help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pru, J. K.
Right arrow Articles by Rueda, B. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pru, J. K.
Right arrow Articles by Rueda, B. R.
Endocrinology Vol. 143, No. 11 4350-4357
Copyright © 2002 by The Endocrine Society

ARTICLE

Soluble Fas Ligand Activates the Sphingomyelin Pathway and Induces Apoptosis in Luteal Steroidogenic Cells Independently of Stress-Activated p38MAPK

James K. Pru, Isabel R. Hendry, John S. Davis and Bo R. Rueda

Vincent Center for Reproductive Biology, Massachusetts General Hospital (J.K.P., B.R.R.), Boston, Massachusetts 02114; The Women’s Research Institute (I.R.H., J.S.D.), Wichita, Kansas 67214-3199; Department of Obstetrics and Gynecology, University of Kansas School of Medicine (J.S.D.), Wichita, Kansas 67214-3199; and Veterans Affairs Medical Center (J.S.D.), and Olson Center for Women’s Health, Department of Obstetrics and Gynecology, University of Nebraska Medical Center (J.S.D.), Omaha, Nebraska 68105

Address all correspondence and requests for reprints to: Bo R. Rueda, Ph.D., Vincent Center for Reproductive Biology, Massachusetts General Hospital, VBK137E-GYN, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: brueda{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fas ligand (FasL) is implicated as a mediator of luteolysis. However, a gap exists in our understanding of the Fas-mediated signaling mechanisms that are involved in either the loss of progesterone production or the structural regression of the corpus luteum. In the present study we investigated the acute and chronic effects of FasL with respect to activation of cytokine/stress-induced signaling pathways and apoptosis in bovine steroidogenic cells. More specifically, we investigated soluble FasL (sFasL)-activated production of ceramide, a second messenger of the sphingomyelin pathway, and activation of p38MAPK, a member of the MAPK family. sFasL activated the sphingomyelin pathway, as evidenced by a 2-fold increase (P < 0.05) in the production of ceramide. Pretreatment with imipramine (50 µM), an inhibitor of acid sphingomyelinase activity, attenuated (75%; P < 0.05) sFasL-induced ceramide production, suggesting that the increase in ceramide was partially the result of acid sphingomyelinase-mediated hydrolysis of sphingomyelin. Treatment of luteal cells with sFasL or a cell-permeable ceramide analog (C8) for 24–48 h resulted in a significant increase (P < 0.05) in apoptosis. Western blot analysis revealed that sFasL had little effect on the activation of p38MAPK in primary bovine luteal steroidogenic cells. Furthermore, pretreatment with the p38MAPK inhibitor SB203580 failed (P > 0.05) to inhibit sFasL- or C8-induced death. Although sFasL did not alter basal progesterone levels detected in the culture medium, C8 caused a significant increase (P < 0.05) in progesterone concentrations within the medium. Collectively, these data suggest that the role of FasL in luteolysis may be to activate the stress-induced sphingomyelin pathway that, in turn, serves as a mediator of apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE ABSENCE of pregnancy, the corpus luteum (CL) undergoes luteolysis, ending the menstrual/estrous cycle. Luteolysis is characterized by a decline in circulating progesterone levels and involution of the CL tissue (1). It is generally accepted that apoptosis mediates the involution of luteal tissue (2, 3, 4), a process commonly referred to as structural regression. In many species, prostaglandin F2{alpha} (PGF2{alpha}) initiates luteolysis by binding its cognate receptor on luteal steroidogenic cells, causing a decline in progesterone production (5, 6). The decline in progesterone production in response to PGF2{alpha} has been demonstrated in vivo and in vitro (5, 6). Interestingly, however, PGF2{alpha} initiates apoptosis only in vivo. PGF2{alpha} does not reduce basal levels of progesterone, nor does it initiate cell death in bovine steroidogenic cells in vitro (7, 8, 9), suggesting that the apoptotic action(s) of PGF2{alpha} can be mediated indirectly by other factors or cells not present in culture conditions (e.g. immune cells). Several studies have demonstrated that cytokines (9, 10, 11) and other stressors can disrupt gonadotropin-stimulated and/or basal progesterone synthesis and/or initiate apoptosis of luteal cells, potentially through conserved signaling pathways (12, 13). It is conceivable that cytokines generated by resident immune cells (14, 15, 16), act as intermediary signals for PGF2{alpha} to disrupt steroidogenesis or initiate luteal cell apoptosis.

Numerous characterization studies provide evidence that Fas (CD95/APO-1) and Fas ligand (FasL) are present in the ovary. Furthermore, binding of FasL to its receptor coordinates various aspects of ovarian function, including atresia (17, 18, 19, 20, 21, 22) and luteolysis (14, 23). In contrast to the studies published on PGF2{alpha}-mediated signaling in luteal cells (24, 25, 26), the downstream signaling pathway(s) mediating activated Fas actions in the ovary has not been delineated. Previous studies performed in nonovarian cells suggest that FasL-activated Fas initiates multiple signal transduction pathways, including the sphingomyelin pathway (27, 28, 29, 30, 31). Within the ovary, Fas activation of the sphingomyelin pathway has been demonstrated in granulosa and thecal cells of the rat ovarian follicle (19).

The sphingomyelin pathway is an evolutionarily conserved stress response pathway (30) activated by diverse signals, including cytokines (27, 28, 29, 30), oxidative stress (32), and environmental (31) or pharmacological (33) stressors. Upon activation of the sphingomyelin pathway, the second messenger ceramide is generated by one of several enzymes. Acute production of ceramide is thought to result from activation of neutral or acid (a) sphingomyelinase (SMase) (27, 28, 30, 31). Alternatively, ceramide may be generated de novo by ceramide synthase several hours or days after stimulation (34). Although production of ceramide results in diverse biological responses (30), it is primarily thought to be a messenger of apoptosis (35, 36).

Ceramide is an endogenous mediator of Fas-induced cytotoxicity (37). Moreover, in vivo Fas-mediated apoptosis involves activation of aSMase (37) in a cell autonomous fashion (38, 39). Production of ceramide via aSMase can cause the formation of ceramide-rich membrane rafts (40) that enables Fas to cap and kill (41). Facilitation of capping by ceramide suggests that ceramide, rather than aSMase, is obligatory for the induction of apoptosis (38, 39). Once generated, ceramide has the potential, depending again on the cell type and form of stimulus, to suppress pro-survival signaling pathways (42) and to activate stress-induced pro-death pathways (31). Within the ovary, ceramide induces the apoptosis of avian granulosa cells (43), rat thecal cells (19), and presumably murine oocytes (44). There is little or no information about the role of ceramide in luteal function or in response to cytokine-induced cell stress in luteal cells.

Others have demonstrated that FasL or a Fas-activating antibody stimulates p38MAPK activity, and subsequently apoptosis, by a ceramide-dependent mechanism (45). p38MAPK, a member of the MAPK superfamily, is implicated in stress-related signaling, inflammation, and apoptosis (46). With respect to stress-related signaling, p38MAPK is activated in a multitude of cell types in response to many insults, including biomechanical stress, nerve growth factor, heavy metals, UV irradiation, and various activated death receptors besides Fas (46, 47). The requirement for p38MAPK activation in most of these paradigms has been confirmed through the use of chemical inhibitors of p38MAPK activity as well as antisense and dominant negative approaches. Intriguingly, p38MAPK also participates as a component of cell survival pathways in many cell types. Hence, an understanding of the involvement of p38MAPK in pro-life or pro-death pathways must be considered in a cell-specific fashion. With respect to luteal cells of the ovary, p38MAPK is rapidly activated in response to TNF{alpha} (another cytokine linked to luteolysis) and UV light (12). However, whether p38MAPK is a critical mediator of FasL-induced cell death in luteal cells is not known. The objectives of the present study were to 1) determine whether soluble FasL (sFasL) activates the sphingomyelin pathway, 2) establish a link between the sphingomyelin and p38MAPK stress-related signaling pathways as has been demonstrated in other cells, 3) determine whether sFasL or a cell-permeable ceramide analog reduces basal progesterone synthesis by luteal steroidogenic cells, and 4) provide evidence that sFasL and a cell-permeable ceramide analog induce apoptosis, as determined by classical morphological and biochemical parameters.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary luteal steroidogenic cell culture conditions
CL were obtained from cows in early pregnancy (fetal crown rump length, >9 and <30 cm) at a local slaughter facility and dispersed with collagenase as previously described (8, 12). The cultures were established to promote enrichment with steroidogenic cells by using medium supplemented with 0.1% BSA and 5 µg/ml insulin-5 µg/ml transferrin-5 ng/ml selenium (BD Biosciences, Mountain View, CA). After culturing steroidogenic cells for 24 h, the medium was replaced with fresh medium 199 supplemented with 0.1% BSA and 5 µg/ml insulin-5 µg/ml transferrin-5 ng/ml selenium. Cells were allowed to equilibrate for 3 h before the addition of prescribed treatments. All experiments were repeated at least four times using steroidogenic cells isolated from individual CL derived from different animals unless otherwise stated.

Evaluation of ceramide production in response to sFasL
Treatments.
To determine the optimal time to measure ceramide production, steroidogenic cells were untreated (time zero control) or were treated for 5, 10, 30, and 60 min with sFasL (50 ng/ml). To determine whether aSMase or ceramide synthase was actively involved in acute sFasL-induced ceramide production, steroidogenic cells were pretreated with the aSMase inhibitor imipramine (48) (50 µM, 15 min; Sigma, St. Louis, MO) or the ceramide synthase inhibitor fumonisin B1 (49) (FB1; 100 µM, 15 min; Sigma, St. Louis, MO), followed by treatment with sFasL (50 ng/ml) for 10 min. To compare the ceramide response to treatment with sFasL to that with PGF2{alpha}, some cells were treated with PGF2{alpha} (1 µM) for up to 60 min. The medium was aspirated and discarded immediately after the treatment period, and lipids were extracted. To determine whether ceramide production required p38MAPK activity, steroidogenic cells were pretreated with the p38MAPK inhibitor SB203580 (10 µM, 30 min; Calbiochem, San Diego, CA), followed by treatment with sFasL (50 ng/ml) for 10 min. At the end of the treatment period the medium was aspirated and discarded, and cells were subjected to lipid extraction.

Ceramide levels in response to sFasL.
The amount of ceramide generated by steroidogenic cells in response to treatment with sFasL or PGF2{alpha} was determined in three sets of experiments via the diacylglycerol kinase assay (50). To accomplish this, the membrane lipids were extracted (1 ml methanol, 10 min, -80 C) from steroidogenic cells immediately after treatment and dried under nitrogen gas. Lipids were subjected to mild alkaline hydrolysis (500 µl 0.1 N KOH for 1 h at 37 C); reextracted with 500 µl chloroform, 270 µl saline, and 30 µl EDTA (1.5 mM); and dried under nitrogen gas. A reaction mixture containing 6 µl cardiolipin [25 mg/ml; Avanti Polar Lipids (Alabaster, AL)] and 20 µl diethylenetriaminepentaacetic acid (1 mM) was vortexed vigorously, followed by the addition of 6.2 µl octyl-ß-D-glucopyranoside, 50 µl 2x reaction buffer (100 mM NaCl, 100 mM imidazole, 2 mM EDTA, and 25 mM MgCl2), 8 µl imidazole/DETAPAC (10 mM/1 mM), 2 µl dithiothreitol (100 mM), 1 µl AMP (100 mM), 8 µl water, and 3.5 µl diacylglycerol kinase (1 mg/ml; Calbiochem). The mixture was vortexed briefly and preincubated for 30 min at 22 C. After the addition of 1 µl [{gamma}-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL), samples were incubated for 30 min at 22 C. The reaction was stopped with chloroform (500 µl) to extract lipids. The samples were vortexed and centrifuged briefly to separate the phases, and the organic phase was dried under nitrogen gas. The final residue of lipids was reconstituted in 50 µl chloroform, and 40 µl of this were loaded onto thin layer chromatography plates (LK6D silica gel, Fisher Scientific, Pittsburgh, PA). The resulting product of the kinase reaction, ceramide-1-phosphate, was resolved in 65% chloroform/15% methanol/5% acetic acid. Plates were air-dried and exposed to film for 2 h. The resolved band in each lane corresponding to ceramide-1-phosphate was cut from the chromatography plate and counted in a scintillation counter. Changes in ceramide production in response to cytokine treatment were presented as a percentage of the untreated control value.

Analysis of sFasL effects on p38MAPK
Treatment.
After a 3-h equilibration period in serum-free medium, steroidogenic cells were untreated (control, 0 and 60 min) or treated with sFasL (50 ng/ml) for 5, 15, 30, or 60 min to determine whether p38MAPK was phosphorylated. Some cells were treated with TNF{alpha} (100 ng/ml) as a positive control.

Western blot analysis.
Protein lysates were collected for Western blot analysis as described previously (8, 12). Briefly, after separation by SDS-PAGE, the proteins (20 µg/lane) were transferred (100 V, 1 h) to polyvinylidene difluoride membranes. Nonspecific binding was blocked with 5% fat-free milk in TBST [50 mM Tris-HCl (pH 7.5), 0.15 NaCl, and 0.05% Tween 20] for 1 h at 4 C. Previous studies completed in our laboratory as well as by others have demonstrated that the phosphorylated (active) form of p38MAPK can be detected by Western blot analysis with commercial polyclonal antibody (p38MAPK, 1:1000; New England Biolabs, Inc., Beverly, MA). Membranes were then washed three times (5 min each time) with TBST buffer and incubated with antirabbit IgG horseradish peroxidase conjugate (1:2000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature. The membranes were washed with TBST as before, and the bound antibody was detected using ECL reagents based on the manufacturer’s recommendations (Amersham Pharmacia Biotech). X-Ray films were scanned, and band intensity was determined using a Kodak-1D software package (Rochester, NY). Membranes were then stripped and reprobed with the Pan-Erk antibody (Transduction Laboratories, Inc., Lexington, KY), which recognizes the constitutively expressed ERKs in bovine luteal cells as previously described (12, 24, 25).

Morphological and biochemical assessment of apoptosis
Luteal steroidogenic cells were exposed to increasing concentrations of C8 (0, 50, or 100 µM) for 24 h. Genomic DNA was prepared as previously described and analyzed for internucleosomal DNA fragmentation (51). The quantity and purity of nucleic acid preparations were estimated by measuring the OD of each sample (A260:A280). An equivalent amount of genomic DNA from each treatment group was radiolabeled via 3'-end labeling with [{alpha}-32P]dideoxy-ATP (3000 Ci/mmol; Amersham Pharmacia Biotech) using terminal transferase (25 IU; Roche Molecular Biochemicals, Indianapolis, IN). DNA samples (500 ng DNA/well) were separated by electrophoresis (3.5 h at 65 V) in 2% agarose gels. The gels were dried without heat in a slab dryer and exposed to film (Kodak X-OMAT films) at -70 C. To provide further biochemical evidence that C8-induced apoptosis, a fluorogenic substrate (10 µM, PhiPhi Lux; OncoImmunin, Inc., College Park, MD) was administered at 12 h post treatment and allowed to incubate for 30 min. After the incubation the cells were rinsed three times with medium and analyzed by fluorescent microscopy as described previously (52).

To compare treatment effects of sFasL (50 ng/ml) and C8 (50 µM), luteal steroidogenic cell cultures were treated with vehicle (control; culture medium for sFasL and dimethylsulfoxide for C8), sFasL, or C8 for 24, 48, or 72 h. Culture medium was aspirated after treatment and frozen at –20 C for progesterone assay. Culture dishes were rinsed with PBS, and the cells were fixed for 10 min in 4% paraformaldehyde buffered with PBS (pH 7.2). The nuclei were stained by placing 200 µl glycerol (80% in 1x PBS) containing 2 µg/ml Hoechst 33258 in each well. Cell death was determined by counting the number of pyknotic nuclei and the total number of cells per field of view as previously described (12). The average number of live cells in 10 fields was determined and presented as a percentage of the control value.

Measurement of secreted and cellular progesterone levels
Aliquots of medium were collected and stored at -20 C for progesterone analysis. For determination of cellular progesterone concentrations, medium was aspirated, and then 1 ml ice-cold methanol was added to extract the cellular progesterone content. After 20 min methanol was collected into glass tubes. The methanol was evaporated under a stream of air, and the dried samples were stored at 4 C. Samples were reconstituted in PBS before analysis. Progesterone concentrations were determined by RIA in accordance with standard procedures used in our laboratory (53).

Data analysis and quantitation
Assignment to treatments was made at random. Raw data were subjected to least squares ANOVA, followed by Duncan’s new multiple range test or t tests for paired comparisons. The results were expressed as the mean ± SEM, and P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
sFasL activates the sphingomyelin pathway in luteal steroidogenic cells
Treatment of luteal steroidogenic cell cultures with sFasL resulted in activation of the sphingomyelin pathway, as evidenced by an increase in ceramide production. sFasL caused a 67 ± 3.79% increase in ceramide by 5 min (Fig. 1AGo), and this was essentially sustained through 60 min. Pretreatment with the aSMase inhibitor imipramine completely blocked sFasL-induced ceramide production at 10 min (Fig. 1BGo) and interestingly, imipramine attenuated basal levels of ceramide. Pretreatment of steroidogenic cells with the ceramide synthase inhibitor FB1 had no acute (10 min) effect on ceramide production (not shown). Treatment with PGF2{alpha} had no significant effect on ceramide levels (data not shown).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. sFasL induces ceramide production in primary luteal steroidogenic cell cultures. A, To determine the appropriate time for analysis of sFasL-induced ceramide production, cultured luteal cells were treated with sFasL (50 ng/ml) for 0, 5, 10, 30, and 60 min. At the completion of each treatment the medium was aspirated, the cells were rinsed with PBS, and the lipids were extracted with methanol as described in Materials and Methods. The levels of ceramide-1-phosphate were determined by the diacylglycerol kinase assay as a determinant of cellular levels of ceramide. The upper panel represents an autoradiograph obtained after exposure of thin layer chromatographic plates to film. The lower panel represents quantitative analysis of ceramide-1-phosphate levels as an indirect measure of ceramide in lipid extracts derived from luteal cells after treatment with sFasL. All experiments were repeated at three separate times with luteal cell preparations derived from different animals (*, P = 0.004). B, Luteal steroidogenic cells were treated with vehicle (control), sFasL (50 ng/ml), imipramine (Imip; 50 µM), or sFasL in the presence of imipramine (50 µM) as described in Materials and Methods. Ceramide levels in lipid extracts derived from luteal cells were determined by the diacylglycerol kinase assay and quantitative analysis of the product ceramide-1-phosphate. Data are the mean and SEM after expression, as a percentage of the levels of ceramide in the controls in each experiment. Different letters represent significant differences (P < 0.05).

 
sFasL and C8 induce apoptosis
Using Hoechst nuclear stain in luteal steroidogenic cell cultures, the onset of apoptosis was determined under a variety of treatment conditions to better understand how these cells respond to sFasL or ceramide. Treatment with sFasL caused a 35% loss of steroidogenic cells within the first 24 h compared with the vehicle-treated control (Fig. 2AGo). In contrast, steroidogenic cells were extremely vulnerable to treatment with C8 in that apoptosis was evident in more than 90% of the cells by 24 h (Fig. 2BGo). Culture conditions were determined to be appropriate for these experiments because there was no significant loss of cells detected over the time course of 48 h in vehicle-treated control cultures. The form of cell death initiated by C8 is presumably apoptosis, because caspase-3 activity (Fig. 2CGo) and internucleosomal DNA fragmentation (Fig. 2DGo) were evident.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. sFasL or C8 ceramide induces cell death in primary luteal steroidogenic cell cultures. A, Quantitation of sFasL-induced apoptosis of cultured steroidogenic luteal cells. Dissociated steroidogenic cells were cultured in the presence or absence of sFasL (50 ng/ml) for 24 or 48 h. After the culture period the medium was removed, and the cells were fixed to assess the number of live cells based on nuclear morphology (pyknotic vs. nonpyknotic). Data are represented as the percentage of live cells compared with the control. *, Difference compared with its control within time. B, Identification of nonapoptotic cells after treatment with or without C8. Dissociated steroidogenic cells were cultured in the presence or absence of C8 (50 µM) for 24 or 48 h. Data (number of viable cells) are represented as the percentage of live cells compared with the control. *, Difference compared with its control within time. C, Detection of active caspase-3-like activity after C8 treatment. Luteal steroidogenic cells were treated in the absence or presence of 50 µM C8 for 12 h. Thirty minutes before termination of the experiment a cell-permeable fluorescent caspase-3 substrate linked to rhodamine (PhiPhiLux) was added to the medium. Qualitative analysis was performed using light microscopy equipped with fluorescent filters. D, Analysis of low molecular weight (<15 kb) DNA labeling after treatment with increasing concentrations of C8 (0, 50, and 100 µM). To provide evidence that C8 induced apoptosis and not necrosis, genomic DNA was isolated and analyzed for oligonucleosomal DNA fragmentation as previously described (51 ). The individual results from two separate experiments and their mean value for each treatment group are shown.

 
sFasL does not activate the p38MAPK pathway
Multiple experiments were performed to consider the involvement of p38MAPK in sFasL-induced signaling. Western blot analysis using an antibody that recognizes the phosphorylated (active) form of p38MAPK confirmed that p38MAPK is not activated in steroidogenic cells in response to a 10-min treatment with sFasL (Fig. 3AGo). In contrast, treatment with TNF{alpha} induced a 4.1-fold increase in phosphorylated p38MAPK. In experiments performed over a 60-min time course with sFasL, we observed no significant changes in p38MAPK activation. Pretreatment of steroidogenic cells with the p38MAPK inhibitor SB203580 did not alter sFasL-induced ceramide production (not shown). Furthermore, pretreatment of steroidogenic cells with the p38MAPK inhibitor SB203580 did not alter sFasL-induced apoptosis at 24, 48, or 72 h (Fig. 3CGo).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. p38MAPK is not involved in the actions of sFasL in primary luteal steroidogenic cell cultures. A, Dissociated steroidogenic luteal cells were cultured in the presence or absence of sFasL (50 ng/ml) or TNF{alpha} (100 ng/ml) for 10 min. Luteal cell lysates (20 µg total protein/lane) were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and probed with an antibody for phospho-p38MAPK. The blots were reprobed with pan-ERK antibodies for comparison of equal loading. The blot depicted is a representative sample of a minimum of three different cell preparations from three different animals. B, Quantitative assessment of phosphorylated p38MAPK in response to treatment of steroidogenic luteal cells with TNF{alpha} or sFasL. *, P = 0.007 compared with untreated control. C, The p38MAPK inhibitor SB203580 does not block sFasL-induced apoptosis. Dissociated steroidogenic luteal cells were cultured in the presence or absence of the p38MAPK inhibitor SB203580 (10 µM) and/or sFasL (50 ng/ml). After the culture period the medium was removed, and the cells were fixed to assess the number of live cells based on nuclear morphology (pyknotic vs. nonpyknotic). Data are represented as the percentage of live cells compared with the control. Soluble FasL initiated a significant level of apoptosis in luteal steroidogenic cells at each time point. SB203580 failed to inhibit the level of apoptosis caused by treatment with sFasL. *, Significant (P < 0.05) decrease in the number of viable cells compared with the control.

 
sFasL and C8 effects on progesterone production
Treatment with sFasL had no effect on progesterone concentrations in the medium, whereas treatment with C8 resulted in a modest increase (~2-fold) in progesterone levels in the medium within the first 24 h (Fig. 4Go). The p38MAPK inhibitor SB203580 did not alter the concentrations of progesterone in response to sFasL or C8 (data not shown). Additional experiments (n = 3) were performed to verify this finding. It was determined that C8 treatment elevated (P < 0.5) progesterone secretion (534.7 ± 51.4 ng/ml) compared with untreated controls (306.3 ± 58.6 ng/ml). Analysis of the cellular progesterone content yielded no significant decline in progesterone in response to C8 (64.3 ± 16.2 ng/well) compared with the control cells (80.6 ± 20.1 ng/well).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Effects of sFasL or C8 on progesterone levels in primary luteal steroidogenic cell cultures. Shown are progesterone levels in medium after treatment of steroidogenic luteal cells with vehicle, sFasL (50 ng/ml), or C8 (50 µM). At the end of each treatment period (24 or 48 h) the medium was removed and assayed for progesterone as described in Materials and Methods. Data are represented as the mean ± SEM of three separate experiments (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous studies have been reported to provide evidence that luteolysis is mediated by a multitude of factors (e.g. PGF2{alpha}, nitric oxide, endothelin-1, cytokines, and monocyte chemoattractant protein-1) (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) rather than by a single luteolysin. The effects of these factors are often overlapping and temporally defined (e.g. decline in steroidogenesis, infiltration of immune cells, increase in reactive oxygen species, and onset of apoptosis). At present, essentially nothing is known about the cytokine-initiated signaling events immediately distal to activated receptors of the TNF receptor superfamily in luteal cells. This is especially true for Fas, despite many gene expression studies demonstrating the presence of the Fas or its ligand in the CL of many species. In the present studies activation of Fas by sFasL caused an increase in the levels of the second messenger, ceramide, in luteal steroidogenic cells, suggesting that the general enzymatic machinery of the sphingomyelin pathway is present in steroidogenic cells of the CL. Our results support the observations of Carlson et al. (54), who more than 20 yr ago used wide angle x-ray diffraction to demonstrate that during regression of the CL, a liquid-crystalline to gel phase transition occurred in the lipid molecules of cellular membranes. It was proposed that a decrease in membrane fluidity during luteolysis stemmed from changes in the abundance of the membrane lipid sphingomyelin (55). The change observed in the luteal sphingomyelin content coincident with luteolysis may be speculatively explained in part by the conversion of sphingomyelin to ceramide in response to cytokines (e.g. sFasL). Indeed, the extreme hydrophobicity of ceramide adds rigidity to cell membranes (40, 41).

The acute increase in ceramide in steroidogenic cells is probably not derived from de novo synthesis from ceramide synthase, but conceivably is the result of aSMase hydrolysis of sphingomyelin. These findings are based on results obtained from experiments using the chemical inhibitors imipramine (which inhibits aSMase activity) and FB1 (which inhibits de novo synthesis of ceramide) under specified conditions.

In the present study primary bovine luteal steroidogenic cells undergo apoptosis in response to treatment with sFasL or the cell-permeable ceramide analog C8. Treatment with sFasL resulted in the death of 35% of the cells within the first 24 h. The limited extent of cell death may be explained if Fas is not expressed on the surface of all cells. This concept is supported by studies by Quirk et al. (18), who, using flow cytometry, demonstrated that only 54% of luteinized human granulosa cells express Fas. It is possible that cytokine support required for maintaining the lethal expression of Fas is deficient or absent in culture. Indeed, several groups have demonstrated in nonovarian cells (56) and steroidogenic cells (21) that the expression of Fas is up-regulated in part by interferon-{gamma}. TNF{alpha} also increases the expression of Fas in cultured murine luteal cells (57). Not surprisingly, we (8) and others (19) have shown that treatment of luteal cells with combinations of cytokines is very effective in disrupting steroidogenesis and inducing apoptosis of steroidogenic cells. Additional studies are required to determine whether TNF{alpha} or interferon-{gamma} alters Fas expression or FasL-induced death of luteal cells.

Treatment with the cell-permeable ceramide C8 induces apoptosis in steroidogenic cells. The degree of and rate at which sFasL and C8 induce cell death were different. A possible explanation is that exogenous ceramide affects all cells, whereas only cells with Fas respond to sFasL. As the intracellular target of C8 activity is not known, any effect observed by the use of C8 must be interpreted with caution. Cytokines such as sFasL have been shown to cause an accumulation of natural ceramide in the outer leaflet of the plasma membrane, which resulted in hypermultimerization of Fas (i.e. receptor capping) (30). This phenomenon is thought to serve as an amplification step for signal transduction. Furthermore, an increase in the level of ceramide within caveolae, which form within the inner leaflet of the plasma membrane, is believed to disrupt pro-survival signaling pathways (30). Ceramide may also disrupt membrane potential in the outer mitochondrial membrane by forming pores (32). The addition of sFasL to bovine steroidogenic cells had no effect on basal levels of progesterone found in the culture medium. In contrast, the addition of C8 resulted in a modest increase in progesterone in the medium. Previous studies performed in steroidogenic cells have demonstrated that cell-permeable ceramide analogs can have disparate effects (58, 59). The differences observed (even within the same cell types) may be attributed in part to culture conditions or the source and/or type of ceramide analog used. However, most intriguing is the fact that ceramide can regulate steroidogenesis in addition to apoptosis.

It is possible that the role of sFasL and endogenously produced ceramide may be to facilitate apoptosis and not to inhibit steroidogenesis. Administration of Fas-activating antibody to pseudopregnant mice caused a decline in progesterone production (60). However, this same study also reported that the CL completely involuted, unlike that observed during a normal luteolytic process, in which it takes several cycles to remove luteal remnants from the previous cycle. Therefore, any decrease in progesterone in treated mice was probably an indirect interruption of steroidogenesis due to the cytolytic nature of the activating antibody. Furthermore, Fas is expressed most robustly after a decline in steroidogenesis, and elevated progesterone suppresses the expression of Fas in the rat CL (61). Many features of apoptosis, including an increase in caspase-3 activity, changes in the gene expression of several key regulators of apoptosis (e.g. Bax, Bcl-2, caspase-3, IL-1ß-converting enzyme, and internucleosomal DNA fragmentation), appear after the decline in progesterone in several species (2, 3, 4). Therefore, it appears that distinct signaling events coordinate the decline in steroidogenesis and the initiation of apoptosis.

Knowing that cytokines generally activate multiple signaling pathways, we were interested in determining whether sFasL activated other stress-related pathways in addition to the sphingomyelin pathway. Clearly, p38MAPK has the potential to regulate cell stress and/or apoptosis (46). This member of the MAPK family has been shown to phosphorylate Bcl-2, thereby inactivating it in cycling cells (46). The p38MAPK-mediated phosphorylation of effector caspases may also serve to amplify an apoptotic stimulus (46). Based on knowledge that the activation of p38MAPK occurs very rapidly in response to cytokines, and that p38MAPK is critical for FasL-induced apoptosis in some cell types (45, 46), we suspected that sFasL would activate p38MAPK, and its inhibition would reduce sFasL-induced luteal cell death. However, sFasL had little effect on the activation of p38MAPK in luteal cells. Moreover, treatment of luteal cells with SB203580, an inhibitor of p38MAPK, failed to inhibit sFasL-induced activation of the sphingomyelin pathway or sFasL-induced apoptosis. Together these data suggest that sFasL-initiated apoptosis does not involve p38MAPK in steroidogenic luteal cells. Based on other studies it is likely the FasL or complimentary cytokines activate death receptors and caspase activation (7, 12).

From these data we conclude that sFasL functions as a signal to induce apoptosis of cultured bovine luteal steroidogenic cells via a p38MAPK-independent mechanism. Likewise, treatment of steroidogenic cells with ceramide, a second messenger that increases in steroidogenic cells in response to treatment with sFasL, also induces apoptosis. Although not directly tested in these studies, we propose that the evolutionarily conserved stress-activated sphingomyelin pathway, via production of ceramide, may be critically involved in the onset of apoptosis in steroidogenic cells. As PGF2{alpha} does not induce ceramide accumulation (present study) or apoptosis of steroidogenic cells in vitro (7, 8, 9), the data presented here support and extend the growing list of reports demonstrating that cytokines coordinate many aspects of CL function and, in particular, structural regression. Our results extend what is known about Fas activation of the sphingomyelin pathway in nonovarian and ovarian cells. Future studies that employ genetic models will allow us to continue to explore the molecular basis of how key paracrine factors and components of intracellular signaling cascades contribute to structural involution of the CL.


   Acknowledgments
 
We are indebted to Dr. Richard N. Kolesnick and Desiree Ehleiter for technical assistance with the establishment and validation of the DG kinase assay in our laboratory and for helpful comments during the preparation of this manuscript. We also would like to acknowledge Dr. Seema Dubey for her assistance with the progesterone assays.


   Footnotes
 
This work was supported in part by NIH Grant R01-HD-35934 (to B.R.R. and J.S.D.) and the Veterans Affairs Medical Center (to J.S.D.).

J.K.P. is a Lalor Foundation fellow.

Abbreviations: aSMase, Acid sphingomyelinase; CL, corpus luteum; FasL, Fas ligand; FB1, fumonisin B1; PGF2{alpha}, prostaglandin F2{alpha}; sFasL, soluble Fas ligand.

Received February 27, 2002.

Accepted for publication July 19, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Malven PV 1969 Hypophysial regulation of luteolysis in the rat. In: McKerns KW, ed. Hyphophysial regulation of luteolysis in the rat. New York: Meredith; 367–382
  2. Rueda BR, Hendry IR, Tilly JL, Hamernik DL 1999 Accumulation of caspase-3 messenger ribonucleic acid and induction of caspase activity in the ovine corpus luteum following prostaglandin F2{alpha} treatment in vivo. Biol Reprod 60:1087–1092[Abstract/Free Full Text]
  3. Rueda BR, Tilly KL, Botros IW, Jolly PD, Hansen TR, Hoyer PB, Tilly JL 1997 Increased bax and interleukin-1ß-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression. Biol Reprod 56:186–193[Abstract]
  4. Juengel JL, Garverick HA, Johnson AL, Youngquist RS, Smith MF 1993 Apoptosis during luteal regression in cattle. Endocrinology 132:249–254[Abstract]
  5. Niswender GD, Juengel JL, Silvia PJ, Rollyson MK, McIntush EW 2000 Mechanisms controlling the function and life span of the corpus luteum. Physiol Rev 80:1–29[Abstract/Free Full Text]
  6. McCracken JA, Custer EE, Lamsa JC 1999 Luteolysis: a neuroendocrine-mediated event. Physiol Rev 79:263–323[Abstract/Free Full Text]
  7. Pate JL 1994 Cellular components involved in luteolysis. J Anim Sci 72:1884–1890[Abstract]
  8. Suter J, Hendry IR, Ndjountche L, Obholz K, Pru JK, Davis JS, Rueda BR 2001 Mediators of interferon-{gamma} signaling in bovine luteal cells. Biol Reprod 64:1481–1486[Abstract/Free Full Text]
  9. Matsubara H, Ikuta K, Ozaki Y, Suzuki Y, Suzuki N, Sato T, Suzumori K 2000 Gonadotropins and cytokines affect luteal function through control of apoptosis in human luteinized granulosa cells. J Clin Endocrinol Metab 85:1620–1626[Abstract/Free Full Text]
  10. Friedman A, Weiss S, Levy N, Meidan R 2000 Role of tumor necrosis factor {alpha} and its type I receptor in luteal regression: induction of programmed cell death in bovine corpus luteum-derived endothelial cells. Biol Reprod 63:1905–1912[Abstract/Free Full Text]
  11. Endo T, Aten RF, Wang F, Behrman H 1993 Coordinate induction and activation of metalloproteinase and ascorbate depletion in structural luteolysis. Endocrinology 133:690–698[Abstract]
  12. Rueda BR, Hendry IR, Ndjountche L, Suter J, Davis JS 2000 Stress-induced mitogen-activated protein kinase signaling in the corpus luteum. Mol Cell Endocrinol 164:59–67[CrossRef][Medline]
  13. Karanaga E, Kanuka H, Furuhata Y, Yonezawa T, Suzuki M, Nishihara M, Yakahashi M 2000 Requirement of the Fas ligand-expressing luteal immune cells for regression of corpus luteum. FEBS Lett 472:137–142[CrossRef][Medline]
  14. Kuranaga E, Kanuka H, Bannai M, Suzuki M, Nishahara M, Takahashi M 1999 Fas/Fas ligand system in prolactin-induced apoptosis in rat corpus luteum: possible role of luteal immune cells. Biochem Biophys Res Commun 260:167–173[CrossRef][Medline]
  15. Bowen JM, Towns R, Warren JS, Keyes PL 1999 Luteal regression in the normally cycling rat: apoptosis, monocyte chemoattractant protein-1, and inflammatory cell involvement. Biol Reprod 60:740–746[Abstract/Free Full Text]
  16. Mori T, Xu JP, Mori E, Sato E, Saito S, Guo MW 1997 Expression of Fas-Fas ligand system associated with atresia through apoptosis in murine ovary. Horm Res 48:11–19
  17. Porter DA, Vickers SL, Cowan RG, Huber SC, Quirk SM 2000 Expression and function of fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia. Biol Reprod 62:62–66[Abstract/Free Full Text]
  18. Quirk SM, Cowan RG, Joshi SG, Henrikson KP 1995 Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biol Reprod 52:279–287[Abstract]
  19. Foghi A, Ravandi A, Teerds KJ, van der Donk H, Kuksis A, Dorrington J 1998 Fas-induced apoptosis in rat thecal/interstitial cells signals through sphingomyelin-ceramide pathway. Endocrinology 139:2041–2047[Abstract/Free Full Text]
  20. Vickers SL, Cowan RG, Harman RM, Porter DA, Quirk SM 2000 Expression and activity of the Fas antigen in bovine ovarian follicle cells. Biol Reprod 62:54–61[Abstract/Free Full Text]
  21. Quirk SM, Harman RM, Cowan RG 2000 Regulation of Fas antigen (Fas, CD95)-mediated apoptosis in bovine granulosa cells by serum and growth factors. Biol Reprod 63:1278–1284[Abstract/Free Full Text]
  22. Kuranaga E, Kanuka H, Hirabayashi K, Suzuki M, Nishihara M, Takahashi M 2000 Progesterone is a cell death suppressor that down regulates Fas expression in rat corpus luteum. FEBS Lett 466:279–282[CrossRef][Medline]
  23. Roughton SA, Lareu RR, Bittles AH, Dharmarajan AM 1999 Fas and Fas ligand messenger ribonucleic acid and protein expression in the rat corpus luteum during apoptosis-mediated luteolysis. Biol Reprod 60:797–804[Abstract/Free Full Text]
  24. Chen D, Fong HW, Davis JS 2001 Induction of c-fos and c-jun messenger ribonucleic acid expression by prostaglandin F2{alpha} is mediated by a protein kinase C-dependent extracellular signal-regulated kinase mitogen-activated protein kinase pathway in bovine luteal cells. Endocrinology 142:887–895[Abstract/Free Full Text]
  25. Chen DB, Westfall SD, Fong HW, Roberson MS, Davis JS 1998 Prostaglandin F2{alpha} stimulates the Raf/MEK1/mitogen-activated protein kinase signaling cascade in bovine luteal cells. Endocrinology 139:3876–3885[Abstract/Free Full Text]
  26. Davis JS May JV, Keel BA 1996 Mechanisms of hormone and growth factor action in the bovine corpus luteum. Theriogenology 45:1351–1380
  27. Kirschnek S, Paris F, Weller M, Grassme H, Ferlinz K, Riehle A, Fuks Z, Kolesnick R, Gulbins E 2000 CD95-mediated apoptosis in vivo involves acid sphingomyelinase. J Biol Chem 275:27316–27323[Abstract/Free Full Text]
  28. Grullich C, Sullards MC, Fuks Z, Merrill AH, Kolesnick R 2000 CD95 (Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis. J Biol Chem 275:8650–8656[Abstract/Free Full Text]
  29. Lin T, Genestier L, Pinkoski MJ, Castro A, Nicholas S, Mogil R, Paris F, Fuks Z, Schuchman EH, Kolesnick RN, Green DR 2000 Role of acidic sphingomyelinase in Fas/CD95-mediated cell death. J Biol Chem 275:8657–8663[Abstract/Free Full Text]
  30. Riboni L, Viani P, Bassi R, Prinetti A, Tettamanti G 1997 The role of sphingolipids in the process of signal transduction. Prog Lipid Res 36:153–195[CrossRef][Medline]
  31. Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz-Friedman A, Fuks Z, Kolesnick RN 1996 Requirement of ceramide-initiated SAPK/JNK signaling in stress-induced apoptosis. Nature 380:77–79
  32. Andrieu-Abadie N, Gouaze V, Salvayre R, Levade T 2001 Ceramide in apoptosis signaling: relationship with oxidative stress. Free Rad Biol Med 31:717–728[CrossRef][Medline]
  33. Claus R, Russwurm S, Meisner M, Kinscherf R, Deigner HP 2000 Modulation of the ceramide level, a novel therapeutic concept? Curr Drug Targets 1:185–205[CrossRef][Medline]
  34. Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, Kolesnick R 1995 Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 82:405–414[CrossRef][Medline]
  35. Obeid LM, Linardic CM, Karolak LA, Hannun YA 1993 Programmed cell death induced by ceramide. Science 259:1769–1771[Abstract/Free Full Text]
  36. Tepper CG, Jayadev S, Liu B, Bielawska A, Wolff R, Hannan YA, Seldin MF 1995 Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity. Proc Natl Acad Sci USA 92:8443–8447[Abstract/Free Full Text]
  37. Cifone MG, De Maria R, Roncaioli P, Rippo MR, Azuma M, Lanier LL, Santoni A, Testi R 1993 Apoptotic signaling through CD95 (Fas/Apo-1) acitivates an acid sphingomyelinase. J Exp Med 177:1547–1552
  38. Lozano J, Menendez S, Morales A, Ehleiter D, Liao WC, Wagman R, Haimovitz-Friedman A, Fuks Z, Kolesnick R 2001 Cell autonomous apoptosis defects in acid sphingomyelinase knockout fibroblasts. J Biol Chem 276:442–448[Abstract/Free Full Text]
  39. Paris F, Fuks Z, Kang A, Capodieci P, Juan G, Ehleiter D, Haimovitz-Friedman A, Cordon-Cardo C, Kolesnick R 2001 Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science 293:293–297[Abstract/Free Full Text]
  40. Grassme H, Jekle A, Riehle A, Schwarz H, Berger J, Sandhoff K, Kolesnick R, Gulbins E 2001 CD95 signaling via ceramide-rich membrane rafts. J Biol Chem 276:20589–20596[Abstract/Free Full Text]
  41. Cremesti A, Paris F, Grassme H, Holler N, Tschopp J, Fuks Z, Gulbins E, Kolesnick R 2001 Ceramide enables FAS to cap and kill. J Biol Chem 276:23954–23961[Abstract/Free Full Text]
  42. Zhou H, Summers SA, Birnbaum MJ, Pittman RN 1998 Inhibition of Akt kinase by cell-permeable ceramide and its implications for ceramide-induced apoptosis. J Biol Chem 273:16568–16575[Abstract/Free Full Text]
  43. Witty JP, Bridgham JT, Johnson AL 1996 Induction of apoptotic cell death in hen granulosa cells by ceramide. Endocrinology 137:5269–5277[Abstract]
  44. Morita Y, Perez GI, Paris F, Miranda SR, Ehleiter D, Haimovitz-Friedman A, Fuks Z, Xie Z, Reed JC, Schuchman EH, Kolesnick RN, Tilly JL 2000 Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphase therapy. Nat Med 6:1109–1104[CrossRef][Medline]
  45. Brenner B, Ferlinz K, Grassme H, Weller M, Koppenhoefer U, Dichga J, Sandhoff K, Lang F, Gulbins E 1998 Fas/CD95/Apo-I activates the acidic sphingomyelinase via caspases. Cell Death Differ 5:29–37[CrossRef][Medline]
  46. Martin-Blanco E 2000 p38 MAPK signaling cascades: ancient roles and new functions. Bioessays 22:637–645[CrossRef][Medline]
  47. Kummer JL, Rao PK, Heidenreich KA 1997 Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase. J Biol Chem 272:20490–20494[Abstract/Free Full Text]
  48. Hauck CR, Grassme H, Bock J, Jendrossek V, Ferlinz K, Meyer TF, Gulbins E 2000 Acid sphingomyelinase is involved in CEACAM receptor-mediated phagocytosis of Neisseria gonorrhoeae. FEBS Lett 478:260–266[CrossRef][Medline]
  49. Garzotto M, Haimovitz-Friedman A, Liao WC, White-Jones M, Huryk R, Heston WD, Cardon-Cardo C, Kolesnick R, Fuks Z 1999 Reversal of radiation resistance in LNCaP cells by targeting apoptosis through ceramide synthase. Cancer Res 59:5194–5201[Abstract/Free Full Text]
  50. Bose R, Kolesnick R 2000 Measurement of ceramide levels by the diacylglycerol kinase reaction and by high-performance liquid chromatography-fluorescent spectrometry. Methods Enzymol 322:373–378[Medline]
  51. Tilly JL, Hsueh AJ 1993 Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J Cell Physiol 154:519–526[CrossRef][Medline]
  52. Peluso JJ, Pappalardo A, Fernandez G 2001 E-Cadherin-mediated cell contact prevents apoptosis of spontaneously immortalized granulosa cells by regulating Akt kinase activity. Biol Reprod 64:1183–1190[Abstract/Free Full Text]
  53. Davis JS 1992 Modulation of luteinizing hormone-stimulated inositol phosphate accumulation by phorbol esters in bovine luteal cells. Endocrinology 131:749–757[Abstract]
  54. Carlson JC, Buhr MM, Gruber MY, Thompson JE 1981 Composition and physical properties of microsomal membrane lipids from regressing rat corpora lutea. Endocrinology 108:2124–2128[Abstract]
  55. Goodsaid-Zaldouondo F, Rintoul DA, Carlson JC, Hansel W 1982 Luteolysis-induced changes in phase composition and fluidity of bovine luteal cell membranes. Proc Natl Acad Sci USA 79:4332–4336[Abstract/Free Full Text]
  56. Yonehara S, Ishii A, Yonehara M 1989 A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-down regulated with the receptor of tumor necrosis factor. J Exp Med 169:1747–1756[Abstract/Free Full Text]
  57. Quirk SM, Harman RM, Huber SC, Cowan RG 2000 Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis. Biol Reprod 63:49–56[Abstract/Free Full Text]
  58. Meroni SB, Pellizzari EH, Canepa DF, Cigorraga SB 2000 Possible involvement of ceramide in the regulation of rat Leydig cell function. J Steroid Biochem Mol Biol 75:307–313[CrossRef][Medline]
  59. Kwuan C, Patel A, Pletcher S, Lyons B, Abdelrahim M, Nicholson D, Morris E, Salata K, Francis GL 1999 Ceramide increases steroid hormone production in MA-10 Leydig cells. Steroids 64:499–509
  60. Sakamaki K, Yoshida H, Nishimura Y, Nishikawa S-I, Manabe N, Yonehara S 1997 Involvement of Fas antigen in ovarian follicular atresia and luteolysis. Mol Reprod Dev 47:11–18[CrossRef][Medline]
  61. Kuranaga E, Kanuka H, Hirabayashi K, Suzuki M, Nishihara M, Takahashi M 2000 Progesterone is a cell death suppressor that downregulates Fas expression in rat corpus luteum. FEBS Lett 466:279–282



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
L. E. Henkes, B. T. Sullivan, M. P. Lynch, R. Kolesnick, D. Arsenault, M. Puder, J. S. Davis, and B. R. Rueda
Acid sphingomyelinase involvement in tumor necrosis factor {alpha}-regulated vascular and steroid disruption during luteolysis in vivo
PNAS, June 3, 2008; 105(22): 7670 - 7675.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
C. Stocco, C. Telleria, and G. Gibori
The Molecular Control of Corpus Luteum Formation, Function, and Regression
Endocr. Rev., February 1, 2007; 28(1): 117 - 149.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. E. Skaznik-Wikiel, T. Kaneko-Tarui, A. Kashiwagi, and J. K. Pru
Sphingosine-1-Phosphate Receptor Expression and Signaling Correlate with Uterine Prostaglandin-Endoperoxide Synthase 2 Expression and Angiogenesis During Early Pregnancy
Biol Reprod, March 1, 2006; 74(3): 569 - 576.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pru, J. K.
Right arrow Articles by Rueda, B. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pru, J. K.
Right arrow Articles by Rueda, B. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals