Fas-Induced Apoptosis in Rat Thecal/Interstitial Cells Signals Through ...

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 4 Printed in U.S.A.

Fas-Induced Apoptosis in Rat Thecal/Interstitial Cells Signals Through Sphingomyelin-Ceramide Pathway* ARMIN FOGHI, AMIR RAVANDI, KATJA J. TEERDS, HANS VAN DER DONK, ARNIS KUKSIS AND JENNIFER DORRINGTON Banting & Best Department of Medical Research (A.F., A.R., A.K., J.D.), University of Toronto, Toronto, Ontario, Canada; Department of Cell Biology and Histology (K.J.T., H.V.D.D.), School of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; Department of Cell Biology, Medical School, Utrecht University, Utrecht, The Netherlands ABSTRACT Of the ovarian follicles that develop during reproductive life, more than 99% do not ovulate and are eliminated from the ovary by follicular atresia. Atresia is achieved by the self destruction of thecal and granulosa cells that comprise the follicle, by the process of apoptosis. The objective of this study was to determine if activation of the Fas receptor could enact apoptosis of thecal cells, and to explore the signal transduction pathway involved. Primary cultures of thecal/interstitial cells isolated from immature rat ovaries were treated with antiFas monoclonal antibody (anti-Fas mAb) (2.5 mg/ml). Morphological changes indicative of apoptosis, such as, condensation of chromatin, nucleoplasmic segmentation and formation of apoptotic bodies, were observed by fluorescence microscopy following nucleic acid staining with Hoechst 33342 dye and propidium iodide. DNA analysis of cells after 10 h of treatment with anti-Fas mAb showed that DNA had been cleaved into fragments that were multiples of 180 –300 bp in length; biochemical evidence of apoptosis. The sphingomyelin (N-acylsphingosine-1-phosphocholine, SM) pathway that is initiated by the hydrolysis of SM to ceramide (Cer) has been shown previously to be activated by the Fas ligand/receptor system in a number of different cell types. It was therefore possible that the intracellular transduction

F

OLLICULAR growth in the mammalian ovary is initiated by proliferation of the granulosa cells around the oocyte (1). This is followed by differentiation of the stromal/ interstitial cells in close proximity to developing follicles to form the initial theca layer (2). Both granulosa and thecal cell populations proliferate during the subsequent growth of follicules to generate a pool of antral follicles from which dominant follicles are selected (3). Those follicles that are not selected are eliminated by a process known as atresia (4). The elimination of these follicles is essential for the maintenance of the cell mass and homeostasis of the adult ovary. The process is strictly regulated and results in the initial rapid loss of granulosa cells by apoptosis (5, 6), followed by the loss of thecal cells at a slower rate (7, 8). Studies in rat, porcine, and avian ovaries have shown that apoptosis is the underlying mechanism by which, not only granulosa cells, but also thecal cells are eliminated from atretic follicles (8 –10). Received July 11, 1997. Address all correspondence and requests for reprints to: Amir Ravandi, Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, 112 College Street, Toronto, Ontario, M5G 1L6, Canada. E-mail: [email protected]. * This study was supported by the Medical Research Council of Canada and a NATO Colleborative Research Grant.

of Fas receptor activation of thecal/interstitial cells could also involve the SM-Cer pathway. Hence, we have measured the SM levels in control and treated thecal/interstitial cells. Extracts of untreated thecal/interstitial cells contained six major species of SM identified as d18:1/16:0 (sphingosine base/fatty acid), d18:1/18:0, d18:1/20:0, d18: 1/22:0, d18:1/24:1, d18:1/24:0 by normal phase high performance liquid chromatography interfaced with electrospray mass spectrometry. Treatment with anti-Fas mAb (2.5 mg/ml) for 30 min caused significant hydrolysis of only two of the SM species, d18:1/16:0 and d18: 1/24:1. The involvement of ceramide, the central lipid in this phospholipid second messenger system, was tested using the synthetic cell permeable Cer analog (N-acetyl-N-sphingosine, C2-Cer). C2-Cer (10 mM). This analog induced both morphological and biochemical changes in thecal/interstitial cells, that were characteristic of apoptosis, and the same as those induced by anti-Fas mAb. C2-dihydroceramide (10 mM), an inactive analog of C2-Cer, failed to induce apoptosis of thecal/interstitial cells. In conclusion, the sphingomyelinceramide cycle that can lead to cell suicide by apoptosis is functional and activated through the Fas ligand/receptor signal transduction pathway, not only in the immune system, but also in thecal/interstitial cells of the ovarian follicle. (Endocrinology 139: 2041–2047, 1998)

Recently, we have shown that simultaneous treatment with transforming growth factor-a (TGFa) and transforming growth factor-b (TGFb) induced apoptosis in thecal/interstitial cells, whereas in the presence of either growth factor alone cells maintained their viability (11). Exploring the possible signal transduction pathways by which TGFa plus TGFb could trigger apoptosis, we have found that this treatment caused a rapid hydrolysis of sphingomyelin (12). Historically, sphingomyelin was recognized as a structural component of cellular membranes; however, recently it has been shown to be involved in a signal transduction pathway that can lead to apoptosis (13). This pathway involves the activation of the enzyme sphingomyelinase that hydrolyzes sphingomyelin to phosphocholine and ceramide, the latter being a proposed intracellular mediator of apoptosis (14, 15). Most of the information on the Fas system has been obtained from lymphocytes where the SM-Cer pathway is triggered by activation of Fas/APO-1/CD-95 to induce apoptosis. The Fas receptor belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family and binds Fas ligand, a 31-KDa transmembrane protein (16 –19). Fas and APO-1 cell surface antigens were identified independently based on the ability of specific monoclonal antibodies to induce apoptosis. Subsequently, Fas and APO-1 were shown

2041

2042

Fas-INDUCED APOPTOSIS THROUGH SPHINGOMYELIN-CERAMIDE PATHWAY

to be identical (20, 21). Human Fas/APO-1 cell surface receptor is a 48-kDa transmembrane glycoprotein with a cytoplasmic domain that has sequence homology with the TNF receptor p55 (22). In addition to thymus, liver, and heart, the ovary also expresses abundant levels of Fas messenger RNA (23). The presence of high levels of Fas in the ovary in vivo prompted the present study to determine if an agonistic Fas antibody (anti-Fas mAb), known to activate the Fas signal transduction pathway, would induce apoptosis in thecal/ interstitial cells in vitro. We explored the possibility that activation of Fas could trigger the sphingomyelin-ceramide pathway previously identified in rat thecal/interstitial cells (12).

Endo • 1998 Vol 139 • No 4

solution of 50 mg/ml in PBS. Thecal/interstitial cells were plated on glass cover slips placed at the bottom of 24-multiwell culture dishes. Cells were treated with anti-Fas mAb (2.5 mg/ml), C2-ceramide (10 mm), or C2-dihydroceramide (10 mm) for 10 h. The cultures then, were incubated simultaneously with 10 mm Hoechst 33342, staining healthy and early apoptotic cells, and 4.5 mg/ml propidium iodide, staining late apoptotic and necrotic cells, for 10 min in a waterbath at 37 C and then placed on ice (27). The cells were examined under a fluorescent microscope.

DNA purification and separation

Immature Wistar Crl: (W1) BR rats, 22 days of age, were obtained from Charles River Canada (Montreal, Canada) and maintained under conditions of controlled light and temperature.

Thecal/interstitial cells were plated in 75-ml tissue culture flasks, washed after 2 h, and treated with anti-Fas mAb (2.5 mg/ml), C2-ceramide (10 mm) or C2-dihydroceramide (10 mm) for 10 h. DNA from thecal/interstitial cells was prepared as described previously (11). The length of time of treatment with anti-Fas mAb was selected based on the time at which pronounced morphological changes were observed in thecal/interstitial cells. DNA (100 ng) was loaded in each well of the gel. Samples were run on a 1.5% agarose gel containing 15 ml ethidium bromide at 200 V for 1.5 h, and DNA fragments were visualized under UV light. A 1-kb DNA ladder marker (Gibco BRL) was run simultaneously with the samples as an indicator of the band size of the DNA fragments.

Isolation and treatment of primary cultures of thecal/interstitial cells

Isolation of phospholipids from cultured thecal/interstitial cells

Thecal/interstitial cells were isolated from 25-day-old rats by the method of Lobb et al. (24) with modifications (11). Following the release of granulosa cells from the ovaries, the remaining ovarian tissue was washed once in culture medium and chopped into approximately 0.25mm2 pieces with a tissue chopper. Tissue fragments were digested in culture medium containing 0.25% trypsin (Gibco BRL, Grand Island, NY) and 5 mg DNase/ml at 37 C for 30 min (Sigma Chemical Co., St. Louis, MO) to release residual granulosa cells. Tissue fragments were allowed to settle under unit gravity. The supernatant containing contaminating granulosa cells was discarded and the remaining tissue was digested in culture medium containing 1 mg collagenase (Sigma) and 5 mg DNase/ml at 37 C for 30 min. Digested pieces of ovarian tissue were further dispersed by twenty passages through a Pasteur pipette and centrifuged at 90 3 g for 5 min. The pellet of this centrifugation step was resuspended, and large cell aggregates and vascular elements were removed by sedimentation under unit gravity for 5 min. The supernatant containing small cell aggregates and single cells was centrifuged at 90 3 g for 5 min, resuspended, and plated in culture dishes in culture medium containing 0.1% calf serum (Gibco BRL) at a density that generated subconfluent cultures under control conditions. The culture medium consisted of Eagle’s MEM with Earle’s salts and the following amino acid supplements at a 0.1 mm concentration: l-alanine, l-asparagine, l-aspartic acid, l-glutamic acid, l-proline, l-serine and glycine. The medium also contained 4 mm l-glutamine, 2.5 g/liter NaHCO3, 1.5 mm HEPES, and antibiotics (50 U/ml penicillin, 50 mg/ml streptomycin, and 50 mg/ml gentamycin). Thecal/interstitial cells, prepared as described, have been characterized morphologically and biochemically under a variety of culture conditions (24 –26). Analysis of aromatase activity, exclusively localized in granulosa cells, indicated that the degree of contamination of thecal/interstitial cell cultures with granulosa cells was less than 5% (24). Furthermore, thecal/interstitial cells secreted undetectable levels of fibronectin, which is another marker for the presence of granulosa cells (24). Two hours after plating, the medium was removed and the attached cells were gently washed twice in serum-free medium. Serum-free medium was added, and the cultures were treated immediately after washing with anti-Fas monoclonal antibody (mAb) (2.5 mg/ml) (Transduction Laboratories, Lexington, KY). This agonistic anti-Fas mAb acts like the natural Fas ligand and binds to the Fas receptor to activate the intracellular transduction pathway. Cells were also treated with C2-ceramide (N-acetyl-d-sphingosine) (10 mm) (Calbiochem, La Jolla, CA) or C2-dihydroceramide (10 mm) (Calbiochem).

Thecal/interstitial cells were cultured in 75-ml flasks for 2 h, after which they were washed and treated with anti-Fas mAb (2.5 mg/ml) for 30 min. The media from control and treated cultures were collected after 30 min, and attached cells were removed by 3 ml 0.25% trypsin containing 1 mm EDTA. Medium and detached cells were subjected to total lipid extraction with chloroform:methanol 2:1 (28).

Materials and Methods Animals

Apoptosis assay Hoechst 33342 (Sigma) was prepared as a 2 mm stock solution in deionized water. Propidium iodide (Sigma) was prepared as a stock

Mass spectrometry Normal phase high performance liquid chromatography with on-line electrospray mass spectrometry (LC/ES/MS) of phospholipids. Normal phase HPLC separation of phospholipids was performed on Spherisorb 3 micron columns (100 mm 3 4.6 mm ID, Analtech, Deerfield, IL) installed into a Hewlett-Packard (Palo Alto, CA) model 1090 liquid chromatograph connected to a Hewlett-Packard model 5988B quadruple mass spectrometer equipped with a nebulizer assisted electrospray interface, ESI (HP 59987A). N-palmitoyl and N-nervonoyl-d-sphingomyelins (semisynthetic from bovine brain cerebrosides) were used as LC/ES/MS standards. The column was eluted with a linear gradient of 100% solvent A (chloroform/methanol/30% ammonium hydroxide, 80: 19.5: 0.5, by volume) to 100% solvent B (chloroform/methanol/water/30% ammonium hydroxide, 60: 34.5: 5.0: 0.5, by volume) in 14 min, then, continued at 100% B for 10 min (29). Positive ESI spectra were taken in the mass range 400-1100 (30). Full mass spectra were averaged over the entire SM peak (13.807–14.346 min). Single ion spectra were retrieved from the total ion profile by computer. The masses of phospholipids given are the actual masses observed for the [M1H]1 ions. The nominal masses are one unit lower. The molecular species of the SM were quantitated on the basis of the peak areas recorded by single ion chromatograms and were expressed as abundances per 5 3 106 cells. The species were assumed to give identical responses in positive ion LC/ES/MS.

Data analysis Results are plotted as the mean results (6sd) of triplicate determinations in a representative experiment. The significance of the difference between nontreated controls and treated experiments was analyzed using Student’s t test.

Results Induction of apoptosis with anti-Fas mAb

Morphological evidence of apoptosis. Thecal/interstitial cells were stained simultaneously with Hoechst 33342 and propidium iodide to visualize morphological changes in the


2043

nucleus. Untreated thecal/interstitial cells did not show signs of nuclear fragmentation and cytoplasmic segmentation throughout the study (Fig. 1A). After 10 h of treatment with anti-Fas mAb (2.5 mg/ml), hallmarks of apoptosis, including nuclear compaction, cytoplasmic blebbing, and fragmentation into dense particles called apoptotic bodies were observed (Fig. 1B). Following these changes treated thecal/ interstitial cells rounded up and detached from the culture plate. Biochemical evidence of apoptosis

DNA was extracted from untreated thecal/interstitial cells and from cells treated with anti-Fas mAb (2.5 mg/ml) for 10 h and subsequently analyzed for fragmentation using gel electrophoresis. DNA of untreated cells remained intact and showed no sign of DNA fragmentation (Fig. 2, lane A). AntiFas mAb treatment resulted in a DNA ladder characteristic of apoptosis (Fig. 2, lane B). The ladder consisted of inter-

FIG. 2. Induction of apoptotic internucleosomal DNA fragmentation in cultured thecal/interstitial cells by anti-Fas mAb (2.5 mg/ml) or C2-ceramide (10 mM). Thecal/interstitial cells were prepared and treated as described in the legend to Fig. 1. DNA was extracted and analyzed by gel electrophoresis. In untreated cells (A), and C2-dihydroceramide (C) treated cells the DNA remained intact. Treatment of cells with anti-Fas mAb (B) or C2-ceramide (D) caused DNA fragmentation generating a ladder consisting of internucleosomal DNA fragments, multiples of 180 –300 bp in size. The data were reproducible in three separate experiments.

nucleosomal DNA fragments, multiples of 180 –300 bp in size (Fig. 2, lane B). Involvement of the sphingomyelin-ceramide signal transduction pathway in the induction of apoptosis

FIG. 1. Morphological features of apoptosis induced in thecal/interstitial cells by anti-Fas mAb and C2-ceramide showing chromatin condensation, nucleoplasmic segmentation, and formation of apoptotic bodies. Thecal/interstitial cells were prepared from 25-day-old rat ovaries and plated at a density that generated subconfluent cultures. Two hours after plating, cells were washed and treated immediately for 10 h. (A) control, (B) anti-Fas mAb (2.5 mg/ml), (C) C2-ceramide (10 mM) and (D) C2-dihydroceramide (10 mM). Cells were stained with 10 mM Hoechst 33342 and 4.5 mg/ml propidium iodide (3400). The observed morphological changes induced by anti-Fas mAb and C2-ceramide were reproducible in five separate experiments. (Magnification, 4003)

Sphingomyelin hydrolysis. Hydrolysis of SM, as the result of apoptotic stimuli, has been shown to be a rapid process localized within the plasma membrane (31). Hence, thecal/ interstitial cells were exposed to anti-Fas mAb for 30 min and subsequently analyzed for phospholipid contents. The total lipid extracts were resolved by HPLC based on their polarity. The neutral lipids (NL) were eluted first followed by the more polar phospholipids phosphatidyethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and lyso-phosphatidylcholine (LPC) (chromatogram not shown). Figure 3 shows the total positive ion current profile for the cholinecontaining phospholipids (PC and SM) along with a low response for PE. The SM peak was eluted over 13.807–14.346 min after injection and was analyzed further to identify its most abundant molecular species. Structural identification of these molecules was based on the [M11]1, representing the pseudomolecular ion and on chromatographic retention time. The most abundant species of sphingomyelin were identified by number of carbons:number of double bonds as 34:1 (d18:1–16:0, m/z 703); 36:1 (d18:1–18:0, m/z 731); 38:1 (d18:1–20:0, m/z 759); 40:1 (d18:1–22:0, m/z 787); 42:2 (d18: 1–24:1, m/z 813) and 42:1 (d18:1–24:0, m/z 815) (Fig. 4, A and B). Levels of SM species identified in control extracts were compared with extracts from anti-Fas mAb treated thecal/ interstitial cells (Fig. 5). Treatment of cells with anti-Fas mAb for 30 min caused a 32% and 47% decrease (P , 0.05) in the levels of two species of SM, 34:1 (d18:1–16:0, m/z 703) and

2044


Endo • 1998 Vol 139 • No 4

FIG. 3. Total positive ion profile of the LC/ES/MS of phospholipids present in untreated thecal/interstitial cells. Cells were isolated from 25-day-old rat ovaries and cultured for 30 min under control conditions. NL, Neutral lipids; DET, detergent; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SM, sphingomyelin. HPLC conditions: column, Spherisorb 3 micron (100 mm 3 4.6 mm ID, Analtech, Deerfield, IL); solvents, linear gradient of 100% solvent A to 100% solvent B for 14 min, then solvent B for 10 min. Solvent A chloroform/methanol/30% ammonium hydroxide, 80:19.5:0.5, by vol, solvent B chloroform/methanol/water/30% ammonium hydroxide, 60:34.5:5.5:0.5, by vol.

42:2 (d18:1–24:1, m/z 813), respectively (Fig. 5). There was no significant change in the levels of the other 4 species after 30 min of treatment. The total SM content was reduced by 23% in the treated cells compared with the control. Induction of apoptosis with ceramide Morphological evidence of apoptosis

Ceramide is the central lipid in the sphingomyelin metabolic pathway (32). Natural ceramides contain long chain saturated or monounsaturated fatty acids and are poorly soluble in aqueous solutions. Therefore, the cell permeable analog, C2-ceramide, synthesized and tested for its ability to induce apoptosis (33), was used in this study. Thecal/interstitial cells treated with C2-ceramide for 10 h showed chromatin condensation, nucleoplasmic segmentation, and apoptotic body formation, indicative of apoptosis, followed by rounding up and detachment of cells from the culture plate (Fig. 1C). The cytotoxic effects of C2-ceramide were specific because a closely related structural and stereoisomer, C2dihydroceramide (33), failed to induce apoptosis (Fig. 1D). Biochemical evidence of apoptosis

DNA extracted from thecal/interstitial cells treated with C2-ceramide (10 mm) and C2-dihydroceramide (10 mm) was analyzed by gel electrophoresis. Treatment with C2-ceramide resulted in DNA fragmentation that was characteristic of apoptosis (Fig. 2, lane D) whereas the DNA extracted from C2-dihydroceramide treated thecal/interstitial cells remained intact (Fig. 2, lane C). Discussion

The data presented in this paper give new insights into the molecular mechanisms by which thecal cells can be induced to undergo apoptosis in vitro. The novel findings are: 1) anti-Fas mAb, which activates Fas, induces apoptosis in the-

cal/interstitial cells; 2) anti-Fas mAb causes the rapid hydrolysis of sphingomyelin: six abundant species of sphingomyelin were identified in total cell lipid extracts, of which the two major ones could be determined to be hydrolyzed after 30 min of treatment; and 3) C2-ceramide, a synthetic water soluble analog of ceramide, induced apoptosis in thecal/interstitial cells in vitro. Exposure of thecal/interstitial cells to anti-Fas mAb induced changes, characteristic of apoptosis (11). These changes consisted of morphological changes, such as nuclear condensation, fragmentation of the nucleus and the formation of apoptotic bodies, and biochemical changes, such as DNA fragmentation into pieces that were multiples of 180 – 300 bp in length. Fas has been localized by immunohistochemistry in granulosa cells of the rat ovary where it has also been implicated in the apoptotic process (34). The temporal expression of Fas in rat granulosa cells suggested a possible role for the Fas ligand/receptor system in their elimination during follicular atresia. Oocytes stained intensely for Fas ligand, and apoptosis of granulosa cells was enhanced by coculture with zona-free oocytes (34). This study pointed to potential interactions between oocytes and granulosa cells that could lead to the activation of the apoptotic process through a Fas-induced signal transduction pathway. Fas messenger RNA has also been detected in granulosa/luteal cells isolated from patients at the time of ovum retrieval for in vitro fertilization (IVF) (35). Treatment with anti-Fas mAb induced apoptosis in these cells (35). It appears therefore that ovarian cells in the follicle and the developing corpus luteum that express Fas undergo apoptosis when the receptor is activated by exposure to Fas ligand or anti-Fas mAb. Since its discovery, most of the research into the apoptotic inducing activity of Fas has been devoted to the immune system, where its activation in T cells induces suicide. Recently, the physiological role of Fas was extended to the


2045

FIG. 4. LC/ES/MS of thecal/interstitial cell phospholipids. A, Single ion plots of the masses corresponding to the [M11]1 ions of the major molecular species of SM present in thecal interstitial cells. B, Mass spectra averaged over the SM peak (13.807 to 14.346 min). The masses in panel B correspond to the [M11]1 ions of the d18:1-16:0 (m/z 703); d18:1-18:0 (m/z 731); d18:1-20:0 (m/z 759); d18:1-22:0 (m/z 787); d18:1-24:1 (m/z 813); d18:1-240:0 (m/z 815) of sphingomyelins. Conditions are given in legend to Fig. 3.

female reproductive system. Fas was shown to be directly involved in the regression of vaginal epithelium induced by ovariectomy, establishing a role for Fas outside the immune system (36). Our present studies on thecal/interstitial cells, together with those on rat and human granulosa cells (34, 35) identify Fas as a transmitter of an apoptotic signal in ovarian cells, adding further support to actions of Fas in the female reproductive system. With regard to the molecular mechanisms by which the Fas ligand/receptor system induces apoptosis in ovarian cells, the present study points to the involvement of the sphingomyelin signal transduction pathway. This was based initially on the observation that treatment of thecal/interstitial cells with anti-Fas mAb caused a significant increase in the hydrolysis of sphingomyelin within 30 min. Sphingomyelin is concentrated in the outer leaflet of the cellular membrane and consists of a sphingoid base backbone, a fatty acid and a phosphocholine head group. Initially sphingomyelin was considered to act only as a structural component of membranes; however, recently it has been shown to be a critical component in a mechanistic pathway that signals apoptosis. The sphingomyelin pathway was first described

by Okazaki et al. (37) in human leukemia cell line (HL-60) activated by vitamin D3. Since that time, the initiation of the sphingomyelin transduction pathway has been coupled to receptors that are members of the TNF superfamily, including the Mr 55-kDa receptor (38, 39) and Fas. Fas and TNF are external apoptotic stimuli that activate the enzyme sphingomyelinase to hydrolyze sphingomyelin into phosphocholine and ceramide. Generation of the lipid second messenger ceramide represents a step in the sphingomyelin pathway that can provide a signal for apoptotic cell death also in lymphocytes and other cell lines (33). A novel finding in this study was the identification of the two sphingomyelin species that are hydrolyzed in response to anti-Fas mAb. We have recently determined the six most abundant species of sphingomyelin in thecal/interstitial cells (12). This differential response to apoptotic stimuli could represent an as yet unrecognized specificity of each apoptotic factor in triggering the second lipid messenger pathway. Recent studies have shown (40) that different tissues and cell membranes exhibit great variation in sphingomyelin composition, but the specific role of each SM species is yet to be

2046


Endo • 1998 Vol 139 • No 4

FIG. 5. Levels of most abundant molecular species of sphingomyelin in cultured rat thecal/interstitial cells after treatment for 30 min with anti-Fas mAb (2.5 mg/ml) compared with untreated sample. Data are representative of three separate experiments. Abundances were calculated as peak areas under the curves of the single ion plots that have been shown in Fig. 4A.

investigated. The specificity of hydrolysis of SM species may be related to their submembrane distribution, the specificity of sphingomyelinase, and the biosynthetic enzymes involved in recycling the ceramide to the original SM. The anti-Fas mAb has been shown (41) to be a “rapid” activator of SM-Cer cycle and points to the hydrolysis of the SM in the plasma membranes as the main source of ceramide. Treatment with the hydrolytic enzyme, sphingomyelinase, also induced rapid apoptosis in thecal/interstitial cells (data not shown) strengthening the idea of cell membrane sphingomyelin as the hydrolytic substrate pool for initiation of the apoptotic signaling pathway (42– 44). In conclusion, this is the first study in an ovarian cell system (i.e. thecal/interstitial cells) to demonstrate that the sphingomyelin/ceramide cycle of apoptosis is functional and triggered by the Fas ligand/ receptor signal transduction pathway. Hence, this pathway of cell suicide is no longer restricted to the immune system but also functions in nonimmune tissues. We also have identified two major species of sphingomyelin which are involved in mediating the Fas-apoptotic signal in thecal/ interstitial cells. Acknowledgments We are grateful to Rosa Jara for her skillful secretarial aid as well as Elzbieta Matysiak-Zablocki for her technical assistance.

References 1. Zamboni L 1974 Fine morphology of the follicle wall and follicle cell-oocyte association. Biol Reprod 10:125–129 2. Amsterdam A 1987 Structure function relationships during granulosa cell differentiation. Endocr Rev 8:309 –337 3. Peters H 1979 Some aspects of early follicular development. In: Midgley AR, Sadler AW (eds) Ovarian Follicular Development and Function. Raven Press, NY, pp 1–13 4. Byskov AG 1978 Follicular atresia. In: Jones RE (ed) The Vertebrate Ovary. Plenum Press, NY, pp 533–562 5. Hughes FM Jr, Gorospe WC 1991 Biochemical identification of apoptosis (programmed cell death) in granulosa cell: evidence for a potential mechanism underlying follicular atresia. Endocrinology 129:2415–2422 6. Tilly JL, Kowalski KI, Johnson, AL, Hsueh AJW 1991 Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 129:2799 –2801 7. Logothetopoulos J, Dorrington J, Baily D, Stratis M 1995 Dynamics of follicular growth and atresia of large follicles during the ovarian cycle of the guinea pig. Fate of the degenerating follicles. Anat Rec 243:37– 48

8. Palumbo A, Yeh J 1994 In situ localization of apoptosis in the rat ovary during follicular atresia. Biol Reprod 51:888 – 895 9. Tilly JL, Kowalski KI, Schomberg DW, Hsueh AJW 1992 Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase. Endocrinology 131:1670 –1676 10. Tilly JL, Kowalski KI, Johnson AL, Hsueh AJW 1991 Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 129:2799 –2801 11. Foghi A, Teerds KJ, Van der Donk H, Dorrington J 1996 Induction of apoptosis in rat thecal/interstitial cells by transforming growth factor a plus transforming growth factor b. J Endocrin 153:169 –178 12. Foghi A, Teerds KJ, van der Donk H, Dorrington J, Program of the 11th Ovarian Workshop: Ovarian Cell Growth, Apoptosis and Cancer; Serono Symposia USA. London, Ontario, Canada, 1996, p 17 (Abstract) 13. Hannun YA 1994 The sphingomyelin cycle and the second messenger function of ceramide.J Biol Chem 269:3125–3128 14. Hannun YA, Obeid LM 1995 Ceramide: an intracellular signal for apoptosis. Trends Biochem Sci 20:73–77 15. Jarvis DW, Fornari FA, Traylor RS, Martin HA, Kramer LB, Erukulla RV, Bittman R, Grant S 1996 Induction of apoptosis and potentiation of ceramidemediated cytotoxicity by sphingoid bases in human myeloid leukemia cells. J Biol Chem 271:8275– 8284 16. Suda T, Nagata S 1994 Purification and characterization of the Fas-ligand that induces apoptosis. J Exp Med 179:873– 879 17. Yonehara S, Ishii, A, Yonehara M 1989 A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med 169:1747–1756 18. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima SI, Sameshina M, Hase A, Seto Y, Nagata S 1991 The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66:233–243 19. Suda T, Takahashi T, Golstein P, Nagata S 1993 Molecular cloning and expression of the Fas Ligand, a novel member of the tumor necrosis factor family. Cell 75:1169 –1178 20. Trauth BC, Klas C, Peters AMJ, Matzuku S, Moller P, Falk W, Debatin K-M, Krammer PH 1989 Monoclonal antibody-mediated tumor regression by induction of apoptosis Science 245:301–305 21. Yonehara S, Ishii A, Yonehara M 1989 A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med 169:1747–1756 22. Oehm A, Behrmann I, Falk W, Pawlita M, Maier G, Klas C, Li-Weber M, Richards S, Dhein J, Trauth BC, Ponstingl H, Krammer PH 1992 Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen. J Biol Chem 267:10709 –10715 23. Watanabe-Fukunaga R, Brannan CI, Itah N, Yonehara S, Copeland NG, Jenkins NA, Nagata S 1992 The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J Immunol 148:1274 –1279 24. Lobb DK, Skinner MK, Dorrington JH 1988 Rat thecal/interstitial cells produce a mitogenic activity that promotes the growth of granulosa cells. Mol Cell Endocrinol 55:209 –217 25. Skinner MK, Lobb D, Dorrington JH 1987 Ovarian thecal/interstitial cells produce an epidermal growth factor-like substance. Endocrinology 121:1892–1899 26. Bendell JJ, Dorrington J 1988 Rat thecal/interstitial cells secrete a transform-


27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

ing growth factor-b-like factor that promotes growth and differentiation in rat granulosa cells. Endocrinology 123:941–948 Telford WG, King LE, Fraker PJ 1994 Rapid quantitation of apoptosis in pure and heterogeneous cell populations using flow cytometry. J Immunol Meth 172:1–16 Folch J, Lees M, Sloane-Stanley GH 1957 A simple method for isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509 Becart J, Chevalier C, Biesse JP 1990 Quantitative analysis of phospholipids by HPLC with a light scattering evaporating detector-application to raw materials for cosmetic use. J High Res Chromatogr 10:126 –129 Ravandi A, Kuksis A, Marai L, Myher JJ, Steiner G, Lewisa G, Kamido H 1996 Isolation and identification of glycated aminophospholipids from red cells and plasma of diabetic blood. FEBS Lett 381:77– 81 Zhang Y, Kolesnick R 1995 Signaling through the sphingomyelin pathway. Endocrinology 136:4157– 4160 Jayadev S, Liu B, Bielawska AE, Lee JY, Nazaire F, Pushkareva MY, Obied LM, Hannun YA 1995 Role for ceramide in cell cycle arrest. J Biol Chem 270:2047–2052 Spiegel S, Foster D, Kolesnick RN 1996 Signal transduction through lipid second messengers. Curr Opin Cell Biol 8:159 –167 Hakuno N, Koji T, Yano T, Kobayashi N, Tsutsumi O, Takatani Y, Nakane PK 1996 Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia. Endocrinology 137:1938 –1948 Quirk SM, Cowan RG, Joshi SG, Henrikson KP 1995 Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biol Reprod 52:279 –287 Suzuki A, Enari M, Eguchi Y, Matsuzawa A, Nagata S, Tsujimoto Y, Iguchi T 1996 Involvement of Fas in regression of vaginal epithelia after ovariectomy and during an estrous cycle. EMBO J 15:211–215

2047

37. Okazaki T, Bell RM, Hannun YA 1989 Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation. J Biol Chem 264:19076 –19080 38. Dressler KA, Mathias S, Kolesnick RN 1992 Tumor necrosis factor-alpha activates the sphingomyelin signal transduction pathway in a cell-free system. Science 255:1715–1718 39. Kim MY, Linardic C, Obied L, Hannun Y 1991 Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation. J Biol Chem 266:484 40. Valeur A, Olsson NU, Kaufmann P, Wada S, Kroon CG, Westerdahl G, Odham G 1994 Quantification and comparison of some natural sphingomyelins by on line high-performance liquid chromatography/discharge-assisted thermospray mass spectrometry. Biol Mass Spectrom 23:313–319 41. 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) activates an acidic sphingomyelinase. J Exp Med 180:1547–1552 42. Jarvis DW, Fornari FA, Traylor RS, Martin HA, Kramer LB, Erukulla RV, Bittman R, Grant S 1996 Induction of apoptosis and potentiation of ceramidemediated cytotoxicity by sphingoid bases in human myeloid leukemia cells. J Biol Chem 271:8275– 8284 43. Riboni L, Prinetti A, Bassi R, Caminiti A, Tettamanti G 1995 A mediator role of ceramide in the regulation of neuroblastoma Neuro2a cell differentiation. J Biol Chem 270:26868 –28875 44. Bettaieb A, Record M, Come MG, Bras AC, Chap H, Laurent G, Jafferzou JP 1996 Opposite effects of tumor necrosis factor alpha on the sphingomyelinceramide pathway in two myeloid leukemia cell lines: role of 3 transverse sphingomyelin distribution in the plasma membrane. Blood 88:1465–1472