosis and progesterone production are two independent processes. In rat granulosa cells, both C2- and C6-ceramide inhibited LH-induced progesterone production [13, 14] without ceramide-induced increases in basal progesterone production (Fig. 4). TNF-{alpha}-induced ceramide production in rats is believed to mediate the inhibition of gonadotropin-induced steroidogenesis by TNF-{alpha} [13, 14]. Since TNF-{alpha} did not induce the production of ceramide in hen granulosa cells (Fig. 2), it is not surprising that TNF-{alpha} did not inhibit gonadotropin-induced steroidogenesis. In the rat theca, TNF-{alpha} stimulates basal progesterone production [4] and inhibits hCG binding [52] in vitro. Unlike the situation in mammalian systems, granulosa cells in the hen are the primary producers of progesterone and androstenedione, while the theca produces estrogens [53]. Consequently, ceramide-induced changes in hen granulosa cell steroidogenesis may better reflect the theca of mammalian follicles. Irrespective of the implications of ceramide-induced ovarian progesterone production in other species, the current study suggests that ceramide is a key signaling molecule in the regulation of granulosa cell progesterone production. Although several different activators of the SMase pathway have been identified in other systems, the activators of this signal transduction pathway in hen granulosa cells have not been identified. Nonetheless, the fact that ceramide regulates both granulosa cell progesterone secretion and [Ca2+]i reveals the presence of ceramide-regulated signal transduction mechanisms, suggesting that activators of this pathway are likely to exist.

Although ceramide-induced changes in Cch-induced Ca2+ transients occurred throughout follicular development, differences in progesterone production by F5,6 and F1 granulosa cells were observed. Specifically, C8-ceramide stimulated LH-induced progesterone production by F5,6 but not F1 cells. Consistent with prior studies [54], progesterone production by F1 cells was approximately 10 times higher than that observed in F5,6 cells. Furthermore, 10 ng/ml LH was nearly maximally stimulatory for F1 cells but was near the threshold for stimulation of F5,6 cells (Fig. 3). Consequently, this follicular stage-dependent difference in C8-ceramide-induced progesterone production in the presence of LH may reflect the fact that F1 granulosa cells reached their maximum for progesterone secretion while F5,6 cells did not. This concept is supported by the spare receptor theory, which demonstrates that exposure of granulosa cells to pharmacological levels of LH or forskolin results in concomitant increases in cAMP production but no additional increases in steroid secretion.

Activation of the SMase pathway resulted in changes in both basal and Cch-induced [Ca2+]i (Fig. 1). Since TNF-{alpha} did not activate SMase (Fig. 2), the similarities between these observations and our previous findings of TNF-{alpha}-induced Ca2+ transients [25] may merely be coincidental. Nonetheless, although sphingosine-induced Ca2+ transients have been previously reported to occur via mobilization of Ca2+ from intracellular stores [33, 34], this study represents the first demonstration that ceramide regulates [Ca2+]i. Cch-induced Ca2+ transients occur via IP3-mediated mobilization of Ca2+ from intracellular stores and/or transmembrane influx of Ca2+ [22, 24]. Similar to results with TNF-{alpha}, pretreatment with either C8-ceramide or SMase stimulated slow Cch-induced Ca2+ transients, raising the interesting possibility that ceramide may facilitate the filling of Ca2+ stores and/or Cch-induced IP3 production. Unlike treatment with the cytokine, however, SMase treatment resulted in increases in fast Cch-induced Ca2+ transients. IP3-induced Ca2+ efflux occurs in an all-or-none fashion [55]. These studies are consistent with the concept that ceramide-induced stimulation of Cch-induced Ca2+ transients occurs as a result of increased loading of Ca2+ stores; however, additional studies are required to confirm this conclusion.

Although sphingosine-induced changes in progesterone concentration were not observed, challenge with sphingosine resulted in increases in [Ca2+]i (Fig. 1E). TNF-{alpha}-induced sphingosine production resulting from activation of both SMase and ceramidase has been demonstrated in cardiac myocytes [56]. If similar TNF-{alpha}-regulated mechanisms occur in granulosa cells, TNF-{alpha}-induced ceramidase activity could result in rapid degradation of ceramide, masking the SMase activity. Nonetheless, since decreases in sphingomyelin concentration would still occur, it is unlikely that sphingosine is produced in hen granulosa cells. Consequently, the similarities between sphingosine-induced Ca2+ transients (Fig. 1E) and TNF-{alpha}-induced Ca2+ transients [25] are likely only coincidental.

The role of Ca2+ in steroidogenesis has been extensively studied. There have been numerous studies showing that either the removal of extracellular Ca2+ with the Ca2+ chelator EGTA, or the addition of Ca2+ channel blockers (verapamil, Mn2+, or Co2+), can markedly inhibit FSH-, LH-, GnRH-, or cAMP-induced steroidogenesis in hen, rat, and swine granulosa cells (see [57]). Nonetheless, since Cch [22], ATP [58], and cholecystokinin [59] induce Ca2+ transients but not progesterone production, the role of Ca2+ in steroidogenesis is facilitatory rather than direct. Similarly, since ceramide and Cch interacted to induce Ca2+ transients (Fig. 1) but not progesterone production (Fig. 5), it can be concluded that Ca2+ has no role in ceramide-induced changes in steroidogenesis.

Although several studies have demonstrated that ceramide responses are acyl chain length dependent, the present study is the first to show that ceramides of differing acyl chain length can elicit different responses within the same cell type. C8-ceramide induces apoptosis in hen granulosa cells while C2-ceramide is ineffective [10]. In addition, activation of heterotrimeric protein phosphatase 2A (PP2A) from either bovine brain or heart by ceramide was dependent on acyl chain length in a cell-free system: C10- > C6- > C2-ceramide while C18-ceramide had no effect [60]. The acyl chain of sphingomyelin, the substrate for SMase, is normally 22 or 26 carbons in length. Consequently, the fact that C18-ceramide did not activate PP2A is surprising, since it is the ceramide analogue most similar to membrane ceramide. Nonetheless, the presence of C2-ceramide in HL-60 cells was recently reported [61], suggesting that short-chain ceramides may also be produced in vivo. If this is true, the differences in granulosa cell responses to different ceramide analogues noted in the present study may be physiologically relevant, irrespective of their relationship to the SMase pathway.

Although there is no evidence to date showing the differential subcellular accumulation of ceramides of various acyl chain lengths, different cellular sites of ceramide production have been demonstrated. Linardic and Hannun [62] have shown that only sphingomyelin in the inner leaflet of the plasma membrane is degraded by the neutral SMase. Although exogenous bacterial SMase was unable to induce apoptosis, it is apoptogenic when cloned into a mammalian expression vector and induced in the same cell line [63]. In addition, TNF-{alpha}-induced acidic SMase activity is present in endosomal/lysosomal compartments [64], and ceramide produced from the acidic, but not neutral, SMase can activate NF-{kappa}B [64–66]. This suggests the possibility that the acyl chain length dependence of ceramide responses may be due to differences in their intracellular location, resulting in the activation of different ceramide-induced signal transduction pathways.

In conclusion, although TNF-{alpha} does not induce ceramide production, ceramide analogues regulate hen granulosa cell progesterone production and [Ca2+]i during follicular development in an acyl chain length-dependent manner. Considering the established differences between ceramide responses in different conditions and cell types, the acyl chain length dependence of ceramide responses may reflect distinct differences in their abilities to access, bind to, and/or activate ceramide-dependent signal transduction mechanisms. Prior studies have shown a role for ceramide as an inducer of cell death in hen granulosa cells; however, the current studies demonstrate the ability of ceramide to modulate granulosa cell [Ca2+]i and progesterone production. As such, these studies provide novel insight into a role for ceramide as a regulator of granulosa cell differentiation.


   FOOTNOTES
 
1 This work was supported by the Medical Research Council of Canada (MRC grants MT-10369 to B.K.T. and MA-9690 to M.D.). J.S. is a recipient of a Natural Science and Engineering Research Council Studentship. Back

2 Correspondence: Benjamin K. Tsang, Reproductive Biology Unit, Department of Obstetrics&Gynaecology, The Ottawa Hospital (Civic Campus), 1053 Carling Ave., Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5365; ben{at}civich.ottawa.on.ca Back

Accepted: September 3, 1998.

Received: June 10, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ackland JF, Schwartz NB, Mayo KE, Dodson RE. Nonsteroidal signals originating in the gonads. Physiol Rev 1992; 72:731–787.[Abstract/Free Full Text]
  2. Leung PCK, Steele GL. Intracellular signalling in the gonads. Endocr Rev 1992; 13:476–497.[Abstract]
  3. Chen H, Marcinkiewicz JL, Sancho-Tello M, Hunt JS, Terranova PF. Tumor necrosis factor-{alpha} gene expression in mouse oocytes and follicular cells. Biol Reprod 1993; 48:707–714.[Abstract]
  4. Roby KF, Terranova PF. Effects of tumor necrosis factor-{alpha} in vitro on steroidogenesis of healthy and atretic follicles of the rat: theca as a target. Endocrinology 1990; 126:2711–2718.[Abstract]
  5. Adashi EY, Resnick CE, Croft CS, Payne DW. Tumor necrosis factor {alpha} inhibits gonadotropin hormonal action in nontransformed ovarian granulosa cells. A modulatory non-cytotoxic property. J Biol Chem 1989; 264:11591–11597.[Abstract/Free Full Text]
  6. Andreani CL, Payne DW, Packman JN, Resnick CE, Hurwitz A, Adashi EY. Cytokine-mediated regulation of ovarian function. Tumor necrosis factor {alpha} inhibits gonadotropin-supported ovarian androgen biosynthesis. J Biol Chem 1991; 266:6761–6766.[Abstract/Free Full Text]
  7. Karakji EG, Tsang BK. Tumor necrosis factor alpha inhibits rat granulosa cell plasminogen activator activity in vitro during follicular development. Biol Reprod 1995; 52:745–752.[Abstract]
  8. Veldhuis JD, Garmey JC, Urban RJ, Demers LM, Aggarwal BB. Ovarian actions of tumor necrosis factor-{alpha} (TNF{alpha}): pleiotropic effects of TNF{alpha} on differentiated functions of untransformed swine granulosa cells. Endocrinology 1991; 129:641–648.[Abstract]
  9. Zolti M, Meirom R, Shemesh M, Wollach D, Mashiach S, Sore L, Rafael ZB. Granulosa cells as a source and target organ for tumor necrosis factor-alpha. FEBS Lett 1990; 261:253–255.[CrossRef][Medline]
  10. Witty JP, Bridgham JT, Johnson AL. Induction of apoptotic cell death in hen granulosa cells by ceramide. Endocrinology 1996; 137:5269–5267.[Abstract]
  11. Baker SJ, Reddy EP. Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 1996; 12:1–9.[Medline]
  12. Pushkareva M, Obeid L, Hannun YA. Ceramide: an endogenous regulator of apoptosis and growth suppression. Immunol Today 1995; 16:294–297.[CrossRef][Medline]
  13. Santana P, Llanes L, Hernandez I, Gonzalezrobayna I, Tabraue C, Gonzalezreyes J, Quintana J, Estevez F, Degalarreta CMR, Fanjul LF. Interleukin-1-beta stimulates sphingomyelin hydrolysis in cultured granulosa cells—evidence for a regulatory role of ceramide on progesterone and prostaglandin biosynthesis. Endocrinology 1996; 137:2480–2489.[Abstract]
  14. Santana P, Llanes L, Hernandez I, Gallardo G, Quintana J, Gonzalez J, Estevez F, Ruiz de Galarreta C, Fanjul LF. Ceramide mediates tumor necrosis factor effects on P450-aromatase activity in cultured granulosa cells. Endocrinology 1995; 136:2345–2348.[Abstract]
  15. Brink CI, Grob HS. Response of the denervated mouse ovary to exogenous gonadotropins. Biol Reprod 1972; 9:108.
  16. Burden HW, Lawrence IE Jr. The effect of denervation on compensatory ovarian hypertrophy. Neuroendocrinology 1977; 23:368:378.[Medline]
  17. Bahr JM, Kao L, Nalgandov AV. The role of catecholamines and nerves in ovulation. Biol Reprod 1974; 10:273–290.[CrossRef][Medline]
  18. Gibson WR, Roche PJ. Blood flow in the ovary and oviduct after sympathetic denervation. J Reprod Fertil 1986; 78:193–199.[Abstract]
  19. Kawakami M, Kubo K, Nagase M, Hayani R. Involvement of ovarian innervation in steroid secretion. Endocrinology 1981; 109:136–145.[Abstract]
  20. Heap RB, Fleet IR, Davis AJ, Goode JA, Hamon MH, Walters DE, Flint APF. Neurotransmitters and lymphatic-vascular transfer of prostaglandin F2{alpha} stimulate ovarian oxytocin output in sheep. J Endocrinol 1989; 122:147–159.[Abstract]
  21. Luck MR. Cholinergic stimulation through muscarinic receptors of oxytocin and progesterone secretion from bovine granulosa cells undergoing spontaneous luteinization in serum-free culture. Endocrinology 1990; 126:1256–1263.[Abstract]
  22. Morley P, Tsang BK, Whitfield JF, Schwartz J-L. The effect of muscarinic cholinergic agonists on intracellular calcium and progesterone production by chicken granulosa cells. Endocrinology 1992; 130:663–670.[Abstract]
  23. Li M, Morley P, Schwartz J-L, Whitfield JF, Tsang BK. Muscarinic cholinergic stimulation elevates intracellular pH in chicken granulosa cells by a Ca2+-dependent, Na+-independent mechanism. Endocrinology 1992; 131:235–239.[Abstract]
  24. Soboloff J, Wade MG, Wells G, Désilets M, Tsang BK. Influence of the muscarinic agonist carbachol on intracellular Ca2+ in chicken granulosa cells: I. Dependence on follicular maturation. Biol Reprod 1995; 52:721–728.[Abstract]
  25. Soboloff J, Désilets M, Tsang BK. Influence of tumor necrosis factor alpha on intracellular Ca2+ in hen granulosa cells in vitro during follicular development. Biol Reprod 1995; 53:546–552.[Abstract]
  26. Lee KH, White KJ, Robinson JP, Kim KH. Ca2+ redistribution from bound to free form is required for tumor necrosis factor actions in 30A5 preadipocytes. Mol Endocrinol 1990; 90:1671–1678.
  27. Bouchelouche PN, Bendtzen K, Bak S, Nielsen OH. Recombinant human tumor necrosis factor increases cytosolic free calcium in murine fibroblasts and stimulates inositol phosphate formation in L-M and arachidonic acid release in 3T3 cells. Cell Signalling 1990; 2:479–487.[CrossRef][Medline]
  28. Corkey BE, Geschwind J-F, Deeney JT, Hale DE, Douglas SD, Kilpatrick L. Ca2+ responses to interleukin 1 and tumor necrosis factor in cultured human skin fibroblasts. Possible implications for reye syndrome. J Clin Invest 1991; 87:778–786.
  29. Richter J, Ng-Sikorski J, Olsson I, Andersson T. Tumor necrosis factor-induced degranulation in adherent human neutrophils is dependent on CD11b/CD18-integrin-triggered oscillations of free Ca2+. Proc Natl Acad Sci USA 1990; 87:9472–9476.[Abstract/Free Full Text]
  30. Schumann MA, Gardner P, Raffin TA. Recombinant human tumor necrosis factor {alpha} induces calcium oscillation and calcium-activated chloride current in human neutrophils. The role of calcium/calmodulin-dependent protein kinase. J Biol Chem 1993; 268:2134–2140.[Abstract/Free Full Text]
  31. Koike K, Masumoto N, Kasahara K, Yamaguchi M, Tasaka K, Hirota K, Miyake A, Tanizawa O. Tumor necrosis factor-{alpha} stimulates prolactin release from anterior pituitary cells: a possible involvement of intracellular calcium mobilization. Endocrinology 1991; 128:2785–2790.[Abstract]
  32. Soliven B, Albert J. Tumor necrosis factor modulates Ca2+ currents in cultured sympathetic neurons. J Neurosci 1992; 12:2665–2671.[Abstract]
  33. Ghosh TK, Bian J, Gill DL. Intracellular calcium release mediated by sphingosine derivatives generated in cells. Science 1990; 248:1653–1656.[Abstract/Free Full Text]
  34. Zhang H, Desai NN, Olivera A, Sedi T, Brooker G, Spiegel S. Sphingosine-1-phosphate, a novel lipid involved in cellular proliferation. J Cell Biol 1991; 114:155–167.[Abstract/Free Full Text]
  35. Asem EK, Zakar T, Bieller HV, Hertelendy F. Progesterone production in granulosa cells of the domestic fowl: effects of incubation media, pH, cell density and some other factors. Domest Anim Endocrinol 1984; 1:235–249.
  36. Jayadev S, Linardic CM, Hannun YA. Identification of arachidonic acid as a mediator of sphingomyelin hydrolysis in response to tumor necrosis factor. J Biol Chem 1994; 267:5757–5763.
  37. Smith ER, Merrill AH. Differential roles of de novo sphingolipid biosynthesis and turnover in the "burst" of free sphingosine and sphinganine and their 1-phosphates and N-acyl-derivatives, that occurs upon changing the medium of cells in culture. J Biol Chem 1995; 270:18749–18758.[Abstract/Free Full Text]
  38. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37:911.
  39. Grynkiewcz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260:3440–3450.[Abstract/Free Full Text]
  40. Roe MW, Lemasters JJ, Herman B. Assessment of Fura-2 for measurements of cytosolic free calcium. Cell Calcium 1990; 11:63–73.[CrossRef][Medline]
  41. Obeid L, Linardic CM, Karolak LA, Hannun YA. Programmed cell death induced by ceramide. Science 1993; 259:1769–1771.[Abstract/Free Full Text]
  42. Ji L, Zhang G, Uematsu S, Akahori Y, Hirabayashi Y. Induction of apoptotic DNA fragmentation and cell death by natural ceramide. FEBS Lett 1995; 358:211–214.[CrossRef][Medline]
  43. Kaipia A, Chun SY, Eisenhauer K, Hsueh AJ. Tumor necrosis factor-{alpha} and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles. Endocrinology 1996; 137:4864–4670.[Abstract]
  44. Okazaki T, Bielawska A, Bell RM, Hannun YA. Role of ceramide as a lipid mediator of 1 alpha,25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J Biol Chem 1990; 265:15823–15831.[Abstract/Free Full Text]
  45. Jayadev S, Liu B, Bielawska AE, Lee JY, Naizaire F, Pushkareva M, Obeid LM, Hannun YA. Role for ceramide in cell cycle arrest. J Biol Chem 1995; 270:2047–2052.[Abstract/Free Full Text]
  46. Gomez-Munoz, Martin A, O'Brien L, Brindley DN. Cell-permeable ceramides inhibit the stimulation of DNA synthesis and phospholipase D activity by phosphatidate and lysophosphatidate in rat fibroblasts. J Biol Chem 1994; 269:8937–8943.[Abstract/Free Full Text]
  47. Venable ME, Lee JY, Smyth MJ, Bielawska AE, Obeid LM. Role of ceramide in cellular senescence. J Biol Chem 1995; 270:30701–30708.[Abstract/Free Full Text]
  48. Yao B, Zhang Y, Delikat S, Mathias S, Basu S, Kolesnick RN. Phosphorylation of RAF by ceramide-activated protein kinase. Nature 1995; 378:307–310.[CrossRef][Medline]
  49. Andrieu N, Salvayre R, Levade T. Evidence against involvement of the acid lysosomal sphingomyelinase in the tumor-necrosis-factor and interleukin-1-induced sphingomyelin cycle and cell proliferation in human fibroblasts. Biochem J 1994; 303:341–345.
  50. Lozano J, Berra E, Municio MM, Diaz-Meco MT, Dominguez I, Sanz L, Moscat J. Protein kinase C{varsigma} isoform is critical for {kappa}B-dependent promoter activation by sphingomyelinase. J Biol Chem 1994; 269:19200–19202.[Abstract/Free Full Text]
  51. Foghi A, Ravandi A, Teerds KJ, Van DD, Kuksis A, Dorrington J. Fas-induced apoptosis in rat thecal/interstitial cells signals through sphingomyelin-ceramide pathway. Endocrinology 1998; 139:2041–2047.[Abstract/Free Full Text]
  52. Zachow RJ, Tash JS, Terranova PF. Tumor necrosis factor-{alpha} attenuation of luteinizing hormone-stimulated androstenedione production by ovarian theca-interstitial cells: inhibition at loci within the adenosine 3',5'-monophosphate-dependent signaling pathway. Endocrinology 1993; 133:2269–2276.[Abstract]
  53. Nitta H, Mason IJ, Bahr JA. Localization of 3ß-hydroxysteroid dehydrogenase in the chicken ovarian follicle shifts from the theca layer to granulosa layer with follicular maturation. Biol Reprod 1993; 48:110–116.[Abstract]
  54. Asem EK, Hertelendy F. Influence of follicular maturation on luteinizing hormone-, cyclic 3',5'-adenosine monophosphate-, forskolin- and cholesterol-stimulated progesterone production in hen granulosa cells. Biol Reprod 1985; 32:257–268.[Abstract]
  55. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature 1993; 361:315–325.[CrossRef][Medline]
  56. Oral H, Dorn GW 2nd, Mann DL. Sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian cardiac myocyte. J Biol Chem 1997; 272:4836–4842.[Abstract/Free Full Text]
  57. Soboloff J, Désilets M, Tsang BK. Ca2+ signalling in avian granulosa cells during ovarian follicular development. In: Etches R, Harvey S (eds.), Perspectives in Avian Endocrinology. London, UK: Journal of Endocrinology Ltd.; 1994: 225–240.
  58. Morley P, Vanderhyden BC, Tremblay R, Mealing GAR, Durkin JP, Whitfield JF. Purinergic receptor-mediated intracellular Ca2+ oscillations in chicken granulosa cells. Endocrinology 1994; 134:1269–1276.[Abstract]
  59. Morley P, Wang J, Vanderhyden BC, Chakravarthy B, Durkin J, Whitfield JH. The effect of cholecystokinin on [Ca2+]i membrane-associated protein kinase C activity and progesterone production by chicken granulosa cells. Endocrinology 1993; 133:1956–1962.[Abstract]
  60. Dobrowsky RT, Hannun YA. Ceramide stimulates a cytosolic protein phosphatase. J Biol Chem 1992; 267:5048–5051.[Abstract/Free Full Text]
  61. Lee T-C, Ou M-C, Shinozaki K, Malone B, Snyder F. Biosynthesis of n-acetylsphingosine by platelet-activating factor: sphingosine CoA-independent transacetylase in HL-60 cells. J Biol Chem 1996; 271:209–217.[Abstract/Free Full Text]
  62. Linardic CM, Hannun YA. Identification of a distinct pool of sphingomyelin involved in the sphingomyelin cycle. J Biol Chem 1994; 269:23530–23537.[Abstract/Free Full Text]
  63. Zhang P, Liu B, Jenkins GM, Hannun YA, Obeid LM. Expression of neutral sphingomyelinase identifies a distinct pool of sphingomyelin involved in apoptosis. J Biol Chem 1997; 272:9609–9612.[Abstract/Free Full Text]
  64. Wiegmann K, Schütze S, Machleidt T, Witte D, Krönke M. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 1994; 78:1005–1015.[CrossRef][Medline]
  65. Reddy SAG, Chaturvedi MM, Darnay BG, Chan H, Higuchi M, Aggarwal BB. Reconstitution of nuclear factor {kappa}B activation induced by tumor necrosis factor requires membrane-associated components. Comparison with pathway activated by ceramide. J Biol Chem 1994; 269:25369–25372.[Abstract/Free Full Text]
  66. Schütze S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K, Krönke M. TNF activates NF-{kappa}B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 1992; 71:765–776.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
A Hourvitz, E Gershon, J D Hennebold, S Elizur, E Maman, C Brendle, E Y Adashi, and N Dekel
Ovulation-selective genes: the generation and characterization of an ovulatory-selective cDNA library.
J. Endocrinol., March 1, 2006; 188(3): 531 - 548.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. J. Fields and M. Shemesh
Extragonadal Luteinizing Hormone Receptors in the Reproductive Tract of Domestic Animals
Biol Reprod, November 1, 2004; 71(5): 1412 - 1418.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited